Large Amino Acid Transporter 1 Selective ... - ACS Publications

Subscriber access provided by UNIV OF ESSEX

Article

Large Aminoacid Transporter 1 Selective Liposomes of LDOPA 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 Downloaded from http://pubs.acs.org on October 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Large Aminoacid Transporter 1 Selective Liposomes of L-DOPA Functionalized Amphiphile for Combating Glioblastoma 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, CSIR-Indian Institute of Chemical Technology, Uppal Road,

Hyderabad- 500007, India. 2

Academy of Scientific & Innovative Research (AcSIR), 2 Rafi Marg, New Delhi, India.

3

Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical

4

University of Queensland, School of Veterinary Science, Gatton, Brisbane, QLD, AUS 4343

Technology, Uppal Road, Hyderabad- 500007, India. *To whom correspondence should be addressed. E-mail: [email protected] KEYWORDS: Glioblastoma therapy, STAT3 inhibitor, large amino acid transporter 1,liposomal drug delivery, DNA vaccination

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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, sub-optimal drug accumulation in glioma tissue. To this end, use of large amino acid transporter-1 (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 (100-135 nm) promising LAT1 selective liposomal drug carrier prepared

from

a novelL-3,4-dihydroxyphenylalanine (L-DOPA) functionalized

amphiphile (Amphi-DOPA). In vitro studies using Rh-PE labeled liposomes of Amphi-DOPA in both untreated glioma (GL261) cells and in GL261cells pre-incubated with LAT1 antibody revealedLAT1 mediated cellular uptake. Intravenously administered NIR-dye labeled liposomes of Amphi-DOPA in glioblastoma bearing mice showed preferential accumulation of the dye in brain tissue. Notably i.v. administration of WP1066-loaded liposomes of Amphi-DOPA 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 WP1066-loaded liposomes of Amphi-DOPA is combined with in vivo DC-targeted DNA vaccination using a survivin (a glioblastoma antigen) encoded DNA 2 ACS Paragon Plus Environment

Page 2 of 48

Page 3 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


vaccine. The present findings open a new door for LAT1 mediated systemic chemotherapy of glioblastoma.

1. INTRODUCTION. Glioblastoma (GBM) is one of the most aggressive and deadliest malignant brain tumors with a median survival of 14-17 months post diagnosis and only 3-5% 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 self-repair 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 off-target 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.5-7 Endogenous receptors and transporters over expressed on BCECs including transferrin receptor (TfR),8,9 nicotinic acetylcholine receptors (nAChR),10,11 integrin receptor12-14glucose transporter (GLUT),15-17 have been successfully exploited in overcoming the BBB. Nano-carriers with suitable pharmacokinetic properties are being fabricated with specific ligands of such receptors or transporters on their exo-surface to help the nanoparticles associated drugs cross BBB via receptor or transporter mediated transcytosis. To this end use of large amino acid transporter-1 (LAT1) over expressed both on BCECs and glioma cells have begun.18,19LAT1, a sodium-independent transporter, transports a wide range of substrate including large amino acids (e.g. L-Tyrosine), neurotherapeutics (e.g. L3,4-dihydroxyphenylalanine (L-DOPA) and gabapentin) and thyroid hormones across the 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 cells24-26 including glioblastoma.18,27Thus, high expression level of LAT1 on BCECs& glioma cells makes it a ‘dual target’ for anti-glioma therapy. 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 AmphiDOPA (Scheme 1), a novel cationic amphiphile containing L-DOPA (a substrate for LAT1) in its polar head-group region, for efficient targeting of potent chemotherapeutics to orthotopically established mouse glioblastoma. L-DOPA, 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 Amphi-DOPA 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 Amphi-DOPA containing encapsulated WP1066, 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.29-32 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 4 ACS Paragon Plus Environment

Page 4 of 48

Page 5 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 DC-based DNA vaccination, autologous DCs are often isolated from recipient’s bodies, the isolated DCs are ex vivo pulsed/transduced with tumor-associated 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 DC-transfection based DNA vaccination protocol, recently we demonstrated that cationic liposomes of SHIK-1 (Scheme 1) containing a mannose-mimicking shikimoyl moiety (mannose receptors are over expressed on DC-surfaces) and a transfection enhancing guanidinyl functionality in its polar head-group 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 DC-targeted DNA vaccination (using SHIK-1 liposomes in electrostatic complexation with a DNA vaccine encoding glioblastoma antigen) in combination with targeted chemotherapy (using nicotinic acetylcholine receptor (nAChR) selectiveWP1066loaded 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 WP1066-loaded LAT1 selective liposomes of Amphi-DOPA in combination with in vivo DC-targeted DNA vaccination using liposomes of SHIK-1 complexed with survivin (a well known glioblastoma antigen35,36) encoded DNA vaccine.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 48

pCMV-survivin Cl

O

in vivo DC-targeting lipoplex

H N

HO

O

OH OH

N

HN

H N

(CH2)14CH3 (CH2)14CH3 NH2 NH2 Cl

in vivo DC-targeting lipid SHIK-1

s.c. immunization with in vivo DC-targeted lipoplex

Chemotherapy

Enhancement of Overall Survivability

WP1066-loaded Glioblastoma targeting liposome HO

NH3

HO

H N

2Cl NH

O

Amphi-DOPA WP1066

(CH2)14CH3 (CH2)14CH3

Mice bearing Orthotopic Glioblastoma

DSPE-(PEG)27-Amine

Scheme 1. Schematic representation of combination therapy approach used in the present study

