Targeted Nanoparticles for the Delivery of Novel Bioactive Molecules to Pancreatic Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.5b01571

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor prognosis and limited therapeutic options. Therefore, there is an urgent need to identify new, safe, and targeted therapeutics for effective treatment of late as well as early stage disease. Plectin-1 (Plec-1) was recently identified as specific biomarker for detecting PDAC at an early stage. We envisioned that multivalent attachment of nanocarriers incorporating certain drugs to Plec-1-derived peptide would increase specific binding affinity and impart high specificity for PDAC cells. Previously, we discovered a novel class of compounds (e.g., quinazolinediones, QDs) that exert their cytotoxic effects by modulating ROS-mediated cell signaling. Herein, we prepared novel QD242-encapsulated polymeric nanoparticles (NPs) functionalized with a peptide to selectively bind to Plec-1. Similarly, we prepared QD-based NPs densely decorated with an isatoic anhydride derivative. Furthermore, we evaluated their impact on ligand binding and antiproliferative activity against PDAC cells. The targeted NPs were more potent than the nontargeted constructs in PDAC cells warranting further development.

Full text of DOI:10.1021/acs.jmedchem.5b01571

Subscriber access provided by TULANE UNIVERSITY
Article
Targeted Nanoparticles for the Delivery of Novel
Bioactive Molecules to Pancreatic Cancer Cells
Vanna Sanna, Salvatore Nurra, Nicolino Pala, Salvatore
Marceddu, Divya Pathania, Nouri Neamati, and Mario Sechi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01571 • Publication Date (Web): 03 May 2016
Downloaded from http://pubs.acs.org on May 3, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 48
Journal of Medicinal Chemistry
1
2
3
Journal of Medicinal Chemistry
4
5
6
7
8
9
Targeted Nanoparticles for the Delivery of Novel Bioactive Molecules
to Pancreatic Cancer Cells
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Vanna Sanna,†, Salvatore Nurra,† Nicolino Pala,† Salvatore Marceddu,§ Divya Pathania,┴
Nouri Neamati,‡,* and Mario Sechi†,,
*
†Department of Chemistry and Pharmacy, University of Sassari, Italy, and Laboratory of
§
Nanomedicine, University of Sassari, c/c Porto Conte Ricerche, Alghero, Italy, and Istituto di
Scienze delle Produzioni Alimentari (ISPA), CNR, sez. di Sassari, Italy, and ┴Department of
Pharmacology and Pharmaceutical Sciences, University of Southern California, School of
Pharmacy, Los Angeles, CA, USA, and ‡Department of Medicinal Chemistry, College of Pharmacy,
Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA
KEYWORDS: Pancreatic Cancer; Ductal Adenocarcinoma; Anticancer Therapeutics;
Quinazolinediones; Plectinꢀ1; Polymeric Nanoparticles; Targeted Nanoparticles; Targeted Delivery
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 48
1
2
ABSTRACT
3
4
5
6
7
8
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor prognosis and limited
therapeutic options. Therefore, there is an urgent need to identify new, safe and targeted
therapeutics for effective treatment of late as well as early stage disease. Plectinꢀ1 (Plecꢀ1) was
recently identified as specific biomarker for detecting PDAC at an early stage. We envisioned that
multivalent attachment of nanocarriers incorporating certain drugs to Plecꢀ1ꢀderived peptide would
increase specific binding affinity and impart high specificity for PDAC cells. Previously, we
discovered a novel class of compounds (e.g. quinazolinediones, QDs) that exert their cytotoxic
effects by modulating ROSꢀmediated cell signaling. Herein, we prepared novel QD242ꢀ
encapsulated polymeric nanoparticles (NPs) functionalized with a peptide (CysꢀPTP) to selectively
bind to Plecꢀ1. Similarly, we prepared QDꢀbased NPs densely decorated with an isatoic anhydride
derivative (2ABA). Furthermore, we evaluated their impact on ligand binding and antiproliferative
activity against PDAC cells. The targeted NPs resulted more potent than the nontargeted constructs
in PDAC cells warranting further development.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 48
Journal of Medicinal Chemistry
1
2
INTRODUCTION
3
4
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer worldwide.1 It
has been estimated that in 2015 about 53,000 people will be diagnosed with PDAC, and about
41,800 people will die of this disease in the United States,2 with similar statistics in the European
Union.3 The high mortality rate of PDAC patients is linked to an aggressive malignant character and
extensive metastasis and late onset of clinical symptoms, leading to a lateꢀstage detection and
chemoresistance.1,4,5 In addition to radiotherapy and surgery, chemotherapy represents a significant
therapeutic option, however with only modest benefits.1,5 Firstꢀline chemotherapy includes
combination of folinic acid, 5ꢀfluorouracil, irinotecan and oxaliplatin (FOLFIRINOX).6 The other
option includes gemcitabine7 in combination with albuminꢀbound paclitaxel (nabꢀpaclitaxel).8
Similar to nabꢀpaclitaxel, other strategies consisting of targeting therapeutic payload to cancer cells
through conjugation to a tumourꢀcellꢀspecific ligands can be equally effective in treating PDAC.9ꢀ11
Recently, we identified novel quinazolinedioneꢀbased compounds (QDs)12,13 that target metabolic
reprogramming and oxidative stress, the emerging hallmarks of cancer cells.14ꢀ18 These novel QD
compounds alter the cellular bioenergetic characteristic of cancer cells resulting in cytotoxicity by
Aktꢀdirected generation of reactive oxygen species (ROS),12 and inhibited the growth of tumors in a
mouse xenograft model of PDAC.13 The lead compound effectively blocked the activation of three
critical signaling pathways: Src, FAK, and STAT3.13
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Due to limited therapeutic options for PDAC we designed a novel delivery system to increase
bioavailability and efficacy of the lead compound, the 6ꢀ[(3ꢀacetylphenyl)amino]quinazolineꢀ5,8ꢀ
dione.12,13 Recently, we developed PEGylated ligand targeted polymeric biocompatible
nanoparticles (NPs) recognizing the prostate cancer cells.19 Active targeting by NPs “decorated”
with specific ligands such as antibodies, aptamers, peptides, and small molecules, is envisioned to
provide the most effective therapy.9,20ꢀ28 Several ligandꢀtargeted nanotherapeutics are either
approved or under clinical evaluation.11,25,28 Membrane receptors with significantly higher
expression on cancer versus normal cells are suitable targets for therapeutic intervention.29
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 48
1
2
3
4
5
Functionalization of NPs with ligands targeting the extracellular domain of these receptors can
enable selective delivery of therapeutic payload to tumor cells.23ꢀ28
6
7
8
9
For example, Plectinꢀ1 (Plecꢀ1), a high molecular weight protein (~500 kDa) that links intermediate
filaments to microtubules and microfilaments, has an important function in tumor pathogenesis.30 It
has been shown that Plecꢀ1 levels are low in normal pancreatic ductal cells, but its expression is
upregulated in PDAC.31 Importantly, Plecꢀ1 exhibits distinct cytoplasmic and nuclear localization in
normal fibroblasts, whereas an aberrant expression on the cell membrane with cell surface
localization is observed in PDAC. Moreover, Plecꢀ1 was suggested as a useful biomarker for
PDAC.31
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Previously, Weissleder et al. developed proofꢀofꢀconcept targeted magnetofluorescent NPs
composed of Plecꢀ1 targeted peptides (PTP) for imaging of PDAC.32 The same group reported on
the development of magnetofluorescent reporters as a novel nanomaterial for biomedical
application.33 Moreover, a nonspecific targeting ligand, the isatoic anhydride with its corresponding
isatoic nanoconjugate (i.e., 2ꢀaminobenzoicꢀcarboxylicꢀcoated magnetic NPs), showed higher
uptake in cancer versus normal cells.33
Here, we report on design and preparation of novel biocompatible NPs encapsulating QD242 (1),12
covered by small organic molecules (2ABA, 11) or peptides (CysꢀPTP, 12) as targeting ligands
(Figure 1) in their polymeric shell surface to selectively bind to Plecꢀ1 or to nonspecifically target
PDAC cells. We used pseudoꢀdiꢀblockꢀcopolymer PLGAꢀ11 as well as the parent PLGAꢀA, and
pseudoꢀtriꢀblockꢀcopolymer PLGAꢀPEGꢀ12 and the parent PLGAꢀPEGꢀNH2 to generate two sets of
200ꢀ300 nm size targeted and nontargeted (1)ꢀencapsulated NPs (Figure 1). These NPs were
characterized for drugꢀcontent, drugꢀrelease, binding affinity, and cytotoxicity in MIA PaCaꢀ2 cells.
4
ACS Paragon Plus Environment

