Targeted biocompatible nanoparticles for the delivery of (-)-epigallocatechin 3-gallate to prostate cancer cells.

Abstract

Molecular targeted cancer therapy mediated by nanoparticles (NPs) is a promising strategy to overcome the lack of specificity of conventional chemotherapeutic agents. In this context, the prostate-specific membrane antigen (PSMA) has demonstrated a powerful potential for the management of prostate cancer (PCa). Cancer chemoprevention by phytochemicals is emerging as a suitable approach for the treatment of early carcinogenic processes. Since (-)-epigallocatechin 3-gallate (EGCG) has shown potent chemopreventive efficacy for PCa, we designed and developed novel targeted NPs in order to selectively deliver EGCG to cancer cells. Herein, to explore the recent concept of "nanochemoprevention", we present a study on EGCG-loaded NPs consisting of biocompatible polymers, functionalized with small molecules targeting PSMA, that exhibited a selective in vitro efficacy against PSMA-expressing PCa cells. This approach could be beneficial for high risk patients and would fulfill a significant therapeutic need, thus opening new perspectives for novel and effective treatment for PCa.

Full text

Page 1

rXXXX American Chemical Society
A
dx.doi.org/10.1021/jm1013715|J. Med. Chem. XXXX, XXX, 000–000
ARTICLE
pubs.acs.org/jmc
Targeted Biocompatible Nanoparticles for the Delivery of
(-)-Epigallocatechin 3-Gallate to Prostate Cancer Cells
Vanna Sanna,*,†Gianfranco Pintus,*,‡Anna Maria Roggio,†Stefania Punzoni,‡Anna Maria Posadino,†,‡
Alessandro Arca,§,^Salvatore Marceddu,
||Pasquale Bandiera,‡Sergio Uzzau,†,‡and Mario Sechi*,§,^
†Porto Conte Ricerche, Localit? a Tramariglio, 07041, Alghero, Sassari, Italy
‡Department of Biomedical Sciences, Centre of Excellence for Biotechnology Development and Biodiversity Research,
University of Sassari, Sassari, Italy
§Dipartimento Farmaco Chimico Tossicologico, Universit? a di Sassari, 07100 Sassari, Italy
)
Istituto di Scienze delle Produzioni Alimentari (ISPA), CNR, Sezione di Sassari, Italy
ABSTRACT: Molecular targeted cancer therapy mediated by nano-
particles (NPs) is a promising strategy to overcome the lack of
specificity of conventional chemotherapeutic agents. In this context,
the prostate-specific membrane antigen (PSMA) has demonstrated a
powerful potential for the management of prostate cancer (PCa).
Cancerchemopreventionbyphytochemicalsisemergingasasuitable
approach for the treatment of early carcinogenic processes. Since
(-)-epigallocatechin 3-gallate (EGCG) has shown potent chemo-
preventive efficacy for PCa, we designed and developed novel
targeted NPs in order to selectively deliver EGCG to cancer cells.
Herein, to explore the recent concept of “nanochemoprevention”, we
present a study on EGCG-loaded NPs consisting of biocompatible
polymers, functionalized with small molecules targeting PSMA, that exhibited a selective in vitro efficacy against PSMA-expressing
PCa cells. This approach could be beneficial for high risk patients and would fulfill a significant therapeutic need, thus opening new
perspectives for novel and effective treatment for PCa.
’INTRODUCTION
Drug-encapsulated nanoparticles (NPs) have the potential to
improve current cancer chemotherapies by increasing drug
efficacy, lowering drug toxicity, and maintaining a relatively high
concentration of drug at the site of interest.1-6This is owed to
more specific targeting to tumor tissues via improved pharma-
cokinetics and pharmacodynamics and active intracellular
uptake.1,7,8These properties depend on the size and surface
characteristics of NPs, as well as on the presence of targeting
ligands, which enable NPs to bind to cell-surface receptors and
enter cells by receptor-mediated endocytosis.9,10Ultimately,
active targeting via the inclusion of a specific ligand on the NPs
is envisioned to provide the most effective therapy, and some
ligand-targeted nanotherapeutics are either approved or under
clinicalevaluation.4Asfar asthebiologicaltargets areconcerned,
tumor-associated antigens appeared to be suitable biological
targets for therapeutic intervention.11,12To this end, functiona-
lization of NPs with ligands that bind the extracellular domain of
these receptors to selectively target drugs to diseased cells can
represent a powerfultherapeutic strategy. For example, prostate-
specific membrane antigen (PSMA), a well-known transmem-
brane protein that is overexpressed on prostate cancer (PCa)
epithelialcells,13hasdemonstratedapromisingpotentialforPCa
therapy.12,14-16Recently, in a pioneering work, Farokhzad and
Langer developed proof of concept drug delivery vehicles that
were composed of biocompatible polymeric [poly(D,L-lactic-co-
glycolic acid)-block-poly(ethylene glycol), PLGA-PEG)] NPs
and aptamers to target PSMA and validated them by in vitro
and in vivo studies for targeted delivery of docetaxel and uptake
by PCa cells.17,18The translation of these bioconjugates into
clinicalpracticeisexpectedsoon,eventhoughthereiscontinuing
interest in developing a localized therapeutic option for treat-
ment of early stage PCa that have reduced toxicity. Thus, pre-
vention and/or chemoprevention, possibly through the use of
naturally occurring substances, such as dietary nontoxic phyto-
chemicals, may be the best approach to fight this frequent
diseases.19-21
On the other hand, green tea, a popular beverage consumed
worldwide, has been known to have protective effects against
some common types of cancers,22-26and green tea catechins
(GTCs), expecially (-)-epigallocatechin 3-gallate (EGCG,
Figure 1), have been shown to be potent chemopreventive
agents in vitro and in many in vivo animal models of induced
carcinogenesis.23,24,27More recently, clinical trial reports sug-
gested that oral administration of GTCs might be beneficial in
Received:October 21, 2010

Page 2

B
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
theearlystageofPCa(onHG-PIN,themainpremalignantlesion
of PCa), thus preventing almost definitively the cell transforma-
tion, leading to a long-lasting inhibition of cancer progres-
sion.28,29On this basis, because of inefficient or difficult systemic
delivery and bioavailability of these promising chemopreventive
agents, an improvement of their pharmacological profile using a
cell-specific targeting approach is a major challenge, and nano-
technology can represent a powerful strategy.30In spite of this,
very recently Siddiqui et al. introduced the concept of “nano-
chemoprevention”whichusesnanotechnologyforenhancingthe
outcome of chemopreventive intervention.20,21This new ap-
proach was used for the first time in 2009 to determine the
efficacyofEGCGencapsulatedinapolylacticacid-polyethylene
glycol (PLA-PEG) NPs in preclinical setting.27
In this scenario, by focusing on PCa as a target disease and
combining the above-mentioned insights, in particular on recent
advances in targeting approaches, we aimed to develop novel
targeted EGCG-NPs to be used as nanochemopreventive agents
against early stage PCa. According to the following rationale, we
report on the design and the development of novel EGCG-
encapsulated biocompatible NPs, densely decorated by low-
molecular weight organic molecules as targeting ligands in their
polymericshellsurface,toselectivelybindtoPSMA.Thegoalsof
the present studies are the following: (1) to develop novel and
original polymeric EGCG-encapsulated NPs targeted with small
anticancer molecular entities and (2) to evaluate their antipro-
liferative efficacy with respect to the nontargeted ones.
Wechose PLGA-PEG(-COOH)as apolymersystembecause
of its well-established safety profile in clinical use and by
considering its biocompatible properties.6,10,31-33Moreover, as
a further extension of another published study,34we selected the
pseudomimeticdipeptideN-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-
(S)-lysine (DCL, Figure 1) [IUPAC name: 2-{[(5-amino-1-
carboxypentyl)carbamoyl]amino}pentanedioic acid] as the PSMA
targetingligand,previouslyreportedbyPomperetal.,35,36froma
series of urea-based PSMA-inhibitors,36,37capable of targeting
PSMAwithasimilarhighaffinityandspecificitytoantibodiesand
aptamers. In this work, the synthetic route to obtain DCL has
been revisited and detailed. We therefore synthesized the copo-
lymer PLGA-PEG-DCL, on which PEGylation is considered not
onlytopreservetheantibiofoulingpropertiesofNPsbutalsoasa
spacer in order to maintain optimal distance between the ligand
and the NP surface (see below). To obtain a better control of
DCL functionalization, we strategically envisioned preconju-
gating DCL to the polymer system before nanoformulation.
Subsequently, by use of this pseudo-tri-block-copolymer and
the parent PLGA-PEG(-COOH), a set of <100 nm targeted
(andnontargeted)EGCG-loadedNPswereobtained(Figure1),
which were first submitted to drug-content and drug-release
characterization and then evaluated for their ability to selectively
inhibit the growth of PSMA positive PCa (LNCaP) cells.
’RESULTS AND DISCUSSION
Design of Targeted NPs and Recognition on PSMA Active
Site. PSMA(alsotermed asNAALADaseorglutamatecarboxy-
peptidase II) is highly expressed in PCa cells and in nonprostatic
tumor neovasculature, and it is currently suitable as a target for
anticancer imaging and therapeutic agents.36,37Recently, a 3.5 Å
solvedcrystalstructureofthePSMAectodomain38,39andseveral
other high-resolution X-ray crystal structures of PSMA with
glutamate-containing PSMA inhibitors have been reported.40
Structural studies established that the PSMA binding site con-
tains two zinc ions located on the amino acid catalytic site.
Moreover, a funnel-shaped tunnel with a depth of approximately
20 Å and a width of about 9 Å, near a narrow cavity, is located in
proximity of this amino acid binding pocket.38-41
Inthecurrentstudy,wesoughttosynthesizeanewpolymerby
conjugating PLGA-PEG-COOH with a glutamate-containing
urea-based inhibitor. We selected an inhibitor containing lysine
(DCL, Figure 1), as it contains a primary amine that allows for
amide bond formation with carboxyl terminal groups of PEG
monomer and also for its high affinity for PSMA (IC50s= 498,
from competitive binding assays).35To enable high-affinity bin-
ding of DCL to PSMA, a methylene chain as spacer (>20 Å) to
connecttheinhibitortotheNPsurfaceshouldbeenvisioned.On
thisbasis,partofthelinkerportionofPEGwouldremainoutside
thetunnelaslongastheglutamatemoietyisanchoredwithinthe
binding pocket. Accordingly, we designed a model of conjugated
compound PEG-DCL with a linker length of >20 Å.
To investigate the impact of DCL-PEGylation on ligand bind-
ing, a comparative docking study was performed. First, free DCL
was docked to the PSMA active site, and crucial protein-ligand
Figure 1. Chemical structure of (-)-epigallocatechin 3-gallate, the anti-PSMA ligand (DCL), and schematic representation of the designed targeted NPs.