2. Materials and Methods. 2.1. Materials. DOPC, L-DOPA, DMEM, cholesterol, BCIP/NBT, AmberlystIRA400 chloride ion exchange resin, FITC-Annexin-V and Propidium Iodide (PI) were purchased from Sigma, St. Louis, USA. WP1066 was obtained from Merck-Millipore, USA. RPMI media, Pen-strep solutions, and fetal bovine serum (South American origin) were obtained from PAN Biotech, Aidenbach, Germany. Mouse monoclonal anti-STAT3, Rabbit polyclonal anti-p-STAT3 (Tyr 705), Rabbit polyclonal anti-β-actin antibodies, Goat anti-mouse-alkaline phosphatase conjugate antibody, and Goat anti-rabbit-alkaline phosphatase conjugate antibody were procured from Cell 6 ACS Paragon Plus Environment

Page 7 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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. Dylight-730 NIR dye,IFN-γ, and IL-4 assay kits were procured from Thermo Scientific, USA. pCMV-survivin plasmid DNA was purchased from OriGene, USA (catalog no MC208134) and pCMV-SPORT-β-gal 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. 2.2. Synthesis of Amphi-DOPA. (Figure 1.) Step-1. 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. L-DOPA (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. 1

H NMR of Ia(300 MHz, CD3OD + CDCl3): δ/ppm = 2.8-3.0 (m, 2H, C6H5O2-CH2-CH(NH3+)-

COOCH3), 3.6 (s, 3H, -CH2-CH(NH3+)-COOCH3), 3.9 (t, 1H, -CH2-CH(NH3+)-COOCH3), 6.36.6 (m, 3H, (OH)2C6H3-CH2-). ESI-MS of Ia: 212 [M]+, for C10H14NO4+. Step-2. 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. ̊ BOC-anhydride (2.3 mL, 0.01 moles) was added slowly



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

to the mixture at 0̊̊C and stirred for 5 h at room temperature. THF was removed by rotavapor, 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 60-120 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. 1

H NMR of Ib(300 MHz, CDCl3): δ/ppm = 1.4 (s, 9H, (CH3)3OCO-), 2.9-3.0 (m, 2H,

(OH)2C6H3O2-CH2-CH(NH-BOC)-COOCH3), 3.7 (s, 3H, -CH2-CH(NH-BOC)-COOCH3), 4.5 (t, 1H, -CH2-CH(NH-BOC)-COOCH3), 6.5-6.8 (m, 3H, (OH)2C6H3-CH2-). ESI-MS of Ib: 312 [M+1]+, 334 [M+23]+ for C15H21NO6+. Step-3. 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 (60-120 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.


Page 8 of 48

Page 9 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

H NMR of Ic(300 MHz, CDCl3): δ/ppm = 1.4 (s, 9H, (CH3)3OCO- ), 3.0 (m, 2H, -(OH)2C6H3-

CH2-CH(NH-BOC)-COOCH3), 3.7 (s, 3H, -CH2-CH(NH-BOC)-COOCH3), 4.5 (t, 1H, -CH2CH(NH-BOC)-COOCH3), 5.1 (s, 4H, (C6H5-CH2)2-C6H3O2-CH2-CH(NH-BOC)-),6.6-6.9 (m, 3H, -C6H3O2-CH2-),7.3-7.5 (m, 10H, (C6H5-CH2)2-C6H3O2-CH2-CH(NH-BOC)-). ESI-MS of Ic: 514 [M+23]+ for C29H33NO6+. Step-4. 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,N-dihexadecyl-N-2-aminoethylamine (2.95g, 0.006 moles, freshly prepared from the BOC-protected 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 60-120 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).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

H NMR of Id (300 MHz, CDCl3): δ/ppm = 0.9 (t, 6H, CH3-(CH2)13-), 1.2-1.4 (m, 52H, CH3-

(CH2)13-CH2-CH2-) & s, 9H, (CH3)3OCO-), 1.7 (m, 4H, CH3-(CH2)13-CH2-CH2-), 2.3-2.5 [(m, 4H, CH3-(CH2)13-CH2-CH2-& m, 2H,-CH2-CH2-NHCO-)], 3.0 (m, 2H, -C6H3O2-CH2-CH(NHBOC)-CONH-), 3.2 (m, 2H, -CH2-CH2-NHCO-), 4.3 (t, 1H, -CH2-CH(NH-BOC)-CONH-), 5.2 (s, 4H, (C6H5-CH2)2-C6H3O2-CH2-),6.6-6.9 (m, 3H, -C6H3O2-CH2-),7.3-7.5 (m, 10H, (C6H5CH2)2-C6H3O2-CH2-). ESI-MS of Id: 969 [M]+ for C62H101N3O5+. Step-5.Synthesis of Amphi-DOPA: Id (2.58 g, 0.003 moles) was subjected to BOC deprotection by stirring with 20% TFA in DCM at 0̊̊C 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/C-catalyst (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 IRA-400 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 Amphi-DOPA (1.22 g, 67%, Rf = 0.1 in 5% MeOH/CHCl3, v/v). 1