Page 5 of 48
Journal of Medicinal Chemistry
1
2
RESULTS AND DISCUSSION
Design of targeted NPs
3
4
5
6
7
8
We
incorporated
a
representative
ROSꢀinducing
compound,
6ꢀ{[4ꢀ
9
(phenylcarbonyl)phenyl]amino}quinazolineꢀ5,8ꢀdione (1, Figure 1),12 into a polymeric NPs
conjugated to 2ꢀaminoꢀNꢀ(3ꢀaminopropyl)benzamide (11) as well as a targeting peptide (12, amino
acid sequence: KTLLPTPC, Figure 1).32,33 Compound 1 produced IC50 value of 1.6 ± 0.2 ꢁM in
MIA PaCaꢀ2 cells12 and is well suited as a model drug for nanoformulation. It carries a lipophilic
benzoyl substituent instead of an acetyl group on the lead compound (i.e., 6ꢀ[(3ꢀ
acetylphenyl)amino]quinazolineꢀ5,8ꢀdione) on the aminophenyl side chain. We chose poly(D,Lꢀ
lactideꢀcoꢀglycolide) carboxylic acid (PLGAꢀCOOH, namely PLGAꢀA) and a poly(ethylene glycol)
derivative, PLGAꢀPEGꢀNH2, as polymer systems because of their wellꢀestablished safety
profile.34,35
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Initially, we used the 2ꢀaminobenzoicꢀcarboxylate conjugated with the aminopropane linker (11,
Figure 1) as nonspecific PDAC targeting ligand, to obtain an intermediate to react with activated
PLGAꢀNHS. The 2ꢀaminobenzoicꢀcarboxylic was derived from isatoic anhydride (Scheme 2).
The PTP peptide (amino acid sequence: KTLLPTP), identified using peptide phage display, targets
Plecꢀ1 with a high affinity and specificity.32 We decided to use the cysteineꢀcontaining PTP (12,
Figure 1) because it can react with maleimide terminal groups of PEG in the PLGAꢀPEGꢀmaleimide
(i.e., PLGAꢀPEGꢀmal). PEGylation confers immune shielding and “stealth” properties to NPs and is
a suitable spacer to maintain an optimal distance between the targeting ligand and the NP surface.
We also efficiently synthesized the targeting ligand 11 as well as PLGAꢀ11, PLGAꢀPEGꢀNH2,
PLGAꢀPEGꢀmal, and PLGAꢀPEGꢀ12 that are required for the NP formulation. To obtain a better
control of 11 and 12 functionalization, we conjugated these ligands to their respective polymeric
systems (i.e., PLGAꢀ11 and PLGAꢀPEGꢀ12) prior to nanoformulation.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 48
1
2
Synthesis of 1
3
4
5
6
7
8
Compound 1 (Scheme 1) was synthesized using Bracher's methodology with slight modifications to
our previously reported procedure.12 Regioselective substitution of quinazolineꢀ5,8ꢀdione (2) with
4ꢀaminobenzophenone (3) in the presence of Ce(III) ions gave 1 as expected. The key synthone 2
was synthesized starting from the readily available 2,5ꢀdimethoxybenzaldehyde (4), which was
nitrated with concentrated nitric acid in the presence of acetic anhydride under magnetic stirring
affording the 3,6ꢀdimethoxyꢀ2ꢀnitrobenzaldehyde (5). The acidꢀcatalyzed cyclization of this
regioisomer by exposing it to gaseous HCl led to the formation of the diformamidoꢀderivative 6,
which was then cyclized to 5,8ꢀdimethoxyquinazoline (7) by treatment with zinc powder and acetic
acid. Final oxidation by cerium ammonium nitrate gave the compound 2 (Scheme 1B). It is worth
noting that, the nitration reaction, using nitric acid/acetic anhydride in mild condition, gives higher
yield than a previously reported condition12 using a molar excess of silica gel supported nitric acid
(68% vs 50ꢀ58%, respectively). Moreover, similar results were observed by the oxidation of 7 to 2
with cerium ammonium nitrate in acetonitrile/water solution. These modifications led to a 2ꢀfold
improvement in overall yield of the key synthone 2. Compound 1 and the intermediates were fully
characterized by NMR, MS, and elemental analyses.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of 11
The intermediate tertꢀbutyl (3ꢀ(2ꢀaminobenzamido)propyl)carbamate (10) was prepared by treating
isatoic anhydride (9) with tertꢀbutyl (3ꢀaminopropyl)carbamate (8) in the presence of potassium
carbonate and THF at 70 °C with 74% yield (Scheme 2). The tertꢀbutyloxycarbonyl (Boc) group of
10 was removed with 50% trifluoroacetic acid (TFA) in CH2Cl2 solution at room temperature to
obtain the desired ligand 11, which was characterized by NMR, MS, and elemental analyses.
Synthesis of pseudo-di-block-copolymer PLGA-11
Blockꢀcopolymers are effectively used as preconjugated starting materials for the preparation of
6
ACS Paragon Plus Environment

Page 7 of 48
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
precisely engineered NPs targeted nanoconjugates for cancer therapy.24,36ꢀ38 Conjugation of the
terminal amine group of 11 ligand with the activated electrophilic NHS esters of PLGA carboxylic
acids (e.g., PLGAꢀA) allows the formation of physiologically stable amide bonds. The designed
PLGAꢀ11 copolymer and diꢀblockꢀcopolymers were synthesized in a twoꢀstep reaction.
Specifically, the amine functional groups of 11 were reacted with the carboxyꢀcapped PLGAꢀNHS
in DMF in the presence of DIPEA (Scheme 3, stage 1). The activated amineꢀreactive NHSꢀester of
PLGA (PLGAꢀNHS) was prepared by treating PLGAꢀA with EDC/NHS in an organic solvent at
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
room temperature (Scheme 3). As expected, the HꢀNMR analysis revealed aromatic peaks in the
range 6.5ꢀ8.0 ppm, corresponding to proton pattern of 11 (S25). The successful conjugation of 11 to
PLGA polymer also was supported by two absorption bands in 190 and 255 nm regions as depicted
in the UV chromatogram (Figure 2).
Synthesis of di-block-copolymer of PLGA-PEG-NH2 and PLGA-PEG-mal
NPs should ideally have a favourable hydrophobic/hydrophilic balance on their surface in order to
a) have a minimal selfꢀself and selfꢀnonself interaction, b) escape capture by the reticuloendothelial
system (RES) to ensure immune evasion, c) reduce opsonization, d) facilitate efficient clearance of
materials from the body, and d) prevent nanoparticle loss to undesired location. PEGylation
constitutes a suitable strategy for this purpose, and the preparation of PEGylated NPs is well
documented.39ꢀ41 PLGAꢀPEGs are usually synthesized by conjugation or polymerization of PEG to
PLGA via standard carbodiimide/NHSꢀmediated chemistry.37,41,42
The starting polymer PLGAꢀPEGꢀNH2 and the carboxylateꢀfunctionalized diꢀblockꢀcopolymer
PLGAꢀPEGꢀmal (Scheme 4 and Scheme 5, stage 1, respectively) were prepared by conjugating
bifunctional PEGs, NH2ꢀPEGꢀNH2 or NH2ꢀPEGꢀmal, to activated PLGAꢀA. The structure of
copolymers was confirmed by 1HꢀNMR spectroscopy. Specifically, signals centered at 1.58 ppm for
both polymers are attributed to the lactide methyl repeat units (resonance shift a, Schemes 4 and 5).
Overlapping multiplets observed in the range 5.10ꢀ5.32 and 4.63ꢀ4.96 indicate the lactide methine
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 48
1
2
3
4
5
6
7
8
9
(b, Schemes 4 and 5) and the glycolide protons (c, Schemes 4 and 5), respectively. In addition to the
defined signals for PLGA, the presence of the peak at 3.64/3.65 ppm (for NH2ꢀPEGꢀmal and NH2ꢀ
PEGꢀNH2, respectively), corresponding to the PEG methylene protons (d, Schemes 4 and 5),
confirmed the successful preparation of both polymers.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of pseudo-tri-block-copolymer PLGA-PEG-12
We obtained a prefunctionalized biopolymer, PLGAꢀPEGꢀ12, to avoid the postparticle
modifications. The peptide 12 was prepared by fluorenylmethyloxycarbonylꢀ (Fmoc)ꢀbased solidꢀ
phase peptide synthesis (Supporting Information, S29ꢀS32). Conjugation of the targeting agent, i.e.
12, to carboxylate PEG terminal group was performed using the electrophilic maleimide
functionality of PEG activated carboxylic acids. Reaction between the terminal SH group of 12 with
PEGꢀmal resulted in the formation of physiologically stable SꢀC bonds. The designed pseudoꢀtriꢀ
blockꢀcopolymer, PLGAꢀPEGꢀ12 copolymer, was synthesized using a twoꢀstep reaction (Scheme 5,
1
stage 1) as described above for the preparation of the diꢀblockꢀcopolymer. The HꢀNMR spectrum
recorded for PLGAꢀPEGꢀ12 revealed resonance shifts at 1.58, 3.64, 4.64ꢀ4.88, and 5.10ꢀ5.28 ppm
characteristics of PLGAꢀPEG backbone. Moreover, it also shared a series of partially overlapped
multiple peaks (Figure S27), especially in the aliphatic zone, attributable to the 12 signal pattern
(Figure S32). Furthermore, the strong absorption bands in 245 nm region observed in the UV
chromatogram (Figure 3), support the successful conjugation of 12 to PLGAꢀPEG copolymer.
Further characterization analyses such as molecular weights, refractive and polydispersity indexes,
were assessed by gel permeation chromatography (GPC) ꢀ size exclusion chromatography (SEC)
(see in Supporting Information for details).
Nanoformulation and Characterization of Nontargeted and Targeted NPs
In the present study, we used PLGAꢀA, PLGAꢀPEGꢀNH2, PLGAꢀ11, and PLGAꢀPEGꢀ12 polymer
systems to fabricate nontargeted and targeted NPs as novel constructs and suitable carriers for
8
ACS Paragon Plus Environment

