Intracellular trafficking of nuclear localization signal conjugated nanoparticles for cancer therapy

Article abstract of DOI:10.1016/j.ejps.2009.11.010

Doxorubicin (DOX) is an anticancer drug with an intracellular site of action in the nucleus. For high antitumour activity, it should be effectively internalized into the cancer cells and accumulate in the nucleus. In this study, we have prepared a nuclear localization signal conjugated doxorubicin loaded Poly (d,l-lactide-co-glycolide) nanoparticles (NPs), to deliver doxorubicin to the nucleus efficiently. Physico-chemical characterization of these NPs showed that the drug is molecularly dispersed in spherical and smooth surfaced nanoparticles. NPs (～226 nm in diameter, 46% encapsulation efficiency) under in vitro conditions exhibited sustained release of the encapsulated drug (63% release in 60 days). Cell cytotoxicity results showed that NLS conjugated NPs exhibited comparatively lower IC₅₀ value (2.3 μM/ml) than drug in solution (17.6 μM/ml) and unconjugated NPs (7.9 μM/ml) in breast cancer cell line MCF-7 as studied by MTT assay. Cellular uptake studies by confocal laser scanning microscopy (CLSM) and fluorescence spectrophotometer showed that greater amount of drug is targeted to the nucleus with NLS conjugated NPs as compared to drug in solution or unconjugated NPs. Flow cytometry experiments results showed that NLS conjugated NPs are showing greater cell cycle (G2/M phase) blocking and apoptosis than native DOX and unconjugated NPs. In conclusion, these results suggested that NLS conjugated doxorubicin loaded NPs could be potentially useful as novel drug delivery system for breast cancer therapy.

Full text of DOI:10.1016/j.ejps.2009.11.010

European Journal of Pharmaceutical Sciences 39 (2010) 152–163
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Intracellular trafficking of nuclear localization signal conjugated nanoparticles
for cancer therapy
Ranjita Misra, Sanjeeb K. Sahoo∗
Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Orissa, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 April 2009
Received in revised form 14 October 2009
Accepted 26 November 2009
Available online 2 December 2009
Doxorubicin (DOX) is an anticancer drug with an intracellular site of action in the nucleus. For high
antitumour activity, it should be effectively internalized into the cancer cells and accumulate in the
nucleus. In this study, we have prepared a nuclear localization signal conjugated doxorubicin loaded Poly
(d,l-lactide-co-glycolide) nanoparticles (NPs), to deliver doxorubicin to the nucleus efficiently. Physico-
chemical characterization of these NPs showed that the drug is molecularly dispersed in spherical and
smooth surfaced nanoparticles. NPs (∼226 nm in diameter, 46% encapsulation efficiency) under in vitro
conditions exhibited sustained release of the encapsulated drug (63% release in 60 days). Cell cytotoxicity
results showed that NLS conjugated NPs exhibited comparatively lower IC50 value (2.3 ␮M/ml) than drug
in solution (17.6 ␮M/ml) and unconjugated NPs (7.9 ␮M/ml) in breast cancer cell line MCF-7 as studied
by MTT assay. Cellular uptake studies by confocal laser scanning microscopy (CLSM) and fluorescence
spectrophotometer showed that greater amount of drug is targeted to the nucleus with NLS conjugated
NPs as compared to drug in solution or unconjugated NPs. Flow cytometry experiments results showed
that NLS conjugated NPs are showing greater cell cycle (G2/M phase) blocking and apoptosis than native
DOX andunconjugatedNPs. Inconclusion, theseresultssuggested thatNLSconjugateddoxorubicin loaded
NPs could be potentially useful as novel drug delivery system for breast cancer therapy.
Keywords:
Intracellular drug delivery
PLGA nanoparticles
Nuclear localization signal
Sustained release
MCF-7 cells
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
as proteins and phospholipids inside the body during its course
of action (Sokolovskaya et al., 2001) and also it interacts with
Cancer has been declared as one of the most deadly disease in
the world and many experimental treatment methods are being
explored to fight this fatal disease despite the obstacles that have
surfaced along the way. Chemotherapy is a major therapeutic
approach for the treatment of localized and metastasized cancer.
The sites of action of these cancer drugs are intracellular com-
partments which include nucleus or cytoplasm (Adcock and Ito,
2000; Brana et al., 2001). However, these current chemotherapeu-
tical agents have notorious undesirable side effects like limited
accessibility to tumor tissues, systemic toxicity, development of
multiple drug resistance and non-specific targeting which limit
their further clinical applications (Parveen and Sahoo, 2008). Dox-
orubicin (DOX) is one of the foremost drugs having its action
inside the nucleus and is currently used in the treatment of a
number of solid malignancies (Kalaria et al., 2009). DOX induces
apoptosis in cancer cells by binding to DNA but it undergoes
many non-selective reactions with a variety of biomolecules, such
both healthy and cancerous tissues giving rise to dose limiting
adverse effects such as cardiac myopathy, alopecia, etc. (Tan et
al., 2009). Thus, in an attempt to circumvent these limitations
and to improve therapeutic efficacy of anticancer agents like DOX
alternative approaches are being investigated. Nanotechnology is
one such approach where the premise behind the nanoparticular
delivery system is principally to increase drug activity by max-
imizing drug availability leading to reduction of noxious effect
of drug by minimizing drug exposure to healthy sites (Sahoo et
al., 2007). Particulate drug carrier systems encapsulating drugs
have emerged as promising approach in anticancer treatment by
improving the therapeutic index of drugs by preferential local-
ization at target sites (Bisht and Maitra, 2009). Nanoparticles
formulated from the biocompatible and biodegradable polymers
such as poly(d,l-lactide-co-glycolide) (PLGA) and polylactide (PLA)
have shown the potential for various drug delivery applica-
tions (Sahoo and Labhasetwar, 2003). These nanoparticles can be
designed to slip between intercellular spaces, enter cells, or trans-
port directly through biological barriers to access disease sites
through modifying the surface characteristics or by attaching any
suitable ligand on the surface of these nanoparticles (Das et al.,
2009).
∗
Corresponding author. Tel.: +91 674 2302094; fax: +91 674 2300728.
E-mail address: sanjeebsahoo2005@gmail.com (S.K. Sahoo).
0928-0987/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2009.11.010

