Large Amino Acid Transporter 1 Selective Liposomes of l -DOPA Functionalized Amphiphile for Combating Glioblastoma

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00569

Despite significant progress in neurosurgery and radiation therapy during the past decade, overall survivability (OS) of glioblastoma patients continues to be less than 2 years. The scope of systemic chemotherapy is greatly limited by poor drug transport

Full text of DOI:10.1021/acs.molpharmaceut.7b00569

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Article
Large Aminoacid Transporter 1 Selective Liposomes of L-
DOPA Functionalized Amphiphile for Combating Glioblastoma
Sukanya Bhunia, Venugopal Vangala, Dwaipayan Bhattacharya, HALLEY GORA RAVURI,
Madhusudana Kuncha, Sumana Chakravarty, Ramakrishna Sistla, and Arabinda Chaudhuri
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00569 • Publication Date (Web): 28 Sep 2017
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Large Aminoacid Transporter 1 Selective Liposomes
of LꢀDOPA Functionalized Amphiphile for
Combating Glioblastoma
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Sukanya Bhunia,1,2Venugopal Vangala,1,2Dwaipayan Bhattacharya,1,2 Halley Gora
Ravuri,4Madhusudana Kuncha,3 Sumana Chakravarty,1,2 Ramakrishna Sistla,2,3 Arabinda
Chaudhuri*1,2
1 Department of Chemical Biology, CSIRꢀIndian Institute of Chemical Technology, Uppal Road,
Hyderabadꢀ 500007, India.
2
Academy of Scientific & Innovative Research (AcSIR), 2 Rafi Marg, New Delhi, India.
3 Medicinal Chemistry and Pharmacology Division, CSIRꢀIndian Institute of Chemical
4University of Queensland, School of Veterinary Science, Gatton, Brisbane, QLD, AUS 4343
Technology, Uppal Road, Hyderabadꢀ 500007, India.
*To whom correspondence should be addressed.
Eꢀmail: arabinda@iict.res.in
KEYWORDS: Glioblastoma therapy, STAT3 inhibitor, large amino acid transporter 1,liposomal
drug delivery, DNA vaccination
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ABSTRACT. Despite significant progresses in neurosurgery and radiation therapy during the
past decade, overall survivability (OS)of glioblastoma patients continues to be less than 2 years.
Scope of systemic chemotherapy is greatly limited by poor drug transport across blood brain
barrier (BBB), and thereby, subꢀoptimal drug accumulation in glioma tissue. To this end, use of
large amino acid transporterꢀ1 (LAT1) over expressed both on brain capillary endothelial cells
(BCECs) and glioma cells have begun. Prior reports on the use of LAT1 mediated delivery of
model drugs showed their brain accumulations. However, in depth in vivo glioblastoma
regression studies aimed at examining the therapeutic potential of LAT1 mediated delivery of
potent chemotherapeutics to brain tumor tissues have not yet been undertaken. Herein, we report
on the development of a nanometric (100ꢀ135 nm) promising LAT1 selective liposomal drug
carrier prepared from a novelLꢀ3,4ꢀdihydroxyphenylalanine (LꢀDOPA) functionalized
amphiphile (AmphiꢀDOPA). In vitro studies using RhꢀPE labeled liposomes of AmphiꢀDOPA in
both untreated glioma (GL261) cells and in GL261cells preꢀincubated with LAT1 antibody
revealedLAT1 mediated cellular uptake. Intravenously administered NIRꢀdye labeled liposomes
of AmphiꢀDOPA in glioblastoma bearing mice showed preferential accumulation of the dye in
brain tissue. Notably i.v. administration of WP1066ꢀloaded liposomes of AmphiꢀDOPA
enhanced the overall survivability of C57BL/6J mice bearing orthotopically established mouse
glioblastoma by ~60% compared to that for the untreated mice group. Furthermore, we show that
the OS of established glioblastoma bearing mice can be significantly enhanced (by >300%
compared to that for the untreated mice group) when the presently described LAT1 mediated
targeted chemotherapy with WP1066ꢀloaded liposomes of AmphiꢀDOPA is combined with in
vivo DCꢀtargeted DNA vaccination using a survivin (a glioblastoma antigen) encoded DNA
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vaccine. The present findings open a new door for LAT1 mediated systemic chemotherapy of
glioblastoma.
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1. INTRODUCTION. Glioblastoma (GBM) is one of the most aggressive and deadliest
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malignant brain tumors with a median survival of 14ꢀ17 months post diagnosis and only 3ꢀ5%
survivability beyond 5 years.1 Current standard therapy includes maximal safe surgical removal
of tumor followed by radiotherapy and temozolomide based chemotherapy.2,3 However, highly
infiltrative nature of glioma cells and poor selfꢀrepair capability of healthy brain cells make
selective removal or killing of tumor tissue (without affecting healthy brain tissue) an uphill
challenge.4More often than not rapid recurrence of tumor ensues. Poor drug accumulation in
glioblastoma tissue, unfavorable pharmacokinetic behavior and toxicity to offꢀtarget organs are
retarding the clinical success of systemic chemotherapy of glioblastoma. Blood brain barrier
(BBB), the tight junction of brain capillary endothelial cell (BCEC) covered with pericytes and
astrocytes, greatly prevents the entry of drugs circulating in the blood from reaching
glioblastoma tissue in brain parenchyma.5ꢀ7 Endogenous receptors and transporters over
expressed on BCECs including transferrin receptor (TfR),8,9 nicotinic acetylcholine receptors
(nAChR),10,11 integrin receptor12ꢀ14glucose transporter (GLUT),15ꢀ17 have been successfully
exploited in overcoming the BBB. Nanoꢀcarriers with suitable pharmacokinetic properties are
being fabricated with specific ligands of such receptors or transporters on their exoꢀsurface to
help the nanoparticles associated drugs cross BBB via receptor or transporter mediated
transcytosis. To this end use of large amino acid transporterꢀ1 (LAT1) over expressed both on
BCECs and glioma cells have begun.18,19LAT1, a sodiumꢀindependent transporter, transports a
wide range of substrate including large amino acids (e.g. LꢀTyrosine), neurotherapeutics (e.g. Lꢀ
3,4ꢀdihydroxyphenylalanine (LꢀDOPA) and gabapentin) and thyroid hormones across the
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BBB.20Abundant and selective expression on both luminal and abluminal membrane sides of
BBB21,22combined with markedly enhanced substrate affinity (by ~1000 times compared to that
on peripheral tissues)23 makes LAT1 mediated drug delivery to brain an attractive therapeutic
strategy. Furthermore, LAT1 is over expressed in many types of cancer cells24ꢀ26 including
glioblastoma.18,27Thus, high expression level of LAT1 on BCECs& glioma cells makes it a ‘dual
target’ for antiꢀglioma therapy.
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Prior reports on the use of LAT1 mediated delivery of model drugs showed significant brain
accumulations.18,19 However, in depth in vivo studies aimed at exploring the therapeutic potential
of LAT1 mediated delivery of potent chemotherapeutics in inhibiting glioblastoma growth have
not yet been undertaken. To this end, herein we envisioned exploiting liposomes of Amphiꢀ
DOPA (Scheme 1), a novel cationic amphiphile containing LꢀDOPA (a substrate for LAT1) in
its polar headꢀgroup region, for efficient targeting of potent chemotherapeutics to orthotopically
established mouse glioblastoma. LꢀDOPA, a well known drug to treat Parkinson’s disease, is
previously reported to cross BBB via LAT1.28Based on this prior report, we hypothesized that
i.v. administered liposomes of AmphiꢀDOPA containing potent cytotoxic drugs will be targeted
to brain tumors after crossing BBB presumably via transcytosis. Herein we report on the
therapeutic promise of the liposomal formulation of AmphiꢀDOPA containing encapsulated WPꢀ
1066, a potent STAT3 inhibitor, in inhibiting orthotopically implanted mouse glioblastoma
growth.