H NMR of Amphi-DOPA(300 MHz, CDCl3): δ/ppm = 0.9 (t, 6H, CH3-(CH2)13-), 1.1-1.4 (m,

52H, CH3-(CH2)13-CH2-CH2-), 1.7 (m, 4H, CH3-(CH2)13-CH2-CH2-), 2.8-3.2 [(m, 4H, (CH3(CH2)13-CH2-CH2-) & m, 2H, -CH2-CH2-NHCO-) & 3.0 (m, 2H, C6H5O2-CH2-CH(NH3+)-], 3.43.6 (m, 2H, -CH2-CH2-NHCO-), 4.1-4.2 (t, 1H, -CH2-CH(NH3+)-CONH-), 6.4-6.8 (br s, 1H; br s, 2H; C6H3), 8.0-8.2 ( br s, 1H, -CH2-CH2-NHCO-). ESI-MS of Amphi-DOPA: 689 [M+1]+ for C43H81N3O3+. 10 ACS Paragon Plus Environment

Page 10 of 48

Page 11 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.3. Preparation of Amphi-ALA. (Figure S1,Supporting Information) Step-1. Synthesis of [2-(2-Dihexadecylamino-ethylcarbamoyl)-ethyl]-carbamic acid tert-butyl ester (Ie): BOC protected β-alanine (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, N-di-hexadecyl-N-2-aminoethylamine (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 60-120 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). 1

H NMR of Ie(300 MHz, CDCl3): δ/ppm = 0.9 (t, 6H, CH3-(CH2)13-), 1.2-1.3 (m, 52H, CH3-

(CH2)13-CH2-CH2-), 1.4 (s, 9H, (CH3)3OCO-), 1.7(m, 4H, CH3-(CH2)13-CH2-CH2-), 2.4-2.6 [(m, 4H, CH3-(CH2)13-CH2-CH2-); m, 2H, {CH3-(CH2)13-CH2-CH2}2N-CH2-CH2-NHCO-) & m, 2H, CO-HN-CH2-CH2-NH-COO-C(CH3)3)], 3.2-3.5 [(m, 2H, {CH3-(CH2)13-CH2-CH2}2N-CH2-CH2NHCO-) & -CO-HN-CH2-CH2-NH-COO-C(CH3)3] ESI-MS of Ie: 681 [M + 1]+ for C42H85N3O3+. Step-2. Synthesis of Amphi-ALA: Ie(1g, 0.001 moles) was subjected to BOC-deprotection 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 IRA-400 chloride ion exchange resin for anion exchange using MeOH as eluting solvent. MeOH was removed on a rotavapor and 11 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

residue upon 5 times reprecipitation from CHCl3/Pentane (1:30, v/v) afforded pure white AmphiALA (0.423 g, 73 %, Rf = 0.1 in 5% MeOH/CHCl3, v/v). 1

H NMR of Amphi-ALA(300 MHz, CDCl3): δ/ppm = 0.9 (t, 6H, CH3-(CH2)13-), 1.2-1.3 (m,

52H, CH3-(CH2)13-CH2-CH2-), 1.7(m, 4H, CH3-(CH2)13-CH2-CH2-), 2.7-2.9 (m, 4H, {CH3(CH2)13-CH2-CH2}2N-CH2-) & m, 2H, {CH3-(CH2)13-CH2-CH2}2N-CH2-CH2-NHCO-), 3.1-3.2 (m, 2H, {CH3-(CH2)13-CH2-CH2}2N-CH2-CH2-NHCO-), 3.3-3.4 (m, 2H, -CO-HN-CH2-CH2NH3+), 3.7 (m, 2H, -CO-HN-CH2-CH2-NH3+). ESI-MS of Ie: 580 [M]+ for C37H77N3O+. 2.4. Preparation of liposome.The brain tumor targeting liposomes of Amphi-DOPA were prepared by conventional thin film hydration method using 1:1:1:0.05 mole ratio of DOPC:Amphi-DOPA:Chol:DSPE-(PEG)27-amine.Similarly, the non-targeting control liposomes of Amphi-ALA were prepared by replacing Amphi-DOPA with equivalent amount of AmphiALA. WP1066-loaded liposomes were prepared using 10:1 w/w ratio of total lipid:drug. Rhodamine-PE (0.2 mole%) and NIR-dye (3 mg/kgBW of mice) were mixed with lipid ingredients to prepare Rh-PE labeled and NIR-dye labeled liposomes, respectively. Liposomes of SHIK-1were prepared using 1:1 mole ratio of DOPE: SHIK-1.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 2-3 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). WP1066-loaded liposomal solution was centrifuged at 10000 rpm


