EGFR-Targeted Cationic Polymeric Mixed Micelles for Codelivery of Gemcitabine and miR-205 for Treating Advanced Pancreatic Cancer

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00355

Gemcitabine (GEM), a first-line chemotherapy for pancreatic cancer undergoes rapid metabolism and develops chemoresistance after repeated administration. We previously demonstrated that the combination of GEM and miR-205 provides an effective therapeutic strategy to sensitize GEM-resistant pancreatic cancer cells. Since epidermal growth factor receptor (EGFR) is overexpressed in pancreatic cancer cells, in this study, we aimed to deliver mixed micelles containing GEM and miR-205 decorated with EGFR-targeting cetuximab (C225) monoclonal antibody for targeted therapy. Cetuximab C225 was conjugated to malemido-poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol (C225-PEG-PCD) to prepare mixed micelles with mPEG-b-PCC-g-GEM-g-DC-g-TEPA for targeted codelivery of GEM and miR-205. This mixed micelle formulation showed a significant enhancement in EGFR-mediated cellular uptake in GEM-resistant MIA PaCa-2^R cells. Further, an enhanced tumor accumulation of C225-micelles conjugated with near-infrared fluorescent Cy7.5 dye and Dy677-labeled miR-205 in orthotopic pancreatic tumor bearing NSG mice was evident after systemic administration. In addition, inhibition of tumor growth was also observed with increased apoptosis and reduced EMT after treatment with C225-micelles containing GEM and miR-205. Therefore, we believe that the targeted delivery of GEM and miR-205 in combination could be a novel strategy for treating advanced pancreatic cancer.

Full text of DOI:10.1021/acs.molpharmaceut.7b00355

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Article
EGFR-Targeted Cationic Polymeric Mixed Micelles for Co-delivery of
Gemcitabine and miR-205 for treating Advanced Pancreatic Cancer
Goutam Mondal, Saud Almawash, Amit Kumar Chaudhary, and Ram I. Mahato
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00355 • Publication Date (Web): 18 Jul 2017
Downloaded from http://pubs.acs.org on July 19, 2017
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EGFR-Targeted Cationic Polymeric Mixed Micelles for Co-delivery of Gemcitabine
and miR-205 for treating Advanced Pancreatic Cancer
Goutam Mondal, Saud Almawash, Amit Kumar Chaudhary and Ram I. Mahato*
Department of Pharmaceutical Sciences, University of Nebraska Medical Center,
Omaha, NE 68198
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*
Corresponding Author
Ram I. Mahato, Ph.D.
Department of Pharmaceutical Sciences,
University of Nebraska Medical Center,
9
86025 Nebraska Medical Center
Omaha, NE 68198
Tel: 402ꢀ559ꢀ5422; Fax: 402ꢀ559ꢀ9543
Eꢀmail: ram.mahato@unmc.edu
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ABSTRACT
Gemcitabine (GEM), a firstꢀline chemotherapy for pancreatic cancer undergoes
rapid metabolism and develops chemoresistance after repeated administration. We
previously demonstrated that the combination of GEM and miRꢀ205 provides an
effective therapeutic strategy to sensitize GEMꢀresistant pancreatic cancer cells. Since
epidermal growth factor receptor (EGFR) is overexpressed in pancreatic cancer cells, in
this study, we aimed to deliver mixed micelles containing GEM and miRꢀ205 decorated
with EGFRꢀtargeting cetuximab (C225) monoclonal antibody for targeted therapy.
Cetuximab C225 was conjugated to malemidoꢀpoly(ethylene glycol)ꢀblockꢀpoly(2ꢀ
methylꢀ2ꢀcarboxylꢀpropylene carbonateꢀgraftꢀdodecanol (C225ꢀPEGꢀPCD) to prepare
mixed micelles with mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA for targeted coꢀdelivery of
GEM and miRꢀ205. This mixed micelle formulation showed a significant enhancement in
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EGFRꢀmediated cellular uptake in GEMꢀresistant MIA PaCaꢀ2 cells. Further, an
enhanced tumor accumulation of C225ꢀmicelles conjugated with nearꢀinfrared
fluorescent Cy7.5 dye and Dy677ꢀlabeled miRꢀ205 in orthotopic pancreatic tumor
bearing NSG mice was evident after systemic administration. In addition, inhibition of
tumor growth was also observed with increased apoptosis and reduced EMT after
treatment with C225ꢀmicelles containing GEM and miRꢀ205. Therefore, we believe that
the targeted delivery of GEM and miRꢀ205 in combination could be a novel strategy for
treating advanced pancreatic cancer.
KEY WORDS: pancreatic cancer, gemcitabine, EGFR, targeted drug delivery, polymeric
mixed micelles, C225
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INTRODUCTION
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer
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related deaths in the United States, with a fiveꢀyear survival rate of less than ~8%.
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Pancreatic cancer treatment faces multiple challenges due to the presence of
desmoplasia and emergence of chemoresistance resulting in recurrence and metastatic
spread. Gemcitabine (Gemzar®, GEM), a nucleoside analogue, is the firstꢀline
chemotherapy to treat pancreatic cancer, with a mere 6.8 month survival rate. However,
GEM is rapidly deaminated by cytidine deaminase (CDA) to its inactive metabolite 2, 2′ꢀ
2ꢀ4
difluorodeoxyuridine (dFdU), resulting in its short in vivo halfꢀlife (8−17 min). Further,
dense desmoplastic pancreatic tumor tissue impedes effective GEM delivery to the
cancer cells. GEM was encapsulated into nanoparticles and liposomes to overcome
these limitations, which exhibited greater anticancer activity than GEM alone. However,
poor encapsulation efficiency, rapid leakage and reticuloendothelial system (RES)
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mediated uptake of these particles limit their effectiveness.
Therefore, GEM was
conjugated to stearic acid and formulated into PEG2000ꢀhydrazineꢀC18 pHꢀsensitive
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micelles, which showed improved efficacy. Recently, Chitkara et al. conjugated GEM to
poly(ethylene glycol)ꢀblockꢀpoly(2ꢀmethylꢀ2ꢀcarboxylꢀpropylene carbonate) (PEGꢀPCC).
This drugꢀpolymer conjugate could selfꢀassemble into micelles and show sustained drug
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release and negligible plasma metabolism of the drug . Intravenous administration of
these micelles into tumorꢀbearing NSG mice showed significant inhibition of tumor
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growth. Moreover, Kattel et al. demonstrated that GEM and dodecanol (DC)
conjugated mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDC formed micelles increased GEM accumulation
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in the orthotopic pancreatic tumor by 2.5 fold compared to free GEM after systemic
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administration.
Repeated administration of GEM at a high dose is known to result in
chemoresistance and tumor relapse. A similar limitation is also evident for GEM
derivatives/conjugates, and even in clinical setꢀup. Further, several phase II trials have
been conducted to improve the overall survival of the patients using GEM in
combination with cisplatin, 5ꢀfluorouracil (5ꢀFU), irinotecan, and oxaliplatin. Although
phase II results were encouraging, phase III trials failed to improve the survival rate over
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GEM.
