EGFR-Targeted Polymeric Mixed Micelles Carrying Gemcitabine for Treating Pancreatic Cancer

Article abstract of DOI:10.1021/acs.biomac.5b01419

The objective of this study was to design GE11 peptide (YHWYGYTPQNVI) linked micelles of poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-gemcitabine-graft-dodecanol (PEG-b-PCC-g-GEM-g-DC) for enhanced stability and target sp

Full text of DOI:10.1021/acs.biomac.5b01419

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Article
EGFR Targeted Polymeric Mixed Micelles carrying
Gemcitabine for treating Pancreatic Cancer
Goutam Mondal, Virender Kumar, surendra K Shukla, Pankaj K Singh, and Ram I. Mahato
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01419 • Publication Date (Web): 01 Dec 2015
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EGFR Targeted Polymeric Mixed Micelles carrying Gemcitabine for treating
Pancreatic Cancer
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Goutam Mondal1, Virender Kumar1, Surendra K. Shukla2, Pankaj K. Singh2 and Ram I.
Mahato 1*
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1Department of Pharmaceutical Sciences, University of Nebraska Medical Center,
Omaha, NE 68198
2Department of Biochemistry and Molecular Biology, University of Nebraska Medical
Center, Omaha, Nebraska, USA
*Corresponding Author
Ram I. Mahato, Ph.D.
Department of Pharmaceutical Sciences
University of Nebraska Medical Center
986025 Nebraska Medical Center
Omaha, NE 68198
Tel: (402)559ꢀ5422, Fax: (402)559ꢀ9543
Eꢀmail: ram.mahato@unmc.edu
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ABSTRACT
The objective of this study was to design GE11 peptide (YHWYGYTPQNVI)
micelles of poly(ethyleneglycol)ꢀblockꢀpoly(2ꢀmethylꢀ2ꢀcarboxylꢀ
linked
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propylenecarbonateꢀgraftꢀgemcitabineꢀgraftꢀdodecanol (PEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC) for
enhanced stability and target specificity of gemcitabine (GEM) to EGFR positive
pancreatic cancer cells. GE11ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC mixed micelles
showed EGFR dependent enhanced cellular uptake, and cytotoxicity as compared to
scrambled peptide HW12ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC mixed micelles and
unꢀmodified mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC micelles. Importantly, GE11ꢀlinked mixed
micelles preferentially accumulated in orthotopic pancreatic tumor and tumor
vasculature at 24 h post systemic administration. GE11ꢀlinked mixed micelles inhibited
orthotopic pancreatic tumor growth compared to HW12ꢀlinked mixed micelles, unꢀ
modified mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC micelles and free GEM formulations. Tumor
growth inhibition was mediated by apoptosis of tumor cells and endothelial cells as
determined by immunohistochemical staining. In summary, GE11ꢀlinked mixed micelles
is a promising approach to treat EGFR overexpressing cancers.
Keywords: pancreatic cancer, epidermal growth factor receptor, EGFR, polymeric
mixed micelles, gemcitabine.
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1. INTRODUCTION
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Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer
related death in the United States due to poor prognosis and high metastasis.1ꢀ3
Conventional chemotherapy often fails to treat pancreatic cancer and often causes nonꢀ
specific toxicity to healthy tissues. Gemcitabine (GEM) a nucleoside analogue is the first
line chemotherapy to treat PDAC with a mere 6.8 month survival rate, and up to 11.1
months as combination therapy.4ꢀ6 GEM exhibits its anticancer activity through blocking
DNA replication and inducing Sꢀphase cell cycle arrest. However, due to its rapid
metabolism into inactive form 2’ꢀdeoxyꢀ2’, 2’ꢀdifluorodeoxyuridine (dFdU) and nonꢀ
specific toxicity reduces its therapeutic potential.7, 8
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Various drug delivery systems like liposomes, nanoparticles and micelles fail to
improve GEM plasma stability due to poor drug loading and premature release.9ꢀ13
Chemical conjugation of GEM to lipid or polymeric carriers has been shown to improve
its loading but has lower aqueous solubility.14, 15 To overcome these challenges,
squalenoyl derivatives of GEM were developed, however their efficacy was limited due
to the rapid uptake by the reticuloendothelial system (RES).16 Since the incorporation of
polyethylene glycol (PEG) onto polymeric systems provides stealth properties and
prolonged circulation, we recently synthesized methoxy poly(ethyleneglycol)ꢀblockꢀ
poly(2ꢀmethylꢀ2ꢀcarboxylꢀpropylenecarbonateꢀgraftꢀgemcitabineꢀgraftꢀdodecanol
(mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC), where PEG corona ensures stealth properties and the
presence of carboxyl groups in polymeric backbone enables higher GEM loading. This
polymeric system selfꢀassembled into micelles and inhibited subcutaneous xenograft
tumor after intravenous administration.17 However, passive targeting is effective only in
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highly vascularized and rapidly growing tumors, but it is not effective in poorly
vascularized and metastatic tumors.18 Since tumor progression, invasion and
metastasis critically depend on endothelial cells derived angiogenesis, selective delivery
of anticancer agents to the tumor endothelial cells is an attractive approach.19, 20 To
date, a very few targeted nanoparticles are reported for drug delivery to the tumor and
its vasculature for pancreatic cancer treatment.21
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Epidermal growth factor receptor (EGFR) is overexpressed in the tumor derived
neovasculature of pancreatic tumor.22 Full length EGFR natural ligand, such as EGF
and monoclonal antibody cetuximab conjugated nanoparticles have been shown to
enhance the receptorꢀmediated endocytosis of GEM by pancreatic tumor cells.23
However, high mitogenic and proꢀangiogenic capability of full length EGFR ligands
retard their clinical translation. Alternatively, GE11 peptide (YHWYGYTPQNVI) ligand
has been shown its specific binding to EGFR with low mitogenic activity.24 Moreover,
GE11 peptide decorated delivery systems efficiently deliver drugs/genes to EGFR
overexpressing cancer cells.25ꢀ27
In this study, we conjugated GE11 peptide to the surface of malemidoꢀpoly
(ethylene glycol)ꢀblockꢀpoly (2ꢀmethylꢀ2ꢀcarboxylꢀpropylene carbonateꢀgraftꢀdodecanol
(GE11ꢀPEGꢀPCD) and synthesized methoxyꢀpoly (ethylene glycol)ꢀblockꢀpoly (2ꢀ
methylꢀ2ꢀcarboxylpropylene carbonateꢀgraftꢀgemcitabineꢀgraftꢀdodecanol (mPEGꢀbꢀ
PCCꢀgꢀGEMꢀgꢀDC) to prepare mixed micelles for enhanced GEM delivery to EGFR
expressing pancreatic cancer cells. We showed that GE11 linked mixed micelles
exhibited a better therapeutic effect than nonꢀtargeted mixed micelles in vitro and in MIA
PaCaꢀ2 cells derived orthotopic pancreatic tumor bearing mice.