Page 12 of 48

Page 13 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 Amphi-DOPA were used for in vitro and in vivo studies, respectively. 2.5. Characterization of the Liposomes.The hydrodynamic diameters and surface potentials of the liposomal formulations of Amphi-DOPA and Amphi-ALA (both empty and WP1066-loaded) 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 (4-72 h). TEM images of all the liposomes were recorded under a high-resolution transmission electron microscope (JEOL-JEM 2100). Briefly, 5 µL of liposome was placed on a carbon-coated copper grid (glow discharged for 45s using Tolaron Hivac Evaporator) for 10 min and excess sample was blotted away by Whatman filter paper. 5 µL 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. NIH-3T3 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 Pen-Strep solution in a humidified 5% CO2incubatorat 37 ̊C. 2.7. Animals. Female C57BL/6J mice (~4-6 weeks old, each weighing ~18-20 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

performed in IICT animal house following the Institutional Bio-Safety and Ethical Committee Guidelines and using approved animal protocols. 2.8. Cellular uptake experiment.GL261 cells (~8,000 per well) were seeded in 96-well plates in 100 µL of growth medium 18-24 h prior to treatment. Cells were pre-incubated 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 Rh-PE labeled liposomes of Amphi-DOPA or Amphi-ALA (6 µL) diluted in 50 µL serum free RPMI. Another set of GL261 cells were directly treated with 6 µL of Rh-PE labeled liposomes of Amphi-DOPA or Amphi-ALA without any pre-treatment with LAT1 antibodies. After 3 h of incubation, cells were washed with PBS (3 x 100 µL) and images were taken under an inverted fluorescence microscope (Nikon, Japan). Healthy fibroblast cells NIH-3T3 (~12,000 per well) were seeded in 96-well plates in 100 µL of growth medium 18-24 h prior to treatment. Cells were treated with Rh-PE labeled liposomes of Amphi-DOPA or Amphi-ALA (6 µL) in 100 µL of serum free media. After 3 h of incubation images were taken under the same inverted fluorescence microscope (Nikon, Japan). 2.9. In vitro cell-growth inhibition study. The cellular cytotoxicity of WP loaded liposomes of Amphi-DOPA and Amphi-ALA were evaluated by conventional MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay in GL261 (mouse glioblastoma cell line), healthy NIH-3T3 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 96-well tissue culture plates using


Page 14 of 48

Page 15 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100 µL of growth media 12-18 h before treatment. Cells were then separately treated with liposomes of Amphi-DOPA and Amphi-ALA each containing increasing concentration of WP1066 (across the range of 2-8 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. 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 µM of WP1066-loaded liposomes of Amphi-DOPAandAmphi-ALA 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 annexin-V 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 T-25 flask for 18 h prior treatment. Cells were incubated with 2 mM thymidine in RPMI media for 16-18 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 WP1066-loaded liposomes of Amphi-DOPA and Amphi-ALA in 4 mL RPMI for 18 h. The treated cells were collected by trypsinization, fixed with chilled 70% ethanol for 2 h at -20 ̊C and permeabilized with 500 µL of 0.1% triton-X100 (diluted in 1% BSA in PBS) for 5 min at room temperature. The permeabilized cells were resuspended in antibody solution (100 µL, 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 FITC-conjugated 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). 2.12. Western BlotExperiment.~1 x 106 cells were seeded in T25 flask for 18-24 h prior to

treatment. Cells were incubated with 4 µM of WP1066-loaded liposomes of Amphi-DOPA and Amphi-ALA 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 µg of total proteins were fractionated on 10% SDS-PAGE and transferred onto a PVDF membrane (Merck Millipore) using wet blotting. The membrane was blocked for 1 h with 3% BSA solution in TBST (tris-buffer saline containing 0.05% Tween-20) and separately incubated with primary antibody solutions (10 mL, 1:1000 dilutions in 5% w/v BSA/TBS-T) of: Rabbit polyclonal antip-STAT3 (Tyr 705), mouse polyclonal anti-STAT3 and Rabbit polyclonal anti β-actin (as loading control) antibodies for overnight at 4 ̊C. After washing with TBS-T (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% TBS-T) for 1 h. Protein bands were developed using BCIP/NBT chromogen solution (Sigma). Similarly, LAT1 expression levels in GL261 and NIH-3T3cells also were estimated using Rabbit polyclonal LAT1 antibody. 2.13. Tumor implantation.5-6 weeks old female C57BL/6J mice (each weighing ~20 g) were anaesthetized by i.p. administration of xylazine-ketamine mixture. GL261 (10,000 cells/mouse) was intracranially injected as described previously.37Briefly, ~104 GL261 cells suspended in 3 16 ACS Paragon Plus Environment