Page 3

C
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
interactions were analyzed. In the next step, PEG-DCL was
docked and the binding modes of DCL/PEG-DCL were com-
pared.GOLD,version4.0,wasemployedtodockDCLandPEG-
DCL to PSMA (PDB code 2C6C; for details see Experimental
Section). Analysis of the docking results shows that both DCL
and PEG-DCL show a good mutual match within the active site.
PEG-DCL places its polyether within the tunnel leading from
the deeply buried active site to the surface of the PSMA protein
(Figure 2a). Both ligands show a consistent binding mode
with respect to the DCL moiety: the active site metal ions are
well coordinated by the urea oxygen and a carboxylate group
(Figure 2b). Favorable interactions are observed between the
γ-carboxylate group and Arg210 as well as between urea N-H
and CdO of Gly518. The GOLDscore for the DCL binding
mode was 86.95. For PEG-DCL it was higher (113.53), as
expected because of additional interactions by the PEG chain.
To sum up, the comparative docking study showed that intro-
duction of a PEG linker chain most probably has no significant
impact on DCL binding to PSMA. Therefore, DCL conjugate
polymer seems to maintain good affinity for PSMA, and PEGy-
lated inhibitor can be consequently introduced onto the NP
surface.
Synthesis of Di-Block-Copolymer PLGA-PEG-COOH. NPs
should ideally have a hydrophilic surface in order to have a
minimal self-self andself-nonself interaction,toprevent nano-
particle loss to undesired location, and to escape macrophage
capture,enabling“stealth”propertiesforimmuneevasion.42,43In
thisway,PEGiswidelyusedforthispurpose,andthepreparation
ofPEGylatedNPshasbeenpreviously investigated andobtained
by several synthetic strategies.10,31-33One of these involves the
availability of the starting polymer (for example, PLGA-PEG),
which can be conveniently synthesized by conjugation or poly-
merization of PEG to PLGA.10,17,44
Thus, the starting carboxylate-functionalized di-block-copoly-
merPLGA-PEG-COOH(Scheme2;seebelow)waspreparedby
conjugatingheterofunctionalPEG,NH2-PEG-COOHtoPLGA-
COOH using standard carbodiimide/NHS-mediated chemistry
similar to that reported by Farokhzad et al.,10,18,44following a
modified procedure. PLGA-COOH was reacted with EDC and
NHS in an organic solvent at room temperature to activate the
carboxylic acids to the semistable amine-reactive activated NHS-
ester of PLGA (PLGA-NHS). The structure of copolymer was
confirmed by1H NMR spectroscopy. Interestingly, a large peak
centered at 3.65 ppm, corresponding to the PEG methylene
protons, was detected. In fact, analysisof proton intensities, used
fordeterminationofthecompositionofthecopolymers,revealed
an increasedefficacy of PEG conjugation to PLGA (PLGA/PEG
approximately in 1:1.5 molar ratio), as previously described.33
We confirmed that the optimal combination of parameters
resulted when the final coupling reaction was carried out using
CHCl3as a solvent and when the mixture was stirred for 24 h.
Synthesis of Targeting Ligand DCL. In addition to its
remarkable affinity to PSMA, this small organic molecule was
considered for its chemical stability and the relative low cost
of production, especially when produced in scaled-up quantities.
The overall synthetic route for the preparation of DCL is depic-
ted in Scheme 1. In this work, we planned to obtain DCL from
the urea-based intermediate 7, previoulsy reported by Pomper’s
group.45Accordingly, we revisited the preparation of this key
intermediate.7wassynthesizedbycouplingofbis-4-methoxyben-
zyl-L-glutamate hydrochloride (6) with triphosgene and TEA at
0 ?C, followed by the addition of 2-amino-6-tert-butoxycarbonyl-
aminohexanoic acid 4-methoxybenzyl ester (3). Both syn-
thones 3 and 6 were prepared following previously described
procedures.45,46
Commercially available Nε-Boc-NR-Fmoc-L-lysine 1 was re-
actedwith4-methoxybenzyl chlorideandcesiumcarbonateinN,
N-dimethylformamide (DMF) to give the full protected com-
pound 2. Removal of the Fmoc group using 20% piperidine in
DMF provided the desired synthone 3 in good yield. Syntone 6
wassynthesizedbyreactingthebis-glutamicacid(4)withPMBC
via condensation with ethyl acetoacetate in dry DMF in the
presence of N,N,N0,N0-tetramethylguanidine. Purification by
basic extractions gave protected amino acid 5, which was then
converted to chlorhydrate after treatment with ether hydrogen
chloride solution. It is worth noting that when the amount of
TEAwasincreased(molarratiofrom1:1:4to1:1:10for6,3,and
TEA, respectively), repeating the previoulsy described reaction
conditions,45pseudo-dimeric urea 8 was obtained (Scheme 1,
method A). We found better results when free amine 3 in TEA
solution was added to hydrochloride derivative 6 (Scheme 1,
method B). For the final step reaction, both the tert-butyl-
oxycarbonyl (Boc) and the p-methoxybenzyl (PMB) groups of
3 were conveniently removed at room temperature by using
Figure 2. (a) PEG-DCL located in the PSMA tunnel. (b) Docked binding modes of both PEG-DCL and DCL superimposed within the PSMA
active site.