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2. EXPERIMENTAL SECTION
2.1. Materials
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Nꢀterminal cysteine containing GE11 peptide was custom synthesized by
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Shanghai Hanhong Chemical Co. Ltd. (Shanghai, China). Its purity and structure were
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confirmed by HPLC (purity 98.36 %) and HꢀNMR. Nꢀterminal cysteine containing
CHYPYAHPTHPSW (designated as HW12) peptide was custom synthesized by
Biomatik USA, LLC (Delaware, USA). Its purity and structure were confirmed by HPLC
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(purity 97.58 %) and HꢀNMR, respectively. Malemide PEG hydroxyl (MW 5000) was
purchased from JenKem Technology (Beijing, China). Gemcitabine hydrochloride was
purchased from AK Scientific (Union city, CA). Dodecanol (DC), triethylamine (TEA), 1ꢀ
ethylꢀ3ꢀ(3ꢀdimethylaminopropyl) carbodiimide (EDC), hydroxybenzotriazole (HOBT),
1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU), benzyl bromide, 2,2ꢀbis(hydroxymethyl)
propionic acid, methoxy poly(ethylene glycol) (mPEG, Mn = 5000, PDI = 1.03),
puromycin dihydrochloride were purchased from Sigma Aldrich (St. Louis, MO). Matrigel
®
matrix basement membrane was procured from Corning (Chicago, IL). Fluorescein
cadaverine (FC) dihydrobromide salt was purchased from Life Technology (Chicago,
IL). Trisꢀ(2ꢀcarboxyethyl) phosphine hydrochloride (TCEP) was purchased from
Biosynth Chemistry & Biology (Itasca, IL). Polybrene and VEꢀcadherin (Hꢀ72) were
purchased from Santa Cruz Biotechnology (Dallas, TX). Rat antiꢀmouse CD31 was
purchased from BD Biosciences (San Diego, CA). Lentiviral particle LPꢀhLUCꢀLv201ꢀ
0200 was purchased from GeneCopoeia (Rockville, MD). Dead EndTM Fluorometric
TUNEL System was purchased from Promega Corporation (Madison, WI). Mouse
monoclonal antibodies to EGFR (Phycoerythrin) and mouse IgG1 monoclonal
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antibodies (Phycoerythrin)ꢀIsotype control were purchased from Abchem (Cambridge,
MA). Alexa Fluor 594 F(ab’)2 fragment of goat antiꢀrabbit IgG (H+L) was purchased from
Invitrogen (Grand Island, NY). All other chemicals were of analytical grade and
purchased from Sigma Aldrich (St. Louis, MO).
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2.2. Synthesis of polymers
2.2.1. Synthesis of maleimido poly(ethylene glycol)-block-poly(2-methyl-2-
dodecanoxycarbonylpropylene carbonate) (MAL-PEG-PCD)
2ꢀMethylꢀ2ꢀbenzyloxycarbonylpropylene carbonate (MBC) was synthesized and
converted to 2ꢀmethylꢀ2ꢀcarboxylpropylene carbonate (MCC) by hydrogenation reaction
as reported by Li et al.28 DC was conjugated to the carboxyl group of MCC by
carbodiimide chemistry. Briefly, MTCꢀOH (480 mg) was dissolved in 20 mL of DMF
followed by the addition of HOBT (506 mg), EDC (720 mg) and 490 ꢁL of TEA. After 1h,
DC (465 mg) was added to the reaction mixture and allowed the reaction for 18 h under
N2 atmosphere at RT. Then, ethyl acetate (20 mL) was added to the reaction mixture
and the organic layer was washed with distilled water, and organic solvent dried over
Na2SO4. The organic solvent was evaporated under reduced pressure to yield 2ꢀmethylꢀ
2ꢀdodecanoxycarbonylpropylene carbonate (MDC), which was purified by column
chromatography.
2.2.2. Synthesis of MAL-PEG-PCD from copolymerization of MAL-PEG-OH with
MDC.
MALꢀPEGꢀOH and MDC were dissolved in 15 mL anhydrous CH2Cl2 and stirred
under N2 atmosphere at the room temperature (RT). Then, DBU (40 ꢁL) was dissolved
in 1 mL anhydrous CH2Cl2 and added to the mixture and left the reaction under stirring
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for 3 h. Benzoic acid was added to the reaction mixture at the end of the reaction to
neutralize excess DBU and solvent was removed under reduced pressure. Then, the
crude product was dissolved with the minimum amount of CHCl3 and two times
precipitated with cold isopropyl alcohol, followed by diethyl ether. The precipitate was
dried under high vacuum at RT to get purified MALꢀPEGꢀPCD lipopolymer.
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2.2.3. Conjugation of GE11 and HW12 peptide to MAL-PEG-PCD
MALꢀPEGꢀPCD (20 mg) was dissolved in a mixture of DMSO: distilled water (1:1
v/v, 4ml) and stirred under N2 at RT. GE11 peptide (6.57 mg) and trisꢀ(2ꢀcarboxyethyl)
phosphine hydrochloride (TECP, 100 mM, 100 µL) were dissolved in 1 mL DMSO and
added to the reaction. Then, the reaction mixture was left for 24h at RT under N2
atmosphere with gentle stirring. Finally, the reaction mixture was dialyzed against
phosphate buffer saline using 3000 MWCO dialysis tubing to remove unreacted GE11
peptide and after dialysis, lyophilization was performed to get pure GE11ꢀPEGꢀPCD.
The chemical conjugation of GE11 peptide in MALꢀPEGꢀPCD lipopolymer was finally
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confirmed by HꢀNMR analysis. We synthesized HW12ꢀPEGꢀPCD polymer using the
same synthetic route as the synthesis of GE11ꢀPEGꢀPCD and finally this polymer was
confirmed by 1HꢀNMR analysis.
2.2.4. Synthesis of mPEG-b-PCC-graft-fluorescein cadaverine-graft-dodecanol
(mPEG-b-PCC-g-FC-g-DC)
mPEGꢀPCC was synthesized using our previously reported method.17 For FC
and DC conjugation, mPEGꢀPCC (100 mg), HOBT (61 mg) and EDC (77 mg) were
dissolved in 6 mL DMF and allowed for stirring for 2 h at RT under N2 atmosphere. FC
(13 mg) and DC (56 mg) were added to the reaction mixture and followed by the
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addition of 120 µL DIPEA. The reaction was allowed to continue for 48 h at RT under N2
atmosphere. The crude product was precipitated twice with an excess of cold isopropyl
alcohol and then dissolved in chloroform and precipitated in cold diethyl ether. Finally,
the precipitate was dissolved in acetone and dialyzed against water using 2,000 MWCO
dialysis tubing and after dialysis and lyophilized to yield mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀDC.