Page 9 of 48
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
PDAC therapy. The PLGA was chosen owing to its physicochemical properties as as well as its
biocompatibility and biodegradability. Moreover, modification of the NP’ surface with PEG enables
nonspecific interactions reduction, prolonging circulation time, and increasing accumulation in
tumors due to the enhanced permeability and retention (EPR) effect.43ꢀ46 In particular, the
hydrophilic ethylene glycol repeats of PEG confer to NPs enhanced solubility in serum, with
reduced RES uptake and prolonged circulation time with respect to uncoated counterparts.47,48
Moreover, grafting with PEG prevents NP aggregation by increasing hydrophilicity via ether
repeats, which form hydrogen bonds with solvent, and by modulating the steric distance and the
amount of attraction between NPs, as well as those between tissue proteins and NPs. Modulation of
the NP flexibility and the reduced accumulation in the liver are other key factors that influence
extravasation, thus allowing for a favourable modulation of EPR effect.48
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To optimize the fabrication of NPs and to ensure rapid precipitation of batches containing the
hydrophilic PLGAꢀPEG coꢀpolymer, we used poly(epsilonꢀcaprolactone) (PCL) mixture with
PEGylated polymers to prepare PLGAꢀPEGꢀNH2 and PLGAꢀPEGꢀ12 NPs. PCL is useful for
controlled and sustained drug delivery due to its tunable mechanical properties, high permeability
and low toxicity.49ꢀ51
All batches of (1)ꢀencapsulated NPs were successfully prepared using a modified nanoprecipitation
method as described (Figure 1, Schemes 3 and 5, stage 2).19,51 During the precipitation in water, the
polymers selfꢀassembles to form NPs, in which the hydrophobic PLGA blocks would interact with
the PCL into a core, thus minimizing their exposure to aqueous surroundings. Simultaneously, the
hydrophilic COOH/NH2 surface or PEGꢀNH2 and PEGꢀ12 flexible moieties extend from the shell to
stabilize the core by preventing degradation and aggregation.
NPs generated from both polymer systems (i.e. PLGAꢀ and PLGAꢀPEGꢀbased NPs) were
characterized for surface morphology with SEM, particle size, zeta potential and encapsulation
efficiency (Table 1).
Figure 4 shows representative SEM images of PLGAꢀAꢀ (a), PLGAꢀ11ꢀ (b), PLGAꢀPEGꢀNH2/PCLꢀ
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 48
1
2
3
4
5
6
7
8
9
(c), and PLGAꢀPEGꢀ12/PCLꢀ (d) (1)ꢀloaded NPs. All particles were characterized by similar
morphological properties having smooth surface and spherical shape, and unimodal distribution.
However, the NPs have different mean diameters [PLGAꢀ (155.8 and 220.1 nm, for PLGAꢀAꢀ and
PLGAꢀ11 NPs, respectively) and PLGAꢀPEG/PCLꢀ (353.5 and 328.0 nm, for PLGAꢀPEGꢀ
NH2/PCLꢀ PLGAꢀPEGꢀ12/PCL NPs, respectively)] (Table 1 and Figure 4aꢀd).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The zeta potential was negative for PLGAꢀA NPs due to the negatively charged carboxyl groups on
the terminal of PLGAꢀA. PLGAꢀPEGꢀNH2ꢀ, PLGAꢀ11ꢀ and PLGAꢀPEGꢀ12 NPs showed zeta
potential between 10 and 14 mV due to their surface amine or amino acid funtionalities (Table 1).
Our statistical analysis highlighted that the amount of 1 loaded was significantly higher (p < 0.05)
for PLGAꢀPEGꢀNH2ꢀ and PLGAꢀPEGꢀ12 NPs (ranging from 20% to 24%) than that for PLGAꢀAꢀ
and PLGAꢀ11 NPs (Table 1). This difference is perhaps due to the higher hydrophilicity of the PEG
conjugated polymer than that of PLGA counterparts.
We obtained 50 to 69% yield for all preparations over multiple experiments. The PLGAꢀPEG/PCL
polymeric blended NPs produced encapsulation efficiency values ranging from 64% (for PLGAꢀ
PEGꢀNH2/PCL NPs) to 69% (for PLGAꢀPEGꢀ12/PCL NPs). The PLGAꢀbased nanosystems yields
were 50 and 55%, for PLGAꢀAꢀ and PLGAꢀ11 NPs, respectively. These differences can be
attributed to the presence of the hydrophobic PCL and PLGA chains and the hydrophilic PEG
moieties that can facilitate the incorporation of bioactive molecules into the polymeric matrix.
Additionally, to evaluate the cellular uptake of the NPs, four batches (PLGAꢀAꢀ, PLGAꢀPEGꢀNH2ꢀ,
PLGAꢀ11ꢀ and PLGAꢀPEGꢀ12 of fluorescent 6ꢀcoumarin (6C)ꢀencaspulated NPs were obtained and
characterized using the aboveꢀmentioned method (Table S34).
In Vitro Kinetic Release of 1 from NPs
The in vitro release profiles of compound 1 from NPs is depicted in Figure 5. The experiments were
performed in phosphateꢀbuffered saline (PBS) at pH 6.5 and 7.4 to simulate both the slightly acidic
microenvironment of extracellular fluid in most tumors and the physiological conditions in normal
10
ACS Paragon Plus Environment

Page 11 of 48
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
tissues, respectively.19 The experiments were carried out by direct determination of 1 from different
types of NPs in the release medium. The cumulative amount of 1 released from NPs indicated that
the nanoencapsulation resulted in an effective release rate of 1 under both experimental conditions
(i.e., PBS, pH 6.5 and 7.4, Figure 5a, and 5b, respectively). Nanoparticle formulations showed a
similar release behaviour, and this suggests that the pH of the medium did not significantly affect
the release of 1 from the nanoformulations. In particular, PLGAꢀAꢀ, PLGAꢀ11 NPs, and PLGAꢀ
PEGꢀNH2 NPs in both media shared similar kinetic release profiles. We observed a near 90%
release of 1 from PLGAꢀPEGꢀNH2 NPs within the first 2 h and a complete release within 4ꢀ6 h. We
observed only 45ꢀ50% release of 1 from PLGAꢀPEGꢀ12 NPs within 3 h in both acidic and
physiological medium, and about 70ꢀ80% at 6 h. A possible explanation for this difference could be
due to the presence of peptide 12 in PEGylated NPs potentially affecting degradation kinetics. More
specifically, during the generation of NPs, the PLGA block can interact with PCL to produce a
hydrophobic core. This construct might play a major role in stabilizing the core, whereas the other
part of PEGꢀ12 protrude from the particle surface, forming an external hydrophilic pseudoꢀshield,
and thus preventing degradation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Inhibition of cell proliferation
We compared the cytotoxicity of the targeted (1)ꢀencapsulated NPs (PLGAꢀ11 and PLGAꢀPEGꢀ12
NPs) with those of the nontargeted particles (PLGAꢀA and PLGAꢀPEGꢀNH2) in MIA PaCaꢀ2 cells
using MTT assay (Figure 6). The PLGAꢀ11 and PLGAꢀPEGꢀ12 (1)ꢀloaded NPs showed 70% and
87% growth inhibition, respectively. The nontargeted PLGAꢀA and PLGAꢀPEGꢀNH2 NPs showed
<30% and <50% growth inhibition (Figure 6), suggesting that targeted NPs are significantly more
effective than the nontargeted particles. In particular, we observed an improved antiproliferative
activity for targeting Plecꢀ1 by peptide 12 with (1)ꢀloaded NPs. This behaviour can be attributed to
an active targeting to Plecꢀ1 promoting an enhanced binding between NPs and the cells, thus
resulting in increased accumulation and cell uptake through receptor mediated endocytosis.
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 48
1
2
3
4
5
6
Surface plasmon resonance (SPR) binding assays
7
8
9
The ability of the most effective antiꢀPlecꢀ1 targeted NPs (PLGAꢀPEGꢀ12 NPs) to specifically bind
Plecꢀ1 protein, in comparison with the nontargeted ones (PLGAꢀPEGꢀNH2 NPs), was assessed by
realꢀtime interaction analysis using surface plasmon resonance (SPR) experiments.52 Kinetic and
affinity evaluation and determination of binding specificity were conducted by immobilizing the
Plecꢀ1 protein on a sensor surface. The analyte (PLGAꢀPEGꢀ12 NPs or PLGAꢀPEGꢀNH2 NPs) was
injected in solution over the surface. Changes in SPR response were analyzed, providing affinity
binding results, expressed as response units (RU). A difference of about 350ꢀ400 RU was observed
for NPs with and without peptide 12 (Figure 7), with a significant increase in response units (RU)
for PLGAꢀPEGꢀ12 NPs compared to PLGAꢀPEGꢀNH2 NPs for binding to Plecꢀ1. Indeed,
sensorgrams revealed a slow association and dissociation processes. These results demonstrate good
affinity for 12 targeted NPs toward Plecꢀ1 protein, with only a residual interaction provided by the
nontargeted NPs, thus supporting that this specific interaction can significantly contribute to the
growth inhibition of MIA PaCaꢀ2 cells by (1)ꢀloaded NPs.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cellular Uptake
The correlation between antiproliferative activity of the (1)ꢀloaded NPs with cellular uptake by the
cancer cells was further corroborated by internalization of 6ꢀcoumarinꢀloaded NPs, which were
visualized by fluorescence microscopy. Figure 8 shows images (20x magnification) of cellular
uptake efficiency of nontargeted and targeted NPs. In particular, after 1 h incubation, the targeted
PLGAꢀ11 and PLGAꢀPEGꢀ12 NPs showed higher cellular uptake than PLGAꢀA and PLGAꢀPEGꢀ
NH2, respectively, with strong fluorescence in perinuclear space, thus confirming their
accumulation and sustained retention by the cells.
12
ACS Paragon Plus Environment