A recent report showed a median survival of 8.5 months in the combination of
albuminꢀbound (nab)ꢀpaclitaxel and GEM in patients with metastatic pancreatic cancer
compared to 6.7 months for the patients treated with GEM alone and postꢀtreatment
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oneꢀyear survival rate of 35% vs. 22%. Therefore, different combination approaches
are urgently needed to combat with advanced pancreatic cancer.
PDAC harbors a small population (1ꢀ2%) of selfꢀrenewing cancer stem cells
(CSCs) which are responsible for tumor initiation, propagation, chemoresistance and
recurrence. These CSCs are regulated by small nonꢀcoding RNAs (17ꢀ25 nucleotides)
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known as miRNAs.
Singh et al. identified aberrantly expressed miRNAs in CSCs
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isolated from GEMꢀresistant MIA PaCaꢀ2 cancer cells and in tumors of pancreatic
cancer patients, whereby there was significant downregulation of miRꢀ205, which
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controls several genes involved in cancer progression, invasion, and metastasis.
Recently, Chaudhary et al. showed that miRꢀ205 sensitized GEM resistance MIA PaCaꢀ
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cells and inhibited proliferation of pancreatic CSCs by stably overexpressing miRꢀ
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05. Despite its dominant role in cancer, the clinical potential of miRꢀ205 has not
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been realized due to the challenges involved in its in vivo delivery. Thus, combination
of drug with miRꢀ205 could be viable strategy for enhancing the antitumor efficacy of
small drug molecules in treating advanced pancreatic cancer. To coꢀformulate GEM and
miRꢀ205 mimic into micelles, Mittal et al. synthesized GEM conjugated polymer,
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poly(ethylene
glycol)ꢀblockꢀpoly(2ꢀmethylꢀ2ꢀcarboxylꢀpropylene
carbonateꢀgraftꢀ
by
dodecanolꢀgraftꢀtetraethylenepentamine)
(PEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA),
attaching cationic pendant chains. The cationic polymer formed stable micelles with
miRꢀ205 and exhibited high transfection efficiency, with minimal cytotoxicity. The
micelles carrying GEM and miRꢀ205 were effective in chemosensitizing GEMꢀresistant
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MIA PaCaꢀ2 and significantly reduced tumor growth in pancreatic cancer xenograft
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model.
We previously used ectopic pancreatic tumor model to check combination effect
of miRꢀ205 mimic and GEM where tumors were grown under the skin, not in the
pancreas. A major disadvantage of this tumor model is that after tumor formation; tumor
cells do not develop actual tumor microenvironment. In contrast, in the orthotopic
model, tumor cells are implanted in the pancreas where tumor cells experience similar
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tumor microenvironment. Thus, systemic delivery of miRꢀ205 mimic and GEM to
orthotopic pancreatic tumor is an attractive approach for treating advanced pancreatic
cancer.
Micelleꢀbased targeted drug delivery could be the most efficacious approach for
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inhibiting tumor growth with reduced systemic toxicity . Global efforts are being
directed towards developing nanomedicine that can target overexpressed receptors to
facilitate selective drug binding and internalization, resulting in enhanced therapeutic
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efficacy and reduced side effects.
We selected epidermal growth factor receptor
(
EGFR) since it is overexpressed in almost 95% pancreatic cancer patients.
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Furthermore, EGFR overexpression is correlated with disease progression.
In our
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previous studies, we found GE11 peptide (YHWYGYTPQNVI)ꢀlinked mixed micelles
containing GEM showed MIA PaCaꢀ2 cells derived orthotopic tumor growth inhibition by
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~
1.5 fold compared to unmodified micelles. Recently, monoclonal antibody (MAb)
C225 conjugated antiꢀEGFR interleukins (ILs) and MAb C225 conjugated gold
nanoparticles showed excellent in vivo efficacy in breast cancer and advanced
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pancreatic cancer, respectively.
Although fullꢀlength antibody has more mitogenic
and proꢀangiogenic capability than a peptide, we selected cetuximab (C225) for
targeting EGFR to achieve excellent tumor growth inhibition in advanced pancreatic
cancer. C225 was approved by the FDA for the treatment of EGFRꢀpositive colorectal
cancer and has now also been used for the treatment of head and neck, lung and
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pancreatic cancers.
In this study, we conjugated C225 to malemidoꢀpoly(ethylene glycol)ꢀblockꢀ
poly(2ꢀmethylꢀ2ꢀcarboxylꢀpropylene carbonateꢀgraftꢀdodecanol (C225ꢀPEGꢀPCD). We
also synthesized mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA and prepared mixed micelles
using C225ꢀPEGꢀPCD and PEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA for enhanced GEM and
miRꢀ205 delivery to EGFR receptor overexpressing pancreatic tumor cells. We showed
that C225ꢀmicelles containing conjugated GEM and complexed miRꢀ205 exhibited a
better cytotoxic effect compared to nonꢀtargeted IgGꢀmicelles and unmodified micelles.
Systemic administration of GEM conjugated miRꢀ205 complexed C225ꢀmicelles
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exhibited significant tumor growth inhibition in MIA PaCaꢀ2 cells derived orthotopic
pancreatic tumor bearing mice via apoptosis of tumor cells.
EXPERIMENTAL SECTION
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Materials
Gemcitabine hydrochloride was procured from AK Scientific (Union City, CA).
Immunoglobulin
dimethylamino)propyl) carbodiimide (EDC), triethylamine (TEA), dodecanol (DC),
hydroxybenzotriazole (HOBT), benzyl bromide, Tin(II) 2ꢀethylhexanoate (Sn(Oct) ), 1,8ꢀ
G
(IgG),
tetraethylene
pentamine
(TEPA),
1ꢀethylꢀ3ꢀ(3ꢀ
(
2
diazabicyclo[5.4.0]undecꢀ7ꢀene
(DBU),
puromycin
dihydrochloride,
methoxy
poly(ethylene glycol) (mPEG, Mn = 5000, PDI = 1.03), 2,2ꢀbis(hydroxymethyl) propionic
acid, were purchased from SigmaꢀAldrich (St. Louis, MO). C225 was purchased from
UNMC pharmacy. Malemide PEG hydroxyl (MW 5000) was procured from JenKem
Technology (Beijing, China). Matrigel matrix basement membrane was procured from
Corning (Chicago, IL). Tris(2ꢀcarboxyethyl) phosphine hydrochloride (TCEP) was
procured from Biosynth Chemistry & Biology (Itasca, IL). Polybrene was purchased from
Santa Cruz Biotechnology (Dallas, TX). Lentiviral particle encoding luciferase reporter
gene (LPꢀhLUCꢀLv201ꢀ0200) was procured from GeneCopoeia (Rockville, MD).
TM
TM
mirVana miRꢀ205 mimic (hsaꢀmiRꢀ205ꢀ5p) and mirVana miRNA mimic negative
control #1 were purchased from Life Technologies (Carlsbad, CA). Custom miRIDIAN
mimic hsaꢀmiRꢀ205ꢀ5p w/5' Dy677 was purchased from GE Healthcare Dharmacon, Inc.