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2.2.5. Synthesis of GEM and DC conjugated mPEG-PCC-g-GEM-g-DC copolymer
mPEGꢀPCC (300 mg), HOBT (223 mg) and EDC (317 mg) were dissolved in 12
mL DMF and stirred for 2 h, and then GEM and DC were added to the reaction mixture,
followed by addition of 360 µL N,Nꢀdiisopropylethylamine (DIPEA). The reaction was left
for 48 h at RT under N2 atmosphere. The crude product was precipitated twice with
excess of cold isopropyl alcohol, dissolved in chloroform and precipitated again in the
cold diethyl ether. Finally, the precipitate was dissolved in acetone and dialyzed against
water using 2000 MWCO dialysis tubing and lyophilized to yield mPEGꢀbꢀPCCꢀgꢀGEMꢀ
gꢀDC.
2.3. Quantification of GEM payload in mPEG-b-PCC-g-GEM-g-DC copolymer
The amount of GEM conjugated to the copolymer was determined by alkaline
hydrolysis method as described previously.17 Briefly 10 mg of copolymer micelles were
subjected to alkaline hydrolysis in NaOH (1 N) at 40 °C for 1 h and the samples were
subjected to HPLCꢀUV analysis used the HPLC system equipped with photodiode array
detector 996 (Waters Corporation) and data acquisition and processing were performed
with Empower Pro software. A 20 ꢁL volume of the standard and sample was injected
into Inertsil ODS 3 column (4.5 × 250 mm). Analytes were eluted isocratically at the flow
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rate of 1 mL/min with a mobile phase of sodium acetate buffer (20mM, pH 5.5):
methanol (93: 07 v/v) and the column temperature was 40 °C.
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2.4. Preparation of GE11- or HW12-linked mixed micelles
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GE11ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC and HW12ꢀPEGꢀPCD/mPEGꢀbꢀ
PCCꢀgꢀGEMꢀgꢀDC mixed micelles were prepared by thin film hydration as reported with
slight modification.29, 30 Briefly, GE11ꢀPEGꢀPCD (10ꢀ30% weight ratio) and mPEGꢀbꢀ
PCCꢀgꢀGEMꢀgꢀDC (70ꢀ90% weight ratio) were dissolved in 1 ml CHCl3 in a glass vial.
The solvent was evaporated under reduced pressure to form thin film and dried film was
kept under vacuum overnight to remove residual CHCl3. 1 mL PBS (pH 7.4) was added
to the vacuum dried film and vortexed for 5 min at RT. Then resulting solution was
centrifuged at 5000 g for 5 min and filtrated through 0.22 µ filter membrane to obtain the
micelles. FC labeled GE11ꢀlinked mixed micelles were prepared by keeping FC
concentration constant (100 µM) and mixing GE11ꢀPEGꢀPCD (10ꢀ30% weight ratio)
with mPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀDC (70ꢀ 90 w/w). Similarly, HW12ꢀlinked mixed
micelles were prepared by mixing HW12ꢀPEGꢀPCD and mPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀ
FCꢀgꢀDC (70/30 w/w).
2.5. Particle size measurement
Particle size distribution and surface zeta (ζ) potential of GE11ꢀPEGꢀPCD/mPEGꢀ
bꢀPCCꢀgꢀGEMꢀgꢀDC and HW12ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC mixed micelles
(10 mg/mL) were determined by photon correlation spectroscopy at a scattering angle
of 173° (Malvern Zetasizer).
2.6. Stable transfection of MIA PaCa-2 cells with lentiviral particles
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MIA PaCaꢀ2 cells were kindly gifted by Dr. Rakesh Singh (UNMC, Omaha, NE)
and maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal
bovine serum (FBS) and 1% antibiotic in an incubator at 37 °C/5% CO2. MIA PaCaꢀ2
cells were stably transfected with lentiviral vector encoding luciferase and green
fluorescent protein (GFP) (hLUCꢀLv201ꢀ0200) using polybrene according to the
manufacturer’s instructions. Briefly, 2x105 MIA PaCaꢀ2 cells were seeded in each well
in a 6ꢀwell plate for 24 h. For each well, 2 ꢁL of lentiviral particles was mixed with 1 ꢁL
of polybrene (10 mg/mL) and the final volume was made to 2 mL using DMEM. The
transfection mixture was then incubated for 15 min at RT with occasional agitating and
MIA PaCaꢀ2 cells were washed with PBS and were incubated with the transfection
mixture overnight. The medium was replaced with 2 mL fresh DMEM for an additional
48 h. The cells were trypsinized and cultured in Tꢀ75 flasks for 15 days in 10 mL DMEM
containing 2 µg/mL puromycin. Every alternative day during these 15 days, the medium
was removed with a fresh DMEM containing 2 µg/mL puromycin. MIA PaCaꢀ2 cells
were then grown for another four days with fresh DMEM containing a lower
concentration of puromycin (1 µg/mL). Luciferase and GFP expression from stably
transfected cells were confirmed by in vivo IVIS and epifluorescence microscope
respectively. Finally, cells were sorted to obtain luciferase and GFP reporter gene
expressing cell population using fluorescenceꢀactivated cell sorting (FACS). In vitro and
in vivo experiments were performed with stable luciferase and GFP transfected cells
culturing in DMEM without puromycin.
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2.7. Cellular uptake studies
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Cellular uptake study was carried out in MIA PaCaꢀ2 cells using FC labeled
mixed micelles. Briefly, 2×105 cells were seeded per well in sixꢀwell plates for 24 h.
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HW12ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀ
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DC mixed micelles and mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀDC unmodified micelles were added to
each well. After 12 h cells were washed with PBS and observed under an
epifluorescence microscope. Further, for quantitative estimation of FC dye in cellular
uptake study, cells were washed with PBS, trypsinized, again washed with cold PBS
and centrifuged. Then, cells pellet was reꢀsuspended with PBS containing 4%
paraformaldehyde and kept for 10 min at RT. Cells were centrifuged, wash with PBS,
reꢀsuspended with 1 mL PBS and analyzed by a flow cytometer (BD LSRFortessa™ Xꢀ
20). For competition studies, excess of free GE11 peptide was diluted with medium and
added to cells 2 h prior to the addition of GE11ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀDC
mixed micelles. After 12 h, the medium was discarded and cells were washed with PBS
(pH 7.4) and observed under an epifluorescence microscope (Zeiss, Germany).