Aimed at enhancing overall survivability, conventional targeted chemotherapy are now being
combined with dendritic cell (DC) based cancer immunotherapies.29ꢀ32 DCs are the most
professional antigen presenting cells (APCs) in our body. They efficiently capture the
pathogenic antigens in the peripheral tissues, process them into small antigenic fragments and
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present the antigenic fragments (in complexation with major histocompatibility complexes MHC
class I and II) to the resting T lymphocytes in the nearby lymph nodes for mounting immune
response.33 In DCꢀbased DNA vaccination, autologous DCs are often isolated from recipient’s
bodies, the isolated DCs are ex vivo pulsed/transduced with tumorꢀassociated antigens (TAAs)
encoded DNA vaccines and such ex vivo tranduced DCs are finally reimplanted into the
recipient’s body. Aimed at simplifying such complex ex vivo DCꢀtransfection based DNA
vaccination protocol, recently we demonstrated that cationic liposomes of SHIKꢀ1 (Scheme 1)
containing a mannoseꢀmimicking shikimoyl moiety (mannose receptors are over expressed on
DCꢀsurfaces) and a transfection enhancing guanidinyl functionality in its polar headꢀgroup
region can efficiently target DNA vaccines to DCs under in vivo conditions.34More recently, we
have demonstrated markedly enhanced overall survivability(OS) in glioblastoma bearing mice
when treated with such in vivo DCꢀtargeted DNA vaccination (using SHIKꢀ1 liposomes in
electrostatic complexation with a DNA vaccine encoding glioblastoma antigen) in combination
with targeted chemotherapy (using nicotinic acetylcholine receptor (nAChR) selectiveWP1066ꢀ
loaded liposomes of a novel nicotinylated cationic amphiphile).11Herein we also report on further
enhancing OS of glioblastoma bearing mice using the presently described targeted chemotherapy
with WP1066ꢀloaded LAT1 selective liposomes of AmphiꢀDOPA in combination with in vivo
DCꢀtargeted DNA vaccination using liposomes of SHIKꢀ1 complexed with survivin (a well
known glioblastoma antigen35,36) encoded DNA vaccine.
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pCMVꢀsurvivin
Cl
(CH2)14CH3
(CH2)14CH3
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N
HN
H
O
N
O
H
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N
NH2
Cl
NH2
HO
OH
in vivo DCꢀtargeting lipoplex
OH
in vivo DCꢀtargeting lipid
s.c. immunization with
in vivo DCꢀtargeted lipoplex
SHIKꢀ1
Chemotherapy
Enhancement of
Overall Survivability
WP1066ꢀloaded
Glioblastoma targeting liposome
HO
NH3
2Cl
H
(CH2)14CH3
(CH2)14CH3
N
HO
NH
O
AmphiꢀDOPA
WP1066
Mice bearing
OrthotopicGlioblastoma
DSPEꢀ(PEG)27ꢀAmine
Scheme 1. Schematic representation of combination therapy approach used in the present study
2. Materials and Methods.
2.1. Materials. DOPC, LꢀDOPA, DMEM, cholesterol, BCIP/NBT, AmberlystIRA400 chloride
ion exchange resin, FITCꢀAnnexinꢀV and Propidium Iodide (PI) were purchased from Sigma, St.
Louis, USA. WP1066 was obtained from MerckꢀMillipore, USA. RPMI media, Penꢀstrep
solutions, and fetal bovine serum (South American origin) were obtained from PAN Biotech,
Aidenbach, Germany. Mouse monoclonal antiꢀSTAT3, Rabbit polyclonal antiꢀpꢀSTAT3 (Tyr
705), Rabbit polyclonal antiꢀβꢀactin antibodies, Goat antiꢀmouseꢀalkaline phosphatase conjugate
antibody, and Goat antiꢀrabbitꢀalkaline phosphatase conjugate antibody were procured from Cell
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Signalling Technology, Boston, USA. Rabbit polyclonal LAT1 antibody was purchased from
Bioss Antibodies, Boston, USA. TUNEL assay kit was purchased from Bionova, USA. CTL
assay kit was procured from Promega, USA. Dylightꢀ730 NIR dye,IFNꢀγ, and ILꢀ4 assay kits
were procured from Thermo Scientific, USA. pCMVꢀsurvivin plasmid DNA was purchased from
OriGene, USA (catalog no MC208134) and pCMVꢀSPORTꢀβꢀgal plasmid was a generous gifts
from Dr. Nalam Madhusudhana Rao, Centre for Cellular and Molecular Biology, India. Unless
otherwise stated all materials used in this project were obtained from local suppliers.
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2.2. Synthesis of AmphiꢀDOPA. (Figure 1.)
Stepꢀ1. Synthesis of 2-Amino-3-(3,4-dihydroxy-phenyl)-propionic acid methyl ester (Ia): Acetyl
chloride (5.5 g, 0.07 mole) was slowly added into anhydrous methanol (30 mL) at 0 ̊C and the
mixture was stirred for 30 min under N2 atmosphere. LꢀDOPA (2 gm, 0.01 moles) was added to
the mixture and continued to stir for 8 h at room temperature. Solvent was removed from the
reaction mixture by rotavapor. The residue upon reprecipitation at 4
̊C from methanol/diethyl
ether (1:30, v/v) afforded intermediates Ia (2 g, yield 94%) as white precipitate.
1H NMR of Ia(300 MHz, CD3OD + CDCl3): δ/ppm = 2.8ꢀ3.0 (m, 2H, C6H5O2ꢀCH2ꢀCH(NH3 )ꢀ
+
+
+
COOCH3), 3.6 (s, 3H, ꢀCH2ꢀCH(NH3 )ꢀCOOCH3), 3.9 (t, 1H, ꢀCH2ꢀCH(NH3 )ꢀCOOCH3), 6.3ꢀ
6.6 (m, 3H, (OH)2C6H3ꢀCH2ꢀ).
+
ESIꢀMS of Ia: 212 [M]+, for C10H14NO4 .
Stepꢀ2. Synthesis of 2-tert-Butoxycarbonylamino-3-(3, 4-dimethyl-phenyl)-propionic acid methyl
ester (Ib): Ia(2g, 0.0094 mole) was dissolved in 20 mL mixture of THF & 1M aqueous NaHCO3
̊
(1:1, v/v) and stirred for 20 min at 0
̊
C. BOCꢀanhydride (2.3 mL, 0.01 moles) was added slowly
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to the mixture at 0
̊
C and stirred for 5 h at room temperature. THF was removed by rotavapor,
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residue was diluted with 30 mL of ethyl acetate and the diluted mixture was washed with 5%
HCl (3 x 10 mL) & brine solution (3 x 20 mL). Organic layer was collected, dried over
anhydrous sodium sulphate and concentrated in rotavapor. Column chromatographic purification
of the brown residue using 60ꢀ120 mesh silica gel and 18% ethyl acetate in hexane (v/v) as
eluent afforded pure intermediate Ib (2.5 g, 85% yield, Rf = 0.4 in 50% ethyl acetate/hexane, v/v)
as white solid.
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1H NMR of Ib(300 MHz, CDCl3): δ/ppm = 1.4 (s, 9H, (CH3)3OCOꢀ), 2.9ꢀ3.0 (m, 2H,
(OH)2C6H3O2ꢀCH2ꢀCH(NHꢀBOC)ꢀCOOCH3), 3.7 (s, 3H, ꢀCH2ꢀCH(NHꢀBOC)ꢀCOOCH3), 4.5 (t,
1H, ꢀCH2ꢀCH(NHꢀBOC)ꢀCOOCH3), 6.5ꢀ6.8 (m, 3H, (OH)2C6H3ꢀCH2ꢀ).
+
ESIꢀMS of Ib: 312 [M+1]+, 334 [M+23]+ for C15H21NO6 .