Page 16 of 48

Page 17 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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). 2.14. Biodistribution of NIR-labeled liposomes. C57BL/6J mice (n = 3) bearing orthotopic GL261 glioblastoma were intravenously injected with NIR-labeled liposomes (2 mg dye/kg B.W.) of Amphi-DOPA and Amphi-ALA 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 µL of lysis buffer (0.1 M Tris-HCl, 2 mM EDTA and 0.2% Triton X-100, pH 7.8) using a mechanical homogenizer. The NIR-dye 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 µL of the methanolic solution was taken in 96-well plates and concentrations of NIR-dye 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 NIR-labeled 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 WP1066-loaded (10 mg/kg B.W. of mice) liposomes of Amphi-DOPA andAmphi-ALAon 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. 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.16. Immunohistochemical studies. Tumor cryosections (10-micron 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 Ki-67 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 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 TBS-T (0.3% Triton-X in 1X TBS) for 30 min at room temperature and incubated with primary antibody against Ki67 (thermo fisher scientific, 1:200 dilution in TBS-T) at 4 ̊C for overnight. Antibody treated sections were washed (3 x 5 min) with TBS-T and incubated with secondary antibody (goat anti-rabbit IgG, F(ab’)2-PE-Cy5: Santacruz Biotechnology, sc-3844, 1:300 dilution) for 1 h at room temperature. Tissue sections were washed (3 x 5 min) again with TBS-T 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 p-STAT3 and STAT3 proteins in brain tumor lysate were estimated by Western blotting as described under section 2.12. 2.18. Preparation of plasmid DNA. DH5α-strain of Escherichia coli was transformed with pCMV-survivin plasmid using bacterial transformation kit (TransformAid™, Thermo 18 ACS Paragon Plus Environment

Page 18 of 48

Page 19 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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. 2.19. Inhibiting mouse glioblastoma by LAT1 mediated chemotherapy in combination with in vivo DC-targeted 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 WP1066-loaded (10 mg/kg B.W.) liposomes of Amphi-DOPA 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 p-CMVsurvivin in complexation with in vivo DC-targeting liposomes of SHIK-1 on day 6, 8 and 15post tumor implantation. The2nd group was also s.c. immunized with pCMV-β-gal (as non specific control DNA vaccine) in complexation with liposomes of SHIK-1on day 6, 8 and 15. Each mouse was immunized with lipoplexes of 20 µg 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 co-cultured with GL261 cells (~1 x 106 cells) for 72 h in RPMI growth media supplemented with 100U/mL antibiotic solution (Sigma-USA) and 50 U/ml IL-2 (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 19 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with increasing numbers of effector cells (containing effector cells:target cells ratios across the range 10:1 to 100:1) in U-bottomed 96-well plates for 4 h at 37 ̊C in a 5% CO2 incubator. Lactate dehydrogenase (LDH) levels in the culture supernatants were measured following manufacturer’s protocol (Promega,USA). 2.21. Cytokine Assay. Cytokine (IFN-γ and IL-4)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 co-cultured with target GL261 cells for 72 h as described in the previous section. Relative levels of secreted INF-γ and IL-4 in co-culture supernatant were measured by ELISA following manufacturer’s protocol (Mouse IFN-γ Elisa kit and mouse IL-4 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.5-6 weeks old female healthy C57BL/6J mice (each weighing ~20 g) were intravenously administered with 5% aqueous glucose, empty liposomes of AmphiDOPA and WP1066-loaded liposomes of Amphi-DOPA 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. Two-tailed student’s t-test was performed for comparing two treatment groups. *P 300% compared to untreated mice) when the presently described LAT1 mediated targeted chemotherapy with WP1066-loaded liposomes of Amphi-DOPA is combined with in vivo DC-targeted DNA vaccination using a surviving (a glioblastoma antigen) encoded DNA vaccine. The findings described herein open a new door for LAT1 mediated systemic chemotherapy of glioblastoma. ASSOCIATED CONTENT


Page 38 of 48

Page 39 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Supporting Information Synthetic Scheme for preparation of Amphi-ALA, Spectral Characterizations of Amphi-DOPA and Amphi-ALA, HPLC chromatograms, In vivo serum toxicity profiles of Amphi-DOPA liposomes, CTL and Cytokines profile under combination therapy mode are included in Supporting Information part. ABBREVIATIONS BBB: Blood brain barrier; BCA: Bicinchoninic acid; BCEC: Brain capillary endothelial cell; Chol: Cholesterol; CTL: Cytotoxic T lymphocyte; DC: Dendritic cell; DOPC:1,2-Dioleoyl-snglycero-3-phosphocholine ; DOPE: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine; EDCI:1Ethyl-3-(3-dimethylaminopropyl) carbodiimide; FITC: Fluorescein isothiocyanate; HOBT:Hydroxybenzotriazole ; LAT1: Large Amino acid Transporter-1, STAT3: Signal transducer and activator of transcription 3, TAA: Tumor associated antigen AUTHOR INFORMATION Corresponding Author *Arabinda Chaudhuri, E-mail:[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 48