Page 4

D
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
trifluoroacetic acid (TFA)/CH2Cl2 solution, to achieve the
desired ligand DCL in moderate/good yields. A full character-
ization of DCL and intermediates was assessed by means of
NMR, MS, and elemental analyses.
Synthesis of Pseudo-Tri-Block-Copolymer PLGA-PEG-
DCL. The strategy of the block copolymers as preconjugated
starting material has been previously proposed for targeted NPs-
aptamer bioconjugated for cancer therapy.17,18This synthetic
approachresultsinpreciselyengineeredNPs,reducingthebatch-
to-batch variations in their biophysicochemical properties.
In this way, we developed a prefunctionalized biopolymer,
PLGA-PEG-DCL, that has all of the desired NPs components
to avoid the postparticle modifications. Conjugation of targeting
agent DCL to functional PEG termini was performed with the
useofelectrophilicNHSestersofPEGcarboxylicacids.Reaction
between the terminal amine group of DCL and PEG-NHS
resulted in the formation of physiologically stable amide bonds.
The designed PLGA-PEG-DCL copolymer was therefore
synthesized by using a two-step reaction, already described for
the preparation of the di-block-copolymer. The carboxyl-capped
PLGA-PEG-COOH was conjugated to the amine functional
group of the DCL, via activation with NHS, to form the
intermediate PLGA-PEG-NHS, leading to PLGA-PEG-DCL as
a pseudo-tri-block-copolymer (Scheme 2, stage 1). The progress
of reaction was monitored by HPLC. For conjugation, increased
equivalent ratio of PLGA-PEG-NHS and DCL (from 1:2 to 1:4)
improved the conjugation efficiency.
As far as the characterization by
the resonance shifts of the characteristic PLGA-PEG backbone
were detected (steps a-d Scheme 2, stage 1, and Figure 3A).
Overlapping signals revealed at 1.56 ppm are attributed to the
lactide methyl repeat units. The multiplets at 5.23 and 4.78 ppm
correspond to the lactide methine (CH) and the glycolide
protons (CH2), respectively, with a high complexity of peaks
1H NMR is concerned,
resulting from different D-lactic, L-lactic, glycolic acid sequences
in the polymer backbone.1H NMR spectra recorded for PLGA-
PEG-DCLalso exhibited a series of partiallyoverlappedmultiple
peaks attributable to the pattern signals of DCL (Figure 3A).
Moreover, strong absorption bands in both 225 and 270 nm
regions,depicted inUV scan chromatogram (Figure 3B), support
the successful conjugation of DCL to PLGA-PEG copolymer.
Nanoformulation and Characterization of Nontargeted
and Targeted NPs. Previously, PEGylated urea-based PSMA
inhibitor incorporated into a PLA-PEG-PLA derived NPs have
beendeveloped.34Inthiswork,weusedPLGA-PEG-COOHand
PLGA-PEG-DCL polymer systems to generate nontargeted and
targeted NPs, respectively, as a novel construct. We also planned
to obtain NPs of 100 nm lower diameter because of their
potential accessibility to and within many disseminated tumors
when dosed into a circulatory system, as previously discussed.4,9
EGCG encapsulated NPs were prepared using a modified
nanoprecipitationmethod.44ByprecipitationofthePLGA-PEG-
COOH and PLGA-PEG-DCL in water, the polymer self-assem-
bles to form polymeric NPs, in which the hydrophobic PLGA
blocks form a core to minimize their exposure to aqueous
surroundings, whereas the hydrophilic PEG-COOH and PEG-
DCL, as flexible moieties, extend from a shell generated to
stabilize the core. In particular, the hydrophilic DCL blocks are
thrust in aqueous solution onto the NP surface as targeting
moieties (Figure 1, Scheme 2, stage 2).
NPs derived from both polymer systems were characterized
for their size and surface morphology with SEM (representative
imageswereshowninFigure4).Particleswerecharacterizedbya
smooth surface and spherical shape with a narrow size distribu-
tion and showed a similar mean diameter (77.18 ( 16.3 nm for
NP-1 and 80.53 ( 15.0 nm for NP-2, respectively, Table 1).
As for the drug loading, the amount of EGCG encapsul-
ated in NP-1 resulted in 3.09 ( 0.43 μg/mg, whereas NP-2 was
Scheme 1. Synthetic Route for the Preparation of DCLa
aReagents and conditions: (i) Cs2CO3, PMBC, DMF, N2, room temp for 4 h; (ii) 20% soln of piperidine in DMF, room temp for 2 h; (iii) N,N,N0,N0-
tetramethylguanidine,DMF,0?C,30min,ethylacetoacetate,roomtempfor2h,PMBC,roomtempfor24h;(iv)4MHClindiethylether,acetone;(v)
(A)triphosgene, 3, TEA,10 equiv, CH2Cl2, N2,-78?Cfor1 h,then 30 minatroomtemp, f6, room tempfor12h;(B)triphosgene, 6,TEA,2 equiv,
CH2Cl2, N2, 0 ?C for 15 min, f3, TEA, 2 equiv, room temp for 2 h; (vi) TFA, CH2Cl2, room temp for 2 h.

Page 5

E
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
4.81 ( 0.37 μg/mg. Statistical analysis showed that the amount
ofEGCGencapsulatedwassignificantlylarger(p<0.05)forNP-
2withrespect toNP-1 (Table1).Itcanbeascribedtothehigher
hydrophilicity of the DCL conjugated polymer (of NP-2) than
that of NP-1. Furthermore, the low encapsulation efficiency
for both type of NPs (6.18 ( 0.9% and 9.61 ( 0.7% for NP-1
and NP-2, respectively) may be related to the high solubility
ofEGCGinwater,resultinginasignificantlossofEGCGinstead
of being encapsulated by PLGA-PEG blocks. However, interest-
ing yields of production were obtained for both preparations
(in the range of 61.50 ( 9.8% and 44.10 ( 1.6% for NP-1 and
NP-2, respectively) over multiple experiments.
In Vitro Kinetics Release of EGCG from NPs. The in vitro
EGCG release profiles from NPs-1 and NPs-2, compared to the
dissolution behavior of pure EGCG, are depicted in Figure 5.
The experiments were performed in water (at pH 6.4) and
in phosphate buffer solution (PBS) at pH 6.7 and 7.4, chosen to
simulate both the slightly acidic microenvironment of extracel-
lular fluid in most tumors and the physiological conditions in
normal tissues, respectively.47Results show that EGCG alone
dissolves very quickly, being highly soluble in water. Conversely,
the encapsulation into NPs determined a certain control of
EGCG rate release in water, even if about 100% of released
EGCG was reached within 2.5 h. Moreover, when both systems
(NPs-1 and NPs-2) were tested under the above-mentioned
experimental conditions (i.e., PBS, pH 6.7 and 7.4), a similar
behavior, confirmed by the overlapping of their kinetics release
profiles,wasobserved.Theseresultscanbeexpectedconsidering
Scheme 2. Synthesis of Copolymer PLGA-PEG-DCL (Stage 1) and Nanoformulation (Stage 2)a
aReagents and conditions: (i) NHS, CH2Cl2, EDC, N2, room temp for 12 h; (ii) DMF, DCL salt, DIPEA, N2, room temp for 24 h.
Figure 3. Characterization of PLGA-PEG-DCL: (A)1H NMR with pattern signals of PLGA-PEG fragment (a-d) and of DCL (red lines); (B) UV
spectra comparison of PLGA-PEG-NHS (blue line), DCL (red line), and PLGA-PEG-DCL (green line).

Page 6

F
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
the physicochemical properties of the chosen polymers and of
EGCG.
Thereleaseprofileswereobtained bythedeterminationofthe
EGCG residual amount in NPs during the release experiment
(see Experimental Section). To date, only a few studies based
on the direct determination of EGCG (and of catechins),
released from different types of NPs in the release medium, were
reported.48,49Similarly, when release studies were performed
according to these experiments, a steady-state concentration of
EGCG in solution was reached almost immediately, followed by
a decrease of EGCG concentration. This apparent loss of EGCG
indicates a slow system relaxation to a new equilibrium and was
hypothesized to be due to the adsorption of the NPs on the vial
wallsorattributedtocovalentbindingofEGCG(orcatechins)to
the polymer.48,49In particular, after re-collection and redisper-
sion of NPs in water, additional EGCG is released and new
concentration equilibrium is reached. Following this approach,
almost 100% of EGCG was released after three such stages.
This behavior, where nonspecific putative interactions be-
tween polymer systems (such as PLGA-PEG-COOH) and
EGCG were hypothesized, was further confirmed in this study.
In fact, a similar loss of EGCG in solution was observed after
exposure of PLGA-PEG-COOH-based unloaded NPs with
EGCG free. As displayed in Figure 6, the observed decrease of
the band at 275 nm (for EGCG, blue line f black line) and an
increase of band in the same absorption range (for PLGA-PEG-
COOH-based NPs, green line f red line) suggest a possible
EGCG-polymer nanoparticleinteraction bya minimalcovalent
or noncovalent binding of EGCG with the polymer matrix.
In summary, it seems that our indirect method enables detection
of EGCG by overcoming possible interaction between EGCG-
polymer systems.
InVitroCellularCytotoxicityAssays. Inordertoinvestigate
whether functionalization of NPs with the PSMA inhibitor DCL
enhanced the effectiveness of EGCG toward PSMA positive
(LNCaP) cells, chosen as a model of PCa tumor lines, we
compared the antiproliferative activities of the targeted EGCG-
loadedNPs(NPs-2)withthose ofthenontargetedNPs(NPs-1)
in in vitro assays.
LNCaP were exposed toequimolaramounts of EGCG loaded
NPs, andtwoseries of experiments were planned and performed
(Figure7).Toreducetheeffectsofnonspecificendocytosis,cells
were first exposed to NPs samples for 1 or 3 h and then washed
and incubated in NP free medium for 48 and 72 h. To evaluate
cytotoxicity, cell counts were conducted directly on LNCaP cells
and antiproliferative efficacy was calculated.
After 48 h of incubation, the growthof cells was similar tothat
of the control for NPs-1 treated for 1 h while cell growth
was inhibited for 80% by targeted NPs (NPs-2, Figure 7A). On
the other hand, different behavior was found according to the
experiment conducted after a 3 h sample exposure. With about
50% and 65% growth inhibition for NPs-1 and NPs-2, respec-
tively, significant antiproliferative activities were detected for
both samples. These results clearly demonstrated that targeted
NPs weremoreeffectivethanthenontargetedonesinbothassay
conditions. Probably when cells were pulsed for only 1 h with
Figure 4. SEM images of nontargeted (NPs-1, A) and targeted (NPs-2, B) EGGC-loaded NPs.
Table 1. Average Diameter, EGCG Content, Encapsulation Efficiency, and Yield of Production of EGCG-Loaded NPsa
formulation average diameter (nm)EGCG loading (μg/mg)encapsulation efficiency (%) yield of production (%)
NP-1
NP-2
77.18 ( 16.3
80.53 ( 15.0
3.09 ( 0.43*
4.81 ( 0.37*
6.18 ( 0.9*
9.61 ( 0.7*
61.50 ( 9.8*
44.10 ( 1.6*
aValues presented are the mean ( SD of three preparations. (/) Significant difference (p < 0.05).
Figure 5. In vitro release profiles of EGCG from NPs (NPs-1 and
NPs-2), detected at different pH values and conditions, in comparison
with the dissolution rate of the EGCG alone.