Stepꢀ3. Synthesis of 3-(3,4-Bis-benzyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic acid
methyl ester (Ic): K2CO3 (3.29 g, 0.024 moles), benzyl bromide (6.42 g, 0.024 moles) and
catalytic amount of NaI were added to Ib (2.5 g, 0.0079 moles) in 40 mL of dry acetone and the
mixture was refluxed for 18 h under N2 atmosphere. K2CO3 was filtered off from the reaction
mixture and filtrate was concentrated by rotavapor. The residue was diluted in 30 mL DCM and
washed with 0.5(N) HCl (3 x 10 mL) and brine solution (3 x 30 mL). The brown organic layer
was dried over anhydrous sodium sulphate and concentrated in rotavapor. The oily brown
residue upon column chromatographic (60ꢀ120 mesh silica gel) purification using 8% ethyl
acetate in hexane (v/v) as eluent afforded pure Ic (3.42 g, 87% yield, Rf = 0.8 in 50% ethyl
acetate/hexane, v/v) as white solid.
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1H NMR of Ic(300 MHz, CDCl3): δ/ppm = 1.4 (s, 9H, (CH3)3OCOꢀ ), 3.0 (m, 2H, ꢀ(OH)2C6H3ꢀ
CH2ꢀCH(NHꢀBOC)ꢀCOOCH3), 3.7 (s, 3H, ꢀCH2ꢀCH(NHꢀBOC)ꢀCOOCH3), 4.5 (t, 1H, ꢀCH2ꢀ
CH(NHꢀBOC)ꢀCOOCH3), 5.1 (s, 4H, (C6H5ꢀCH2)2ꢀC6H3O2ꢀCH2ꢀCH(NHꢀBOC)ꢀ),6.6ꢀ6.9 (m,
3H, ꢀC6H3O2ꢀCH2ꢀ),7.3ꢀ7.5 (m, 10H, (C6H5ꢀCH2)2ꢀC6H3O2ꢀCH2ꢀCH(NHꢀBOC)ꢀ).
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ESIꢀMS of Ic: 514 [M+23]+ for C29H33NO6 .
Stepꢀ4. Synthesis of [2-(3,4-Bis-benzyloxy-phenyl)-1-(2-dihexadecylamino-ethylcarbamoyl)-
ethyl]-carbamic acid tert-butyl ester (Id): Aqueous LiOH (2.89 g, 0.069 moles, 4 mL) was
added toIc(3.42 g, 0.007 moles) dissolved in THF & MeOH (3:1, v/v, 16 mL) and stirred for 2 h
at 0 ̊C. THF and MeOH solvents were removed by rotavapor and the resulting reaction mixture
was acidified with saturated KHSO4 solution till pH became acidic (confirmed by pH paper).
Ethyl acetate (50 mL) was added to the acidified mixture. Organic layer was collected, dried
over sodium sulphate and concentrated on a rotavapor. The resulting white solid (2.32 g, 0.005
moles) was dissolved in dry DCM (20 mL) and stirred with EDCI (0.925 g, 0.005 moles) and
HOBT (0.741 g, 0.005 moles) for 30 min at room temperature under N2 atmosphere. N,Nꢀdiꢀ
hexadecylꢀNꢀ2ꢀaminoethylamine (2.95g, 0.006 moles, freshly prepared from the BOCꢀprotected
precursor) was added to the mixture and stirred for 8 h at room temperature. The reaction
mixture was diluted with CHCl3 (30 mL) and washed sequentially with water (30 mL), 1(N) HCl
(30 mL), saturated aqueous NaHCO3 (30 mL) and brine (3 x 30 mL). The organic layer was dried
over anhydrous sodium sulphate, filtered and concentrated by rotavapor. Column
chromatographic purification of the residue using 60ꢀ120 mesh silica gel and 1% methanol in
chloroform (v/v) as eluent afforded pure Id (2.58 g, 55%, yield, Rf = 0.6 in 5% MeOH/CHCl3,
v/v).
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1H NMR of Id (300 MHz, CDCl3): δ/ppm = 0.9 (t, 6H, CH3ꢀ(CH2)13ꢀ), 1.2ꢀ1.4 (m, 52H, CH3ꢀ
(CH2)13ꢀCH2ꢀCH2ꢀ) & s, 9H, (CH3)3OCOꢀ), 1.7 (m, 4H, CH3ꢀ(CH2)13ꢀCH2ꢀCH2ꢀ), 2.3ꢀ2.5 [(m,
4H, CH3ꢀ(CH2)13ꢀCH2ꢀCH2ꢀ& m, 2H,ꢀCH2ꢀCH2ꢀNHCOꢀ)], 3.0 (m, 2H, ꢀC6H3O2ꢀCH2ꢀCH(NHꢀ
BOC)ꢀCONHꢀ), 3.2 (m, 2H, ꢀCH2ꢀCH2ꢀNHCOꢀ), 4.3 (t, 1H, ꢀCH2ꢀCH(NHꢀBOC)ꢀCONHꢀ), 5.2
(s, 4H, (C6H5ꢀCH2)2ꢀC6H3O2ꢀCH2ꢀ),6.6ꢀ6.9 (m, 3H, ꢀC6H3O2ꢀCH2ꢀ),7.3ꢀ7.5 (m, 10H, (C6H5ꢀ
CH2)2ꢀC6H3O2ꢀCH2ꢀ).
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+
ESIꢀMS of Id: 969 [M]+ for C62H101N3O5 .
Stepꢀ5.Synthesis of AmphiꢀDOPA: Id (2.58 g, 0.003 moles) was subjected to BOC deprotection
̊
by stirring with 20% TFA in DCM at 0̊C for 3 h. Reaction mixture was concentrated by
rotavapor and subjected to catalytic debenzylation in 10 mL solvent mixture (MeOH:ethyl
acetate 5:1, v/v) using H2 over Pd/Cꢀcatalyst (catalytic amount) for 6 h at room temperature.
Pd/C was removed by filtration and filtrate was concentrated by rotavapor. The residue was
subjected to chloride ion exchange chromatography over Amberlyst IRAꢀ400 resin using
methanol as eluent. Methanol was removed and the concentrated residue upon five times
recrystalization from CHCl3/Pentane (1:30, v/v) afforded pure white AmphiꢀDOPA (1.22 g,
67%, Rf = 0.1 in 5% MeOH/CHCl3, v/v).
1H NMR of AmphiꢀDOPA(300 MHz, CDCl3): δ/ppm = 0.9 (t, 6H, CH3ꢀ(CH2)13ꢀ), 1.1ꢀ1.4 (m,
52H, CH3ꢀ(CH2)13ꢀCH2ꢀCH2ꢀ), 1.7 (m, 4H, CH3ꢀ(CH2)13ꢀCH2ꢀCH2ꢀ), 2.8ꢀ3.2 [(m, 4H, (CH3ꢀ
+
(CH2)13ꢀCH2ꢀCH2ꢀ) & m, 2H, ꢀCH2ꢀCH2ꢀNHCOꢀ) & 3.0 (m, 2H, C6H5O2ꢀCH2ꢀCH(NH3 )ꢀ], 3.4ꢀ
+
3.6 (m, 2H, ꢀCH2ꢀCH2ꢀNHCOꢀ), 4.1ꢀ4.2 (t, 1H, ꢀCH2ꢀCH(NH3 )ꢀCONHꢀ), 6.4ꢀ6.8 (br s, 1H; br s,
2H; C6H3), 8.0ꢀ8.2 ( br s, 1H, ꢀCH2ꢀCH2ꢀNHCOꢀ).
+
ESIꢀMS of AmphiꢀDOPA: 689 [M+1]+ for C43H81N3O3 .