We thank Mr. Harikrishna from CSIR-Centre for Cellular & Molecular Biology (CSIR-CCMB), Hyderabad, India for his kind help in taking TEM images of our liposomes. We thank Dr. N. M. Rao at CSIR-CCMB for providing us with p-CMV-SPORT-β-Gal plasmid. S.B. thanks Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, for her doctoral research fellowship. VV thanks University Grant Commission, New Delhi, Government of India, for his doctoral research fellowship. A.C. thanks CSIR for sponsoring this research work (Project Code: CSC0302). REFERENCES 1. Ostrom, Q. T.; Gittleman, H.; Fulop, J.; Liu, M.; Blanda, R.; Kromer, C.; Wolinsky, Y.; Kruchko, C.; Barnholtz-Sloan, J. S. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012. Neuro Oncol. 2015, 17, iv1 –iv62. 2. Young, R. M.; Jamshidi, A.; Davis, G.; Sherman, J. H. Current trends in the surgical management and treatment of adult glioblastoma. Ann. Transl. Med. 2015, 3, 121-135. 3. Stupp, R.; Mason, W. P.; van den Bent, M. J.; Weller, M.; Fisher, B.; Taphoorn, M. J.; Belanger, K.; Brandes, A. A.; Marosi, C.; Bogdahn, U.; Curschmann, J.; Janzer, R. C.; Ludwin, S. K.; Gorlia, T.; Allgeier, A.; Lacombe, D.; Cairncross, J. G.; Eisenhauer, E.; Mirimanoff, R. O. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 2005, 352, 987-96. 4. Rao, J. S. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat. Rev. Cancer 2003, 3, 489-501. 5.Daneman, R. The blood-brain barrier in health and disease.Ann. Neurol. 2012, 72, 648-672. 40 ACS Paragon Plus Environment

Page 41 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6.Boer, A. G.; Gaillard, P. J. Drug Targeting to the Brain. Ann. Rev. Pharmacol. Toxicol.2007, 47, 323–355. 7. Pardridge, W. M. Drug targeting to the brain. Pharm. Res. 2007, 24, 1733–1744. 8. Dixit, S.; Novak, T.; Miller, K.; Zhu, Y.; Kenney, M. E.; Broome, A. M.Transferrin receptortargeted theranostic gold nanoparticles for photosensitizer delivery in brain tumors. Nanoscale2015, 5,1782-1790. 9.

Zhang,

P.; Hu,

L.; Yin,

Q.; Zhang,

Z.; Feng,

L.; Li,

Y.Transferrin-conjugated

polyphosphoester hybrid micelle loading paclitaxel for brain-targeting delivery: synthesis, preparation and in vivo evaluation. J. Control. Release 2012, 159, 429-34. 10. Kumar, P.; Wu, H.; McBride, J. L.; Jung, K. E.; Kim, M. H.; Davidson, B. L.; Lee, S. K.; Shankar, P.; Manjunath, N.Transvascular delivery of small interfering RNA to the central nervous system. Nature 2007, 448, 39-43. 11. Saha, S.; Yakati, V.; Bhattacharya, V.; Kompella, S. D.; Kuncha, M.; Chakravarty, S.; Ramakrishna, S.; Chaudhuri, A. Combating Established Mouse Glioblastoma through Nicotinylated-Liposomes-Mediated Targeted Chemotherapy in Combination with DendriticCell-Based Genetic Immunization. Advanced Biosystems2017, 1, 1600009-15. 12. Chen, C.; Duan, Z.;Yuan, Y.;Li, R.;Pang, L.;Liang, J.;Xu, X.;Wang, J. Peptide-22 and Cyclic RGD Functionalized Liposomes for Glioma Targeting Drug Delivery Overcoming BBB and BBTB. ACS Appl. Mater.Interfaces2017, 9, 5864−5873. 13. Miura, Y.; Takenaka, T.; Toh, K.; Wu, S.; Nishihara, H.; Kano, M. R.; Ino, Y.; Nomoto, T.; Matsumoto, Y.; Koyama, H.; Cabral, H.; Nishiyama, N.; Kataoka, K. Cyclic RGD-linked



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 48

polymeric micelles for targeted delivery of platinum anticancer drugs to glioblastoma through the blood-brain tumor barrier. ACS Nano. 2013, 7, 8583-92. 14. Wang, K.; Zhang, X.; Liu, Y.; Liu, C.; Jiang, B.; Jiang, Y.Tumor penetrability and antiangiogenesis

using

iRGD-mediated

delivery

of

doxorubicin-polymer

conjugates.

Biomaterials 2014,35, 8735-47. 15.Umezawa, F.; Eto, Y. Liposome targeting to mouse brain: mannose as a recognition marker. BiochemBiophys Res Commun. 1988,153, 1038-44. 16. Qin, Y.; Fan, W.; Chen, H. L.; Yao, N.; Tang, W. W.; Tang, J. In vitro and in vivo investigation of glucose-mediated brain-targeting liposomes. J Drug Target. 2010, 18, 536– 549 17. Jiang, X.; Xin, H.; Ren, Q.; Gu, J.; Zhu, L.; Du, F.; Feng, C.; Xie, Y.; Sha, X.; Fang, X. Nanoparticles of 2-deoxy-D-glucose functionalized poly(ethylene glycol)-co poly(trimethylene carbonate) for dual-targeted drug delivery in glioma treatment. Biomaterials2014, 35, 518-529. 18. Li, L.; Di, X.; Zhang, S.; Kan, Q.; Liu, H.; Lu, T.; Wang, Y.; Fu, Q.; Sun, J.; He, Z. Large amino acid transporter 1 mediated glutamate modifieddocetaxel-loaded liposomes for glioma targeting.Colloids and Surfaces B: Biointerfaces2016,141, 260–267. 19.Kharya, P.; Jain, A.; Gulbake, A.; Shilpi, S.; Jain, A.; Hurkat, P.; Majumdar, S.; Jain, S. K. Phenylalanine-coupled solid lipid nanoparticles for brain tumor targeting. J. Nanopart. Res.2013, 15, 2022-2033.