2.8. Cytotoxicity study
Cytotoxicity of GE11ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC, HW12ꢀPEGꢀ
PCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC
mixed
micelles,
mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC
unmodified micelles and free GEM were evaluated by 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT) assay. MIA PaCaꢀ2 cells were seeded at a density
of ~5000 cells per well in 96ꢀwell plates for 18 h and were treated with GE11ꢀPEGꢀ
PCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC, HW12ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC mixed
micelles, mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC unmodified micelles and free GEM having 60ꢀ500
nM concentration of GEM. The final volume in each well was adjusted to 200 µL using
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complete media and left for 72 h, 10 µL of MTT solution (5mg/ mL in PBS) were added
to each well and incubated for 4 h. The formazan crystals were solubilized with 200 µL
of DMSO and measured the absorbance in each well with a microplate reader at 560
nm wavelength and background correction was done by subtracting absorbance at 655
nm. Results were calculated as percent cell viability = [A560 (treated cells)ꢀ
background/A560 (untreated cells)ꢀbackground] x 100.
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2.9. In vivo distribution and targeting study
All animal experiments were performed in accordance with the NIH animal use
guidelines and protocol approved by the Institutional Animal Care and Use Committee
(IACUC) at the University of Nebraska Medical Center, Omaha, NE. Orthotopic
pancreatic cancer mouse model was generated by implantation of MIA PaCaꢀ2 cells
using PBS: matrigel (1:1 v/v) into pancreas of 6ꢀ8 weeks old athymic nude mice (n=3).
GE11ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀDC,
HW12ꢀPEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀ
DC mixed micelles and mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀDC unmodified micelles were injected to
mice via the tail vein. Mice were sacrificed 24 h post injection of mixed micellar
formulations, tumor were harvested. 5 µM tumor cryosections were made using cryostat
instrument (Leica), fixed with 10% formaldehyde in PBS and fixed tumor sections were
washed with PBS and visualized under a fluorescent microscope. For tumor vasculature
targeting study, same tissue sections were again fixed in 4% paraformaldehyde in PBS
and stained for rabbit polyclonal antiꢀVEꢀcadherin antibody (endothelial cells, red) (1:50
in 0.5% bovine serum albumin in PBS). Then, sections were counterstained with Alexa
Fluor 594 F(ab’)2 fragment of goat antiꢀrabbit IgG (H+L) secondary antibody (10 µg/mL
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in PBS). Finally, sections were mounted with SlowFade Gold antifade reagent with
DAPI and snaps were taken under a inverted fluorescence microscope.
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2.10. Efficacy of mixed micelle formulations in orthotopic pancreatic tumor
bearing mice
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After 12 days of post implantation of luciferase and GFP stably expressing 3x106
MIA PaCaꢀ2 into athymic nude mice, 100 mg/kg of Dꢀluciferin was administered into
each animal via intraperitoneal injection and bioluminescent was measured using the
IVIS Imaging System (Caliper Life Sciences). These tumor bearing mice were then
randomly sorted into five groups (n = 5): 1) vehicle control i.e. normal saline, 2) GEꢀ
linked mixed micelles, 3) HW12ꢀlinked mixed micelles, 4) mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC
un modified micelles, and 5) free GEM at an equivalent dose of 40 mg/kg GEM.
Treatment was started at days 12 via intravenous administration once a week for three
weeks. Bioluminescent was measured at day 12, 16, 20, 24, 26 and 30. Mouse body
weight was measured at day 12, 15, 18, 21, 24, 27, and 30. After final bioluminescent
measurement, mice were sacrificed and tumors were excised. Then excised tumors
were frozen in a cryostat at ꢀ30 °C for 2 h, cryosections were fixed with 10%
formaldehyde in PBS. Fixed tumor sections were washed with PBS and immunostained
for cell proliferation marker (Kiꢀ67), apoptosis (cleaved caspase 3) and H&E. To detect
tumor cell as well as tumor vasculature apoptosis induced by GEM treatment, orthotopic
MIA PaCaꢀ2 bearing mice (n = 3) were intravenously administered with GE11ꢀlinked
mixed micelles, HW12ꢀlinked mixed micelles, mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC unmodified
micelles and free GEM once a week for three weeks post tumor inoculation. Mice were
sacrificed 72 h post last injection, tumors excised. Tumors were cryosectioned and fixed
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cryosections were immunostained with TUNEL assay kit according to the
manufacturer’s instructions and counterstained with DAPI and imaged under
fluorescence microscope. To measure the tumor endothelial microvessels density,
tumor sections were immunostained with CD31 primary antibody.
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2.11. Statistical analysis
Data are represented as means ± SEM. The statistical comparisons of the
experiments were performed by twoꢀtailed Student’s test. P <0.05 was considered
statistically significant.
3. RESULTS AND DISCUSSION
Bioconjugation to a polymeric carrier is an attractive strategy to enhance the in vivo
stability and delivery of GEM to the tumor. Several PEGylated conjugates, lipid
conjugates and squalenoyl derivatives of GEM have been demonstrated to enhance its
bioavailability.14ꢀ16 However, the clinical translation of these delivery systems is limited
by poor solubility, uptake by RES, and lower GEM payload. We synthesized mPEGꢀ
PCC copolymer having several carboxyl pendant groups for conjugating GEM.17 This
coꢀpolymer could selfꢀassemble into micelles and significantly inhibited subcutaneous
MIA PaCaꢀ2 cells implanted xenograft tumor after systemic administration. In the
present study, we used GE11 peptide as a targeting ligand that efficiently binds to
EGFR and has low mitogenic activity.24 We synthesized GE11ꢀPEGꢀPCD using GE11
peptide, MalꢀPEGꢀPCD, and TCEP (Fig. 1A). To confirm GE11 and not
HYPYAHPTHPSW (designated as HW12) is an EGFR ligand, HW12ꢀPEGꢀPCD was
synthesized using the same synthetic route as mentioned for synthesizing GE11ꢀPEGꢀ
PCD (Fig. 1B). We also synthesized mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC and mPEGꢀbꢀPCCꢀgꢀ
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A
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B
Figure 1
FCꢀgꢀDC copolymers (Fig. 2 A&B). It is important to note that the two components of
this copolymer are biocompatible PEG block and a biodegradable polycarbonate (PCC)
block. Polycarbonates are biodegradable and have low toxicity, as their degradation
products are carbon dioxide and alcohol, which are less acidic, have less effect on
microenvironment pH and thus will not result in local inflammation.31
3.1. Synthesis and characterization of GE11-PEG-PCD, HW12-PEG-PCD, mPEG-b-
PCC-g-GEM-g-DC and mPEG-b-PCC-g-FC-g-DC copolymers
1
MBC monomer was characterized by H NMR (500 MHz, DMSOꢀd6), which
showed peaks corresponding to phenyl proton at δ7.5, methylene protons present in the
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carbonate ring at δ4.2 and δ4.7. Methyl and benzyl protons were present at δ1.3 and
δ5.2, respectively (data not shown). MCC was obtained by hydrogenation of MBC and
characterized by 1H NMR (500 MHz, chloroformꢀd) δ13.35 (s, 1H), 4.57 (d, 2H), 4.32 (d,
2H) and 1.18 (s, 3H) (data not shown).