Page 7

G
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
NPs and following 48 h of exposure, there was not sufficient
time to operate by a classical endocytosis mechanism. However,
further investigation to explain this behavior is currently in
progress.
In contrast, following 72 h of exposure to NPs-1 and NPs-2,
similar antiproliferative profiles, with different inhibition values
in the overall cell growth, were observed. In particular, with
respect tocontrol,20%and ∼40% (for 1 hexposure)and ∼40%
and60%(for3hexposure)ofgrowthinhibitionvaluesforNPs-1
andNPs-2,respectively,werecalculated(Figure7B).Alsointhis
case, NPs-1 demonstrated lower efficacy with cells pulsed for 1 h
thanthoseevaluatedfrom3hofexposurewhileenhancedpotency
was observed for NPs-2. By use of this experimental model, an
improvedantiproliferativeactivityfortargetedPSMAwithrespect
to the nontargeted EGCG loaded NPs was observed.
To confirm that the difference in cytotoxicity is due to the
DCL affinity to PSMA and to assess the selectivity of these
prototypes against PCa cell lines, the in vitro inhibitory effects
of NP-1 and NPs-2 on the proliferation of normal cells were
also carried out. Thus, growth inhibition of HUVECs (human
umbilicalveinendothelialcells),asmodelsystemofnormalcells,
wasmeasuredbyfollowingthesameprotocolasdescribedabove.
FromtheresultsshowninFigure8,withabout100%cellsurvival,
no significant cell growth suppression was observed for both
nanosystems at a EGCG concentration of 30 μM after 48 and
72 hof exposure. Thesedatasuggest that EGCG-loadedNPs are
equally ineffective in inhibiting HUVEC proliferation, whereas
they selectively inhibit proliferation of PCa cells.
These results support the hypothesis that the primary role of
target-specific ligands is to enhance cellular uptake of NPs and
their load into cancer cells.
’CONCLUSIONS
The fact that PCa onset and progression take considerable
timetooccurmaybeconsideredasanimportantopportunityfor
treatingpremalignantlesions.Therefore,sincechemoprevention
Figure 6. UV measurements of interaction between PLGA-PEG-
COOH-based unloaded NPs (green line) and EGCG (black line).
Putative polymer-EGCG interaction is depicted by a red line, while
the decrease of EGCG is indicated by a blue line.
Figure 7. In vitro cytotoxicity of nontargeted (NPs-1) and targeted
(NPs-2) nanoparticles on LNCaP cells pulsed for 1 and 3 h at a EGCG
concentration of 30 μM after 48 h (A) and 72 h (B) incubation:
(/) significantly different from control; (O) significantly different from
NP-1; n = 3.
Figure 8. Inhibition of HUVECs proliferation by NPs-1 and NPs-2.
Cells were pulsedfor1 and3h ataEGCG concentration of30μM after
48 h (A) and 72 h (B) of incubation.

Page 8

H
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
by the use of EGCG proved to be an emerging approach for the
treatment of early carcinogenic processes, we thought that the
potent chemopreventive efficacy of this phytochemical for PCa
could be improved by using targeted nanotechnology strategy.
Moreover, this approach could also be exploited to enhance the
bioavailability of EGCG by changing the pharmacokinetics and
biodistribution.
In this work, we indirectly demonstrated that an EGCG-
loaded NP system functionalized with a small organic molecule
(PSMA inhibitor) on the surface significantly enhances bind-
ing to PSMA with respect to the nonfunctionalized ones, thus
leading to an increased antiproliferative activity in in vitro assays
toward PSMA-positive PCa cells, without affecting normal cell
viability. From a comparison of EGCG encapsulated PSMA-
targeted NPs to the nontargeted NPs, we demonstrated that the
viability of LNCaP cells exposed to the PSMA-targeted particles
wasingenerallowerthanthesameparticleswithoutthetargeting
DCL. The cytotoxicity of EGCG loaded into the nontargeted
NPs can be ascribed to a combination of nonspecific uptake and
to the expected nonspecific release of EGCG into the medium
over the exposure period. Meanwhile, the enhanced potency
shared by EGCG encapsulated into targeted NPs can be attrib-
uted to the maximized binding between NPs and cells, which
presumably would promote an active targeting to PSMA, result-
ing in enhanced accumulation and cell uptake through receptor
(antigen) mediated endocytosis.
In summary, these results demonstrate that the effectiveness
ofEGCGencapsulatedNPs,intermsofantiproliferativeefficacy,
can be significantly improved by incorporating specific ligands,
such as small organic molecules, onto the NP surface in order to
bind PSMA antigen present on PCa cells. The translational
potential of these PSMA-targeted ECGC-loaded NPs warrants
furtherinvivostudiesinanimalmodel.Weexpectthattheinsights
obtainedfrom thisstudycanbe useful to connectnanochemopre-
vention and targeting strategy toward management of PCa as a
primary focus but also to be pursued as a validated model in a
larger context.
’EXPERIMENTAL SECTION
Materials. For preparation of NPs, poly(D,L-lactide-co-glycolide)
(PLGA) (lactide/glycolide ratio of 50:50, inherent viscosity of 0.20 dL
g-1in hexafluoroisopropanol) with acid end groups were purchased
from Lactel Absorbable Polymers (Pelham, AL, U.S.). The heterofunc-
tionalPEGpolymerwithaterminalamineandcarboxylicacidfunctional
group, NH2-PEG-COOH (MW = 3400), was purchased from JenKem
Technology USA. All solvents and other chemicals were obtained from
Sigma-Aldrich, Carlo Erba, or ORPEGEN Peptide Chemicals GmbH.
All reagents of commercial quality were used without further puri-
fication. Melting points (mp) were determined using an Electrothermal
melting point or a K€ ofler apparatus and are uncorrected. Nuclear
magnetic resonance (1H NMR,13C NMR,1H-1H COSY,1H-13C
HMBC,1H-13C HSQC, and1H-1H TOCSY) spectra were deter-
mined in CDCl3, DMSO-d6or CDCl3/DMSO-d6(in 3/1 ratio) and
were recorded at 200, 500, and 600 MHz on a Varian XL-200, a Bruker
Avance 500, and a Bruker AMX-600, respectively. Chemical shifts are
reported in parts per million (ppm) downfield from tetramethylsilane
(TMS),usedasaninternalstandard. Splittingpatternsaredesignatedas
follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; brs,
broad singlet; dd, double doublet. The assignment of exchangeable
protons (OH and NH) was confirmed by the addition of D2O. Electron
ionization and MALDI TOF mass spectra (70 eV) were recorded on a
Hewlett-Packard5989MSEnginespectrometerandbyaMALDImicro
MX (Waters, micromass) equipped with a reflectron analyzer. For
MALDI TOF mass spectrometry (MS) analysis, the sample was mixed
with an equal volume of matrix 2,5-dihydroxybenzoic acid (20 mg/mL
in EtOH/H2O (90:10, v/v), applied to the metallic sample plate, and
air-dried. Mass calibration was carried out by using as a standard the
antocyan mixture provided by the manufacturer. Analytical thin-layer
chromatography(TLC)wascarriedoutonMercksilicagelF-254plates.
Flash chromatography purifications were performed on Merck silica
gel 60 (230-400 mesh ASTM) as a stationary phase. The purity
of copolymers was determined by high performance liquid chromatog-
raphy (HPLC) using an HP 1200 (Agilent Technologies, U.S.) system
equipped with a Hypersil BDS C18 column (Alltech Italy, 250 mm ?
4.6 mm i.d., 5 μm particle size); these materials were found to be >95%
pure. Elemental analyses for 7 and DCL were performed on a Perkin-
Elmer 2400 spectrometer at Laboratorio di Microanalisi, Dipartimento
di Chimica, Universit? a di Sassari (Italy), and were within (0.4% of the
theoretical values.
Molecular Modeling Studies. The X-ray crystal structure of
the PSMA (GCPII) protein complexed with GPI-18431 inhibitor was
retrievedfromtheProteinDataBank(PDBcode2C6C)39andservedas
inputstructureforthedockingstudies.TheligandsDCLandPEG-DCL
were generated as follows: SMILES codes of the ligands were converted
to a three-dimensional structure using the MOE program suite. The
initial ligand structures were minimized, and the protonation states of
amine and carboxy moieties were set properly. Rotatable bonds were
kept flexible throughout the docking procedure.
Forliganddocking,thegeneticalgorithm-baseddockingtoolGOLD,
version 4.0 (with GOLDscore), was employed. First, the target protein
was loaded and protonated using the HERMES suite. Then 10 docking
runs were performed for each ligand. For DCL, the following genetic
algorithm parameters were employed: maximum number of operations =
85000,populationsize=100,selectionpressure=1.1,numberofislands=
5, niche size = 2, cross weight = 95, mutation weight = 95, and migration
weight = 10. For PEG-DCL, the same parameters were applied except for
the maximum number of operations (150000).
Synthesis of PLGA-PEG-COOH Block Copolymer. To
a solution of PLGA-COOH (1.5 g, 0.083 mmol) in anhydrous methy-
lene chloride (6 mL), N-hydroxysuccinimide (NHS, 38 mg, 0.33 mmol,
4 equiv) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC,
70 mg, 0.36 mmol, 4.3 equiv) were added, and the reaction mixture was
magnetically stirred at room temperature for 12 h under nitrogen
atmosphere.PLGA-NHSwasobtainedbyprecipitationwithcolddiethyl
ether (5 mL) as a white solid, which was filtered and repeatedly washed
in a cold mixture of diethyl ether and methanol (few drops) to remove
residualNHS,thendriedwithnitrogenandputundervacuumtoremove
solvent(yield,97%).TheintermediatePLGA-NHS(1.5g,0.085mmol)
was dissolved in anhydrous chloroform (5 mL). NH2-PEG-COOH
(0.375 g, 0.11 mmol, 1.3 equiv) and N,N-diisopropylethylamine
(DIPEA) (42 mg, 0.325 mmol, 3.8 equiv) were then added under
magneticstirring.Thereactionmixturewasmagneticallystirredatroom
temperature for 24 h. The desired copolymer was precipitated with cold
diethyl ether and treated with the same solvents to remove unreacted
PEG as described above (yield, 88%). The resulting PLGA-PEG block
copolymerwasdriedundervacuum,characterizedby1HNMR(200and
600 MHz), and used for NP preparation without further treatment.1H
NMR (500 MHz, CDCl3) δ 5.23 (m, -OC-CH(CH3)O-, PLGA), 4.78
(m, -OC-CH2O-, PLGA), 3.65 (s, -CH2CH2O-, PEG), 1.56 (brs, -OC-
CHCH3O-, PLGA).
Synthesis of DCL and Intermediates. 2-{[(5-Amino-1-
carboxypentyl)carbamoyl]amino}pentanedioicAcid(DCL). The
intermediate (S)-2-[3-(5-amino-1-tert-butoxycarbonylpentyl)ureido]-
pentanedioicaciddi-tert-butylester7(130mg,0.17mmol) wasdissolved
in 5 mL of 1:1 trifluoroacetic acid (TFA)/methylene chloride solution
and stirred at room temperature for 3 h. Then the resulting solution was