Page 13 of 48
Journal of Medicinal Chemistry
1
2
CONCLUSIONS
3
4
5
6
7
8
Previously, we discovered a novel class of compounds displaying ROSꢀmediated decrease in
Src/FAK and STAT3 phosphorylation resulting in efficient inhibition of cell proliferation, adhesion,
and migration, ultimately leading to apoptosis. In an attempt to improve their pharmacological, PK,
and distribution properties we used a novel nanotechnology platform to selectively deliver our
novel ROSꢀinducing compounds to PDAC cells.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We demonstrated that the MIA PaCaꢀ2 cells are more sensitive to the targeted than the nontargeted
particles. Furthermore, we observed that the targeted (1)ꢀloaded NPs (PLGAꢀ11 and PLGAꢀPEGꢀ
12) efficiently bind to MIA PaCaꢀ2 cells. In particular, these data indicate that functionalization of
the NPs surface with peptides to selectively bind to Plecꢀ1 or with nonspecific small molecules as
targeting ligands, efficiently increase their adhesion and uptake in PDAC cells in comparison to the
nonfunctionalized particles. As expected, the NPs targeting Plecꢀ1 demonstrated to be the most
effective prototypes. These results also support the hypothesis that the primary role of targeting
agents is to increase cellular uptake of NPs and their loading into tumor cells. These insights might
constitute a suitable translational platform for development of powerful therapeutic tools to treat
pancreatic cancer.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 48
1
2
EXPERIMENTAL SECTION
Materials
3
4
5
6
7
8
9
Poly(D,Lꢀlactideꢀcoꢀglycolide) carboxylic acid end group, (PLGAꢀA, Purasorb Polymer PDLG
5002A, Mw ~17,000) (lactide/glycolide ratio of 50:50, viscosity range: 0.20 dL/g in CHCl3) was
kindly provided by Corbion Purac (Gorinchem, The Netherlands). Poly(epsilonꢀcaprolactone)
(PCL, Mw ~80,000), polyvinyl alcohol (PVA, Mw Mw 31,000–50,000), and 6ꢀcoumarin were
purchased from SigmaꢀAldrich (Steinheim, Germany). The heterobifunctional PEG polymer with a
terminal amine and carboxylic acid functional group, NH2ꢀPEGꢀCOOH (Mw = 3400), the
homobifunctional PEG polymers with both terminal amines NH2ꢀPEGꢀNH2 (HCl salt, Mw = 3500),
and with a terminal amine and maleimide functionality NH2ꢀPEGꢀmal (TFA salt, Mw = 3500), were
purchased from JenKem Technology USA. The anti Plecꢀ1 peptide (KTLLPTPC, 12, lyophilized
trifluoroacetate salt) was purchased from CASLO Laboratory ApS, c/o Scion Denmark Technical
University, Lyngby, Denmark. The peptide was prepared by (Fmoc)ꢀbased solidꢀphase peptide
synthesis using HꢀCys(Trt)ꢀCTC Resin (Supporting Information, S29). The peptide 12 was purified
by preparative reversedꢀphase high performance liquid chromatography (RPꢀHPLC) and quality
controlled by analytical HPLC and mass spectrometry (see in Supporting Information for details,
S29ꢀS32).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
All solvents and other chemicals (used for the preparation of 1) were purchased from Sigmaꢀ
Aldrich or Carlo Erba (analytical grade and were used without further purification). Melting points
(mp) were determined using an Electrothermal melting point or a Köfler apparatus and are
uncorrected. Nuclear magnetic resonance (1HꢀNMR, 13CꢀNMR) spectra were determined in CDCl3,
DMSOꢀd6 or CDCl3/DMSOꢀd6 (in 3/1 ratio) and were recorded on 400 MHz Bruker Avance III.
Chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane (TMS),
used as an internal standard. Splitting patterns are designated as follows: s, singlet; d, doublet; t,
triplet; q, quadruplet; m, multiplet; brs, broad singlet; dd, double doublet. The assignment of
14
ACS Paragon Plus Environment

Page 15 of 48
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
exchangeable protons (OH and NH) was confirmed by the addition of D2O. Mass spectra were
obtained on a tripleꢀquadrupole QqQ Varian 310ꢀMS mass spectrometer, a HewlettꢀPackard 5989
Mass Engine Spectrometer, or a MALDI micro MX (Waters, Micromass) equipped with a
reflectron analyser. Analytical thinꢀlayer chromatography (TLC) was carried out on Merck silica
gel Fꢀ254 plates. Flash chromatography purifications were performed on Merck Silica gel 60 (230ꢀ
400 mesh ASTM) as a stationary phase. Molecular weights, polydispersity and refractive indexes of
polymers were determined by GPCꢀSEC using the instrument Viscotek 270 (for SECꢀTDA,
Alfatest, Italy), and were performed by Alfatestlab s.r.l. (Cinisello Balsamo, Mi, Italy). The purity
of copolymers was determined by HPLC using an HP 1200 (Agilent Technologies, USA) system,
equipped with a Hypersil BDS C18 column (Alltech Italy, 250 × 4.6 mm i.d., 5 µm particle size);
these materials were found to be >95% pure. Elemental analyses for 1, 11, and for all intermediates,
were performed on a PerkinꢀElmer 2400 spectrometer at Laboratorio di Microanalisi, Dipartimento
di Chimica, Università di Sassari (Italy), and were within ±0.4% of the theoretical values.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of 6-{[4-(phenylcarbonyl)phenyl]amino}quinazoline-5,8-dione (1)
A solution of quinazolineꢀ5,8ꢀdione (2, 0.22 g, 1.37 mmol), cerium (III) chloride heptahydrate
(CeCl3·7H2O, 1.1 eq), and 4ꢀaminobenzophenone (3, 1.1 eq.) in absolute ethanol (20 mL) was
stirred at room temperature for 3 h. Next, most of the ethanol was removed under vacuum, and
water was added, followed by extraction with CH2Cl2. The organic layer was washed with water
and brine, dried over anhydrous sodium sulfate, and concentrated to dryness. The crude product was
purified by flash chromatography (eluent: ethyl acetate) to give the expected product. Redꢀbrown
solid. Yield: 79%; mp 233ꢀ234 °C (lit. mp 233ꢀ236 °C).12 1HꢀNMR (400 MHz, CDCl3): δ 9.70 (s,
1H, H4), 9.54 (s, 1H, H2), 7.93 (d, 2H, Ar–H), 7.80 (d, 2H, Ar–H), 7.75 (s, 1H, NH), 7.64–7.61 (m,
13
1H, Ar–H), 7.54–7.51 (m, 2H, Ar–H), 7.41 (d, 2H, Ar–H), 6.85 (s, 1H, H8). CꢀNMR (100 MHz,
CDCl3): δ 195.04, 180.77, 180.42, 163.87, 156.59, 153.99, 143.44, 140.25, 137.27, 132.71, 132.08,
129.93, 128.48, 123.29, 121.69, 106.37. MALDIꢀTOF: m/z 354 [M]+.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 48
1
2
3
4
5
6
Quinazoline-5,8-dione (2)
7
8
9
A solution of 5,8ꢀdimethoxyquinazoline (7, 0.50 g, 2.63 mmol) in (7:3) acetonitrile:water (13 mL)
was cooled at 0 °C in an ice bath and a solution of ceric ammonium nitrate [(NH4)2Ce(NO3)6 (2.7
eq) (9:1) acetonitrile:water (13 mL) was added dropwise. The reaction mixture was stirred for 20
min and, after monitoring with TLC (CHCl3/CH3OH 9.5:0.5), poured into ice/water and extracted
with CH2Cl2. Then, the organic layer was washed with water, dried over anhydrous sodium sulfate,
and concentrated to dryness to give 2 as brown powder. Yield: 69%. mp: > 320 °C. (lit. mp: > 320
°C).13 1HꢀNMR (400 MHz, DMSOꢀd6): δ 9.68 (s, 1H), 9.43 (s, 1H), 7.28 (d, 1H), 7.18 (d, 1H). 13Cꢀ
NMR (100 MHz, DMSOꢀd6): δ 184.07, 182.88, 162.08, 156.27, 152.60, 139.46, 137.74, 124.61.
ESI: m/z 161 [M+1]+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5,8-dimethoxyquinazoline (7)
Zinc powder (8.18 g, 125.13 mmol, 15.15 eq) was added to a suspension of N,N’ꢀ[(3,6ꢀdimethoxyꢀ
2ꢀnitrophenyl)methylene)]diformamide (6, 2.5 g, 8.26 mmol) in triturated ice (30 g) and glacial
acetic acid (11.5 mL), under constant magnetic stirring. The reaction mixture was stirred for 2 h in
ice bath, and for 4 h at room temperature. Next, the reaction mixture was dropped (through filter
paper) on cooled 50% NaOH (45 mL), and the yellow colored suspension thus formed was left
without stirring for 1 h. Then, the suspension was filtered to give a yellow powder, which was
solubilized in ethyl acetate, filtered, dried over anhydrous sodium sulfate, and concentrated to
dryness yielding the desired compound 7. Yield: 91%. mp: 116ꢀ117 °C. (lit. mp 116ꢀ119 °C).12 1Hꢀ
NMR (400 MHz, DMSOꢀd6): δ 9.64 (s, 1H), 9.28 (s, 1H), 7.38 (d, 1H), 7.09 (d, 1H), 3.98 (s, 3H),
13
3.94 (s, 3H). CꢀNMR (100 MHz, DMSOꢀd6): δ 154.88, 154.49, 148.35, 148.02, 141.60, 117.16,
113.59, 106.14, 56.11. GCꢀMS: m/z 190 [M]+.
16
ACS Paragon Plus Environment