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O
O
OH OH
OH OH
A
O
iii
i
ii
O
O
O
O
H
y
CH3
O
O
O
x
O
O
O
OH
O
O
O
v
iv
O
H
y
CH3
O
O
O
x
O
OH
O
O
O
H
CH3
O
O
O
O
O
x
y
z
O
OC12H25
O
NH
5
NH
S
NH
B
HO
O
O
O
OH
Figure 2
DC was conjugated to the carboxyl group of MCC by EDC/HOBT coupling and final
1
product was purified by column chromatography and characterized by H NMR (500
MHz, chloroformꢀd), with the following characteristic peaks: δ4.50 (d, 2H), 4.2 (d, 4H),
1.65 (m, 2H), 1.4ꢀ1.2 (m, 21H) and 0.85 (t, 3H) (data not shown). Ring opening
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polymerization of MDC and MalꢀPEG in the presence of 1,8ꢀdiazabicycloundecꢀ7ꢀene
(DBU) catalyst at RT for 3 h under N2 atmosphere yielded MALꢀPEGꢀPDC copolymer.
1H NMR (500 MHz, DMSOꢀd6) spectra of MalꢀPEGꢀPDC showed peaks corresponding
to PEG (−CH2−CH2−O) at δ3.5, PCC (−COꢀOꢀCH2ꢀ & CH3ꢀC(CO)ꢀCH2) at δ4.2 (m, 4H),
CH3ꢀ(CH2)9ꢀ at δ0.9 (t, 3H), CH3ꢀ(CH2)9ꢀ at δ1.2ꢀ1.45 (bs, 18H), CH3ꢀ(CH2)9ꢀCH2 & ꢀNHꢀ
CH2ꢀCH2 at δ1.5ꢀ1.8 (m, 4H), CH3ꢀ(CH2)9ꢀCH2ꢀCH2ꢀ at δ4.02 (m, 2H), ꢀNHꢀCH2ꢀCH2 & ꢀ
NHꢀCOꢀCH2ꢀCH2 at δ2.5ꢀ3.2 (m, 4H), ꢀNHꢀCOꢀCH2ꢀCH2 at δ4.6 (m, 2H), ꢀNHꢀCOꢀCH2ꢀ
CH2 at δ8.4 (m, 1H), ꢀCOꢀCH=CHꢀCOꢀ at δ7.2ꢀ7.5 (t, 2H) (Fig. S1).
GE11 peptide was conjugated to MALꢀPEGꢀPDC via the addition reaction. GE11ꢀPEGꢀ
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PCD copolymer was lyophilized and characterized using H NMR (500 MHz, DMSOꢀ
d6), which showed peaks corresponding to PEG (−CH2−CH2−O) at δ3.5, PCC (−COꢀOꢀ
CH2ꢀ & CH3ꢀC(CO)ꢀCH2) at δ4.2 (m, 4H), CH3ꢀ(CH2)9ꢀ at δ0.9 (t, 3H), CH3ꢀ(CH2)9ꢀ at
δ1.2ꢀ1.45 (bs, 18H), Ile CβꢀH at δ 2.6 (m, 1H), CαꢀH of (Cys,Tyr, His, Trp, Thr, Pro, Gln,
Asn, Val & Ile) amino acids at δ4.2ꢀ4.9 (m, 12H), Gly –CH2ꢀNH at δ 4.4 (m, 2H), Ile Cγꢀ
H2 at δ 1.5 (m, 2H), Ile CδꢀH3 at δ 0.9 (t, 3H), Ile CβꢀHꢀCH3 at δ 1.1 (m, 3H), Val CβꢀH at
δ 2.5 (m, 1H), Val CβHꢀCH3 and Val CγꢀH3 at δ 0.9 (t, 6H), Asn CβꢀH2 at δ 2.5ꢀ2.9 (m,
2H), Gln CβH2 and Gln CγꢀH2 at δ 2.1ꢀ2.3 (m, 4H), Pro CβꢀH2 at δ 2.0ꢀ2.2 (m, 2H), Pro
CγꢀH2 at δ 1.9ꢀ2.1 (m, 2H), Pro CδꢀH2 at δ 3.3ꢀ3.5 (m, 2H), Thr –CHꢀCH(CH3)ꢀOH at δ
4.6 (m, 1H), Thr –CHꢀCH(CH3)ꢀOH at δ 1.2 (m, 3H), Tyr CβꢀH2 at δ 3.2ꢀ3.45 (m, 2H),
Tyr–CH2ꢀC6H4ꢀOH at δ 6.5ꢀ6.9 (m, 4H), Trp CβꢀH2 at δ 3.0ꢀ3.4 (m, 2H), Trp –CH2ꢀ
C8H5NH at δ 7.1ꢀ8.3 (m, 5H), His CβꢀH2 at δ 2.9ꢀ3.15 (m, 2H), His –CH2ꢀC3H2N2H at δ
8.2ꢀ8.7 (m, 2H), Cys CβꢀH2 at δ 2.9ꢀ3.2 (m, 2H) (Fig. S2). The peaks at 6.5–9.3 ppm
confirmed the successful conjugation of GE11 peptide to the copolymer.25 Similarly,
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HW12ꢀPEGꢀPCD was synthesized and characterized using 1H NMR (500 MHz, DMSOꢀ
d6), which showed peaks corresponding to PEG (−CH2−CH2−O) at δ3.5, PCC (−COꢀOꢀ
CH2ꢀ & CH3ꢀC(CO)ꢀCH2) at δ4.2 (m, 4H), CH3ꢀ(CH2)9ꢀ at δ0.9 (t, 3H), CH3ꢀ(CH2)9ꢀ at
δ1.2ꢀ1.45 (bs, 18H), Ala CαHꢀCH3 at δ1.3 (s, 3H), CαꢀH of (Cys, Tyr, His, Trp, Thr, Pro,
Ser, & Ala) amino acids at δ3.4ꢀ4.9 (m, 12H), Ser CβꢀH2 at δ 3.2ꢀ4.12 (m, 2H), Pro Cβꢀ
H2 at δ 2.0ꢀ2.2 (m, 2H), Pro CγꢀH2 at δ 1.9ꢀ2.1 (m, 2H), Pro CδꢀH2 at δ 3.3ꢀ3.5 (m, 2H),
Thr –CHꢀCH(CH3)ꢀOH at δ 4.6 (m, 1H), Thr –CHꢀCH(CH3)ꢀOH at δ 1.2 (m, 3H), Tyr Cβꢀ
H2 at δ 3.2ꢀ3.45 (m, 2H), Tyr –CH2ꢀC6H4ꢀOH at δ 6.5ꢀ6.9 (m, 4H), Trp CβꢀH2 at δ 3.0ꢀ3.4
(m, 2H), Trp –CH2ꢀC8H5NH at δ 7.1ꢀ8.3 (m, 5H), His CβꢀH2 at δ 2.9ꢀ3.15 (m, 2H), His –
CH2ꢀC3H2N2H at δ 8.2ꢀ8.7 (m, 2H), Cys CβꢀH2 at δ 2.9ꢀ3.2 (m, 2H) (Fig. S3).