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2.3. Preparation of AmphiꢀALA. (Figure S1,Supporting Information)
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Stepꢀ1. Synthesis of [2-(2-Dihexadecylamino-ethylcarbamoyl)-ethyl]-carbamic acid tert-butyl
ester (Ie): BOC protected βꢀalanine (1 g, 0.005 moles) was dissolved in dry DCM (20 mL) and
stirred with EDCI (0.96 g, 0.005 moles) and HOBT (0.77 g, 0.005 moles) for 30 min at room
temperature under N2 atmosphere. N, NꢀdiꢀhexadecylꢀNꢀ2ꢀaminoethylamine (2.08 g, 0.004
moles) was added to it and the reaction mixture was continued to stir for 8 h at room
temperature. CHCl3 (30 mL)was added to the reaction mixture and the diluted reaction mixture
was sequentially washed with water (30 mL), 1(N) HCl (30 mL), saturated aqueous NaHCO3 (30
mL) and brine (3x 30 mL). The organic layer was concentrated by rotavapor. The residue was
purified by column chromatography technique using 60ꢀ120 mesh silica gel and 1.2% methanol
in chloroform (v/v) as eluting solvent to get pure Ie(1.89 g, 68%, yield, Rf = 0.5 in 5%
MeOH/CHCl3, v/v).
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1H NMR of Ie(300 MHz, CDCl3): δ/ppm = 0.9 (t, 6H, CH3ꢀ(CH2)13ꢀ), 1.2ꢀ1.3 (m, 52H, CH3ꢀ
(CH2)13ꢀCH2ꢀCH2ꢀ), 1.4 (s, 9H, (CH3)3OCOꢀ), 1.7(m, 4H, CH3ꢀ(CH2)13ꢀCH2ꢀCH2ꢀ), 2.4ꢀ2.6 [(m,
4H, CH3ꢀ(CH2)13ꢀCH2ꢀCH2ꢀ); m, 2H, {CH3ꢀ(CH2)13ꢀCH2ꢀCH2}2NꢀCH2ꢀCH2ꢀNHCOꢀ) & m, 2H, ꢀ
COꢀHNꢀCH2ꢀCH2ꢀNHꢀCOOꢀC(CH3)3)], 3.2ꢀ3.5 [(m, 2H, {CH3ꢀ(CH2)13ꢀCH2ꢀCH2}2NꢀCH2ꢀCH2ꢀ
NHCOꢀ) & ꢀCOꢀHNꢀCH2ꢀCH2ꢀNHꢀCOOꢀC(CH3)3]
+
ESIꢀMS of Ie: 681 [M + 1]+ for C42H85N3O3 .
Stepꢀ2. Synthesis of AmphiꢀALA: Ie(1g, 0.001 moles) was subjected to BOCꢀdeprotection by
stirring with 20% TFA in DCM (10 mL) at 0 ̊C for 3 h. Reaction mixture was concentrated by
rotavapor and the residue was passed through Amberlyst IRAꢀ400 chloride ion exchange resin
for anion exchange using MeOH as eluting solvent. MeOH was removed on a rotavapor and
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residue upon 5 times reprecipitation from CHCl3/Pentane (1:30, v/v) afforded pure white Amphiꢀ
ALA (0.423 g, 73 %, Rf = 0.1 in 5% MeOH/CHCl3, v/v).
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1H NMR of AmphiꢀALA(300 MHz, CDCl3): δ/ppm = 0.9 (t, 6H, CH3ꢀ(CH2)13ꢀ), 1.2ꢀ1.3 (m,
52H, CH3ꢀ(CH2)13ꢀCH2ꢀCH2ꢀ), 1.7(m, 4H, CH3ꢀ(CH2)13ꢀCH2ꢀCH2ꢀ), 2.7ꢀ2.9 (m, 4H, {CH3ꢀ
(CH2)13ꢀCH2ꢀCH2}2NꢀCH2ꢀ) & m, 2H, {CH3ꢀ(CH2)13ꢀCH2ꢀCH2}2NꢀCH2ꢀCH2ꢀNHCOꢀ), 3.1ꢀ3.2
(m, 2H, {CH3ꢀ(CH2)13ꢀCH2ꢀCH2}2NꢀCH2ꢀCH2ꢀNHCOꢀ), 3.3ꢀ3.4 (m, 2H, ꢀCOꢀHNꢀCH2ꢀCH2ꢀ
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+
+
NH3 ), 3.7 (m, 2H, ꢀCOꢀHNꢀCH2ꢀCH2ꢀNH3 ).
ESIꢀMS of Ie: 580 [M]+ for C37H77N3O+.
2.4. Preparation of liposome.The brain tumor targeting liposomes of AmphiꢀDOPA were
prepared by conventional thin film hydration method using 1:1:1:0.05 mole ratio of
DOPC:AmphiꢀDOPA:Chol:DSPEꢀ(PEG)27ꢀamine.Similarly, the nonꢀtargeting control liposomes
of AmphiꢀALA were prepared by replacing AmphiꢀDOPA with equivalent amount of Amphiꢀ
ALA. WP1066ꢀloaded liposomes were prepared using 10:1 w/w ratio of total lipid:drug.
RhodamineꢀPE (0.2 mole%) and NIRꢀdye (3 mg/kgBW of mice) were mixed with lipid
ingredients to prepare RhꢀPE labeled and NIRꢀdye labeled liposomes, respectively. Liposomes of
SHIKꢀ1were prepared using 1:1 mole ratio of DOPE: SHIKꢀ1.Briefly, appropriate amounts of
the liposomal ingredients were dissolved in 3:1 (v/v) chloroform:methanolin a glass vial and the
solvent was evaporated with a thin flow of nitrogen gas. The residue was dried under high
vacuum for 5 h and the dried lipid film was allowed to swell in sterile deionized water for 8 h.
The resulting lipid suspensions were vortexed for 2ꢀ3 min and sonicated in an ice bath until
clarity using a Branson 450 sonifier at 100% duty cycle and 25 W output power to obtain small
unilamellar vesicles (SLVs). WP1066ꢀloaded liposomal solution was centrifuged at 10000 rpm
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for 20 min to remove unentrapped drug. The percent entrapment efficiency (ee) was calculated
using the relation ee = {(weight of WP added – weight of unentrapped WP)/weight of WP
added} × 100. Liposomes with 1mM and 5 mM AmphiꢀDOPA were used for in vitro and in vivo
studies, respectively.
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2.5. Characterization of the Liposomes.The hydrodynamic diameters and surface potentials of
the liposomal formulations of AmphiꢀDOPA and AmphiꢀALA (both empty and WP1066ꢀloaded)
were measured by photon correlation spectroscopy using a Zetasizer (Malvern, UK). Serum
stability of liposomes was evaluated by measuring the variation of its hydrodynamic diameters in
presence of 10% or 20% added fetal bovine serum (v/v) for different time points (4ꢀ72 h). TEM
images of all the liposomes were recorded under a highꢀresolution transmission electron
microscope (JEOLꢀJEM 2100). Briefly, 5 ꢁL of liposome was placed on a carbonꢀcoated copper
grid (glow discharged for 45s using Tolaron Hivac Evaporator) for 10 min and excess sample
was blotted away by Whatman filter paper. 5 ꢁL of 2% uranyl acetate solution was added to the
grid, incubated for 2 min, air dried and analyzed at 120 KV.
2.6. Cells and Culture Media. Mouse glioblastoma cells GL261were procured from National
Cancer Institute USA and maintained in RPMI media. NIHꢀ3T3 cells were procured from
National Centre for Cell Science, Pune, India and maintained in DMEM media. All the cells
were cultured in corresponding media supplemented with 10% fetal bovine serum (FBS) and 1X
PenꢀStrep solution in a humidified 5% CO2incubatorat 37 ̊C.
2.7. Animals. Female C57BL/6J mice (~4ꢀ6 weeks old, each weighing ~18ꢀ20 g) were procured
from Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India andmaintained in
IICT animal house under standard housing conditions. All the in vivo experiments were
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performed in IICT animal house following the Institutional BioꢀSafety and Ethical Committee
Guidelines and using approved animal protocols.
