Entrapment of hydrophobic drugs in nanoparticle monolayers with efficient release into cancer cells

Full text of DOI:10.1021/ja808137c

Published on Web 01/09/2009
Entrapment of Hydrophobic Drugs in Nanoparticle Monolayers with Efficient
Release into Cancer Cells
Chae Kyu Kim,† Partha Ghosh,† Chiara Pagliuca,‡ Zheng-Jiang Zhu,† Stefano Menichetti,‡ and
Vincent M. Rotello*,†
Department of Chemistry, UniVersity of Massachusetts at Amherst, Amherst, Massachusetts 01003, and
Dipartimento di Chimica Organica “U. Schiff”, UniVersita` di Firenze, 50019, Italy
Received October 15, 2008; E-mail: rotello@chem.umass.edu
Drug delivery systems (DDSs) provide an important tool for
increasing the efficacy of pharmaceuticals through improved
pharmacokinetics and biodistribution.1 A wide variety of nanoscale
materials such as liposomes, polymeric micelles, and dendrimers
have been employed as drug carriers.2 Both covalent and nonco-
valent approaches can be applied to the conjugation of drugs into/
onto these DDSs.3 Noncovalent approaches have the capability of
employing active drugs, whereas covalent attachment generally
requires chemical modification which can cause reduced efficiency
of drug release or incomplete intracellular processing of a prodrug.4
Recently, gold nanoparticle (AuNP) based drug/gene delivery
systems have attracted attention due to their functional versatility,5
biocompatibility,6 and low toxicity.7 Recent studies have demon-
strated a controlled release of payload by intracellular thiols.8
However, controlled dissociation of drugs in active form from
covalent AuNP-drug conjugates remains a challenge for clinical
applications.2
Noncovalent incorporation of drugs into AuNP monolayers
provides an alternative delivery strategy with the potential for
avoiding drug release and prodrug processing issues. The structure
of commonly used water-soluble AuNPs is similar to that of
unimolecular micelles such as dendrimers, featuring a hydrophobic
interior and a hydrophilic exterior.9 The alkanethiol monolayer of
the nanoparticle coupled with the radial nature of the ligands10
creates “hydrophobic pockets” inside the monolayer of the AuNP
where organic solutes can be partitioned, as demonstrated by
Lucarini and Pasquato.11 We report here the use of these pockets
to encapsulate drugs and deliver them with high efficiency to cells.
The biocompatible AuNPs used in this study feature two
functional domains: a hydrophobic alkanethiol interior and a
hydrophilic shell composed of a tetra(ethylene glycol) (TEG) unit
terminated with a zwitterionic headgroup. Particles with this general
Figure 1. (a) Delivery of payload to cell through monolayer-membrane
structure have been shown to minimize nonspecific binding with
interactions. (b) Structure of particles and guest compounds: Bodipy, TAF,
biomacromolecules.12
and LAP, the number of encapsulated guests per particle, and log P of the
We chose three different hydrophobic guest compounds: 4,4-
difluoro-4-bora-3a,4a-diaza-s-indacene (Bodipy) as a fluorescent
probe13 and the highly hydrophobic therapeutics tamoxifen (TAF)
andꢀ-lapachone(LAP)asdrugs(Figure1).Thenanoparticle-payload
conjugates (AuNPZwit-Bodipy, TAF, and LAP) were prepared
by a solvent displacement method.14 First, AuNPZwit (Au core:
2.5 ( 0.4 nm) and guest were dissolved in acetone/water, and the
solvent slowly evaporated. The bulk of the excess guest precipitated
out and was removed by filtration; the particles were further purified
by multiple filtrations through a molecular weight cutoff filter until
no free guest was observed, followed by dialysis against buffer.
The number of entrapped guest molecules per particle was
guests. (c) Release of Bodipy from AuNPZwit-Bodipy in DCM-aqueous
solution two-phase systems (λex) 499 nm, λem) 517 nm). (d) PL intensity
of AuNPZwit-Bodipy in cell culture medium and 100% serum, indicating
little or no release relative to AuNPZwit-Bodipy in PBS after NaCN-
induced release of guest molecules (λex) 499 nm, λem) 510 nm).
determined from 1H NMR spectra and NaCN-induced decomposi-
tion experiments (see Supporting Information) and varied depending
on size, hydrophobicity (log P), and molecular structure of
hydrophobic molecules (Figure 1). The particle/guest complexes
are stable in buffer for >1 month and to extended dialysis, a level
of kinetic entrapment greater than that observed with dendrimers.4
The ability of the delivery systems to release their payload was
first explored in Vitro using AuNPZwit-Bodipy in a two-phase
dichloromethane (DCM)-water system,15 where the dye is quenched
by the AuNP and photoluminescence (PL) was only observed upon
† University of Massachusetts at Amherst.