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Fluorescence cadaverine (FC) and dodecanol (DC) were conjugated to the
carboxyl groups of mPEGꢀPCC by EDC/HOBT coupling reaction. At the end of reaction,
FC conjugated copolymer was purified using isopropanol and diethyl ether and by
1
extensive dialysis and lyophilized. H NMR (500 MHz, DMSOꢀd6) spectra of mPEGꢀbꢀ
PCCꢀgꢀFCꢀgꢀDC showed peaks corresponding to PEG (−CH2−CH2−O) at δ3.5, PCC
(−COꢀOꢀCH2ꢀ & CH3ꢀC(CO)ꢀCH2) at δ4.2 (m, 4H), CH3ꢀ(CH2)9ꢀ at δ0.9 (t, 3H), CH3ꢀ
(CH2)9ꢀ at δ1.2ꢀ1.45 (bs, 18H), ꢀCOꢀNHꢀCH2ꢀ(CH2)3ꢀ at δ1.2ꢀ1.6 (m, 6H), ꢀCSꢀNHꢀCH2ꢀ
& ꢀCOꢀNHꢀCH2ꢀ at δ2.6ꢀ3.2 (m, 4H), ꢀCSꢀNHꢀCH2ꢀ & ꢀCOꢀNHꢀCH2ꢀ at δ (m, 2H), ꢀNHꢀ
C6H3(CO2H)ꢀC13H6O2(OH) at δ6.1ꢀ7.9 (m, 9H) (Fig. S4).
GEM and DC were conjugated to the carboxyl groups of mPEGꢀPCC to get
GEMꢀconjugated copolymers. 1H NMR spectra of mPEGꢀPCC showed peaks
corresponding to PEG (−CH2−CH2−O) at δ3.5 and PCC (−CH2−) at δ4.2. Absence of
benzene protons at δ7.3 confirmed complete hydrogenation along with the presence of ꢀ
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COOH peaks at δ12ꢀ14 as reported earlier.17 Based on the peak integrals of mPEG and
PCC protons, an average molecular weight of mPEGꢀPCC was calculated to be ~9600
Da with 29 PCC units. GEM and DC were conjugated onto the carboxyl groups of
mPEGꢀPCC by EDC/HOBT coupling reaction and GEM conjugated copolymer was
purified by precipitation in isopropanol and diethyl ether followed by extensive dialysis
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and lyophilization. H NMR (500 MHz, DMSOꢀd6) of mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC
showed peaks corresponding to PEG (−CH2−CH2−O) at δ3.5, PCC (−COꢀOꢀCH2ꢀ &
CH3ꢀC(CO)ꢀCH2) at δ4.2 (m, 4H), CH3ꢀ(CH2)9ꢀ at δ0.9 (t, 3H), CH3ꢀ(CH2)9ꢀ at δ1.2ꢀ1.45
(bs, 18H), GEM 5’ꢀ and 3’–OH groups at δ6.18ꢀ6.39 (m, 2H), ꢀCOꢀNHꢀ at δ8.26 (m, 1H),
ꢀCOꢀNꢀCH=CHꢀ at δ8.15 (m, 1H) and ꢀCOꢀNꢀCH=CHꢀ at δ5.35 (m, 1H) (Fig. S5).
3.2. Formulation development and characterization
Mixed micellar formulations have been successfully applied for drug delivery
application.32, 33 Various block copolymers at a certain molar or weight ratios are used
for mixed micelles formation and they formed single polymeric micellar system rather
than two separate micelles.34, 35 In our previous report, we synthesized mPEGꢀbꢀPCCꢀgꢀ
GEMꢀgꢀDC copolymer for GEM delivery by passive targeting only. In the present study,
we have conjugated GE11 peptide to our polymeric micelles for EGFR receptor
mediated active targeting. However, GE11 peptide, GEM and DC conjugation in the
same micellar system created synthetic complexity and reduced drug loading (data not
shown). To avoid these obstacles, we synthesized GE11ꢀPEGꢀPCD and mPEGꢀbꢀPCCꢀ
gꢀGEMꢀgꢀDC copolymers separately and prepared EGFR targeted mixed micelles by
combining GE11ꢀPEGꢀPCD (10ꢀ30% weight ratio) and mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC (70ꢀ
90% weight ratio) copolymers by film hydration. In addition, we used HW12ꢀPEGꢀ
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PCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC (30/70 w/w) mixed micelles. mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀ
DC had a mean particle size of 26 ± 3 nm with a PDI of 0.27. We measured particle size
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HW12ꢀlinked (30/70 w/w ratio) mixed micelles to determine effect of GE11 or HW12
conjugation on size and surface charge of mixed micelles. Unmodified micelles had a
mean particle size of 26 ± 3 nm (PDIꢀ0.27), whereas GE11ꢀlinked mixed micelles (10ꢀ
30% weight ratio) and HW12ꢀlinked mixed micelles (30% weight ratio) had a mean
particle size of 51 ± 5 (PDIꢀ0.229), and 53 ± 0.8 (PDIꢀ0.188), respectively indicating the
conjugation of GE11 or HW12 peptide did not significant change the size of mixed
micelles than mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC micelles. We observed increased in surface
ζ–potential of GE11ꢀlinked mixed micelles (10ꢀ30% weight ratio) ꢀ6.8 ± 2 mV and
HW12ꢀlinked mixed micelles (30% weight ratio) ꢀ5.9± 0.2 mV as compared to
unmodified micelles ꢀ4.6 ± 0.2 mV. GEM loading in mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC was
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calculated by H NMR, which showed GEM 5’ꢀand 3’–OH protons at δ 5.90 and δ 6.2
respectively. The presence of amide proton at δ8.26 confirmed the formation of amine
bond with pendant carboxyl group of mPEGꢀPCC, which is in agreement with the
literatures.7, 36, 37 GEM loading in mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC was ~14.8% w/w by
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integrating and calculating amide protons peak at δ8.26 (Fig. S5). However HꢀNMR
analysis can only confirm the chemical conjugation of GEM to the polymer backbone
but cannot predict accurate drug loading. Therefore, GEM loading in mPEGꢀbꢀPCCꢀgꢀ
GEMꢀgꢀDC was further determined by alkaline hydrolysis and then analyzed by HPLCꢀ
UV method as reported previously.17 GEM loading in micelles was found ~12% w/w
(data not shown). Although, our mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC copolymer (12% w/w drug
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loading) showed higher drug loading than previously reported PEGylated GEM
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derivatives (0.98ꢀ6.79% w/w drug loading range), but it was lower than reported
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GEM−squalene nanoassemblies (GEM loading of 41% w/w).7,
PEGylated
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gemcitabine nanoassemblies were achieved by addition of PEG−squalene or
PEG−cholesterol (1:0.7 w/w) which showed decrease in gemcitabine loading but higher
efficacy than gemcitabine−squalene nanoassemblies. Therefore, our formulation is a
balanced between PEG coating (55%) and GEM loading (12 % w/w). Though our
formulation needs improvement to increase GEM payload but this polymeric drug
conjugate offers numerous advantages over the squalenoylated nanoassemblies.