Page 43 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20. Kanai, Y.; Segawa, H.; Miyamoto, Ki.; Uchino, H.; Takeda, E.; Endou, H. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem. 1998, 273, 23629-32. 21. Boado, R. J.; Li, J. Y.; Nagaya, M.; Zhang, C.; Pardridge, W. M. Selective expression of the large neutral amino acid transporter at the blood-brain barrier. Proc Natl AcadSci U S A. 1999,96, 12079-84. 22. Peura, L.; Malmioja, K.; Laine, K.; Leppänen, J.; Gynther, M.; Isotalo, A.; Rautio, J. Large amino acid transporter 1 (LAT1) prodrugs of valproic acid: new prodrug design ideas for central nervous system delivery.Mol Pharm. 2011, 8, 1857-66. 23.Killian, D. M.; Hermeling, S.; Chikhale, P. J. Targeting the cerebrovascular large neutral amino acid transporter (LAT1) isoform using a novel disulfide-based brain drug delivery system.Drug Deliv. 2007, 14, 25-31. 24. Kaira, K.; Oriuchi, N.; Imai, H.; Shimizu, K.; Yanagitani, N.; Sunaga, N.; Hisada, T.; Tanaka, S.; Ishizuka, T.; Kanai, Y.; Endou, H.; Nakajima, T.; Mori, M. Prognostic significance of L-type amino acid transporter 1 expression in resectable stage I–III nonsmall cell lung cancer. Br J Cancer.2008, 98, 742–748. 25. Sakata, T.; Ferdous, G.; Tsuruta, T.; Satoh, T.; Baba, S.; Muto, T.; Ueno, A.; Kanai, Y.; Endou, H.; Okayasu, I.L-type amino-acid transporter 1 as a novel biomarker for high-grade malignancy in prostate cancer. Pathol Int. 2009,59, 7-18. 26. Kaira, K.; Oriuchi, N.; Imai, H.; Shimizu, K.; Yanagitani, N.; Sunaga, N.; Hisada, T.; Tanaka, S.; Ishizuka, T.; Kanai, Y.; Endou, H.; Nakajima, T.; Mori, M. L-type amino acid



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 48

transporter 1 and CD98 expression in primary and metastatic sites of human neoplasms. Cancer Sci.2008, 99, 2380–2386. 27. Kobayashi, K.; Ohnishi, A.; Promsuk, J.; Shimizu, S.; Kanai, Y.; Shiokawa, Y.; Nagane, M.Enhanced tumor growth elicited by L-type amino acid transporter 1 in human malignant glioma cells. Neurosurgery2008, 62, 493-504. 28. Kageyama, T.; Nakamura, M.; Matsuo, A.; Yamasaki, Y.; Takakura, Y.; Hashida, M.; Kanai, Y.; Naito, M.; Tsuruo, T.; Minato, N.; Shimohama, S. The 4F2hc/LAT1 complex transports LDOPA across the blood-brain barrier.Brain Res.2000, 879, 115-21. 29.Garg, A. D.;Vandenberk, L.; Koks, C.; Verschuere, T.; Boon, L.; Gool, S. w. V.; Agostinis, P. Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell– driven rejection of high-grade glioma. Sci. Transl. Med. 2016, 8, 328. 30. Vanneman, M.; Dranof, G. Combining immunotherapy and targeted therapies in cancer treatment.Nature Reviews Cancer 2012, 12, 237-251. 31. Melero, I.; Berman, D. M.; Aznar, M. A.; Korman, A. J.; Pérez Gracia, J. L.; Haanen, J.Evolving synergistic combinations of targeted immunotherapies to combat cancer.Nat. Rev. Cancer. 2015,15, 457-72. 32.van der Burg, S. H.; Arens, R.; Ossendorp, F.; van Hall, T.; Melief, C. J. Vaccines for established cancer: overcoming the challenges posed by immune evasion. Nat. Rev. Cancer. 2016, 16, 219-33. 33. Steinman R. M.; Banchereau, J. Taking dendritic cells into medicine.Nature2007, 449, 41926.