R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
153
Cumulative evidence supports the fact that doxorubicin has
its action inside the nucleus, thus much effort is being made to
target doxorubicin to nucleus to increase its therapeutic efficacy.
However, the main obstacles for effective nuclear delivery of DOX
include plasma membrane and subcellular membrane bound vesi-
cles (Pouton et al., 2007). The limitations associated with plasma
membrane can be overcome by conjugating any ligand to nanopar-
ticulate systems that cross the plasma membrane by receptor
mediated endocytosis (Harashima et al., 2001;Qaddoumi et al.,
2003). But nuclear membrane acts as a bigger obstacle for the
drugs which are expected to obtain their highest accumulation
in the nuclei. As several vital cellular processes are regulated in
the nucleus, it is guarded by a double-layered membrane known
as the nuclear envelope (NE), which is difficult to break and is a
formidable barrier for the passage of macromolecules or drug car-
riers (Harashima et al., 2001). The NE contains specialized channels
called nuclear pore complex (NPC), which allow passive diffusion
of ions and small molecules of diameter ∼9 nm through aqueous
channels (Paine et al., 1975) while molecules of larger size (∼39 nm
in diameter) are transported across the NPC in an energy dependent
manner (Pante and Kann, 2002). However, entry of larger particles
has also been established by Cheng et al., who have demonstrated
that particles of 72 nm diameter were capable of successful entry
into the nuclei of HeLa cells (Cheng et al., 2008). In another study, it
has been shown by confocal microscopy that arginine peptide con-
jugated PLGA coated iron oxide NPs of around 115 nm size were
effectively adsorbed onto the membrane of stem cells and deliv-
ered into the nucleus without cytotoxicity (Lee et al., 2005). Thus
we can anticipate that particulate systems may help in efficient
delivery of nuclear targeting drugs.
cles over native DOX and unconjugated NPs due to greater cellular
internalization and direct accumulation of drug in nuclear com-
partment. Taken together these results suggest the development of
a novel targeted delivery system which has the potential to open
up new avenues for cancer therapy in near future.
2. Materials and methods
2.1. Materials
Poly (d,l-lactide-co-glycolide) (PLGA, copolymer ratio
50:50, I.V. = 0.55–0.75) was purchased from Birmingham poly-
mers, Inc. (Birmingham, AL). Polyvinyl alcohol (PVA, average
MW 30,000–70,000), Doxorubicin hydrochloride (DOXHCL),
N-hydroxysulfosuccinamide (Sulfo-NHS), 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide (MTT), RNAase and
propidium iodide were obtained from Sigma–Aldrich (St.
Louis, MO, USA). EDC (1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide) was purchased from Fluka Chemie AG (Switzerland).
Sodium chloride, Disodium hydrogen phosphate, Potassium bro-
mide and Potassium chloride were obtained from Sigma–Aldrich
chemicals, Germany. Potassium hydrogen phosphate was
purchased from Qualigens Fine Chemicals (Mumbai, India).
Chloroform, dichloromethane, methanol, acetonitrile and acetic
acid were purchased from E-merk (India). NLS (DRQIKIWFQNR-
RMKWKK) and NLS-FITC (DRQIKIWFQNRRMKWKK-FITC) peptides
were purchased from Imgenex Corporation, San Diego, USA. All
reagents used were of analytical grade from E-merk (India).
However, for efficient nuclear targeting any cell-specific nuclear
probe must satisfy the following requirements: small size for enter-
ing cells and crossing membrane barriers like nuclear membrane
and cell membranes, ability of binding to cell-specific plasma
membrane receptors, capability of escaping endosomal/lysosomal
pathways, or helping importins to pass through the nuclear pore
complex, and limiting toxicity (Tkachenko et al., 2004). Nuclear
localization signals (NLS) are special sequences which are used for
enhancing nuclear delivery by active transport mechanism (Vasir
and Labhasetwar, 2007) and satisfies most of the criteria of an effi-
cient nuclear delivery probe. NLS peptide mediates binding of the
karyophilic’ protein to importin ␣. This complex formation triggers