Gemcitabine−squalene nanoassemblies are rapidly cleared from the bloodstream by the
reticuloꢀendothelial system (RES) due to absence of PEG coating.16 PEG coating in
outer shell needed for stealth effect and to reduce opsonization and blood clearance.
Our micelles had a much smaller particle size (25ꢀ50 nm) than gemcitabine−squalene
nanoassemblies (140 nm) and presence of PEG (~55%) coating in our formulation can
provide stealth effect. In our previous study, we found a controlled release pattern of
GEM (39% GEM release in 10 days) from the micelles even in the presence of
proteolytic enzyme Cathepsin B.17 Based on that we assume that all our formulations
had similar drug release patterns.
3.3. EGFR-mediated uptake of GEM conjugated GE11-modified mixed micelles
To convert mixed micellar system into cell surface probe as well as to verify
receptor mediated GEM delivery in vitro and in vivo, we conjugated FC as fluorescent
tag to carboxyl terminated mPEGꢀPCC copolymer via amide bond. Various fluorescein
molecules are widely used to label nanoparticles for intracellular and intraꢀtumoral
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distribution of nanoparticles.41, 42 We selected FC as a fluorescein substance since prior
reports showed that carboxylic acids of proteins, water soluble biopolymer and
difluorinated cyclooctyne (DIFO) chemically conjugated with the primary aliphatic amine
of FC through EDAC coupling for subcellular distribution, interaction between cell
surface receptor and ligand, and in vivo imaging.43, 44 Therefore, we synthesized FC
conjugated mPEGꢀbꢀPCCꢀgꢀFCꢀgꢀDC copolymer. We prepared FC labeled GE11ꢀlinked
mixed micelles (10ꢀ30% weight ratios) and unmodified micelles. Before cellular uptake
of labeled mixed micelles in MIA PaCaꢀ2 cells, we determined EGFR expression levels
in MIA PaCaꢀ2 cells by flow cytometry. MIA PaCaꢀ2 cells showed high expression of
EGFR (Fig. S6). To determine whether GE11 peptide enhanced the cellular uptake by
EGFR expressing MIA PaCaꢀ2 cells, cells were incubated with FC labeled GE11ꢀlinked
mixed micelles and unmodified micelles for 12 h. The fluorescein images are shown in
Fig. S7 for qualitative analysis of cellular uptake. MIA PaCaꢀ2 cells treated with GE11ꢀ
linked mixed micelles at 30% w/w ratios showed significantly higher fluorescence than
GE11ꢀlinked mixed micelles at 10 and 20% weight ratios and unmodified micelles (Fig.
S7 A, B, C&D). To confirm, GE11 and not HW12 is an EGFR ligand, we also prepared
FC labeled HW12ꢀlinked mixed micelles at 30% weight ratio and cells were incubated
with FC labeled GE11ꢀlinked mixed micelles and HW12ꢀlinked mixed micelles at 30%
weight ratio for 12 h respectively. The fluorescein images of MIA PaCaꢀ2 cells treated
with GE11ꢀlinked mixed micelles at 30% weight ratio showed stronger fluorescence
compared to HW12ꢀlinked mixed micelles, suggesting that mixed micelles containing
GE11 peptide could facilitate cellular delivery of conjugated drugs (Fig. S7E). To
further confirm the ligand receptor interaction and specific role of GE11 peptide, we preꢀ
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saturated MIA PaCaꢀ2 cells with free GE11 peptide prior to incubation with GE11ꢀlinked
mixed micelles at 30% weight ratio. Results depicted in (Fig. S7 F) showed significant
reduction of the fluorescence signal and comparable fluorescence signal of HW12ꢀ
linked mixed micelles treated cells (Fig. S7 E), supporting EGFR receptor mediated
uptake. Cellular uptake results were further analyzed by flow cytometry for quantitative
analysis. Results delineated in Fig. 3 A, B, C & D clearly indicated that the mean
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fluorescence intensity of GE11ꢀlinked mixed micelles (30% w/w) treated cells were 1.2
fold and 3.1 fold higher than unmodified micelles and HW12ꢀlinked mixed micelles
respectively. However, we observed the cellular uptake of HW12ꢀlinked mixed micelles
was lower than unmodified micelles. The exact mechanism for this lower uptake of
peptide linked mixed micelles require additional investigation, but it may be due to the
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increase in hydrophillicity of HW12 peptide. To illustrate this point, we calculated
hydrophobicity for GE11 (YHWYGYTPQNVI) peptide which was 50 whereas for HW12
(HYPYAHPTHPSW) 33.33 suggesting, HW12ꢀlinked micelles more hydrophilic as
compared to GE11ꢀlinked mixed micelles. This increase in hydrophilicity is expected to
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decrease cellular uptake as shown in Fig 3. When preꢀtreated cells with free GE11
peptide prior incubation of cells with GE11ꢀlinked mixed micelles (30% w/w) mixed
micelles, the mean fluorescence intensity decreased significantly (Fig. 3 B & D).