Page 9

I
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
evaporated under reduced pressure. The colorless solid residue was
triturated with dry diethyl ether, filtered, and washed with dry diethyl
ether to yield the desired product as an off beige solid (yield, 70%).1H
NMR(600MHz,DMSO-d6)δ7.80-7.60(brs,3H),6.40-6.30(s,2H),
4.15-4.00 (m, 2H), 3.78-3.55 (m, 2H), 3.46-3.20 (brs, 1H), 2.78-
2.72 (d, 2H), 2.27-2.22 (m, 2H), 2.00-1.85 (m, 2H), 1.76-1.59
(m, 2H), 1.53-1.47 (m, 2H), 1.38-1.19 (m, 2H). MS (MALDI-TOF):
[Mþ319]; [Mþ319 þ Na, 342]. EI: [Mþ319]. Anal. (C12H21N3O73
1.15CF3COOH) C, H, N.
Preparation of 2-{3-[1-p-Methoxybenzylcarboxylate
(5-tert-Butylcarbamylpentyl)]ureido}-di-p-methoxybenzyl-
pentanedioate(7). Triphosgene(0.18g,0.613mmol)wasplacedina
flame-dried flask cooled to 0 ?C, under nitrogen atmosphere, and a
solutionof3(0.68g,1.86mmol,3equiv) andTEA(0.53mL,3.72mmol,
6equiv)dissolvedinCH2Cl2(3mL)wasadded.Thereactionmixturewas
stirredat0?Cfor15min.Amixtureof6(0.79g,1.86mmol,3equiv)and
TEA (0.53 mL, 3.72 mmol, 6 equiv) in CH2Cl2(3 mL) was then added.
The resulting mixture was allowed to warm to room temperature and
stirred for 2 h. Product was extracted by CH2Cl2, washed with water
and brine, and dried over Na2SO4. Purification by flash chromatography
(20/80 EtOAc/CH2Cl2) afforded an oil that solidified upon standing
(yield, 59%).1H NMR (CDCl3) δ 7.26 (d, 6H), 6.87 (d, 6H), 5.52
(d, 2H), 5.13-4.98 (m, 6H), 4.76 (brs, 1H), 4.53-4.48 (m, 1H), 4.50-
4.43 (m, 1H), 3.79 (s, 9H), 3.04-2.96 (m, 2H), 2.40-2.34 (m, 2H),
2.14-2.11 (m, 2H), 1.94-1.70 (m, 2H), 1.60-1.56 (m, 2H), 1.42 (s,
9H),1.25-1.22(m,2H).MS-ESI:780[Mþ 1]þ.Anal.(C41H53N3O12)
C, H, N.
Preparation of 2-Amino-6-tert-butoxycarbonylamino-
hexanoic Acid 4-Methoxybenzyl Ester (3). To a solution of Nε-
Boc-N-Fmoc-L-lysine (1, 8.16 g, 17.41 mmol) in 70 mL of dry DMF,
cesium carbonate (8.14 g, 24.4 mmol, 1.4 equiv) and 4-methoxybenzyl
chloride (PMBC, 3.0 g, 2.6 mL, 19.15 mmol, 1.1 equiv) were added
under nitrogen atmosphere, and the suspension was stirred at room
temperature for 4 h.Thereaction mixture was then filtered, sequentially
washed with ethyl acetate, 5% Na2CO3, water, and dried over Na2SO4.
Purification by recrystallization from 60/40 hexane/EtOAc gave a beige
powder corresponding to compound 2 (yield, 95%). Mp 117-119 ?C
(lit. 118-120 ?C).45 1H NMR(CDCl3) δ 8.12 (s, 1H), 7.76-7.67 (m,
4H),7.40-7.25(m,6H),6.89(d,2H),5.29(s,1H),5.08(s,2H),4.53-
4.42(m,1H),4.09(t,2H),3.80(s,3H),3.07-2.98(m,3H),1.92-1.10
(m, 15H). MS-ESI m/z: 588 [M þ 1]þ. A solution of compound 2
(8.44 g, 14.4 mmol) in 100 mL of a 20% solution of piperidine in DMF
was stirred at room temperature for 2 h. Product was extracted by
CH2Cl2, washed with water, and dried over Na2SO4. The crude residue
was purified by silica gel flash chromatography (5/95 MeOH/CH2Cl2,
20/80 EtOAc/CH2Cl2, 10/90 EtOAc/CH2Cl2). Compound 3 was
obtained as a yellow oil (yield, 71%).1H NMR (CDCl3) δ: 8.02 (brs,
2H), 7.30 (d, 2H), 6.89 (d, 2H), 5.30 (s, 1H), 5.09 (s, 2H), 4.58-4.40
(m, 1H), 3.83 (s, 3H), 3.08-2.28 (m, 2H), 2.05-1.18 (m, 15H). MS-
ESI m/z: 367 [M þ 1]þ.
Bis-4-methoxybenzylglutamate Hydrochloride (6). To an
ice-cooled mixture of amino acid 4 (8.0 g, 54.4 mmol) in dry DMF (10
mL), N,N,N0,N0-tetramethylguanidine (13.4 mL, 108.8 mmol, 2 equiv)
was added. After 30 min under magnetic stirring, ethyl acetoacetate
(13.84 mL, 108.8 mmol, 2 equiv) was added and stirring was continued
atroomtemperatureuntiltheaminoacidhaddissolved.PMBC(16.98g,
7.36mL,108.8mmol,2equiv)inDMF(55mL)wasadded,andstirring
continued at room temperature for 24 h. The reaction mixture was then
diluted with ethyl acetate, washed with water, 1 N NaHCO3, and dried
overNa2SO4toaffordcompound5asayellowoil.1HNMR(CDCl3)δ
8.80-8.72 (d, 2H), 7.27 (d, 4H), 6.87 (d, 4H), 5.10 (s, 2H), 5.03 (s,
2H), 4.24-4.15 (m, 1H), 3.80 (s, 6H), 2.45-2.38 (m, 2H), 2.28-2.00
(m, 2H). The product (compound 5) was treated with hydrogen
chloride 4 N solution in diethyl ether to give chlorohydrate 6 as a white
powder (yield, 84%). Mp 114-116 ?C (lit. 114-115 ?C).50 1H NMR
(CDCl3) δ 8.89 (brs, 3H), 7.20 (d, 4H), 6.81 (d, 4H), 5.08 (brs, 2H),
4.94 (s, 2H), 4.41-4.23 (m, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 2.72-2.50
(m, 2H), 2.49-2.31 (m, 2H).
Preparation of (2S,20S)-1,5-Bis(4-methoxybenzyl)-2,20-
carbonylbis(azanediyl)dipentanedioate (8). To a mixture of
bis-4-methoxybenzyl-L-glutamate3HCl 6 (3.6 g, 8.5 mmol, 3 equiv) in
15mLofCH2Cl2,asolutionoftriphosgene(0.833g,2.8mmol)dissolved
in 3 mL of CH2Cl2was added under nitrogen atmosphere. After the
mixture was cooled to -78 ?C, TEA (12 mL, 85 mmol in 10 mL of
CH2Cl2, 30 equiv) was slowly added. The reaction mixture was stirred at
-78 ?C for 1 h, allowed to warm at room temperature, and stirred for a
further 30 min at room temperature. Compound 3 (3.1 g, 8.5 mmol in
7 mL CH2Cl2, 3 equiv) was then added, and the resulting mixture was
continually stirred overnight. Product was extracted by CH2Cl2, washed
with water and brine, and dried over Na2SO4. Purification by flash
chromatography (20/80 EtOAc/CH2Cl2) afforded an oil that solidified
uponstanding(yield,62%).Mp97-99?C.1HNMR(CDCl3)δ7.25(d,
8H), 6.85 (d, 8H), 5.29 (s, 1H), 5.25 (s, 1H), 5.05 (s, 4H), 5.01 (s, 4H),
4.52-4.46 (m, 2H), 3.79 (s, 12H), 2.41-2.22 (m, 4H), 2.20-1.78 (m,
4H). MS-ESI m/z: 801 [M þ 1]þ.
Synthesis of PLGA-PEG-DCL Pseudo-Tri-Block-Copoly-
mer. To a solution of PLGA-PEG-COOH di-block-copolymer (1.4
g, 0.065 mmol) in anhydrous methylene chloride (5 mL), NHS (30 mg,
0.26 mmol, 4 equiv) and EDC (53 mg, 0.28 mmol, 4.3 equiv) were
added,andthesolutionwasmagneticallystirredatroomtemperaturefor
12 h under nitrogen atmosphere. The activated PLGA-PEG-NHS
copolymer was precipitated in ice-cold diethyl ether and methanol
(few drops), consequently filtered and dried, as described above, to
give a white powder (yield, 92%). The PLGA-PEG-DCL was obtained
bytheadditionofasolutionofDCLsalt(38mg,0.088mmol,4equiv)in
DMF(1mL)andDIPEA(0.72mL,0.0041mol)toasolutionofPLGA-
PEG-NHS(0.45g,0.022mmol)indimethylformamide(DMF,2.5mL),
and the reaction mixture was magnetically stirred at room temperature
for 24 h under nitrogen atmosphere. PLGA-PEG-DCL was analyzed by
high performance liquid chromatography (HPLC) on a Hypersil BDS
C18column(AlltechItaly),(250mm?4.6mmi.d.,5μmparticlesize),
usingaseluentalineargradientofeluentB(95%MeCN,0.07%TFA)in
A (0.1% TFA) from 15% to 100% for 30 min (flow rate of 1 mL/min,
temperature at 25 ?C, and a detector wavelength of 280 nm). The
equipment consisted of an HP 1200 (Agilent Technologies, U.S.)
system controlled by HP ChemStation software, including an autosam-
pler and a diode array detector. After purification, a white solid was
obtained (yield, 65%), which was characterized by1H NMR and UV
analysis.Thepolymerwasthenfreeze-driedandstoredat-20?Cbefore
use.
FormulationandCharacterizationofEGCG-LoadedPLGA-
PEG-COOH and PLGA-PEG-DCL NPs. Preparation of EGCG-
Loaded NPs. NPs were prepared by modification of nanoprecipita-
tion method.44Briefly, polymer (PLGA-PEG or PLGA-PEG-DCL)
(100 mg) and EGCG (5 mg) were co-dissolved in acetonitrile
(10 mL) and added dropwise into 7.5 mL of water. The milky colloidal
suspension was evaporated at room temperature to eliminate residual
organicsolvent.NPswereisolatedbycentrifugation(10min,14000rpm)
and washed three times with water to remove the nonencapsulated
EGCG. The pellets were suspended in 1.5 mL of water and stored
at 4 ?C until use. A part of the nanoparticle aqueous dispersion was
rapidly frozen below -80 ?C in a deep-freezer, lyophilized (5 Pascal
LIO 5P apparatus, Cinquepascal SRL, Milano, Italy), and collected for
other experiments.
Scanning Electron Microscopy (SEM). The morphology (shape and
surface characteristics) of NPs was studied by scanning electron micro-
scopy (SEM) (model DSM 962, Carl Zeiss Inc., Germany). A drop of
NPsuspensionwasplacedonaglasscoverslideanddriedundervacuum