(
Lafayette, CO). Cyanine7.5 amine was purchased from Lumiprobe Corporation
Hallandale Beach, FL). Dead End Fluorometric TUNEL System was procured from
(
Promega Corporation (Madison, WI). Mouse IgG1 MAb (Phycoerythrin)ꢀIsotype control
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and mouse MAb to EGFR (Phycoerythrin) were procured from Abchem (Cambridge,
MA). All other reagents and chemicals were procured from SigmaꢀAldrich (St. Louis,
MO).
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Synthesis of polymers
Synthesis of 2-methyl-2-dodecane oxycarbonylpropylene carbonate (MDC)
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ꢀmethylꢀ2ꢀbenzyloxycarbonylꢀpropylene carbonate (MBC) and 2ꢀmethylꢀ2ꢀ
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carboxylpropylene carbonate (MCC) were synthesized as reported by Li et al. DC was
conjugated to the carboxyl group of MCC using HOBT/EDC coupling reaction. Briefly,
MTCꢀOH (240 mg), HOBT (253 mg) and EDC (360 mg) were dissolved in 20 mL of
DMF followed by the addition of 245 ꢁL of TEA. After 1h, DC (235 mg) was added to the
reaction mixture and left for 18 h under N atmosphere at RT. Then, 150 mL ethyl
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acetate was added to the reaction vessel, and distilled water was used to wash organic
layer, and organic solvent dried over sodium sulfate. The solvent was evaporated by the
rotary evaporator to yield 2ꢀmethylꢀ2ꢀdodecane oxycarbonylpropylene carbonate
(MDC), which was purified by column chromatography.
Synthesis of MAL-PEG-PCD
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MALꢀPEGꢀPCD was synthesized as reported by Mondal et al. Briefly, MDC
(
450 mg) and MALꢀPEGꢀOH (300 mg) were dissolved in 15 mL anhydrous CH Cl and
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2
kept under N atmosphere at RT with continuous stirring. Then, 40 ꢁL DBU was diluted
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in 1 mL anhydrous CH Cl and added to the reaction mixture and left for 3 h under
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2
stirring. At the end of the reaction, 250 mg benzoic acid was added to the reaction
mixture, and solvent was removed. Then, the product was dissolved with CHCl and we
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used diethyl ether and isopropyl alcohol to get the precipitate. Finally, the precipitate
was dried under high vacuum at RT and got ~80% yield of purified MALꢀPEGꢀPCD
lipopolymer.
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Conjugation of C225 and IgG to MAL-PEG-PCD
EGFR targeting C225 (10 mg) was first thiolated by Traut’s reagent (5ꢀfold
excess) in HEPES buffer (pH 8.0) for 1 h at RT. PDꢀ10 column was used to remove
unreacted Traut’s reagent using degassed HEPES buffer (pH 6.5) as an elution buffer.
MALꢀPEGꢀPCD (25 mg) was dissolved in distilled water and stirred under N at RT.
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Thiolated C225 (35 mg) and TECP (100 mM, 100 ꢁL) was added to the reaction and left
for 24 h at RT under N atmosphere with gentle stirring. Then, 1mM MEA (50 ꢁL) was
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added to the reaction mixture and left for 30 min at RT to quench excess maleimide
groups. Sepharose CLꢀ4B gel filtration was used to remove unbound MAb and MEA.
Bicinchoninic acid assay (BCA) was carried out to determine the coupling efficiency as
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reported previously.
synthesized.
Similarly, IgGꢀconjugated MALꢀPEGꢀPCD polymer was
Synthesis of mPEG-b-PCC-g-GEM-g-DC-g-TEPA
mPEGꢀPCC and mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA were synthesized using
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our previously reported method. Briefly, mPEGꢀPBC was synthesized by ringꢀopening
polymerization of mPEG and MBC using stannousꢀ2ꢀethyl hexanoate as a catalyst at
1
00 °C for 18 h under reduced pressure. Then, the polymer was dissolved in
chloroform, precipitated in cold hexane− diethyl ether mixture and dried under vacuum
to get pure polymer with ~65% yield. Debenzylation of mPEGꢀPBC was carried out by
hydrogenation reaction. Briefly, mPEGꢀPBC was tetrahydrofuran and methanol (1:1)
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mixture containing palladium on carbon (Pd/C), charged with hydrogen at a pressure of
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0 psi and left for 18 h. Then, reaction mixture was filtered to remove Pd/C and mPEGꢀ
PCC was obtained by solvent evaporation using a rotary evaporator with ~95% yield. To
synthesize mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA, mPEGꢀPCC (100 mg), HOBT (54 mg)
and EDC (77 mg) were added to reaction vessel containing 10 mL DMF and left for 2 h
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stirring at RT under N atmosphere. GEM (60 mg), DC (37 mg) and TEPA (38 mg) were
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added to the reaction mixture and followed by the addition of 125 ꢁL DIPEA. Then, the
reaction mixture was left for 72 h continuous stirring under N atmosphere at RT. The
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crude product was precipitated using cold isopropyl alcohol and then dissolved in
chloroform and precipitated in cold diethyl ether. Then, the precipitate was purified by
dialysis against water using 2 kDa MWCO dialysis tubing after dissolving in acetone.
Finally, purified dialysis polymer solution was lyophilized to yield mPEGꢀbꢀPCCꢀgꢀGEMꢀ
gꢀDCꢀgꢀTEPA. Similarly, mPEGꢀbꢀPCCꢀgꢀCy7.5ꢀgꢀDCꢀTEPA was synthesized.
Formulation and characterization
C225ꢀ, IgGꢀ and unmodifiedꢀmicelles containing GEM and miRꢀ205, were
prepared by film hydration as reported earlier with slight modification. Briefly, 10 mg of
C225ꢀPEGꢀPCD (10−30% weight ratio) and mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀTEPA
(
70−90% weight ratio) were taken in a glass vial, dissolved in CHCl and followed by
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evaporation under reduced pressure to form a thin film. Similarly, IgGꢀ and unmodifiedꢀ
micelles were prepared using 10 mg of IgGꢀPEGꢀPCD (30% weight ratio) and mPEGꢀbꢀ
PCCꢀgꢀGEMꢀgꢀDCꢀTEPA (70% weight ratio) and 10 mg of mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀ
TEPA respectively. HEPES buffer (10 mM, pH 6.5, 1 ml) containing miRꢀ205 was then
added to hydrate the film and solution was vortexed for 5 min and allowed to stand for
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0 min at 37 °C to enable miRNA complexation. It was then centrifuged at 5000 g for 5
min and filtered through 0.22 µm filter (Millipore). The particle size distribution and zeta
ζ) potential of 1 mg/mL of micelles (C225ꢀ, IgGꢀ and unmodifiedꢀmicelles containing
(
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GEM and miRꢀ205) was determined by dynamic light scatterring using a Malvern
Zetasizer. GEM loading to the copolymer was determined by alkaline hydrolysis as
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described previously. Agarose gel retardation assay was carried out to determine the
appropriate N/P ratio resulting in effective complex formation between miRꢀ205 and
TEPA of the polymer. miRNA dissociation from micelles was carried out by polyanion
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competition assay as reported earlier.