3.4. In vitro cytotoxicity studies
Density of surface ligand is critical to target cell binding in vitro as well as
pharmacokinetic profiles in vivo. Our cellular uptake study clearly indicated that 30%
w/w ratios of GE11ꢀlinked mixed micelles was superior to HW12ꢀlinked mixed micelles
and unmodified micelles. Therefore, we prepared GE11ꢀlinked mixed micelles (GE11ꢀ
PEGꢀPCD/mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC) at 30% w/w ratios to enhance receptor
mediated cellular uptake. Similarly, we prepared HW12ꢀlinked mixed micelles using
HW12ꢀPEGꢀPCD and mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC copolymers at 30% weight ratio. For
cytotoxicity studies, MIA PaCaꢀ2 cells were treated with GE11ꢀlinked mixed micelles,
HW12ꢀlinked mixed micelles, unmodified micelles and free GEM. Free GEM exhibited
highest cytotoxicity within the studied concentration range compared to GE11ꢀlinked
mixed micelles, HW12ꢀlinked mixed micelles, unmodified micelles, indicated by lowering
cell viability (Fig. 3E). Although free GEM showed maximum toxicity but its plasma
instability and lack of target specificity would restrict its in vivo tumor inhibition
capability. GE11ꢀlinked mixed micelles showed a higher cytotoxicity than HW12ꢀlinked
mixed micelles and unmodified micelles at all GEM concentrations tested, further
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demonstrating that GE11 peptide play important role for antiꢀtumor efficacy in GE11
modified mixed micelles (Fig. 3E). The IC50 value was calculated for GE11ꢀlinked mixed
micelles (95.32 nM), which was 1.2 fold and 1.3 fold lower than unmodified micelles
(116.91 nM) and HW12ꢀlinked mixed micelles (126.58 nM) respectively. Our results are
in good agreement with the literature. For example, Chen et al. demonstrated that a
GE11 density of 10% is enough to enable EGFRꢀligand binding.25 In their study, the cell
viability for liposomes with a 15% GE11 density was similar to that for liposomes with a
10% GE11 density.
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3.5. GE11 peptide conjugated mixed micelles for targeting EGFR expressing
pancreatic tumor as well as tumor neovasculature
After in vitro efficacy of GE11ꢀlinked mixed micelles, we determined targeting
efficiency of GE11ꢀlinked mixed micelles in orthotopic pancreatic tumor bearing athymic
nude mice by systemic administration of FC labeled GE11ꢀlinked mixed micelles,
HW12ꢀlinked mixed micelles and unmodified micelles. FC labeled GE11ꢀlinked mixed
micelles injected mouse tumor sections showed higher fluorescence intensity at 24 h
post injection than FC labeled HW12ꢀlinked mixed micelles and unmodified micelles
injected tumor sections (Fig. 4), supporting that GE11 peptide facilitated distribution and
accumulation of drug molecule into the tumor. Our results are in good agreement with
the work of Xu et al. who demonstrated enhanced accumulation of GE11 peptide
conjugated gelatin nanoparticles in subcutaneous Pancꢀ1 pancreatic tumor model
especially at early time points.26 Previous reports showed that tumor cells as well as
tumor derived endothelial cells express EGFR.46 Toward confirming GE11ꢀlinked mixed
micelles can target tumor as well as tumor neovaculature under in vivo condition, we
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stained same tumor sections with rabbit polyclonal antiꢀVEꢀcadherin antibody (markers
of tumor endothelial cells) and counterstained with Alexa Fluor 594 F(ab’)2 fragment of
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goat antiꢀrabbit IgG (H+L) secondary antibody. When tumor sections were observed
under a fluorescent microscope, the merged image of FC labeled GE11ꢀlinked mixed
micelles mouse tumor sections showed that targeted mixed micelles deliver FC dye to
tumor as well as tumor vasculature. In contrast, FC labeled HW12ꢀlinked mixed micelles
and unmodified micelles did not deliver FC dye to tumor vasculature. Therefore, our
result demonstrated in Fig. 4A clearly indicated that GE11ꢀlinked mixed micelles not
only capable of delivering drug to tumor cells but also effective in distributing drug to
tumor vasculature.
3.6. In vivo efficacy in orthotopic pancreatic tumor model
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After demonstrating the ability of GE11ꢀlinked mixed micelles to target EGFR
expressing tumor as well as tumor endothelial cells, we investigated whether GE11ꢀ
linked mixed micelles could inhibit orthotopic pancreatic tumor growth. After
orthotopically implanting MIA PaCaꢀ2/luciferase tumor cells in the pancreas, mice were
intravenously administered GE11ꢀlinked mixed micelles, HW12ꢀlinked mixed micelles,
unmodified micelles and free GEM at an equivalent dose of 40 mg/kg GEM three times
in a week after 12 days of cell implantation. We selected this dose according to our
previous report and this dose is lower than the maximum tolerated dose (MTD) of
GEM.17 In vivo efficacy of GEM in mice numerous reports showed wide range of its
dose ranging from 7mg/kg to 500 mg/kg for intraperitoneal route and intravenous route
of administration.40, 47ꢀ49 At day 12, all mice in different group were noninvasively imaged
for luciferase bioluminescence to determine the tumor growth and randomized for
treatment. Tumor growth inhibitions of different formulations were monitored by
measuring luciferase bioluminescence at different time points during the treatment (day
12, 16, 20, 24, 26 and 30) and photon counts was calculated by Living Image® 3.2
software (Caliper Life Science). Final bioluminescence images were taken at day 30
and images depicted in Fig. 5A clearly showed significant tumor growth inhibition in
mice treated with GE11ꢀlinked mixed micelles compared to HW12ꢀlinked mixed
micelles, unmodified micelles and free GEM. We compared photon counts between
different groups after plotting photon value versus days after treatment (Fig. 5B). This
result further strengthened our in vitro observation and signified GE11 important role for
EGFR targeting. HW12 decorated HW12ꢀlinked mixed micelles showed minimal tumor
growth effect, supporting that GE11 peptide is EGFR targeting ligand not HW12
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peptide. Unmodified micelles showed better tumor growth inhibition than free GEM. We
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removed all tumors from different group, measured tumor volume and weight and taken
images of tumor after sacrificing the mice at the end of the experiment. Tumor images
as shown in Fig. 5C confirmed remarkable tumor growth inhibition capability of GE11ꢀ
linked mixed micelles compared to HW12ꢀlinked mixed micelles, unmodified micelles
and free GEM. This was further confirmed by measuring the tumor volume and weight
(Fig. 6 A&B). Mouse body weight was taken during the treatment course to monitor the
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adverse effect of GEM. Fig. 6C clearly indicated that our in vivo GEM dose (40 mg/kg)
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did not show any abnormal body weight change in all the experimental groups.
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HW12-linked mixed micelles
Free GEM
Unmodified micelles
GE11-linked mixed micelles
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Days after treatment
Figure 6
Immunohistochemical analysis was done for Kiꢀ67, cleaved caspaseꢀ3 and H&E
staining of tumor cryosections obtained from all treated groups. Unmodified micelles
treated mice showed reduction in Kiꢀ67 staining and increase in cleaved caspaseꢀ3
staining compared to the free GEM.
Mice treated with GE11ꢀlinked mixed micelles showed least Kiꢀ67 staining and
highest cleaved caspaseꢀ3 staining (Fig. 7A&B). GE11ꢀlinked mixed micelles and
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