Page 17 of 48
Journal of Medicinal Chemistry
1
2
N,N'-[(3,6-dimethoxy-2-nitrophenyl)methylene]diformamide (6)
3
4
5
6
7
8
A solution of 3,6ꢀdimethoxyꢀ2ꢀnitrobenzaldehyde (5, 4.0 g, 18.94 mmol) in formamide (H2NCHO,
66.5 eq.), heated at 40 °C, was exposed to dry HCl gas (1 h) until the temperature reached 80 °C.
Then, the solution was cooled to room temperature, and water/ice was added. Pale yellow
precipitate was formed, which was filtered, dried and triturated with ethyl acetate and petroleum
ether to yield the desired compound 6. Yield: 90%. mp: 254ꢀ255 °C (lit. mp 254 °C).12 1HꢀNMR
(400 MHz, DMSOꢀd6): δ 8.67 (d, 2H), 7.92 (s, 2H), 7.28 (s, 2H), 6.77 (t, 1H), 3.88 (s, 3H), 3.82 (s,
3H). NMR (100 MHz, DMSOꢀd6): δ 160.17, 150.77, 143.64, 139.60, 119.59, 114.33, 113.94, 57.02,
56.64, 48.51. ESI: m/z 284 [M+1]+.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3,6-Dimethoxy-2-nitrobenzaldehyde (5)
Nitric acid (91.83 mmol, 7.63 eq), acetic anhydride (43.31 mmol, 3.6 eq) and 2,5ꢀ
dimethoxybenzaldehyde (4, 1.3 g, 12.03 mmol) were added at 0 °C with stirring, respectively. After
1.5 h stirring, the mixture was poured onto 10 mL ice/water. The resulting yellow solid was filtered,
washed with cold water and then purified by flash chromatography on silica gel using ethyl acetateꢀ
petroleum ether (1:1) to give first the regioisomer 2,5ꢀdimethoxyꢀ4ꢀnitrobenzaldehyde, and then (by
further elution with only ethyl acetate), the desired compound 5. Yield: 68%. mp 165ꢀ167 °C (lit.
mp 163ꢀ165 °C and 159 °C).12 1HꢀNMR (400 MHz, DMSOꢀd6): δ 10.26 (s, 1H), 7.70 (d, 1H), 7.49
(d, 1H), 3.96 (s, 3H), 3.87 (s, 3H). 1HꢀNMR (400 MHz, CDCl3): δ 10.39 (s, 1H), 7.30 (d, 1H), 7.12
(d, 1H), 3.97 (s, 3H), 3.89 (s, 3H). 13CꢀNMR (100 MHz, DMSOꢀd6): δ 186.69, 166.22, 154.21,
153.42, 148.42, 123.73, 116.48, 57.14, 57.02. GCꢀMS: m/z 211 [M]+.
Synthesis of 2-amino-N-(3-aminopropyl)benzamide (11)
The intermediate tertꢀbutyl (3ꢀ(2ꢀaminobenzamido)propyl)carbamate (10, 0.23 g, 0.78 mmol) was
dissolved in 20 mL of 1:1 TFA and CH2Cl2 solution in an ice bath (0 °C), and stirred at room
temperature for 3 h. Then, the resulting solution was evaporated under reduced pressure. The
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 48
1
2
3
colorless residue was triturated with dry diethyl ether to yield the desired compound 11 as brown oil
4
5
6
7
8
9
1
+
(yield, 100%). HꢀNMR (400 MHz, DMSOꢀd6): δ 8.38 (brs , 1H, COꢀNH), 7.79 (brs , 3H, NH3 ),
7.50 (d, 1H, ArꢀH), 7.18 (t, 1H, ArꢀH), 6.75 (d, 1H, ArꢀH), 6.59 (t, 1H, ArꢀH), 3.29 (d, 2H, NHꢀ
CH2), 2.84 (d, 2H, CH2ꢀNH2), 1.79 (t, 2H, CH2). 13CꢀNMR (100 MHz, DMSOꢀd6): δ 169.02,
148.25, 131.75, 128.04, 116.96, 115.51, 115.38, 36.91, 35.83, 27.42 (CF3COOH, 158.84, 158.49,
158.13, 157.78, 120.26, 117.37, 114.46, 111.55). MALDIꢀTOF: m/z 193 [M+].
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of tert-butyl (3-(2-aminobenzamido)propyl)carbamate (10)
A mixture of isatoic anhydride (9, 0.22 g, 1.38 mmol), tertꢀbutyl (3ꢀaminopropyl)carbamate (8, 0.2
mL, 1.15 mmol), and potassium carbonate (0.32 g, 2.30 mmol, 2.0 eq) in anhydrous THF (3 mL)
was stirred at 70 °C for 20 h under nitrogen atmosphere. After cooling, water was added and stirring
was continued for 4 h. The reaction mixture was concentrated and extracted with ethyl acetate,
dried over Na2SO4, then concentrated and triturated to give a white powder (yield, 74%). mp 80ꢀ81
°C. 1HꢀNMR (400 MHz, DMSOꢀd6): δ 8.15 (brs , 1H, COꢀNH), 7.44 (d, 1H, ArꢀH), 7.12 (t, 1H, Arꢀ
H), 6.80 (brs , 1H, NHꢀCO), 6.68 (d, 1H, ArꢀH), 6.52 (t, 1H, ArꢀH), 6.36 (s, 2H, ArꢀNH2), 3.20ꢀ3.19
13
(m, 2H, NHꢀCH2), 2.97ꢀ2.96 (m, 2H, CH2ꢀNH2), 1.62ꢀ1.58 (m, 2H, CH2), 1.38 (s, 9H, CH3). Cꢀ
NMR (100 MHz, DMSOꢀd6): δ 168.78, 155.57, 149.50, 131.49, 127.90, 116.26, 114.86, 114.49,
77.45, 37.69, 36.44, 29.58, 28.23. MALDIꢀTOF: m/z 293 [M+].
Synthesis of PLGA-NHS
PLGAꢀNHS was prepared (yield, 94%) by using the previoulsy reported procedure,19 and with the
following conditions: PLGAꢀA (3.0 g, 0.166 mmol), Nꢀhydroxysuccinimide (NHS, 0.077 g, 0.714
1
mmol) and 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDC, 0.14 g, 0.664 mmol). HꢀNMR
(400 MHz, CDCl3): δ 5.23ꢀ5.20 (m, 1H, ꢀOCꢀCH(CH3)Oꢀ, PLGA), 4.90ꢀ4.70 (m, 2H, ꢀOCꢀCH2Oꢀ,
PLGA), 1.58 (brs , 3H, ꢀOCꢀCH(CH3)Oꢀ, PLGA).19
18
ACS Paragon Plus Environment