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2.8. Cellular uptake experiment.GL261 cells (~8,000 per well) were seeded in 96ꢀwell plates
in 100 ꢁL of growth medium 18ꢀ24 h prior to treatment. Cells were preꢀincubated with 50 µL of
antibody against the LAT1 diluted in serum free RPMI (1:25, v/v dilution) for 45 min at room
temperature. After 45 min media were taken out and a fresh lot of 50 µL LAT1 antibody
solutions (same dilution) were added to each well followed by addition of RhꢀPE labeled
liposomes of AmphiꢀDOPA or AmphiꢀALA (6 µL) diluted in 50 µL serum free RPMI. Another
set of GL261 cells were directly treated with 6 µL of RhꢀPE labeled liposomes of AmphiꢀDOPA
or AmphiꢀALA without any preꢀtreatment with LAT1 antibodies. After 3 h of incubation, cells
were washed with PBS (3 x 100 ꢁL) and images were taken under an inverted fluorescence
microscope (Nikon, Japan). Healthy fibroblast cells NIHꢀ3T3 (~12,000 per well) were seeded in
96ꢀwell plates in 100 ꢁL of growth medium 18ꢀ24 h prior to treatment. Cells were treated with
RhꢀPE labeled liposomes of AmphiꢀDOPA or AmphiꢀALA (6 µL) in 100 ꢁL of serum free
media. After 3 h of incubation images were taken under the same inverted fluorescence
microscope (Nikon, Japan).
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2.9. In vitro cellꢀgrowth inhibition study.
The cellular cytotoxicity of WP loaded liposomes of AmphiꢀDOPA and AmphiꢀALA were
evaluated by conventional MTT [3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2, 5ꢀdiphenyltetrazolium
bromide] assay in GL261 (mouse glioblastoma cell line), healthy NIHꢀ3T3 cells. The
cytotoxicity of empty liposomes (without WP1066) was also examined in GL261 cell line by
MTT assay. Briefly, cells were seeded (5000 cells per well) in 96ꢀwell tissue culture plates using
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100 µL of growth media 12ꢀ18 h before treatment. Cells were then separately treated with
liposomes of AmphiꢀDOPA and AmphiꢀALA each containing increasing concentration of WPꢀ
1066 (across the range of 2ꢀ8 µM) as well as empty liposomes (only for GL261 cells) in 100 µL
of growth media and incubated for 24h. MTT solution (10 µL from 5 mg/mL stock in PBS) was
added to each well and incubated for 4 hours at 37 °C in an air incubator. Cell viability was
assayed by reading absorbance of formazan at 550 nm using a microplate reader. Results were
expressed as percent viability = [A550 (treated cells) ꢀ background/A550 (untreated cells) ꢀ
background] × 100.
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2.10. Flow cytometric apoptosis assay.GL261 cells were seeded (~1x105 cells/well) in a 6 well
plate 18 h prior to treatment. Cells were incubated with 4 ꢁM of WP1066ꢀloaded liposomes of
AmphiꢀDOPAandAmphiꢀALA in 1mL RPMI media. After 24 h cells were trypsinized, washed
with PBS and centrifuged. The cell pellets were taken in 500 µL binding buffer containing
annexinꢀV FITC (0.25 µg) and PI (1.0 µg). The mixture was incubated for 10 min in dark with
gentle shaking and analyzed by flow cytometer (BD FACS Canto II).
2.11. Flow cytometric Analysis of cell cycle arrest. GL261 cells were seeded (~4x105 cells) in
Tꢀ25 flask for 18 h prior treatment. Cells were incubated with 2 mM thymidine in RPMI media
for 16ꢀ18 h (thymidine block) and then grown in fresh culture media (thymidine release) for 8 h
after washing with PBS. Cells were subjected to a second similar thymidine block for additional
18 h, washed with PBS and incubated in fresh media for another 1 h. Cells were then treated with
4 µM of WP1066ꢀloaded liposomes of AmphiꢀDOPA and AmphiꢀALA in 4 mL RPMI for 18 h.
The treated cells were collected by trypsinization, fixed with chilled 70% ethanol for 2 h at ꢀ20
and permeabilized with 500 µL of 0.1% tritonꢀX100 (diluted in 1% BSA in PBS) for 5 min at
room temperature. The permeabilized cells were resuspended in antibody solution (100 µL,
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dilution 1:100, v/v) of cyclin B1 (marker of G2/M phase of cell cycle) in PBS, vortexed for 1.5 h
at room temperature and centrifuged at 5000 rpm for 5 min. Cell pellets were washed with PBS,
resuspended in FITCꢀconjugated secondary antibody solution (100 µL, dilution 1:50, v/v in PBS)
and vortexed for 40 min. After washing with PBS (2 x 1 mL) cell pellets were taken in 500 µL of
PBS and analyzed by flow cytometry (BD FACS Canto II).
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2.12. Western BlotExperiment.~1 x 106 cells were seeded in T25 flask for 18ꢀ24 h prior to
treatment. Cells were incubated with 4 µM of WP1066ꢀloaded liposomes of AmphiꢀDOPA and
AmphiꢀALA in 4 mL RPMI media for 22 h. Cells were lysed with RIPA buffer (Sigma)
supplemented with protease inhibitor cocktail (Thermo Scientific) at 4 ̊C and total protein
contents of the cell lysates were estimated by BCA based protein estimation assay. 80 ꢁg of total
proteins were fractionated on 10% SDSꢀPAGE and transferred onto a PVDF membrane (Merck
Millipore) using wet blotting. The membrane was blocked for 1 h with 3% BSA solution in TBSꢀ
T (trisꢀbuffer saline containing 0.05% Tweenꢀ20) and separately incubated with primary
antibody solutions (10 mL, 1:1000 dilutions in 5% w/v BSA/TBSꢀT) of: Rabbit polyclonal antiꢀ
pꢀSTAT3 (Tyr 705), mouse polyclonal antiꢀSTAT3 and Rabbit polyclonal anti βꢀactin (as
loading control) antibodies for overnight at 4 ̊C. After washing with TBSꢀT (3 x 10 mL, 10 min
each), the membranes were incubated with the corresponding secondary antibodies conjugated to
alkaline phosphatase (10 mL, 1:5000 dilution in 0.05% TBSꢀT) for 1 h. Protein bands were
developed using BCIP/NBT chromogen solution (Sigma). Similarly, LAT1 expression levels in
GL261 and NIHꢀ3T3cells also were estimated using Rabbit polyclonal LAT1 antibody.
2.13. Tumor implantation.5ꢀ6 weeks old female C57BL/6J mice (each weighing ~20 g) were
anaesthetized by i.p. administration of xylazineꢀketamine mixture. GL261 (10,000 cells/mouse)
was intracranially injected as described previously.37Briefly, ~104 GL261 cells suspended in 3
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µL of PBS were stereotactically injected through an entry site at the bregma (2mm to the right of
the sagittal suture and 3mm below the surface of the skull) using a 10 µL Hamilton syringe and a
stereotactic frame (Harvard apparatus, USA).
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2.14. Biodistribution of NIRꢀlabeled liposomes. C57BL/6J mice (n = 3) bearing orthotopic
GL261 glioblastoma were intravenously injected with NIRꢀlabeled liposomes (2 mg dye/kg
B.W.) of AmphiꢀDOPA and AmphiꢀALA on day 7 post tumor implantation. Mice were
sacrificed after 4h and 24 h post i.v. injections. Organs were collected and lysed with 800 ꢁL of
lysis buffer (0.1 M TrisꢀHCl, 2 mM EDTA and 0.2% Triton Xꢀ100, pH 7.8) using a mechanical
homogenizer. The NIRꢀdye was extracted from the homogenized mixture with EtOAc (2 x 1
mL) by stirring for 4 h followed by centrifugation at 14,000 rpm for 20 min at 4 ̊C. The ethyl
acetate from the combined extract was lyophilized and the residue was dissolved in1 mL MeOH.