Cellular uptake studies
MIA PaCaꢀ2 cells were obtained from ATCC (Manassas, VA) and GEM resistant
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MIA PaCaꢀ2 cells were maintained in DMEM containing 10% FBS and 1% antibiotic in
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an incubator at 37 °C/5% CO . Cellular uptake study was carried out in MIA PaCaꢀ2
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cells treated with free BLOCKꢀiT™ fluorescent Oligo and C225ꢀ, IgGꢀ, and unmodifiedꢀ
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micelles containing BLOCKꢀiT™ fluorescent Oligo. Briefly, 2 × 10 cells were seeded
per well in sixꢀwell plates for 24 h. Free BLOCKꢀiT™ fluorescent Oligo and C225ꢀ, IgGꢀ,
and unmodifiedꢀmicelles containing BLOCKꢀiT™ fluorescent Oligo (50 nM/well) were
added to each well. After 4 h, the cells were washed with PBS and observed under an
epifluorescence microscope. Further, for quantitative analysis of BLOCKꢀiT™
fluorescent oligo, cells were washed with phosphate buffer saline, trypsinized, again
washed with cold PBS, and centrifuged. Then, the cell pellet was resuspended in PBS
containing 4% paraformaldehyde and kept for 10 min at RT. Cells were centrifuged,
washed with PBS, resuspended in 1 mL of PBS, and analyzed by a flow cytometer (BD
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LSRFortessa Xꢀ20). For competition studies, an excess of C225 was diluted with
medium and added to cells 1 h before the addition of C225ꢀmicelles. After 4 h, the cells
were washed with PBS and images were taken under an inverted microscope (Zeiss,
Germany).
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Cytotoxicity studies
For checking transfection efficiency of miRNA in pancreatic cancer cells, MIA
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PaCaꢀ2 cells were seeded into 6ꢀwell plate at a density of 2×10 cells/well in serumꢀ
free DMEM medium. 3'ꢀFluorescein labeled oligonucleotide (50 nM/well) was complexes
with Lipofectamine® RNAiMAX and transfected according to manufacturer’s instructions
(Thermo Fisher Scientific, MA). 4 h postꢀtransfection, cells were observed under a
fluorescent microscope (Zeiss, Germany). Similarly, for cytotoxic effect of GEM and
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miRꢀ205 in combination, 5000 MIA PaCaꢀ2 cells per well were used in 96ꢀwell plate
and cell viability was assessed by MTT assay after 72 h postꢀtreatment with 500 nM
GEM and 50 nM miRꢀ205 mimic. Cytotoxicity of C225ꢀ, IgGꢀ and unmodifiedꢀmicelles
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containing GEM and miRꢀ205 were evaluated by MTT assay by treating MIA PaCaꢀ2
cells with free GEM (abbreviated FꢀGEM), GEM conjugated polymer (mPEGꢀbꢀPCCꢀgꢀ
GEMꢀgꢀDC, abbreviated as PꢀGEM), C225ꢀ, IgGꢀ and unmodifiedꢀmicelles containing
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00 nM GEM and 50 nM miRꢀ205.
Biodistribution studies
All in vivo experiments were carried out following the NIH guidelines and protocol
was approved prior to in vivo study by the Institutional Animal Care and Use Committee
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(
IACUC) at the University of Nebraska Medical Center, Omaha, NE . MIA PaCaꢀ2 cells
were stably transfected with lentiviral vector encoding luciferase and green fluorescent
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protein (GFP) (hLUCꢀLv201ꢀ0200) using polybrene as described previously. The
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orthotopic pancreatic tumor model was generated by implantation of 2 × 10 MIA PaCaꢀ
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ꢀGFPꢀLuciferase cells suspending in PBS:matrigel (1:1 v/v) into the pancreas of 6−8
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weekꢀold (NOD.CgꢀPrkdcscid Il2rgtm1wjl/SzJ) NSG mice (n = 3). C225ꢀ, IgGꢀ, and
unmodifiedꢀmicelles containing Cy7.5 (2 mg/Kg) and hsaꢀmiRꢀ205ꢀ5p w/5' Dy677 (2
mg/Kg) were injected into mice via the tail vein. After 6h and 24 h post injection of mixed
micellar formulations, Dꢀluciferin (150 mg/kg) was intraperitoneally administered to each
animal via injection, and bioluminescent as well as fluorescent was measured by IVIS
System (Caliper Life Sciences). After bioluminescent as well as fluorescent
measurement, mice were sacrificed and all major organs and tumors were removed for
exꢀvivo imaging using the IVIS Imaging System (Caliper Life Sciences) to determine the
organ distribution of Cy7.5 dye, and Dy677 labeled miRꢀ205.
Efficacy of C225-micelles carrying GEM and miR-205 in orthotopic pancreatic
tumor bearing mice
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After 7 days post implantation of luciferase and GFP stably expressing 2 × 10
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MIA PaCaꢀ2 into NSG mice, we started bioluminescent measurment using IVIS
Imaging System (Caliper Life Sciences) by intraperitoneally injection of Dꢀluciferin (150
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mg/kg) into mice. When bioluminescent value of tumor load was ~ 4x10 p/sec/cm /sr,
mice were then randomly sorted into five groups (n = 6): control, C225ꢀ, IgGꢀ,
unmodifiedꢀmicelles containing GEM and miRꢀ205, and C225ꢀmicelles containing GEM
and scrambled miRNA. Treatment was started at that time via intravenous
administration thrice a week for 2 weeks at the dose of 40 mg/ kg GEM and and 2
mg/kg miRꢀ205 mimic or scrambled miRNA. Bioluminescent was measured thrice a
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week for 19 days’ postꢀtreatment. Mice were sacrificed and tumors were removed after
final bioluminescent measurement. Tumors were fixed with 10% formaldehyde in PBS,
and paraffinꢀembedded sections were immunostained for Kiꢀ67 (cell proliferation
marker), cleaved caspase 3 for apoptosis and hematoxylin and eosin (H&E). To detect
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tumor cells apoptosis induced by GEM and miRꢀ205 or scrambled miRNA, MIA PaCaꢀ
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cells derived orthotopic tumor bearing mice (n = 3) were intravenously administered
with C225ꢀ, IgGꢀ, unmodifiedꢀmicelles containing GEM and miRꢀ205, and C225ꢀmicelles
containing GEM and scrambled miRNA thrice a week for 2 weeks after 12 days post
tumor inoculation. Mice were sacrificed 72 h post last injection, tumors were removed.
Frozen tumors were cryosectioned and fixed with 10% paraformaldehyde and
immunostained with a TUNEL kit as stated in the manufacturer’s protocols and
counterstained with DAPI and imaged under an inverted microscope.
Statistical Analysis
Results are represented as the means ± SEM. The statistical analysis of the
results was done using twoꢀtailed Student’s test. *P<0.05 was considered statistically
significant.