Page 19 of 48
Journal of Medicinal Chemistry
1
2
Synthesis of pseudo-di-block-copolymer PLGA-11
3
4
5
6
7
8
To a solution of PLGAꢀNHS (0.50 g, 0.028 mmol) in anhydrous dimethylformamide (DMF, 3 mL),
a solution of 11 (0.034 g, 0.111 mmol) in DMF (1.5 mL) and N,Nꢀdiisopropylethylamine (DIPEA,
0.90 mL, 5.16 mmol) were added. The resulting mixture was magnetically stirred at room
temperature for 24 h under nitrogen atmosphere. The desired pseudoꢀdiꢀblockꢀcopolymer PLGAꢀ11
was obtained by precipitation from cold diethyl ether and subsequent vacuum drying (yield, 93%).
1HꢀNMR (400 MHz, CDCl3): δ 5.30ꢀ5.15 (m, 1H, ꢀOCꢀCH(CH3)Oꢀ, PLGA), 4.95ꢀ4.65 (m, 2H, ꢀ
OCꢀCH2Oꢀ, PLGA), 1.61 (brs , 3H, ꢀOCꢀCH(CH3)Oꢀ, PLGA).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of PLGA-PEG-NH2
To a solution of PLGAꢀNHS (0.50 g, 0.028 mmol) in anhydrous CHCl3 (2 mL), NH2ꢀPEGꢀNH2
(0.49 g, 0.14 mmol) in anhydrous CHCl3 (5 mL) and was added DIPEA (0.9 mL, 5.17 mmol), and
the solution was magnetically stirred at room temperature for 24 h under nitrogen atmosphere. The
PLGAꢀPEGꢀNH2 copolymer was obtained by precipitation with cold diethyl ether, dried under
1
vacuum, and used for NP preparation without further treatment (yield, 89%). HꢀNMR (400 MHz,
CDCl3): δ 5.30ꢀ5.12 (m, 1H, ꢀOCꢀCH(CH3)Oꢀ PLGA), 4.90ꢀ4.62 (m, 2H, ꢀOCꢀCH2Oꢀ PLGA), 3.65
(brs , 2H, ꢀCH2CH2Oꢀ, PEG), 1.58 (brs , 3H, ꢀOCꢀCH(CH3)Oꢀ, PLGA).
Synthesis of PLGA-PEG-12
PLGAꢀPEGꢀmal (0.47 g, 0.022 mmol) and peptide KTLLPTPC (12, 0.02 g, 0.023 mmol) were
dissolved in 15 mL of 1:1 solution of acetonitrile and DMF. The resulting reaction was stirred for
24 h under nitrogen atmosphere. The desired pseudoꢀtriꢀblockꢀcopolymer was obtained by
precipitation with cold diethyl ether, then dried under vacuum, and used for NP preparation without
further treatment (yield, 88%). 1HꢀNMR (400 MHz, CDCl3): δ 5.28ꢀ5.10 (m, 1H, ꢀOCꢀCH(CH3)Oꢀ,
PLGA), 4.88ꢀ4.64 (m, 2H, ꢀOCꢀCH2Oꢀ, PLGA), 3.64 (brs , 2H, ꢀCH2CH2Oꢀ, PEG), 1.58 (brs , 3H, ꢀ
OCꢀCH(CH3)Oꢀ, PLGA).
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 48
1
2
3
4
5
6
Synthesis of PLGA-PEG-mal
7
8
9
To a solution of PLGAꢀNHS (0.51 g, 0.029 mmol) in anhydrous CHCl3 (3 mL), NH2ꢀPEGꢀ
maleimide (0.40 g, 0.11 mmol) in anhydrous CHCl3 (2 mL) and was added DIPEA (0.92 mL, 5.31
mmol), and the solution was magnetically stirred at room temperature for 24 h under nitrogen
atmosphere. The PLGAꢀPEGꢀmal copolymer was obtained by precipitation with cold diethyl ether,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
then dried under vacuum, and used for NP preparation without further treatment (yield, 95%). Hꢀ
NMR (400 MHz, CDCl3): δ 5.32ꢀ5.10 (m, 1H, ꢀOCꢀCH(CH3)Oꢀ PLGA), 4.93ꢀ4.63 (m, 2H, ꢀOCꢀ
CH2Oꢀ PLGA), 3.64 (brs , 2H, ꢀCH2CH2Oꢀ, PEG), 1.58 (brs , 3H, ꢀOCꢀCH(CH3)Oꢀ, PLGA).
Nanoformulation and characterization of non-targeted and 11- and 12-targeted NPs
Preparation of (1)ꢀloaded PLGAꢀA and PLGAꢀ11 NPs
Targeted (PLGAꢀ11) and nontargeted (PLGAꢀA) NPs were prepared using a modified solvent
displacement method as previously described.19 Briefly, polymer (PLGAꢀA or PLGAꢀ11) (99.5 mg)
and 1 (0.5 mg) were coꢀdissolved in acetonitrile (6.0 mL) and added dropwise into 20 mL of PVA
1% (w/v) solution under rapid magnetic stirring. The resulting suspension was treated as reported,19
with minimal modifications (ie, centrifugation was conducted at 13000 rpm, and pellets suspended
in a minimal volume of water before storage). Batches without 1 were prepared and used as
comparison.
Preparation of (1)ꢀloaded PLGAꢀPEGꢀNH2 and PLGAꢀPEGꢀ12 NPs
The PLGAꢀPEGꢀ12 conjugate (or the PLGAꢀPEGꢀNH2 copolymer for nontargeted particles) (19.9
mg), PCL (79.6 mg), and 1 (0.5 mg) were coꢀdissolved in 3 mL of acetonitrile and added dropwise
to 3 mL of distilled water under magnetic stirring. Samples were treated as reported above. Batches
without 1 were prepared and used for comparison.
20
ACS Paragon Plus Environment

Page 21 of 48
Journal of Medicinal Chemistry
1
2
3
Preparation of 6ꢀcoumarinꢀloaded NPs
4
5
6
7
8
The preparation of NPs loaded with 6ꢀcoumarin was the same as that of NPs without dye, except
that 6ꢀcoumarin (0.5 mg) was additionally added to acetonitrile containing polymers (total weight
100 mg) before nanoprecipitation.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Characterization of nanoparticles
Morphology, size and zeta potential
The morphology of NPs was characterized by Scanning Electron Microscopy (SEM) (model DSM
962, Carl Zeiss Inc., Jena, Germany). A drop of NPs aqueous suspension was placed on a glass
cover slide and dried under vacuum for 12 h. Next, the slides were mounted on aluminum stub and
the samples were then analyzed at 20 kV acceleration voltage after gold sputtering, under an argon
atmosphere.
Mean diameter and polydispersity index of NPs were measured by using photon correlation
spectroscopy (Zetasizer Nano ZS, Malvern Instruments, U.K.) at 25 °C, and a scattering angle of
90° after dilution of samples with MilliꢀQ water (1.0 mg of NPs / 2.0 mL). Each sample was
measured in triplicate.
The zeta potential of the NPs was detected at 25 °C with a Zeta Plus analyzer (Brookhaven, USA).
The samples were diluted with distilled water (1.0 mg of NPs / 2.0 mL) and sonicated for several
minutes before measurement. All data were obtained with the average of three measurements.
Determination of 1 content and yield of productions
The amount of the encapsulated 1 into NPs was determined by dissolving a weighted amount (10
mg) of dried (1)ꢀloaded NPs in CH2Cl2 (1 mL), and measured using an HPLC method, according
with a procedure previously reported by us,19 with the following modification: the mobile phase
consisting of solvent A/solvent B (60/40 v/v), where solvent A was TFA in water (0.1/99.9 v/v) and
solvent B was acetonitrile/water/TFA (95/0.43/0.07 v/v), both prepared daily and degassed by
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 48
1
2
3
4
5
6
7
8
9
sonication for 30 min, and filtered before use. Elution was performed at a flow rate of 0.2 mL/min.
The injection volume was 25 ꢁL, and the wavelength for UV detection was 255 nm. The total
analysis time was 20 min, and the retention time of 1 was 8.3 min. The calibration curves were
found to be linear in the range of 2.5ꢀ100 ꢁg/mL (y = 777.56x ꢀ 303.23, R2 = 0.9999). The yields of
production were expressed as the weight percentage of the final product after drying, regarding the
initial total amount of solid materials used for the preparation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The amount of 6ꢀcoumarin encapsulated was determined using the same procedure already
described by us,48 with UV−vis spectroscopy analysis conducted at 450 nm.
In vitro kinetics release of 1 from NPs
Formulation of PLGAꢀA, PLGAꢀ11, PLGAꢀPEGꢀNH2 and PLGAꢀPEGꢀ12 (1)ꢀloaded NPs, were
chosen for the next step of the study. Aliquots of (1)ꢀloaded NPs in original suspensions (containing
about 1.0 ꢀg of 1) were dispersed in 2.0 mL of PBS at different pHs (pH 6.5 and pH 7.4) containing
0.1% w/v Tweenꢀ80 (used to improve the solubility of 1 in PBS), and continuously shaken at 37 °C.
At various time points, the suspension was centrifuged at 13,000 rpm for 2 min, and 1.0 mL
aliquots of medium were taken from supernatant and replaced with fresh solution. The collected
samples were extracted with 0.2 mL dichloromethane, evaporated, and reconstituted in 100 ꢀL
mobile phase. The concentration of 1 released from the NPs was determined by HPLC assay as
described above and corrected for the dilution due to the addition of fresh medium.
Cell culture
Pancreatic cancer cell line (MIA PaCaꢀ2) was purchased from the American Type Culture
Collection (Manassas, VA, USA). Cells were maintained in culture under 35 passages and tested
regularly for Mycoplasma contamination using Plasmo TestTM (InvivoGen, San Diego, CA, USA).
MIA PaCaꢀ2 cell line was maintained in appropriate growth media DMEM (Cellgro, Mediatech,
Manassas, VA, USA) containing 10% heatꢀinactivated FBS (Geminiꢀ Bioproducts, West
22
ACS Paragon Plus Environment