100 ꢁL of the methanolic solution was taken in 96ꢀwell plates and concentrations of NIRꢀdye
were measured fluorometrically. Background fluorescence from tissue extracts, if any, were
corrected by subtracting the fluorescence of the tissue extracts from untreated control mice.
Organs obtained from mice treated with NIRꢀlabeled liposomes 24 h post i.v. injection were
further analyzed by measuring fluorescence intensities using IVIS spectrum imaging system
(Perkin Elmer, serial no: IS1614N6957).
2.15. Tumor Growth Inhibition Study. On day 7, mice bearing orthotopic glioblastoma were
randomly sorted into three groups. Each group (n = 10 per group) was intravenously
administered with 5% aqueous glucose and WP1066ꢀloaded (10 mg/kg B.W. of mice) liposomes
of AmphiꢀDOPA andAmphiꢀALAon day 7, 9, 11, 13 and 15 post tumor inoculation. On day 16,
five mice from each group were sacrificed. Weights and pictures of the brains were recorded.
Remaining five mice in each group were kept under observation for survivability studies.
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2.16. Immunohistochemical studies. Tumor cryosections (10ꢀmicron thickness) were prepared
from differently treated mice group (n = 3 in each group) on day 16 post tumor implantation. The
cryosections were fixed with 4% paraformaldehyde for overnight, washed with PBS and stored
in 30% sucrose solution (in 1X PBS). Fixed cryosections were then immunostained with TUNEL
and Kiꢀ67 staining kits. Briefly, fixed tissue sections were washed with PBS and antigen retrieval
was done by dipping sections in citrate buffer (pH 6) at boiling temperature for 1 min. The
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sections were washed with 1X PBS and permeabilized with 2 NHCl at 62 ̊C for 30 min. Sections
were then blocked with 5% BSA solution in TBSꢀT (0.3% TritonꢀX in 1X TBS) for 30 min at
room temperature and incubated with primary antibody against Ki67 (thermo fisher scientific,
1:200 dilution in TBSꢀT) at 4 ̊C for overnight. Antibody treated sections were washed (3 x 5
min) with TBSꢀT and incubated with secondary antibody (goat antiꢀrabbit IgG, F(ab’)2ꢀPEꢀCy5:
Santacruz Biotechnology, scꢀ3844, 1:300 dilution) for 1 h at room temperature. Tissue sections
were washed (3 x 5 min) again with TBSꢀT and further immunostainedwith TUNEL (apoptosis)
assay kit according to manufacturers’ protocol. The stained cryosections were finally observed
under an inverted fluorescence microscope (Nikon, Japan,10X magnification) using green (for
TUNEL) and red (for Ki67) filters.
2.17. In vivo inhibition of STAT3 phosphorylation. Brain tumors were collected from
differently treated mice groups (n = 2 in each group) on day 16 post tumor implantation. Tumor
lysates were prepared using RIPA buffer (Sigma) supplemented with protease inhibitor cocktail
(Thermo Scientific) at 4 ̊C. Expression levels of pꢀSTAT3 and STAT3 proteins in brain tumor
lysate were estimated by Western blotting as described under section 2.12.
2.18. Preparation of plasmid DNA. DH5αꢀstrain of Escherichia coli was transformed with
pCMVꢀsurvivin plasmid using bacterial transformation kit (TransformAid™, Thermo
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Scientific™). Transformed bacteria was amplified and isolated by using Gene JET Plasmid
Maxiprep Kit (Thermo Fischer) as per the Manufacturer’s protocol. Purity of the plasmid DNAs
was determined by A260/A280 ratio (~1.8) and 1% agarose gel electrophoresis.
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2.19. Inhibiting mouse glioblastoma by LAT1 mediated chemotherapy in combination with
in vivo DCꢀtargeted genetic immunization. On day 6, C57/BL6J mice bearing orthotopic
glioblastoma were randomly sorted into five groups (n = 8). 1st, 2nd and 3rd groups were i.v
.administered with WP1066ꢀloaded (10 mg/kg B.W.) liposomes of AmphiꢀDOPA on day 7, 9,
11, 13 and 15 post tumor inoculation. The 4th group (vehicle control group) was intravenously
injected with 5% aqueous glucose. Both the 3rd& 5th groups were immunized (s.c) with pꢀCMVꢀ
survivin in complexation with in vivo DCꢀtargeting liposomes of SHIKꢀ1 on day 6, 8 and 15post
tumor implantation. The2nd group was also s.c. immunized with pCMVꢀβꢀgal (as non specific
control DNA vaccine) in complexation with liposomes of SHIKꢀ1on day 6, 8 and 15. Each
mouse was immunized with lipoplexes of 20 ꢁg DNA containing4:1lipid:DNA charge ratio.
Three mice from each group were sacrificed on Day 27 for CTL and Cytokine assays. Remaining
five mice in each group were observed for overall survivability study.
2.20. CTL assays. CTL assays were performed as described previously.38 Briefly, on day 12
post last immunization, spleens were excised from each group of mice (for each of the five
treatment groups, n = 3 per group). Splenocytes were isolated by mincing the spleens followed
by lysing the erythrocytes with 1 mL of lysis buffer (0.14 M ammonium chloride in 0.02 M Tris.
HCl, pH =7.2). Cells (~1 x 107 cells) were then coꢀcultured with GL261 cells (~1 x 106 cells) for
72 h in RPMI growth media supplemented with 100U/mL antibiotic solution (SigmaꢀUSA) and
50 U/ml ILꢀ2 (Thermo Scientific, USA) taken in a 100 mm dish. The stimulated splenocytes
were used as effector cells. Fresh target GL261cells (~104 cells/well in triplicate) were incubated
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with increasing numbers of effector cells (containing effector cells:target cells ratios across the
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range 10:1 to 100:1) in Uꢀbottomed 96ꢀwell plates for 4 h at 37 ̊C in a 5% CO2 incubator.
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Lactate dehydrogenase (LDH) levels in the culture supernatants were measured following
manufacturer’s protocol (Promega,USA).
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2.21. Cytokine Assay. Cytokine (IFNꢀγ and ILꢀ4)assays were performed as described
previously.38Briefly, on day 12 post third immunization single cell suspensions of splenocytes
(for each of the five treatment groups, n = 3 per group) were prepared and coꢀcultured with target
GL261 cells for 72 h as described in the previous section. Relative levels of secreted INFꢀγ and
ILꢀ4 in coꢀculture supernatant were measured by ELISA following manufacturer’s protocol
(Mouse IFNꢀγ Elisa kit and mouse ILꢀ4 Elisa kit, Pierce Biotechnology, USA).The results shown
are the average of the triplicated experiments performed on the same day.
2.22. In vivo toxicity studies.5ꢀ6 weeks old female healthy C57BL/6J mice (each weighing ~20
g) were intravenously administered with 5% aqueous glucose, empty liposomes of Amphiꢀ
DOPA and WP1066ꢀloaded liposomes of AmphiꢀDOPA on day 0, 2, 4, 6 & 8. Two weeks after
last injection blood samples were collected from each group of mouse (n = 5). Serum
biochemical parameters were evaluated using siemens kits (Siemens Healthcare Diagnostics Kit,
Newark, USA) on autoblood analyzer (Dimension X pand plus Clinical Chemistry System,
Siemens, Germany). Hematology parameters were estimated using Siemens hematology analyzer
(Siemens advia 2120i).
2.23. Statistical analysis. Error bars represent mean values ± SD. Twoꢀtailed student’s tꢀtest was
performed for comparing two treatment groups. *P <0.05 were considered statistically
significant.