RESULTS
Synthesis and characterization of polymers
MALꢀPEGꢀPDC, mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀTEPA, mPEGꢀbꢀPCCꢀgꢀCy7.5ꢀgꢀ
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DCꢀgꢀTEPA, and MCC were characterized by HꢀNMR (500 MHz, DMSOꢀd6). mPEGꢀ
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PCC was characterized by HꢀNMR (400 MHz, DMSOꢀd6) and MDC was characterized
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by HꢀNMR (500 MHz, chloroformꢀd). MALꢀPEGꢀPDC copolymer was synthesized by
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ring opening polymerization using 2ꢀmethylꢀ2ꢀdodecanoxycarbonylpropylene carbonate
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(
MDC) and MALꢀPEG5000ꢀOH and characterized by H NMR analysis with desired peaks
of PEG (−CH −CH −O) at δ3.5, PCC (−CO−OꢀCH −) at δ4.2, CH −(CH ) − at δ0.9,
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CH −(CH ) − at δ1.2−1.45, CH −(CH ) −CH ꢀCH ꢀ at δ1.5−1.8, CH −(CH ) −CH −CH −
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at δ4.02, −NH−COꢀCH −CH ꢀ at δ2.5−3.2, −NH−COꢀCH −CH ꢀ at δ4.6,
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−
CO−CH=CH−CO− at δ7.2−7.5 and −NH−CO−CH −CH ꢀ at δ8.4 as described before
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(
Figure S1). Since C225 targets EGFR, we next synthesized C225ꢀPEGꢀPCD using
thiolated C225, MALꢀPEGꢀPCD and tris(2ꢀcarboxyethyl) phosphine hydrochloride
TECP) as shown in Figure 1A. For effective combination therapy, GEM, DC and TEPA
(
were conjugated to pendant carboxyl groups of mPEGꢀPCC to get mPEGꢀbꢀPCCꢀgꢀ
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GEMꢀgꢀDCꢀgꢀTEPA (Figure 1B). mPEGꢀPCC polymer showed characteristic H NMR
peaks of PEG (ꢀCH ꢀCH ꢀO) at δ3.5, PCC (−CO−OꢀCH −) at δ4.2 and ꢀCOOH at δ12ꢀ
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4 as described before (Figure S2). By peak integration of mPEG and PCC proton, we
found ~30ꢀ32 PCC units with average molecular weight of 9800 Da. Further, the final
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mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀTEPA showed characteristic H NMR peaks of PEG
(−CH −CH −O) at δ3.5, PCC (−CO−OꢀCH −) at δ4.2, CH −(CH ) − at δ0.9,
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CH −(CH ) − at δ1.2−1.45, CH −(CH ) −CH ꢀCH ꢀ at δ1.5−1.8, CH −(CH ) −CH −CH −
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at δ4.02, GEM 5′ꢀ and 3′ꢀOH groups at δ6.18−6.39, −CO−NH− at δ8.26,
−CO−N−CH=CH− at δ8.15 and −CO−N−CH=CH− at δ5.35, TEPA (−CO−NH−) at
δ7−8, ꢀNHꢀCH ꢀCH ꢀNHꢀCH ꢀCH ꢀNHꢀCH ꢀCH ꢀNHꢀCH ꢀCH ꢀNH at δ2.7ꢀ3.0, ꢀNHꢀ
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CH ꢀCH ꢀNHꢀCH ꢀCH ꢀNHꢀCH ꢀCH ꢀNHꢀCH ꢀCH ꢀNH at δ2.5ꢀ2.7, and ꢀNHꢀCH ꢀCH ꢀ
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NHꢀCH ꢀCH ꢀNHꢀCH ꢀCH ꢀNHꢀCH ꢀCH ꢀNH at δ3ꢀ3.2 (Figure S3). Similarly, mPEGꢀbꢀ
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PCCꢀgꢀCy7.5ꢀgꢀDCꢀgꢀTEPA were synthesized (Figure 1C) and characterized by H
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NMR (Figure S4). MCC was synthesized by hydrogenation of MBC and characterized
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by H NMR peaks of ꢀCOOH at δ13.35, −CO−OꢀCH − at δ4.2 and ꢀOꢀCH ꢀC(CH )ꢀ at
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δ1.5ꢀ1.8 (Figure S5). EDC/HOBT coupling method was used to conjugate DC with the
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carboxyl group of MCC. At the end, MDC was purified and characterized by H NMR
showing characteristic peaks of −CO−OꢀCH − at δ4.2, ꢀOꢀCH ꢀC(CH )ꢀ at δ1.5ꢀ1.8,
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CH −(CH ) − at δ0.9, CH −(CH ) − at δ1.2−1.45, CH −(CH ) −CH ꢀCH ꢀ at δ1.5−1.8,
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CH −(CH ) −CH −CH − at δ4.02 as reported earlier (Figure S6).
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Formulation development and evaluation
The efficiency of C225 conjugation to MalꢀPEGꢀPCD was determined to be ~60%
by BCA using IgG as a standard. The prepared 10ꢀ30% C225ꢀ, IgGꢀ and unmodifiedꢀ
micelles containing GEM and miRꢀ205 showed the mean particle size of 121.7 ± 7,
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22.3 ± 6, 124.6 ± 4 nm, 127.7 ± 4, and 76.6 ± 6 nm with ζ potentials of ꢀ1.05 ± 0.4, ꢀ
1.82 ± 0.7, ꢀ2.36 ± 0.6, ꢀ2.73 ± 0.65, and 4.73 ± 1.65 mV, respectively (Figure S7 &
S8). After preparation of C225ꢀmicelles using 10 mg of C225ꢀPEGꢀPCD (10ꢀ30% w/w)
and mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀTEPAꢀgꢀDC (70ꢀ90% w/w), we determined the antibody
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concentration on micelles surface by BCA as reported previously. We found ~510
µg/mL C225ꢀmicelles (C225ꢀPEGꢀPCD, 30% w/w, total 10 mg polymer), ~340 µg/mL
C225 (C225ꢀPEGꢀPCD, 20% w/w, total 10 mg polymer), and ~170 µg/mL C225 (C225ꢀ
PEGꢀPCD, 10% w/w, total 10 mg polymer). GEM loading of 10% w/w in mPEGꢀbꢀPCCꢀ
gꢀGEMꢀgꢀDCꢀgꢀTEPA was achieved as determined by alkaline hydrolysis. The complex
formation between miRꢀ205 and C225ꢀ, IgGꢀ and unmodifiedꢀmicelles was determined
at N/P ratios ranging from 4:1 to 128:1. At N/P ratio of 32:1, C225ꢀ, IgGꢀ and
unmodifiedꢀmicelles formed complete complex with miRꢀ205 without any interference
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from C225, IgG and GEM (Figure S9A). Further, the dissociation of miRꢀ205 from these
micelle formulations was observed at miRNA: heparin weight ratio of 1:8 (Figure S9B).
C225 mediated cellular uptake of micelles
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Before going into cellular uptake study, we first investigated the cytotoxicity
nature of C225ꢀ, IgGꢀ and unmodifiedꢀmicelles. In our observation, these micelle
formulations carrying BLOCKꢀiT™ fluorescent oligo did not show any cytotoxicity on
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MIA PaCaꢀ2 cells (Figure S10A). Since our objective was to achieve targeted delivery
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of C225 conjugated micelles, cellular uptake was studied on MIA PaCAꢀ2 cells after
confirming EGFR expression (Figure S10B). As a result, higher uptake of 30% w/w
C225ꢀmicelles was observed compared to IgGꢀmicelles and unmodified micelles,
whereas there was no significant differences between 10ꢀ20% w/w C225ꢀmicelles, IgGꢀ
micelles and unmodified micelles indicating C225 density is crucial for targeting EGFR
(Figure 2). Significant decrease in cellular uptake of 30% w/w C225ꢀmicelles was
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observed when MIA PaCAꢀ2 cells were presaturated with C225 (Figure 2). Similar
pattern of cellular uptake was also evident in flow cytometry analysis (Figure 3A & B).