Page 23 of 48
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Sacramento, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2. For subculture and
experiments, cells were washed with 1 × Dulbecco’s PBS (DPBS, Cellgro), detached using 0.025%
trypsinꢀEDTA (Cellgro), collected in growth media and centrifuged. All experiments were
performed in growth media using subꢀconfluent cells in the exponential growth phase.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cytotoxicity assay
Cytotoxicity was assessed by a 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide
(MTT) assay as previously described.12 Briefly, cells were seeded in 96ꢀwell tissue culture treated
plates and allowed to adhere overnight. Cells were subsequently treated with vehicle or 100 ꢁL of a
suspension of (1)ꢀloaded NPs in serumꢀfree culture medium to a final concentration of 2.8 ꢁM 1,
then incubated in medium at 37 °C for required amount of time. After 72 h, MTT (0.3 mg/mL) was
added to each well. Cells were incubated with MTT for 3 h at 37 °C. After removal of the
supernatant, DMSO was added and the absorbance was read at 570 nm. All assays were done in
triplicate. Percent cytotoxicity was calculated by comparing the absorbance from treated wells to
that of the control wells using following formula: % cytotoxicity = 100 x (1 ꢀ [Abs (drug
treated)/Abs (control)]).
Surface plasmon resonance binding assays
Binding experiments were performed using a Biacore Tꢀ200 instrument (Biacore, Uppsala, Sweden)
at 25 °C. 11000 RU of the human Plecꢀ1 partial ORF (4384ꢀ4493 a.a.) recombinant protein with
GSTꢀtag at Nꢀterminal (37.84 kDa, from Abnova Corporation, Taiwan) were directly immobilized
on the NꢀethylꢀNꢀ(3ꢀdimethylaminopropyl) carbodiimide (EDC) and Nꢀhydroxysuccinimide (NHS)
activated flow cell2 of the CM5 chip (GE certified) in water. The flow rate of the 10 mM sodium
acetate buffer (pH 5.0), used for capturing the protein, was 5 ꢁl/min. The unoccupied sites were
blocked with 1M ethanolamine.
NPs were diluted to 5 mg/ml in standard HBSꢀN buffer [10 mM HEPES buffer (pH 7.4), 150 mM
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 48
1
2
3
4
5
6
7
8
9
NaCl, 3 mM EDTA, 0.05% P20 (polyoxyethylenesorbitan), and 1 mg/ml bovine serum albumin
(BSA)], sonicated with 5 sec pulses three times and kept on ice until ready. Comparative binding
analysis was performed using analyte NPs flowed over the chip at single analyte concentration, with
a flow rate of 30 ꢁl/min. Binding of analyte to the immobilized protein was monitored in real time.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fluorescence Microscopy
Cells were seeded in clear bottom black plates and allowed to adhere overnight. Following which
cells were treated with indicated samples for 1h at 37 °C. After this, cells were washed with 1x PBS
to remove excess nanoparticles and dye. Cells were then imaged using BD Pathway 435 Highꢀ
Content Bioimager (BD Biosciences) using 20x objective.
Statistical analysis. The data for preparation and characterization of NPs as well as drug release
studies were processed and analyzed by Origin software (version 7.0 SR0, OriginLab Corporation,
USA). The statistical analysis, evaluated by a Student’s tꢀtest and pꢀvalues < 0.05, was considered
statistically significant. The data obtained from cytotoxicity assays were processed by Oneꢀway
analysis of variance (ANOVA) followed by a postꢀhoc “NewmanꢀKeuls Multiple Comparison Test”
to detect differences of means among treatments with significance defined as p < 0.05.
ASSOCIATED CONTENT
Supporting Information. MS and NMR spectra, elemental analyses, for compounds 1, 2, 5, 6, 7,
1
10, 11. NMR spectra for polymers PLGAꢀ11, PLGAꢀPEGꢀNH2 and PLGAꢀPEGꢀ12. HꢀNMR
spectrum for peptide 12. Synthesis and characterization of 12. Characterization details of 6ꢀ
coumarin (6C)ꢀloaded NPs. GPCꢀSEC data (molecular weights, polydispersity and refractive
indexes) for PLGAꢀPEGꢀNH2 and PLGAꢀPEGꢀ12 polymers.
24
ACS Paragon Plus Environment

Page 25 of 48
Journal of Medicinal Chemistry
1
2
3
AUTHOR INFORMATION
4
5
6
Corresponding Authors:
7
8
9
For M.S.: phone, +39 079ꢀ228ꢀ753; fax: +39 079ꢀ229ꢀ559, eꢀmail: mario.sechi@uniss.it
For N.N.: phone, +1 734 647ꢀ2732; fax, +1 734 647ꢀ8430, eꢀmail: neamati@umich.edu
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the Regione Autonoma della Sardegna for financial support
grant CRP 25920, awarded to MS within the frame of “Legge regionale n. 7/2007, promozione della
ricerca scientifica e dell’innovazione tecnologica in Sardegna, Annualità 2010”, and the Fondazione
Banco di Sardegna for its partial support. The work in NN laboratory was supported by the National
Cancer Institute Grant CA188252. The authors thank Dr. Anna Maria Roggio for her help with
Mass and HPLC analysis, the Purac Biomaterials (Gorinchem, Netherlands) for providing the
PLGA polymer, the Precision AntibodyTM (Columbia, MD, USA), the CASLO Laboratory ApS, c/o
Scion Denmark Technical University (Lyngby, Denmark), and the Alfatestlab s.r.l. (MI, Italia) for
their helpful technical assistance.
ABBREVIATIONS
NPs, nanoparticles; PLGA, polyꢀ(D,Lꢀlactideꢀcoꢀglycolide); PLGAꢀPEG, poly(D,Lꢀlacticꢀcoꢀ
glycolic acid)ꢀblockꢀpoly(ethylene glycol); PCL, poly(epsilonꢀcaprolactone); PaCa, Pancreatic
Cancer; PDAC, ductal adenocarcinoma; QDs, quinazolinedioneꢀbased compounds; ROS, reactive
oxygen species; Plecꢀ1, Plectinꢀ1; PTP, KTLLPTP peptide; CysꢀPTP, KTLLPTPC peptide; 2ABA,
2ꢀaminoꢀNꢀ(3ꢀaminopropyl)benzamide;
TFA,
trifluoroacetic
acid;
Fmoc,
fluorenylmethyloxycarbonyl; SPR, surface plasmon resonance; BSA, bovine serum albumin; GPC,
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 48
1
2
3
4
5
6
7
8
9
gel permeation chromatography; SEC, size exclusion chromatography; MTT, 3ꢀ(4,5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide; Akt, protein kinase B; Src, protoꢀoncogene
tyrosineꢀprotein kinase; FAK, focal adhesion kinase; STAT3, signal transducer and activator of
transcription 3; RES, reticuloendothelial system; PK, pharmacokinetic
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
26
ACS Paragon Plus Environment