‡ Universita` di Firenze.
9
1360 J. AM. CHEM. SOC. 2009, 131, 1360–1361
10.1021/ja808137c CCC: $40.75
2009 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Cytotoxicity of AuNPZwit complexes measured by Alamar blue
assay after 24 h incubation with MCF-7 cells. IC50 of AuNP (NP), equivalent
drugs (Drug), and free drugs are shown in table.
Figure 2. CLSM images of MCF-7 cell treated with AuNPZwit-Bodipy
for 2 h: (a) green channel, (b) bright field, and (c) overlap. TEM images of
fixed cell treated with (d) AuNPZwit-Bodipy and (e) AuNPTTMA as a
positive control. Endosomally trapped AuNPs are marked by arrow. (f) ICP-
MS measurement (200 000 cells/well), indicating low cellular uptake of
AuNPZwit (31 ng/well after 4 h).
accumulation in tumor tissues by the enhanced permeability and
retention (EPR) effect.17 Additionally, the noninteracting nature of
their monolayer should make these systems highly amenable to
targeting strategies. We are currently exploring these applications
as well as the role of monolayer and guest structure in the
encapsulation process.
dye release. In these studies, a rapid increase in PL intensity is
observed along with transfer of Bodipy into the DCM layer (Figure
1c). Significantly, since no release is observed in monophasic
aqueous conditions and no particle was observed in the DCM layer,
payload release occurs interfacially.
Acknowledgment. This research was support by the NIH
(GM077173).
Payload delivery to cells using AuNPZwit-Bodipy was deter-
mined by confocal laser scanning microscopy (CLSM) using human
breast cancer (MCF-7) cells. Efficient delivery of the dye to the
cytosol is observed after 2 h of incubation with AuNPZwit-Bodipy
(Figure 2a-c). Cellular uptake of nanoparticles was studied using
transmission electron microscopy (TEM) and inductively coupled
plasma mass spectrometry (ICP-MS), using the analogous cationic
particle/dye conjugate AuNPTTMA-Bodipy as a positive control.
Little cellular uptake of AuNPZwit was observed by either TEM
(Figure 2d,e) or ICP-MS for AuNPZwit-Bodipy (31 ng/well at
4 h (Figures 2), 71 ng/well at 24 h Figure S3, corresponding to
uptake of 0.06% and 0.14% of available particle, respectively),
whereas substantial particle uptake was observed with AuNPTTMA-
Bodipy (1750 ng/well (4 h), 2150 ng/well (24 h)). Since no free
dye was observed during the 24 h incubation of AuNPZwit-Bodipy
in the medium or serum solution at 37 °C (Figure 1d), Bodipy
delivery presumably occurs via a monolayer-membrane transfer
process, consistent with our in Vitro studies.16
Demonstration of drug delivery to MCF-7 cells through presum-
ably the same mechanism was determined through cytotoxicity
studies of free and encapsulated drugs using an Alamar blue assay
(Figure 3). Notably, AuNPZwit itself was nontoxic at 30 µM. In
contrast, IC50 values of 4 and 4.6 µM were observed using
AuNPZwit-LAP and AuNPZwit-TAF, respectively. The delivery
process was quite efficient, with the per drug molecule IC50 of
AuNPZwit-TAF (46 µM) only 3-fold higher than that of TAF (16
µM), and with that of AuNPZwit-LAP (6.0 µM) essentially
identical to that of LAP (5.2 µM).
Supporting Information Available: Experimental procedures,
synthesis of gold nanoparticles and complexes, and TEM of gold
nanoparticles. This information is available free of charge via the
Internet at http://pubs.acs.org.
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In conclusion, we have demonstrated that hydrophobic dyes/drugs
can be stably entrapped in a hydrophobic pocket of AuNPs and
released into the cell by membrane-mediated diffusion without
uptake of the carrier nanoparticle. Importantly, the small size of
these nanocarriers coupled with their biocompatible surface func-
tionality should provide long circulation lifetimes and preferential
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