We observed negligible cellular uptake in control experiment with free BLOCKꢀiT™
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fluorescent oligo treated MIA PaCaꢀ2 cells (Figure S11).
Restoration of chemosensitivity in resistant pancreatic cancer cells
To determine whether there is a correlation between miRꢀ205 expression level
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and the degree of GEM resistance, MIA PaCaꢀ2 cells were subjected to cytotoxicity
assays. In good agreement with our previous studies, combination of GEM and miRꢀ205
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suppressed the proliferation of MIA PaCaꢀ2 cells by 38.9% compared to monotherapy
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(Figure S12A & B).
These results indicate that the combination of GEM and miRꢀ
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to treat GEM resistant PDAC. Since miRꢀ205 plays an important role in sensitizing GEM
resistant pancreatic cancer cells, we further checked the efficacy of C225ꢀmicelles
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containing 500 nM GEM and 50 nM miRꢀ205. As a result, MIA PaCAꢀ2 cells treated
with C225ꢀmicelles showed significant reversal of chemoresistance compared to IgGꢀ
and unmodifiedꢀmicelles containing GEM and mIRꢀ205 and C225ꢀmicelles containing
GEM and scrambled miRNA (Figure S12C).
In vivo and ex vivo imaging
For whole body imaging, mice were intravenously injected with different micelles.
As a result, 30% w/w C225ꢀmicelles containing conjugated Cy7.5 dye and complexed
Dy677 labeled miRꢀ205 showed significantly higher level of fluorescent signal in whole
body at 6 h and 24 h compared to IgGꢀ and unmodifiedꢀmicelles (Figure 4A & S13A).
An enhanced level of tumor accumulation of C225ꢀmicelles was observed compared to
IgGꢀ and unmodifiedꢀmicelles confirming EGFR mediated uptake of micellar formulation
(Figure 4B & S13B). The quantitative analysis of Cy7.5 dye intensity in tumor tissue at
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h and 24 h were nearly 1.5 and 2ꢀfold higher in C225ꢀmicelles compared to IgGꢀ and
unmodifiedꢀmicelles (Figure 4C). Similarly, fluorescence intensity of Dy677 labelled
miRꢀ205 in tumor tissue at 6 h and 24 h were nearly 1.6 and 3ꢀfold higher in C225ꢀ
micelles compared to IgGꢀ and unmodifiedꢀmicelles (Figure 4C), indicating C225ꢀ
micelles to form stable electrostatic complex with miRꢀ205 and protecting it during in
vivo delivery.
In vivo therapeutic study
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The synergistic effect of GEM and miRꢀ205 after intratumoral administration in
subcutaneous tumor bearing mice in our earlier study prompted us to evaluate the effect
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of this combination in more realistic orthotopic pancreatic tumor model. To continue
this endeavor, we evaluated comparative therapeutic potential between EGFR targeted
C225ꢀ and unmodifiedꢀmicelles containing GEM (40 mg/Kg) and miRꢀ205 (2 mg/Kg)
after systemic administration. A significantly low level of bioluminescence intensity and
smaller tumor size was observed in mice treated with C225ꢀmicelles containing GEM
and miRꢀ205 compared to
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others (Figure 5A, B & C). In addition, mice treated with C225ꢀmicelles containing GEM
and miRꢀ205 showed smallest tumor volume and tumor weight compared to others
(Figure 6A & B) without any changes in body weight (Figure 6C). H & E staining of all
the treated mice showed no obvious pathological change in the major organs such as
heart, liver, spleen and kidney (Figure 7). Immunohistochemical staining of the tumor
tissue from mice treated with C225ꢀmicelles containing GEM and miRꢀ205 showed the
lowest Kiꢀ67 and highest cleaved caspaseꢀ3 expression compared to all other treatment
groups (Figure 8). In addition, mice treated with C225ꢀmicelles containing GEM and
miRꢀ205 showed more TUNEL positive cells compared to all other treatment groups
(Figure 9). Immunohistochemical analysis of tumor specimen of mice treated with
C225ꢀmicelles containing GEM and miRꢀ205 showed significantly enhanced Eꢀcadherin
and reduced ZEB1 expression compared to other treatment groups (Figure 10).
DISCUSSION
GEM based chemotherapy does not completely improve the outcome of
pancreatic cancer. Most patients develop drug resistance after repeated administration
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of GEM at a high dose.
Therefore, to overcome GEM resistance, global effort has
been directed to uncover molecular mechanisms behind this phenomenon. Most of the
study has observed decrease in chemoresistance in preclinical studies, but not in
clinical trials.
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miRNAs modulate several genes in the postꢀtranscriptional level or target mRNA
degradation. Since several oncogenic and tumor suppressor miRNAs are linked to drug
resistance, miRNA replenishment therapy would be an attractive approach to overcome
GEM resistance. However, very few miRNAs (miRꢀ218, miRꢀ1246, miRꢀ15a, miRꢀ214,
miRꢀ29a and miRꢀ200c) have been examined for their roles in GEM resistance in
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pancreatic cancer.
We have previously demonstrated that miRꢀ205 was highly
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downregulated in pancreatic cancer tissues and GEMꢀresistant MIA PaCAꢀ2 cells and
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transient expression of miRꢀ205 mimic reversed GEM resistance in MIA PaCAꢀ2 cells.
The role of miRꢀ205 in many different cancers including melanoma, lung cancer,
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prostate cancer, and breast cancer have been reported.
Thus, a combination of
miRꢀ205 mimic with GEM could be an advantageous therapeutic approach to reverse
GEM resistance and inhibit advanced pancreatic tumor progression.
Effective GEM delivery is hindered due to its hydrophilicity leading to low drug
loading and rapid release. For coꢀdelivery of GEM and miRNA to overcome GEM
resistance needs efficient nanomedicines that can conjugate GEM to enhance its
loading and protect from plasma metabolism as well as a complex with miRNA to
protect it from rapid degradation in circulation and increase membrane penetration.
Recently, therefore, Mittal et al. synthesized amphiphilic copolymer mPEGꢀbꢀPCCꢀgꢀ
GEMꢀgꢀDCꢀgꢀTEPA that has PEG forming the hydrophilic segment and polypropylene
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carbonate forming the hydrophobic segment, and this segment was grafted with GEM,
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DC and cationic TEPA chains, which efficiently complexed with miRꢀ205.
Polyethylenimine (PEI) and poly(Lꢀlysine) (PLL) are widely used as a cationic block
grafted to polyester end groups for gene delivery, but this cationic segment has high
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toxicity in various cell lines.
Xiong et al. used polycaprolactone backbone in cationic
poly(ethylene oxide)ꢀbꢀpoly(εꢀcaprolactoneꢀgꢀpolyamine) for siRNA delivery, but we
used polycarbonate hydrophobic backbone in mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA for
miRꢀ205 delivery which improves biocompatibility as its degradation products are CO2
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and H O instead of acids like polyesters. GEM hydrophilicity creates a problem for its
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encapsulation in nanocarriers. Thus, chemical conjugation approach was used to get
better GEM loading (~10ꢀ12% w/w) and slow release compared to previously reported
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PEGylated GEM prodrugs.