Page 27 of 48
Journal of Medicinal Chemistry
1
2
3
4
5
References
[1] Ryan, D. P.; Hong, T. S.; Bardeesy, N. Pancreatic adenocarcinoma. N. Engl. J. Med. 2014, 371,
1039ꢀ1049.
6
7
8
9
[2] Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer statistics, 2016. CA Cancer J. Clin. 2016, 66, 7ꢀ30.
[3] Malvezzi, M.; Bertuccio, P.; Levi, F.; La Vecchia, C.; Negri, E. European cancer mortality
predictions for the year 2014. Ann. Oncol. 2014, 25, 1650ꢀ1656.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[4] Hidalgo, M. New insights into pancreatic cancer biology. Ann. Oncol. 2012, 23 (Suppl 10), x135ꢀ
x138.
[5] Hidalgo, M. Pancreatic cancer. N. Engl. J. Med. 2010, 362, 1605ꢀ1617.
[6] Conroy, T.; Desseigne, F.; Ychou, M.; Bouche, O.; Guimbaud, R.; Becouarn, Y.; Adenis, A.;
Raoul, J. L.; GourgouꢀBourgade, S.; de la Fouchardiere, C.; Bennouna, J.; Bachet, J. B.; Khemissaꢀ
Akouz, F.; PereꢀVerge, D.; Delbaldo, C.; Assenat, E.; Chauffert, B.; Michel, P.; MontotoꢀGrillot,
C.; Ducreux, M.; Groupe Tumeurs Digestives of, U.; Intergroup, P. FOLFIRINOX versus
gemcitabine for metastatic pancreatic cancer. N. Engl. J. Med. 2011, 364, 1817ꢀ1825.
[7] Di Marco, M.; Di Cicilia, R.; Macchini, M.; Nobili, E.; Vecchiarelli, S.; Brandi, G.; Biasco, G.
Metastatic pancreatic cancer: is gemcitabine still the best standard treatment? (Review). Oncol. Rep.
2010, 23, 1183ꢀ1192.
[8] Von Hoff, D. D.; Ervin, T.; Arena, F. P.; Chiorean, E. G.; Infante, J.; Moore, M.; Seay, T.;
Tjulandin, S. A.; Ma, W. W.; Saleh, M. N.; Harris, M.; Reni, M.; Dowden, S.; Laheru, D.; Bahary,
N.; Ramanathan, R. K.; Tabernero, J.; Hidalgo, M.; Goldstein, D.; Van Cutsem, E.; Wei, X.;
Iglesias, J.; Renschler, M. F. Increased survival in pancreatic cancer with nabꢀpaclitaxel plus
gemcitabine. N. Engl. J. Med. 2013, 369, 1691ꢀ1703.
[9] Srinivasarao, M.; Galliford, C. V.; Low, P. S. Principles in the design of ligandꢀtargeted cancer
therapeutics and imaging agents. Nat. Rev. Drug. Discov. 2015, 14, 203ꢀ219.
[10]
Byrne, J. D.; Betancourt, T.; BrannonꢀPeppas, L. Active targeting schemes for nanoparticle
systems in cancer therapeutics. Adv. Drug. Deliv. Rev. 2008, 60, 1615ꢀ1626.
[11]
Lammers, T.; Kiessling, F.; Hennink, W. E.; Storm, G. Drug targeting to tumors: principles,
pitfalls and (preꢀ) clinical progress. J. Control. Release 2012, 161, 175ꢀ187.
[12]
Pathania, D.; Sechi, M.; Palomba, M.; Sanna, V.; Berrettini, F.; Sias, A.; Taheri, L.;
Neamati, N. Design and discovery of novel quinazolinedioneꢀbased redox modulators as therapies
for pancreatic cancer. Biochim. Biophys. Acta 2014, 1840, 332ꢀ343.
[13]
Pathania, D.; Kuang, Y.; Sechi, M.; Neamati, N. Mechanisms underlying the cytotoxicity of
a novel quinazolinedioneꢀbased redox modulator, QD232, in pancreatic cancer cells. Br. J.
Pharmacol. 2015, 172, 50ꢀ63.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 48
1
2
3
4
5
6
7
[14]
[15]
Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144,
646ꢀ674.
Pathania, D.; Millard, M.; Neamati, N. Opportunities in discovery and delivery of anticancer
drugs targeting mitochondria and cancer cell metabolism. Adv. Drug Deliv. Rev. 2009, 61, 1250ꢀ
8
9
1275.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[16]
[17]
Storz, P. Reactive oxygen species in tumor progression. Front. Biosci. 2005, 10, 1881ꢀ1896.
Shackelford, R. E.; Kaufmann, W. K.; Paules, R. S. Oxidative stress and cell cycle
checkpoint function. Free Radic. Biol. Med. 2000, 28, 1387ꢀ1404.
[18]
Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ROSꢀmediated
mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 2009, 8, 579ꢀ591.
[19]
Sanna, V.; Pintus, G. P.; Roggio, A. M.; Punzoni, S.; Posadino, A. M.; Arca, A.; Marceddu,
S.; Bandiera, P.; Uzzau, S.; Sechi, M. Targeted biocompatible nanoparticles for the delivery of (ꢀ)ꢀ
epigallocatechin 3ꢀgallate to prostate cancer cells. J. Med. Chem. 2011, 54, 1321ꢀ1332.
[20]
Davis, M. E.; Chen, Z. G.; Shin, D. M. Nanoparticle therapeutics: an emerging treatment
modality for cancer. Nat. Rev. Drug Discov. 2008, 7, 771ꢀ782.
[21]
Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as
an emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2, 751ꢀ760.
[22]
3, 16ꢀ20.
[23] Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Design, functionalization
Farokhzad, O. C.; Langer, R. Impact of nanotechnology on drug delivery. ACS Nano 2009,
strategies and biomedical applications of targeted biodegradable/biocompatible polymerꢀbased
nanocarriers for drug delivery. Chem. Soc. Rev. 2013, 42, 1147ꢀ1235.
[24]
Shi, J.; Xiao, Z.; Kamaly, N.; Farokhzad, O. C. Selfꢀassembled targeted nanoparticles:
evolution of technologies and bench to bedside translation. Acc. Chem. Res. 2011, 44, 1123ꢀ1134.
[25]
Sanna, V.; Pala, N.; Sechi, M. Targeted therapy using nanotechnology: focus on cancer. Int.
J. Nanomedicine 2014, 9, 467ꢀ483.
[26]
Gu, F.; Langer, R.; Farokhzad, O. C. Formulation/preparation of functionalized
nanoparticles for in vivo targeted drug delivery. Methods Mol. Biol. 2009, 544, 589ꢀ598.
[27]
Alexis, F.; Pridgen, E. M.; Langer, R.; Farokhzad, O. C. Nanoparticle technologies for
cancer therapy. Handb. Exp. Pharmacol. 2010, 197, 55ꢀ86.
[28]
Kamaly, N.; Xiao, Z.; Valencia, P. M.; RadovicꢀMoreno, A. F.; Farokhzad, O. C. Targeted
polymeric therapeutic nanoparticles: design, development and clinical translation. Chem. Soc. Rev.
2012, 41, 2971ꢀ3010.
28
ACS Paragon Plus Environment

Page 29 of 48
Journal of Medicinal Chemistry
1
2
3
[29]
Siu, D. Cancer therapy using tumorꢀassociated antigens to reduce side effects. Clin. Exp.
4
5
Med. 2009, 9, 181ꢀ198.
6
7
8
[30]
[31]
Steinbock, F. A.; Wiche, G. Plectin: a cytolinker by design. Biol. Chem. 1999, 380, 151ꢀ158.
Bausch, D.; Thomas, S.; MinoꢀKenudson, M.; Fernandezꢀdel, C. C.; Bauer, T. W.; Williams,
9
M.; Warshaw, A. L.; Thayer, S. P.; Kelly, K. A. Plectinꢀ1 as a novel biomarker for pancreatic
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cancer. Clin. Cancer Res. 2011, 17, 302ꢀ309.
[32]
Kelly, K. A.; Bardeesy, N.; Anbazhagan, R.; Gurumurthy, S.; Berger, J.; Alencar, H.;
Depinho, R. A.; Mahmood, U.; Weissleder, R. Targeted nanoparticles for imaging incipient
pancreatic ductal adenocarcinoma. PLoS Med. 2008, 5, e85.
[33]
Weissleder, R.; Kelly, K.; Sun, E. Y.; Shtatland, T.; Josephson, L. Cellꢀspecific targeting of
nanoparticles by multivalent attachment of small molecules. Nat. Biotechnol. 2005, 23, 1418ꢀ1423.
[34]
Langer, R.; Folkman, J. Polymers for the sustained release of proteins and other
macromolecules. Nature 1976, 263, 797–800.
[35]
Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R.
Biodegradable longꢀcirculating polymeric nanospheres. Science 1994, 263, 1600ꢀ1603.
[36]
Farokhzad, O. C.; Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.; Richie, J. P.;
Langer, R. Targeted nanoparticleꢀaptamer bioconjugates for cancer chemotherapy in vivo. Proc.
Natl. Acad. Sci. U.S. A. 2006, 103, 6315ꢀ6320.
[37]
Gu, F.; Zhang, L.; Teply, B. A.; Mann, N.; Wang, A.; Radovicꢀ Moreno, A. F.; Langer, R.;
Farokhzad, O. C. Precise engineering of targeted nanoparticles by using selfꢀassembled
biointegrated block copolymers. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2586ꢀ2591.
[38]
Gu, F.; Langer, R.; Farokhzad, O. C. Formulation/preparation of functionalized
nanoparticles for in vivo targeted drug delivery. Methods Mol Biol. 2009, 544, 589ꢀ598.
[39]
Otsuka, H.; Nagasaki, Y.; Kataoka, K. PEGylated nanoparticles for biological and
pharmaceutical applications. Adv. Drug. Deliv. Rev. 2003, 55, 403ꢀ419.
[40]
Avgoustakis, K. PEGylated poly(lactide) and poly(lactideꢀcoglycolide) nanoparticles:
Preparation, properties and possible applications in drug delivery. Curr. Drug. Deliv. 2004, 1, 321ꢀ
333.
[41]
Betancourt, T.; Byrne, J. D.; Sunaryo, N.; Crowder, S. W.; Kadapakkam, M.; Patel, S.;
Casciato, S.; BrannonꢀPeppas, L. PEGylation strategies for active targeting of PLA/PLGA
nanoparticles. J. Biomed. Mater. Res. A. 2009, 91, 263ꢀ276.
[42]
Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; LevyꢀNissenbaum, E.;
RadovicꢀMoreno, A. F.; Langer, R.; Farokhzad, O. C. Formulation of functionalized PLGAꢀPEG
nanoparticles for in vivo targeted drug delivery. Biomaterials 2007, 28, 869ꢀ876.
29
ACS Paragon Plus Environment