Page 10

J
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
for 12 h. After that, the slides were mounted on an aluminum stub and
the samples were then analyzed at 25 kV acceleration voltage after gold
sputtering under an argon atmosphere. The size of NPs was determined
by measuring more than 100 particles from SEM images.
Determination of EGCG Content in NPs and Yields of Production.
The amount of the encapsulated EGCG was determined by dissolving a
weighted amount (10 mg) of dried EGCG loaded NPs in methylene
chloride (1 mL) and measured using a validated HPLC method.51The
EGCG content was expressed as μg/mg of the NPs mass and the
encapsulationefficiencycalculatedasapercentageratioofmeasuredand
initial amount of EGCG encapsulated into NPs. Chromatographic
analysis was performed on a HP 1200 (Agilent Technologies, U.S.)
liquid chromatography system equipped with a diode array detector of
thesameseries,usingaJupiterC18(5μmporesize)250mm?2.0mm
column (Phenomenex). The mobile phase consisting of water/acetoni-
trile/methanol/ethyl acetate/glacial acetic acid (89:6:1:3:1 v/v/v/v/v)
was prepared daily and degassed by sonication for 30 min and filtered
through a j 55 mm blue ribbon filter paper before use. The columnwas
thermostated at 20 ?C. The method51was modified and delivered
isocraticallywithaflowrateof0.2mL/min.Theinjectionvolumewas25
μL,andthewavelengthforUVdetectionwas 280nm.Thetotalanalysis
time was 30 min, and the retention time of EGCG was 12.86 min. The
calibration curves were found to be linear in the range of 5-50 μg/mL
(y = 925.38x þ 42.047; 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.
In Vitro Release Kinetics of EGCG from NPs. The in vitro
release profile of EGCG from NPs was assessed by the determination of
the residual amount of EGCG present in the NPs. The tests were
performed in water (pH 6.4) at room temperature and in phosphate
buffer solution (PBS) with different pH (pH 6.7 and pH 7.4) at 37 ?C.
For that purpose, several aliquots (100 μL) of the original suspensions
of NPs were diluted with release medium (final volume of 1 mL) in a
1.5 mL vial and continuously shaken at the selected temperature.
At predetermined time intervals, the sample vial was centrifuged at
14000rpmfor5min,andtheprecipitatewaswashedtwicewithdistilled
waterandlyophilized.Thedriedsamplewasthendissolvedin100μLof
methylene chloride and analyzed by HPLC using the previously
described procedure. Every 30 min, all sample vials were centrifuged,
thepelletwaswashedoncewithwater,andthesupernatantwasreplaced
withfreshmediumforthenextreleasedatacollection.Thepercentageof
EGCG release at a specific timing was calculated on the basis of the total
amount measured from sample NPs. The dissolution of EGCG as raw
materialwasmadeinthesameconditionsascomparison.Eachsamplewas
assayed in triplicate. To study NP-EGCG interaction, 30 μL of PLGA-
PEG-COOH-based unloaded NPs (1.34 mg) and 8 μL (1 mg/mL) of
EGCG in 142 μL of water were mixed and stirred at room temperature.
SampleswereanalyzedbyHPLC(after0.5,1,2,5,10h)withadiodearray
detector using the same procedure previously described.
In Vitro Cytotoxicity Assays to PSMA Expressing PCaCells
(LNCaP) and Human Umbilical Veins Endothelial Cells
(HUVEC). LNCaP cell lines (ECACC, Salisbury, U.K.) were grown
in 24-well plates with RPMI 1640 medium containing 100 units/mL
penicillin G, 100 μg/mL streptomycin, and 10% FBS (Invitrogen,
Carlsbad, CA) at a concentration that allows 70% confluence in 48 h.
HUVECs (Cell Applications, San Diego, CA) were cultured in en-
dothelial cell basal medium (Cell Applications) supplemented with
endothelial cell growth supplement (Cell Applications). When conflu-
ent, HUVECs were subcultured at a split ratio of 1:2 and used within
three passages. For proliferation experiments, cells were transferred to
24-well plates at a concentration that allows 70% confluence in 48 h.
On the day of the experiments, similar treatment was applied for both
celltypes.CellswerewashedwithPBSandthenexposedfor1or3hwith
100μLofasuspensionofEGCGloadedNPs(NP-1orNPs-2)inserum-
freeculturemediumtoafinalconcentrationof30μMEGCG.Cellswere
washed three times with PBS (100 μL), and fresh growth medium was
replaced in the plates. Cells were then incubated in medium at 37 ?C,
and attheend of48and 72h,effects oncellgrowth were determined by
automaticcellcounting(Countess Invitrogen)andexpressedaspercent
of the number of cells per mL.
Statistical Analysis. The data for preparation and characteriza-
tion of NPs as well as drug release studies were processed and analyzed
by Origin software (version 7.0 SR0, OriginLab Corporation, U.S.).
The statistical analysis was evaluated by a Student’s t-test, and p < 0.05
was considered statistically significant. The data obtained from cyto-
toxicity assays were processed by one-way analysis of variance
(ANOVA) followed by a post hoc “Newman-Keuls multiple compar-
ison test” to detect differences of mean values among treatments with
significance defined as p < 0.05.
’AUTHOR INFORMATION
Corresponding Author
*For M.S.: phone, þ39 079-228-753; fax, þ39 079-228-720;
e-mail,mario.sechi@uniss.it.ForV.S.:phone,þ39079-998-619;
fax, þ39 079-228-720; e-mail, sannav@portocontericerche.it.
For G.P.: phone, þ39 079-228-121; fax, þ39 079-228-120;
e-mail, gpintus@uniss.it.
Notes
^The department name was changed on January 31, 2011.
Current address is Dipartimento di Scienze del Farmaco, Uni-
versit? a di Sassari, 07100 Sassari, Italy.
’ACKNOWLEDGMENT
The research activities presented were done within the frame
of “Progetto Cluster, Sviluppo ed Utilizzo di Nanodevices”,
funded by Sardinian Technology Park, Porto Conte Ricerche,
Italy. The authors thank Dr. Roberto Anedda, Dr. Maria
Orecchioni,andPaoloFioriforassistancewithNMRspectroscopy.
The authors also thank Dr. Martin Sippel, Dr. Monika Nocker,
and Dr. Nicolino Pala for their generous contribution of the time
spent on the elaboration of the molecular modeling calculation
and images.
’ABBREVIATIONS USED
NPs, nanoparticles;PLGA, poly-(D,L-lactide-co-glicolyde);PLGA-
PEG, poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol);
DCL, N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-lysine;
GTCs, green tea catechin;EGCG, (-)-epigallocatechin 3-gallate;
PSMA,prostate-specificmembraneantigen;PCa,prostatecancer;
HG-PIN, high-grade prostatic intraepithelial neoplasia;GCPII,
glutamate carboxypeptidase;HUVECs, human umbilical vein
endothelial cells
’REFERENCES
(1) Farokhzad, O. C.; Langer, R. Impact of nanotechnology on drug
delivery. ACS Nano 2009, 3, 16–20.
(2) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.;
Langer,R.Nanocarriersasanemergingplatformforcancertherapy.Nat.
Nanotechnol. 2007, 2, 751–760.
(3) Ferrari, M. Cancer nanotechnology: opportunities and chal-
lenges. Nat. Rev. Cancer 2005, 5, 161–171.
(4) Davis,M.E.;Chen,Z.G.;Shin,D.M.Nanoparticletherapeutics:
an emerging treatment modality for cancer. Nat. Rev. Cancer 2008, 7,
771–782.