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3. RESULTS AND DISCUSSION
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3.1. Synthesis. The brain tumor targeting amphiphile AmphiꢀDOPA was prepared starting from
commercially available LꢀDOPA (Figure1). Sequential protection of carboxyl group, amine
group and two hydroxyl groups present in LꢀDOPA provided the protected intermediate Ic
(Figure1). Intermediate Ic upon methyl ester deprotection followed by conventional peptide
coupling with N,NꢀdiꢀnꢀhexadecylꢀNꢀ2ꢀaminoethylamine(16TꢀNH2,Figure 1) provided the
hydrophobic intermediate Id (Figure1). BOCꢀdeprotection of Id followed by debenzylation with
catalytic hydrogenation and chloride ionꢀexchange of the resulting compound over Amberlyst
A400 chloride ion exchange resin afforded the target amphiphile (AmphiꢀDOPA) (Figure1).The
nonꢀtargeting amphiphile AmphiꢀALA was synthesized (FigureS1,Supporting Information)by
conventional peptide coupling of NꢀBOCꢀprotected βꢀalanine with N,NꢀdiꢀnꢀhexadecylꢀNꢀ2ꢀ
aminoethylamine (16TꢀNH2). The resulting intermediate Ie (FigureS1, Supporting Information),
upon BOCꢀdeprotecion followed by chloride ionꢀexchange over Amberlyst A400 chloride ion
exchange resin produced AmphiꢀALA (Figure S1,Supporting Information).The structures of both
glioblastoma targeting lipid AmphiꢀDOPA and nonꢀtargeting control lipid AmphiꢀALA were
confirmed by 1H NMR and ESI mass spectrometry and their purities were confirmed by reversed
phase analytical HPLC using two different mobile phases (Figure S2ꢀS6,Supporting
Information). The OH peaks of AmphiꢀDOPA were not observed in the 1H NMR spectrum
presumably due to extensive hydrogenꢀdeuterium exchange between deuterated solvent (CDCl3)
and the acidic phenolic OH protons.
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O
O
Cl
HO
HO
HO
CH3COCl, MeOH,
6 h, 0 °C, N2
(BOC)2O, THF:1(M) NaHCO3
(1:1, v/v), 6 h, 0 °C,
HO
HO
NH3
O
NH3
O
HN
O
O
O
O
HO
94% Yield
85% Yield
LꢀDOPA
Ia
Ib
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PhCH2Br, K2CO3, NaI (cat),
Acetone, N2, 18 h, reflux
Yield 87%
O
O
O
O
O
i) LiOH, THF:MeOH:water
(3:1:1), 2 h, 0 °C, 70% Yield
HN
H
O
O
(CH2)14CH3
(CH2)14CH3
HN
O
N
N
O
ii) EDCI, HOBT, 16T-NH2,
DCM, 6 h, rt, N2, 55% Yield
O
Id
O
Ic
i) TFA:DCM(1:4), 3 h, 0 °C, N2
67% Yield
ii) H2/Pd, MeOH-EtOAc (5:1, v/v), 8 h, rt
iii) Amberlyst IRA400, Cl-exchange resin;
(CH2)14CH3
(CH2)14CH3
H2N
N
2Cl
HO
HO
NH3
O
H
N, N-di-n-hexadecyl-N-2-aminoethylamine (16T-NH2)
(CH2)14CH3
(CH2)14CH3
N
NH
AmphiꢀDOPA
Figure 1. Synthetic Scheme for preparation of AmphiꢀDOPA.
3.2. Physicochemical characterization of liposomal formulations. The targeting amphiphile
AmphiꢀDOPA and the nonꢀtargeting control amphiphile AmphiꢀALA readily formed liposomes
in presence of 1,2ꢀDioleoylꢀsnꢀglyceroꢀ3ꢀphosphocholine (DOPC) and cholesterol as coꢀlipids. A
small amount of DSPEꢀ(PEG)27ꢀNH2 (5 mole % with respect to DOPC) was used in formulation
to enhance circulation stability of the liposomes. Both the liposomes of AmphiꢀDOPA and
AmphiꢀALA efficiently entrapped hydrophobic anticancer drug WP1066.Hydrodynamic
diameters and zeta potentials of all the liposomal formulations (empty liposomes as well as
WP1066ꢀloaded liposomes) were measured by dynamic light scattering (DLS) technique. Size
and zeta potential of the liposomal formulations were found to vary within the ranges 100ꢀ135
nm and 9ꢀ19 mV, respectively (Figure 2A,B). Polydispersity indexes (PDI) of all those
liposomes were also found to vary in between 0.22ꢀ0.28(Figure 2B). Transmission electron
micrographs of the empty liposomes of AmphiꢀDOPA (Figure 2Cꢀi) & AmphiꢀALA (Figure 2Cꢀ
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ii) as well as that for WPꢀloaded liposomes of AmphiꢀDOPA (Figure 2Cꢀiii) & AmphiꢀALA
(Figure 2Cꢀiv) revealed formation of spherical vessels. No major microscopic change was noted
upon loading of WP1066 in liposomes presumably due to solubilization of the hydrophobic drug
within the lipidꢀbilayer region (Figure 2C). Size of the WP1066ꢀloaded liposomes of Amphiꢀ
DOPA and AmphiꢀALA did not vary significantly upon incubation with 10% and 20% added
serum (Figure 2D) up to 48 h of post incubation. However, size of the liposomes increased after
48 h post incubation with serum (Figure 2D) presumably because of the enhanced interaction of
the cationic liposomes with the negatively charged serum protein with time.
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A
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ii
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iii
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Figure 2. Physicoꢀchemical characterizations of liposomal formulations. Hydrodynamic
diameters and surface potentials of the empty and WP1066ꢀloaded liposomes of AmphiꢀDOPA
(black& blue, respectively) and those of AmphiꢀALA (green & red, respectively) in deionized
water (AꢀB); Transmission electron micrographs of: empty liposomes of AmphiꢀDOPA (Cꢀi),
empty liposomes of AmphiꢀALA (Cꢀii),WPꢀloaded liposomes of AmphiꢀDOPA (Cꢀiii, indicated
by arrow) and WPꢀloaded liposomes of AmphiꢀALA (Cꢀiv). Size variation of WPꢀloaded
liposomes of AmphiꢀDOPA (red & green) and AmphiꢀALA (purple & black) with time in
presence of 10% and 20% added serum (D).
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3.3. LAT1 mediated cellular uptake of AmphiꢀDOPA liposomes. Toward examining whether
or not liposomes of AmphiꢀDOPA enter into cells via LAT1,first we confirmed significantly
higher LAT1 expression level in GL261 (mouse glioblastoma cells) than that in NIHꢀ3T3, nonꢀ
cancerous mouse fibroblast cells(Figure 3A).Next, to confirm LAT1 mediated cellular uptake of
AmphiꢀDOPA liposomes in GL261 cells, we performed a 4 h cellular uptake study.Gl261 cells
were divided into two sets: the first set was preꢀsaturated with LAT1 antibody and the second set
was left untreated. Both antibody treated cells and untreated cells were separately incubated with
RhꢀPE labeled liposomes of AmphiꢀDOPA as well as liposomes of AmphiꢀALA. Importantly,
cellular uptake of RhꢀPE labeled liposomes of AmphiꢀDOPA was found to be significantly less
in cells preꢀsaturated with LAT1antibodythan in untreated cells (Figure 3B, Parts IꢀII).The
corresponding difference in the degree of cellular uptake for RhꢀPE labeled liposomes of Amphiꢀ
ALA was not that significant (Figure 3C, Part IꢀII).These findings are consistent with selectively
LAT1mediated cellular uptake of AmphiꢀDOPA liposomes in GL261cells. It is worth
mentioning that cellular uptake efficiency of RhꢀPE labeled liposomes of both AmphiꢀDOPA
(Figure S7, upper panel, Supporting information) and AmphiꢀAlA (Figure S7, lower panel,
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Supporting information) in healthy NIHꢀ3T3 cells was found to be significantly less than that
observed in GL261 cells.