Passive targeting is based on the enhanced permeation and retention (EPR)
effect, whereas active targeting is based on specific interactions of drug or drug carrier
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with the target cells, known as ligand–receptor interactions. This ligandꢀreceptor
interaction for active targeting is dependent on many factors such as the receptor
overexpression in target cells compared to nonꢀtarget cells and receptor availability in
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the cell surface of the target cells.
EGFR, a cell surface receptor and its
overexpression in tumor cells facilitates selective targeting, and preclinical and clinical
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reports, indicating that EGFR targeting for cancer therapy is a valid strategy. Several
approaches have been developed for EGFR targeting by antibodyꢀbased
immunoconjugates such as immunoliposomes for targeted drug delivery to EGFR
overexpressing cancer cells. Recent studies demonstrated that MAb C225 conjugated
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antiꢀEGFR immunoliposomes exhibit efficient activity in an EGFRꢀoverexpressing MDAꢀ
MBꢀ468 breast cancer cells and Uꢀ87 glioblastoma cells compared to nonꢀtargeted
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liposomes.
Patra et al. proved that C225 conjugated gold nanoparticles exhibit
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effective GEM delivery to EGFR expressing pancreatic cancer and inhibited orthotopic
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pancreatic tumor growth. In our previous studies, we confirmed EGFR expression in
MIA PaCAꢀ2 cells, and we assumed that this expression would not change by GEM
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resistance. Further, we confirmed EGFR expression in MIA PaCaꢀ2 cells by FACS
analysis (Figure S10B). Therefore, we selected C225 as a targeting agent for coꢀ
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delivery of GEM and miRꢀ205 to EGFR expressing MIA PaCaꢀ2 cells in vitro and MIA
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PaCaꢀ2 derived orthotopic pancreatic tumor.
Chemical conjugation is the best strategy for covalent conjugation of targeting
agent with the polymer otherwise, there is a chance for impairment of functional activity
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of targeting agent or reduction of targeting capability. We previously reported GEM
conjugation to the polymer, mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA, but we failed to
conjugate C225 to the polar part of this polymer. To solve this synthetic complexity, we
synthesized C225ꢀPEGꢀPCD and IgGꢀPEGꢀPCD using thiolated C225 or IgG and MALꢀ
PEGꢀPCD. We prepared mixed micelles using mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA and
C225ꢀPEGꢀPCD for EGFR targeting. We also prepared nonꢀtargeted mixed micelles
using mPEGꢀbꢀPCCꢀgꢀGEMꢀgꢀDCꢀgꢀTEPA and IgGꢀPEGꢀPCD. In our previous study,
we found that miRꢀ205 did not affect GEM release in PBS at pH 5.5 and 7.4 from the
micelles as GEM is conjugated to the polymer via an amide bond, and GEM release
critically depends on the cleavage of amide bond that is not disturbed by electrostatic
interaction between miRꢀ205 and cationic TEPA chain. We assume similar release
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pattern of GEM would be achieved from C225ꢀmicelles and IgGꢀmicelles as thiolated
C225 and thiolated IgG have conjugated via thiol bond to MALꢀPEGꢀPCD polymer,
respectively and this thiol bond are stable at pH 5.5 and 7.4 as well as would not disturb
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GEM release. Micelles should protect miRꢀ205 from degradation in a biological
environment. We found that our unmodified ꢀmicelles, C225ꢀmicelles and IgGꢀmicelles
improved miRꢀ205 stability compared to naked miRꢀ205 up to 24 h in serum (data not
shown). This improved stability was observed due to stable electrostatic complex
formation between miRꢀ205 and cationic TEPA chain and steric effect exerted by PEG
corona reduces miRꢀ205 interaction with the exogenous RNases that are present in
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serum.
As EGFR mediated cellular uptake depends on EGFR recognition and
internalization of C225 on the surface of C225ꢀmicelles, we noticed highest uptake of
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BLOCKꢀiT™ of C225ꢀmicelles (C225ꢀPEGꢀPCD, 30%) in MIA PaCAꢀ2 cells compared
to unmodifiedꢀ, C225ꢀ (C225ꢀPEGꢀPCD, 10ꢀ20%) and IgGꢀmicelles (Figure 2& 3A-B).
However, the cellular uptake of C225ꢀmicelles significantly diminished when MIA PaCAꢀ
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cells were presaturated with C225 before transfection, indicating C225ꢀmicelles
specificity towards EGFR (Figure 2A-C). Although all C225ꢀmicelles have the same
C225ꢀtargeting moiety and ζ potential close to zero, the cellular uptake efficacy
improved with increase in C225ꢀPEGꢀPCD weight percentage that may be due to C225
density on the micelle surface. We measured the amount of C225 per mL micelles (10
mg total polymer) using BCA method and found ~510 µg antibody/mL micelle (10 mg
total polymer) in C225ꢀmicelles (C225ꢀPEGꢀPCD, 30% w/w) and ~170ꢀ340 µg antibody/
mL micelle (10 mg total polymer) in C225ꢀmicelles (C225ꢀPEGꢀPCD, 10ꢀ20% w/w),
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respectively. It seems that at density ~170ꢀ340 µg/mL micelle (10 mg total polymer), the
C225 might fail to reach surface saturation and may be responsible for poor EGFR
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targeting. Therefore, we assume that this phenomena could be due to the changes in
valency of C225 that increases EGFR binding affinity which is consistent with earlier
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study.
Previous report also showed that cellular internalization can be increased
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with an optimal density of targeting agents on the surface of micelles. On the other
hand, the higher uptake of C225ꢀmicelles was due to EGFR mediated strong uptake of
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BLOCKꢀiT™ compared to IgGꢀmicelles. Since the combination of GEM and miRꢀ205
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has shown significant inhibition of MIA PaCaꢀ2 cells (Figure S12B) compared to GEM
and miRꢀ205 alone, we concentrated on GEM and miRꢀ205 in combination to further
evaluate its targeted delivery using C225 as a targeting ligand. We observed a
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significant enhancement in cytotoxicity when MIA PaCaꢀ2 cells were treated with
C225ꢀꢀmicelles containing GEM and miRꢀ205 compared to IgGꢀ and unmodifiedꢀmicelle
formulation carrying GEM and miRꢀ205 (Figure S12C). This observation further
confirms EGFR mediated cellular uptake of C225ꢀmicelles indicating the capability of
GEM and miRꢀ205 in combination to reverse GEM resistance. As 30% of C225ꢀmicelles
containing GEM and miRꢀ205 showed better cellular uptake and cytotoxicity profile, we
used this formulation for in vivo study.