Page 11

K
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
(5) Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.;
Farokhzad, O. C. Nanoparticles in medicine: therapeutic applications
and developments. Clin. Pharmacol. Ther. 2008, 83, 761–769.
(6) Shi, J.; Votruba, A. R.; Farokhzad, O. C.; Langer, R. Nanotech-
nology in drug delivery and tissue engineering: from discovery to
applications. Nano Lett. 2010, 10, 3223–3230.
(7) Gu,F.;Langer,R.;Farokhzad,O.C.Formulation/preparationof
functionalized nanoparticles for in vivo targeted drug delivery. Methods
Mol. Biol. 2009, 544, 589–598.
(8) Byrne,J.D.;Betancourt,T.;Brannon-Peppas,L.Activetargeting
schemes for nanoparticle systems in cancer therapeutics. Adv. Drug
Delivery Rev. 2008, 60, 1615–1626.
(9) Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O. C. Factors
affecting the clearance and biodistribution of polymeric nanoparticles.
Mol. Pharmaceutics 2008, 5, 505–515.
(10) Alexis, F.; Pridgen, E. M.; Langer, R.; Farokhzad, O. C.
Nanoparticle technologies for cancer therapy. Handb. Exp. Pharmacol.
2010, 197, 55–86.
(11) Siu, D. Cancer therapy using tumor-associated antigens to
reduce side effects. Clin. Exp. Med. 2009, 9, 181–198.
(12) Cozzi, P. J.; Kearsley, J.; Li, Y. Overview of tumor-associated
antigens (TAAs) as potential therapeutic targets for prostate cancer
therapy. Curr. Cancer Ther. Rev. 2008, 4, 271–284.
(13) Chang,S.S.;O’Keefe,D.S.;Bacich,D.J.;Reuter,V.E.;Heston,
W. D.; Gaudin, P. B. Prostate-specific membrane antigen is produced in
tumor-associated neovasculature. Clin. Cancer Res. 1999, 5, 2674–2681.
(14) Ghosh, A.; Heston, W. D. Tumor target prostate specific
membrane antigen (PSMA) and its regulation in prostate cancer. J. Cell.
Biochem. 2004, 91, 528–539.
(15) Sch€ ulke, N.; Varlamova, O. A.; Donovan, G. P.; Ma, D.;
Gardner, J. P.; Morrissey, D. M.; Arrigale, R. R.; Zhan, C.; Chodera,
A. J.; Surowitz, K. G.; et al. The homodimer of prostate-specific
membrane antigen is a functional target for cancer therapy. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 12590–12595.
(16) Els€ asser-Beile, U.; B€ uhler, P.; Wolf, P. Targeted therapies for
prostate cancer against the prostate specific membrane antigen. Curr.
Drug Targets 2009, 10, 118–125.
(17) Farokhzad, O. C.; Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.;
Kantoff, P. W.; Richie, J. P.; Langer, R. Targeted nanoparticle-aptamer
bioconjugatesforcancerchemotherapyinvivo.Proc.Natl.Acad.Sci.U.S.
A. 2006, 103, 6315–6320.
(18) Gu, F.; Zhang, L.; Teply, B. A.; Mann, N.; Wang, A.; Radovic-
Moreno, A. F.; Langer, R.; Farokhzad, O. C. Precise engineering of
targeted nanoparticles by using self-assembled biointegrated block
copolymers. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2586–2591.
(19) Surh, Y.-J. Cancer chemoprevention with dietary phytochem-
icals. Nat. Rev. Cancer 2003, 3, 768–780.
(20) Siddiqui, I. A.; Mukhtar, H. Nanochemoprevention by bio-
active food components: a perspective. Pharm. Res. 2010, 27, 1054–
1060.
(21) Siddiqui, I. A.; Adhami, V. M.; Ahmad, N.; Mukhtar, H.
Nanochemoprevention: sustained releaseofbioactive foodcomponents
for cancer prevention. Nutr. Cancer 2010, 62, 883–890.
(22) Saleem, M.; Adhami, V. M.; Siddiqui, I. A.; Mukhtar, H. Tea
beverage in chemoprevention of prostate cancer: a mini-review. Nutr.
Cancer 2003, 47, 13–23.
(23) Yang,C.S.;Wang,X.;Lu,G.;Picinich,S.C.Cancerprevention
bytea:animalstudies,molecularmechanismsandhumanrelevance.Nat.
Rev. Cancer 2009, 9, 429–439.
(24) Johnson,J.J.;Bailey,H.H.;Mukhtar,H.Greenteapolyphenols
for prostate cancer chemoprevention: a translational perspective. Phy-
tomedicine 2010, 17, 3–13.
(25) Khan, N.; Adhami, V. M.; Mukhtar, H. Review: green tea
polyphenols in chemoprevention of prostate cancer: preclinical and
clinical studies. Nutr. Cancer 2009, 61, 836–841.
(26) Kurahashi, N.; Sasazuki, S.; Iwasaki, M.; Inoue, M.; Tsugane, S.
Green tea consumption and prostate cancer risk in Japanese Men: a
prospective study. Am. J. Epidemiol. 2008, 167, 71–77.
(27) Siddiqui,I.A.;Adhami,V.M.;Bharali,D.J.;Hafeez,B.B.;Asim,
M.; Khwaja, S. I.; Ahmad, N.; Cui, H.; Mousa, S. A.; Mukhtar, H.
Introducing nanochemoprevention as a novel approach for cancer
control: proof of principle with green tea polyphenol epigallocatechin-
3-gallate. Cancer Res. 2009, 69, 1712–1716.
(28) Bettuzzi, S.; Brausi, M.;Rizzi, F.;Castagnetti, G.;Peracchia, G.;
Corti, A. Chemoprevention of human prostate cancer by oral adminis-
tration of green tea catechins in volunteers with high-grade prostate
intraepithelial neoplasia: a preliminary report from a one-year proof-of-
principle study. Cancer Res. 2006, 66, 1234–1240.
(29) Brausi, M.; Rizzi, F.; Bettuzzi, S. Chemoprevention of human
prostate cancer by green tea catechins: two years later. A follow-up
update. Eur. Urol. 2008, 54, 472–473.
(30) Leonarduzzi, G.; Testa, G.; Sottero, B.; Gamba, P.; Poli, G.
Design and development of nanovehicle-based delivery systems for
preventive or therapeutic supplementation with flavonoids. Curr. Med.
Chem. 2010, 17, 74–95.
(31) Otsuka, H.; Nagasaki, Y.; Kataoka, K. PEGylated nanoparticles