Page 45 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34. Garu, A.; Moku, G.; Gulla, S. K.; Chaudhuri, A. Genetic Immunization With In Vivo Dendritic Cell-targeting Liposomal DNA Vaccine Carrier Induces Long-lasting Antitumor Immune Response. Mol. Ther. 2016, 24, 385-97. 35. Chakravarti, A.; Noll, E.; Black, P. M.; Finkelstein, D. F.; Finkelstein, D. M.; Dyson, N. J.; Loeffler, J. S. Quantitatively determined survivin expression levels are of prognostic value in human gliomas. J.Clin.Oncol. 2002, 20, 1063-8. 36. Kajiwara, Y.; Yamasaki, F.; Hama, S.; Yahara, K.; Yoshioka, H.; Sugiyama, K.; Arita, K.; Kurisu, K. Expression of survivin in astrocytic tumors: correlation with malignant grade and prognosis. Cancer 2003, 97, 1077-83. 37. Okada, H.; Tahara, H.; Shurin, M. R.; Attanucci, J.; Giezeman-Smits, K. M.; Fellows, W. K.; Lotze, M. T.; Chambers, W. H.; Bozik, M. E. Bone marrow-derived dendritic cells pulsed with a tumor-specific peptide elicit effective anti-tumor immunity against intracranial neoplasms. Int. J. Cancer1998, 78, 196-201. 38. Srinivas, R.; Garu, A.; Moku, G.; Agawane, S. B.; Chaudhuri, A. A long-lasting dendritic cell DNA vaccination system using lysinylated amphiphiles with mannose-mimicking headgroups.Biomaterials 2012, 33, 6220-9. 39. Yu, H.; Pardoll, D.; Jove, R. STATs in cancer inflammation and immunity: a leading role for STAT3, Nat Rev Cancer 2009, 9, 798-809. 40. Rahaman, S. O.; Harbor, P. C.; Chernova, O.; Barnett, G. H.; Vogelbaum, M. A.; Haque, S. J. Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells. Oncogene 2002, 21, 8404-13.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 48

41. Weissenberger, J.; Loeffler, S.; Kappeler, A.; Kopf, M.; Lukes, A.; Afanasieva, T. A.; Aguzzi, A.; Weis, J. IL-6 is required for glioma development in a mouse model. Oncogene2004, 23, 3308-16. 42. Iwamaru, A.; Szymanski, S.; Iwado, E.; Aoki, H.; Yokoyama, T.; Fokt, I.; Hess, K.; Conrad, C.; Madden, T.; Sawaya, R.; Kondo, S.; Priebe, W.; Kondo, Y. A novel inhibitor of the STAT3 pathway induces apoptosis in malignant glioma cells both in vitro and in vivo.Oncogene2007, 26, 2435-44. 43. Kong, L. Y.; Abou-Ghazal, M. K.; Wei, J.; Chakraborty, A.; Sun, W.; Qiao, W.; Fuller, G. N.; Fokt, I.; Grimm, E. A.; Schmittling, R. J.; Archer, G. E. Jr.; Sampson, J. H.; Priebe, W.; Heimberger, A. B. A novel inhibitor of signal transducers and activators of transcription 3 activation is efficacious against established central nervous system melanoma and inhibits regulatory T cells. Clin. Cancer Res. 2008, 14, 5759-68. 44. Hussain, S. F.; Kong, L. Y.; Jordan, J.; Conrad, C.; Madden, T.; Fokt, I.; Priebe, W.; Heimberger, A. B. A novel small molecule inhibitor of signal transducers and activators of transcription 3 reverses immune tolerance in malignant glioma patients. Cancer Res. 2007, 67, 9630-6. 45. Satoh, K.; Kaneko, K.; Hirota, M.; Masamune, A.; Satoh, A.; Shimosegawa, T. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer 2001, 92, 271-278. 46. Altieri, D. C. Validating survivin as a cancer therapeutic target. Nat Rev Cancer 2003, 3, 4654.


Page 47 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


47. Ciesielski, M. J.; Apfel, L.; Barone, T. A.; Castro, C. A.; Weiss, T. C.; Fenstermaker, R. A. Antitumor effects of a xenogeneic survivin bone marrow derived dendritic cell vaccine against murine GL261 gliomas. Cancer Immunol.Immunother.2006, 55, 1491-503. 48. Xiang, R.; Mizutani,N.; Luo, Y.; Chiodoni, C.; Zhou, H.; Mizutani, M.; Ba, Y.; Becker, J.C.; Reisfeld, R. A. A DNA Vaccine Targeting survivin Combines Apoptosis with Suppression of Angiogenesis in Lung Tumor Eradication. Cancer Res.2005, 65, 553-61. 49. Yu, H.; Kortylewski, M.; Pardoll, D. Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment. Nat. Rev.Immunol. 2007, 7, 41-51. 50. Ferguson, S. D.; Srinivasan, V. M.; Heimberger A. B. The role of STAT3 in tumor-mediated immune suppression. J. Neurooncol.2015, 123, 385-94. 51. Geiera, E. G.; Schlessingera, A.; Fana, H.; Gablec, J. E.; Irwina, J. J.; Salia, A.; Giacominia, K. M. Structure-based ligand discovery for the Large-neutral Amino Acid Transporter 1, LAT-1. Proceedings of the National Academy of Sciences2013, 110, 5480-5485.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 48

TABLE OF CONTENTS 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, Arabinda Chaudhuri* HO

NH3

HO

H N

2Cl NH

O

(CH2)14CH3 (CH2)14CH3

Amphi-DOPA WP1066 DSPE-(PEG)27 -Amine

A novel LAT-1 selective liposomal system for targeting potent cytotoxic drug to brain tumor


Large Amino Acid Transporter 1 Selective ... - ACS Publications

Recommend Documents