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Figure 3. Cellular uptake of AmphiꢀDOPA liposomes are mediated via LAT1. Protein
expression level of LATꢀ1 in GL261 cells and NIHꢀ3T3 cells measured by western blot analysis
in cultured cells (A), Epifluorescence micrographs (10X) of GL261 cells treated with RhꢀPE
(red) labeled liposomes of: AmphiꢀDOPA (B) and AmphiꢀALA (C): untreated cells (i) and cells
preꢀsaturated with LAT1 antibody (ii).Results shown here are the representative figure of two
independent experiments. Bars = 100 ꢁm.
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3.4. In vitro cytotoxicity study
.
With a view to examine the GL261 selective cell killing
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potential of the WP1066ꢀloaded liposomal formulations we performed conventional MTTꢀbased
cell viability assays in murine glioblastoma cells (GL261) and healthy fibroblast cells (NIHꢀ
3T3). The cell killing efficiency of WPꢀ1066 loaded AmphiꢀDOPA liposomes was significantly
higher than that of WPꢀ1066 loaded AmphiꢀALA liposomes in GL261 cells (Figure S8 A,
Supporting information) whereas both of them minimally affected healthy NIHꢀ3T3 cells (Figure
S8 B, Supporting information) at lower concentration of WP1066. Therefore, we proceeded with
4 µM liposomal WP1066 for subsequent in vitro studies. Notably, around 90% Gl261 cells were
alive when cells were treated with empty (with no WP1066 loaded) liposomes of both Amphiꢀ
DOPA and AmphiꢀALA even up to 40 µM of lipid concentration for 24 h (maximum lipid
concentration used in this study was 30 µM) (Figure S8 C, Supporting information) supporting
the nonꢀcytotoxic natures of the empty liposomes of the presently described lipids. Collectively,
these observations indicate GL261 selective cell killing efficacy of WP1066ꢀloaded liposomes of
AmphiꢀDOPA.
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3.5. Flow cytrometric studies on apoptosis induction by WP1066ꢀloaded liposomes of
AmphiꢀDOPA. With a view to examine whether or not WP1066ꢀloaded liposomes of Amphiꢀ
DOPA can induce apoptosis in GL261 cells, we performed FITCꢀannexin V/Propidium iodide
(PI) binding based flow cytometric apoptosis assay. Incubation of GL261 cells with WP1066ꢀ
loaded liposomes of AmphiꢀDOPA induced enhanced accumulation (~35%) of early and late
apoptotic cells (lower and upper right quadrants, Figure 4A, PartꢀII) compared to those in
untreated cells (Figure 4A, PartꢀI). Consistent with the aboveꢀmentioned poor cellular uptake of
AmphiꢀALA liposomes in GL261 cells (Figure 3C, PartꢀI), significantly less populations of early
and late apoptotic cells (~6%) were observed in cells treated with WP1066ꢀloaded liposomes of
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AmphiꢀALA (Figure 4A, PartꢀIII).Flow cytometry based cell cycle analysis using Cyclin B1(a
marker of G2/M phase in cell cycle) antibody revealed enhanced G2/M phase population in
GL261 cells treated with WPꢀ1006 loaded liposomes of AmphiꢀDOPA (Figure 4B, PartꢀII)
compared to that in untreated cells (Figure 4B, PartꢀI) and in cells treated with WP1066ꢀloaded
nonꢀtargeting liposomes of AmphiꢀALA (Figure 4B, PartꢀIII).
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A
Untreated
I
II
III
AmphiꢀDOPA
AmphiꢀALA
B
I
II
III
AmphiꢀALA
Untreated
AmphiꢀDOPA
Figure 4. WP1066ꢀloaded liposomes of AmphiꢀDOPA induce programmed cell death in
Glioblastoma cells by arresting cell cycle at G2/M phase. Flow cytometric analysis of
apoptosis(A) and cell cycle arrestatG2/M phase (B) in GL261 cells: untreated cells (I),cells
treated with WP1066ꢀloaded liposomes of AmphiꢀDOPA (II) and cells treated with WP1066ꢀ
loaded control liposomes of AmphiꢀALA (III).The vertical & horizontal axes in (C)represent
cells labeled with PI & FITCꢀAnnexin V, respectively.Right shift of Cyclin B1 (marker for
G2/M phase) profiles in (D) indicates enhanced population of cells atG2/M phase. Results
shown are the representative figure of two independent experiments.
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3.6. In vivo tissueꢀdistribution studies. Next, toward examining the in vivo bioꢀdistribution
profile, NIR (near infrared)ꢀdye labeled liposomes of AmphiꢀDOPA &AmphiꢀALA were
intravenously administered in mice bearing orthotopic glioblastoma on day 9 post GL261 cells
implantation. Organs were collected 4 h and 24 h post i.v. injections and NIRꢀdye concentrations
in different organs were estimated fluorometrically. Although NIRꢀdye labeled liposomes of
AmphiꢀDOPA distributed among brain, liver and spleen after 4 h post i.v. administration (Figure
5A), the distribution profile was primarily brain selective after 24 h (Figure 5B). Contrastingly,
the NIRꢀdye labeled control liposomes of AmphiꢀALA mostly accumulated in liver after 4 h
(Figure5A) and more so after 24 h (Figure5B). Consistently, ex vivo imaging of organs collected
24 h post injection also showed brain selective accumulation of NIRꢀdye labeled liposomes of
AmphiꢀDOPA(Figure 5C) and primarily liver selective accumulation of NIRꢀdye labeled
liposomes of AmphiꢀALA (Figure 5D).Thus, the bioꢀdistribution profiles summarized in Figure
5AꢀD prompted us to undertake further in vivo studies aimed at evaluating therapeutic potentials
of the liposomes of AmphiꢀDOPA for combating orthotopically established mouse glioblastoma.
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Figure 5. Liposomes of AmphiꢀDOPA efficiently accumulate in glioblastoma bearing mouse
brain upon intravenous administration. Tissue distribution profiles of NIRꢀdye labeled liposomes
of AmphiꢀDOPA (black bars) and AmphiꢀALA (grey bars) in C57BL/6J mice (n = 3) bearing
orthotopically implanted glioblastoma 4 h (A) and 24 h (B) post i.v. administration.* P<
0.001.Ex vivo imaging of brain (I), lung (II), heart (III), liver (IV), spleen (V) and kidney (VI)
excised from mice intravenously administered with NIRꢀdye labeled liposomes of AmphiꢀDOPA
(C) and AmphiꢀALA (D) 24 h post injection.
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3.7. Mouse glioblastoma growth inhibition studies. After confirming the in vivo glioblastoma
targeting ability of AmphiꢀDOPA liposomes, we set out to evaluate its therapeutic promise in
inhibiting mouse glioblastoma growth using WP1066ꢀloadedliposomal formulation.C57BL/6J
mice bearing orthotopic glioblatoma (GL261) were randomly sorted into three groups (n = 10
mice per group). The first group was intravenously administered with 5% aqueous glucose
solution (vehicle control). The second and third group received WP1066ꢀloaded (10 mg/kg B.W
of mice) liposomal formulations of AmphiꢀDOPA and AmphiꢀALA (nonꢀtargeting),
respectively, on day 7, 9, 11, 13 and 15 post intracranial GL261 implantation. On day 16, five
mice from each group were sacrificed and the remaining five mice in each group were left for
survivability study. Dark tumor spots were observed in brains isolated from mice administered
with vehicle control and nonꢀtargeting WP1066ꢀloaded liposome of AmphiꢀALA(Figure6A,
PartsꢀI & II). Contrastingly, the sizes of the tumor spots observed in brain isolated from mice
treated with WP1066ꢀloaded liposomes of AmphiꢀDOPA were much smaller (Figure 6A, Partꢀ
III).Importantly, the percentage of brain weight with respect to total body weight for the mice
group treated with WP1066ꢀloaded AmphiꢀDOPA liposome was found to be similar to that for
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