for biological and pharmaceutical applications. Adv. Drug Delivery Rev.
2003, 55, 403–419.
(32) Avgoustakis, K. Pegylated poly(lactide) and poly(lactide-
coglycolide)nanoparticles:preparation,propertiesandpossibleapplica-
tions in drug delivery. Curr. Drug Delivery 2004, 1, 321–333.
(33) Betancourt, T.; Byrne, J. D.; Sunaryo, N.; Crowder, S. W.;
Kadapakkam,M.;Patel,S.;Casciato,S.;Brannon-Peppas,L.PEGylation
strategies for active targeting of PLA/PLGA nanoparticles. J. Biomed.
Mater. Res., Part A 2009, 91, 263–276.
(34) Chandran, S. S.; Banerjee, S. R.; Mease, R. C.; Pomper, M. G.;
Denmeade, S. R. Characterization of a targeted nanoparticle functiona-
lized with a urea-based inhibitor of prostate-specific membrane antigen
(PSMA). Cancer Biol. Ther. 2008, 7, 974–982.
(35) Maresca,K.P.;Hillier,S.M.;Femia,F.J.;Keith,D.;Barone,C.;
Joyal, J. L.; Zimmerman, C. N.; Kozikowski, A. P.; Barrett, J. A.;
Eckelman, W. C.; et al. A series of halogenated heterodimeric inhibitors
of prostate specific membrane antigen (PSMA) as radiolabeled probes
for targeting prostate cancer. J. Med. Chem. 2009, 52, 347–357.
(36) Byun, Y.; Mease, R. C.; Lupold, S. E.; Pomper, M. G. In Drug
Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease
Applications; Supuran, C. T., Winum, J.-Y., Eds.; Binghe Wang Wiley
Series in Drug Discovery and Development; John Wiley and Sons:
Hoboken, NJ, 2009; pp 881-910.
(37) Zhou,J.;Neale, J.H.;Pomper, M.G.;Kozikowski, A.P.NAAG
peptidase inhibitors and their potential for diagnosis and therapy. Nat.
Rev. Drug Discovery 2005, 4, 1015–1026.
(38) Davis, M. I.; Bennett, M. J.; Thomas, L. M.; Bjorkman, P. J.
Crystalstructureofprostate-specificmembraneantigen,atumormarker
and peptidase. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5981–5986.
(39) Mesters, J. R.; Barinka, C.; Li, W.; Tsukamoto, T.; Majer, P.;
Slusher, B. S.; Konvalinka, J.; Hilgenfeld, R. Structure of glutamate
carboxypeptidase II, a drug target in neuronal damage and prostate
cancer. EMBO J. 2006, 25, 1375–1384.
(40) Barinka, C.; Byun, Y.; Dusich, C. L.; Banerjee, S. R.; Chen, Y.;
Castanares, M.; Kozikowski, A. P.; Mease, R. C.; Pomper, M. G.;
Lubkowski, J. Interactions between human glutamate carboxypeptidase
II and urea-based inhibitors: structural characterization. J. Med. Chem.
2008, 51, 7737–7743.
(41) Barinka, C.; Rovensk? a, M.; MLcochov? a, P.; Hlouchov? a, K.;
Plechanovov? a, A.; Majer, P.; Tsukamoto, T.; Slusher, B. S.; Konvalinka,
J.; Lubkowski, J. Structural insight into the pharmacophore pocket of
human glutamate carboxypeptidase II. J. Med. Chem. 2007, 50, 3267–
3273.
(42) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.;
Torchilin, V.; Langer, R. Biodegradable long-circulating polymeric
nanospheres. Science 1994, 263, 1600–1603.
(43) Gref, R.; Luck, M.; Quellec, P.; Marchand, M.; Dellacherie, E.;
Harnisch, S.; Blunk, T.; Muller, R. H. “Stealth” corona-core nano-
particles surface modified by polyethylene glycol (PEG): influences
of the corona (PEG chain length and surface density) and of the core

Page 12

L
dx.doi.org/10.1021/jm1013715 |J. Med. Chem. XXXX, XXX, 000–000
Journal of Medicinal Chemistry
ARTICLE
composition on phagocytic uptake and plasma protein adsorption.
Colloids Surf., B 2000, 18, 301–313.
(44) Cheng,J.;Teply,B.A.;Sherifi,I.;Sung,J.;Luther,G.;Gu,F.X.;
Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad,
O. C. Formulation of functionalized PLGA-PEG nanoparticles for in
vivo targeted drug delivery. Biomaterials 2007, 28, 869–876.
(45) Banerjee,S.R.;Foss,A.C.;Castanares,M.;Mease,R.C.;Byun,
Y.; Fox, J. J.; Hilton, J.; Lupold, S. E.; Kozikowski, A. P.; Pomper, M. G.
Synthesis and evaluation of technetium-99m- and rhenium-labeled
inhibitors of the prostate-specific membrane antigen (PSMA). J. Med.
Chem. 2008, 51, 4504–4517.
(46) Maclaren, J. A. Some amino acid esters—an improved pre-
parative method. Aust. J. Chem. 1978, 31, 1865–1868.
(47) Lee, E. S.; Oh, K. T.; Kim, D.; Youn, Y. S.; Bae, Y. H. Tumor
pH-responsive flower-like micelles of poly(L-lactic acid)-b-poly-
(ethylene glycol)-b-poly(L-histidine). J. Controlled Release 2007, 123,
19–26.
(48) Shutava, T. G.; Balkundi, S. S.; Vangala, P.; Steffan, J. J.;
Bigelow, R. L.; Cardelli, J. A.; O’Neal, D. P.; Lvov, Y. M. Layer-by-
layer-coated gelatin nanoparticles as a vehicle for delivery of natural
polyphenols. ACS Nano 2009, 3, 1877–1885.
(49) Hu, B.; Pan, C.; Sun, Y.; Hou, Z.; Ye, Y.; Hu, B.; Zeng, X.
Optimization of fabrication parameters to produce chitosan-tripolypho-
sphate nanoparticles for delivery of tea catechins. J. Agric. Food Chem.
2008, 56, 7451–7458.
(50) Mease,R.C.;Dusich, C.L.; Foss,C.A.;Ravert,H. T.;Dannals,
R. F.; Seidel, J.; Prideaux, A.; Fox, J. J.; Sgouros, G.; Kozikowski, A. P.;
Pomper,M. G.N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-
4-[18F]fluorobenzyl-L-cysteine, [18F]DCFBC: a new imaging probe
for prostate cancer. Clin. Cancer Res. 2008, 14, 3036–3043.
(51) Saito,S.T.;Welzel,A.;Suyenaga, E.S.;Bueno,F.Amethodfor
fast determination of epigallocatechin gallate (EGCG), epicatechin
(EC), catechin (C) and caffeine (CAF) in green tea using HPLC. Cienc.
Tecnol. Aliment. 2006, 26, 394–400.