Cy7.5 labeling of mPEGꢀbꢀPCCꢀgꢀCy7.5ꢀgꢀDCꢀgꢀTEPA and Dyꢀ677 labeling of
miRꢀ205 allowed in vivo and ex vivo near infrared (NIR) fluorescence imaging, as well
as tracking of both micellar carrier and miRꢀ205 in the tumor. Mice treated with C225ꢀ
micelles containing Cy7.5 and Dy677ꢀmiRꢀ205 showed enhanced tumor accumulation
of both components at 6h and 24h compared to unmodified and IgGꢀmicelles, indicating
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the effect of C225 conjugation (Figure 4A-C & S13A-B). Unlike chemically conjugated
Cy7.5, Dy677ꢀmiRꢀ205 might dissociate from micelles or degraded in circulation after
intravenous administration. Interestingly, C225ꢀmicelles protected Dy677ꢀmiRꢀ205 in
circulation and delivered Dy677ꢀmiRꢀ205 to the tumor as Dy677 fluorescence intensity
in tumors increased at 24 h than 6 h (Figure 4C), indicating excellent potential of C225ꢀ
micelles for miRNA and drug delivery. This enhanced tumor accumulation of C225ꢀ
micelles containing Cy7.5 and Dy677ꢀmiRꢀ205 compared to other formulations was due
to the presence of C225 on micelle surface for EGFR targeting and this is in good
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agreement with the work of Xiong et al.
A recent report showed that various miRNAs like miRꢀ21, miRꢀ34a, miRꢀ143/145
cluster, miRꢀ219ꢀ1ꢀ3p, miRꢀ34a, miRꢀ217, miRꢀ29a, 330ꢀ5p and miRꢀ29c can be
administered at doses ranging from 0.5 to 4 mg/Kg for preclinical pancreatic cancer
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therapy. Although in our previous work, we used 1 mg/kg miRꢀ205 dose for
intratumoral injection, we selected 2 mg/kg miRꢀ205 or scrambled miRNA dose for
intravenous administration. Mice treated with C225ꢀployplex containing GEM and miRꢀ
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05 showed significantly low photon value compared to IgGꢀmicelles, and unmodified
micelles containing GEM and miRꢀ205 treated mice. More importantly, mice treated with
C225ꢀmicelles containing GEM and miRꢀ205 showed a pronounced effect compared to
C225ꢀ micelles containing GEM and scrambled miRNA treated mice and control mice
(Figure 5A-C). Our findings clearly indicate that EGFR targeted coꢀdelivery of GEM and
miRꢀ205 has the potential to inhibit advanced pancreatic tumor growth. Tumor
bioluminescent measurement is not corresponding absolute tumor volume but gives an
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Average tumor volume and weight of mice treated with C225ꢀmicelles containing
GEM and miRꢀ205 showed significantly low compared to IgGꢀmicelles, and unmodified
micelles containing GEM and miRꢀ205, C225ꢀmicelles containing GEM and scrambled
miRNA treated mice as well as the control mice. This further confirmed the strong
therapeutic potential of C225ꢀmicelles containing GEM and miRꢀ205 (Figure 6A&B).
There was no change in body weights of mice in all treatment groups, suggesting little
systemic toxicity of these formulations (Figure 6C). H&E staining of the control mice
showed loss of cellular architecture with hypercellularity, whereas mice treated with
C225ꢀployplex containing GEM and miRꢀ205 showed significant pyknosis, compared to
IgGꢀmicelles, and unmodified micelles containing GEM and miRꢀ205 treated mice. This
was more drastic when we compared with C225ꢀployplex containing GEM, and
scrambled miRNA treated mice (Figure 8). Analysis of Kiꢀ67 and cleaved caspase 3
expressions of mice treated with C225ꢀmicelles containing GEM and miRꢀ205 showed
significant reduction in proliferation of tumor cells and the highest level of apoptosis
compared to the control mice as well as all other treatment groups (Figure 8). Also, we
found that treatment with C225ꢀmicelles containing GEM and miRꢀ205 led to significant
increase in dead cells in pancreatic tumors compared to all other treatment groups that
were confirmed by TUNEL assay (Figure 9).
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Wang et al. showed that GEMꢀresistant pancreatic cancer cells were associated
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with EMT and high level expression of ZEB1 and mesenchymal markers. Arumugam
et al. found an inverse correlation between Eꢀcadherin and ZEB1 in GEMꢀresistant
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pancreatic cancer cell lines. Our IHC of control tumor tissue which was developed
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expression of ZEB1 and low expression of Eꢀcadherin that is consistent with a previous
report (Figure 10). A recent report showed that miRꢀ205 expression positively
associated with Eꢀcadherin expression but negatively correlated with ZEB1 expression
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in breast cancer. Gregory et al. showed that ectopic expression of miRꢀ205 in MDCKꢀ
Pez cells, was generated by overexpressing PTPꢀPez in MadinꢀDarby canine kidney
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MDCK) epithelial cells, induced mesenchymal to epithelial transition (MET) with
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upregulating Eꢀcadherin and downregulating ZEB1. Another report showed that
downregulation of miRꢀ205 expression level in esophageal squamous cell carcinoma
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cells reduced Eꢀcadherin. We have previously shown that combination formulation of
GEM and miRꢀ205 enhanced Eꢀcadherin and reduced ZEB1 expression in MIA PaCaꢀ
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cells in vitro. Immunohistochemical staining of C225ꢀmicelles containing GEM and
miRꢀ205 treated mouse tissue showed enhanced Eꢀcadherin and reduced ZEB1
expression compared to IgGꢀmicelles and unmodified micelles containing GEM and
miRꢀ205. More importantly, C225ꢀployplex containing GEM and scrambled miRNA
treated mice showed Eꢀcadherin and ZEB1 expression that is comparable to the control
mice, which further confirmed miRꢀ205 has an important role in regulating Eꢀcadherin
and ZEB1.
Conclusion
In conclusion, we have synthesized C225ꢀPEGꢀPCD and mPEGꢀbꢀPCCꢀgꢀGEMꢀ
gꢀDCꢀgꢀTEPA for preparing micelles containing GEM and miRꢀ205 and decorated with
C225 antibody, which targets EGFR overexpressed on pancreatic cancer cells. There
was significant enhancement in cellular uptake by tumor cells and reversal of GEM
resistance, leading to enhanced tumor accumulation and consequent tumor regression.
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Uncovering other targeting ligands more potent than C225 would be an additional
advantage to enhance the therapeutic outcome of this combination.
Notes
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The authors declare no competing financial interest.
Acknowledgements
We acknowledge the financial support from the National Institutes of Health
(1R01EB017853) and the Faculty Startꢀup fund from the University of Nebraska Medical
Center.
Supporting Information
NMR spectra, particle size distribution and Zeta potentials of micelles, complexation and
dissociation of miRNA from micelles, cytotoxicity of micelles without GEM and miRꢀ205
and EGFR expression in MIA PaCaꢀ2R cells, cellular uptake of free BLOCKꢀiT™
fluorescent oligo in MIA PaCaꢀ2R cells, transfection efficiency of fluorescent labeled
oligonucleotide in MIA PaCaꢀ2R cells, MTT assay of free GEM and miRNA complexed
with Lipofectamine® RNAiMAX into MIA PaCaꢀ2R cells, cytotoxicity effect of micelles
containing GEM and miRꢀ205 or scrambled miRNA in MIA PaCaꢀ2R and NIR
fluorescence imaging of micelles containing conjugated Cy7.5 and DY677ꢀmiRꢀ205
(PDF).
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