binding to cytoplasmic importin ␤ and the ternary complex is then
carried through the nuclear pore with the help of the GTPase Ran
(Zanta et al., 1999). Feldherr and co-workers have used gold parti-
cles modified with the nuclear localization signal (NLS) from SV-40
virus and injected the nanoparticle-peptide complex directly into
the cytoplasm near the nucleus, for investigating nuclear transport.
The KKKRK sequence from SV-40 virus has been shown to bind to
importin-␣, a nuclear transport protein that binds importin-␤ and
enters the nucleus through the nuclear pore complex (Tkachenko
et al., 2004). From above, we hypothesized that by conjugating
NLS peptide to the DOX loaded PLGA NPs, the drug can be tar-
geted directly to the nucleus overcoming the intrinsic side effects
of native drug but with elicited therapeutic effect.
2.2. Preparation of doxorubicin (DOX) loaded PLGA nanoparticles
2.2.1. Preparation of doxorubicin free base
Doxorubicin hydrochloride was converted to the free base
using following procedure (Jain et al., 2005). Gaseous ammo-
nia was produced by bubbling nitrogen gas through ammonium
hydroxide solution. This ammonia gas was bubbled through a
stirred suspension of doxorubicin hydrochloride in a 12.5% (v/v)
methanol in chloroform solution. 5 ml of boric acid solution
(10 mM, pH 8.8) was added to the drug solution to extract the
formed ammonium chloride salt and other water soluble by-
products. The upper aqueous layer was removed by vortexing the
above emulsion. This extraction procedure was repeated five times.
Under nitrogen flow the solvent mixture was evaporated and the
drug solution was lyophilized overnight to get doxorubicin free
base.
2.2.2. Preparation of doxorubicin loaded PLGA nanoparticles
NPs containing DOX were formulated by using single emulsion-
solvent evaporation technique (Sahoo et al., 2004). In brief, a
solution of 100 mg of PLGA polymer and 10 mg of DOX in 3 ml
of 12.5% (v/v) methanol in chloroform solution was emulsified in
12 ml of 2% (w/v) aqueous solution of PVA to form an oil-in-water
emulsion. The emulsification was carried out using a micro-tip
probe sonicator set at 55 W of energy output (VC 505, Vibracell
Sonics, Newton, USA) for 2 min over an ice bath. The emulsion was
stirred overnight at room temperature on a magnetic stir plate to
allow evaporation of organic solvent and formation of NPs. NPs
were recovered by ultracentrifugation at 40,000 rpm for 20 min at
4 ◦C (Sorvall Ultraspeed Centrifuge, Kendro, USA), washed twice
with double distilled water to remove unbound PVA and unencap-
sulated DOX and then lyophilized for 2 days (−80 ◦C and <10 ␮m
mercury pressure, LYPHLOCK 12, Labconco, Kansas City, MO) to get
the powdered NPs.
As our aim is to deliver DOX directly to the nucleus more effi-
ciently, the approach taken in this study is to conjugate NLS peptide
(DRQIKIWFQNRRMKWKK) onto DOX loaded poly (d,l-lactide-co-
glycolide) nanoparticles to fabricate a novel nanoparticulate drug
delivery system for nuclear delivery. DOX loaded PLGA nanopar-
ticles were prepared by single oil-in-water emulsion and NLS was
conjugated onto the PLGA nanoparticles using EDC/NHS chemistry.
The therapeutic efficiency of these surface modified nanopar-
ticulate system were studied on MCF-7 cells. Results confirmed
improved antiproliferative efficacy of these targeted nanoparti-

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2.3. Conjugation of NLS-FITC to DOX–NPs
2.4.3. Scanning electron microscopic (SEM) studies
The surface morphology of NPs was studied by SEM. For this,
NPs were sputtered with gold to make them conductive and placed
on a copper stub. The images were taken using scanning electron
microscope (JSM-T220A, JEOL, MA).
The conjugation procedure was carried out using a surface acti-
vation method (Kocbek et al., 2007). In brief, 10 mg of DOX loaded
NPs were suspended in 5 ml of PBS (0.02 M, pH 7.4). 250 ␮l of EDC
solution (1 mg/ml) and 250 ␮l of NHS solution (1 mg/ml) in 0.02 M
PBS were added drop wise to the above NP suspension. EDC acti-
vation was carried out by stirring the above suspension for 4 h
at room temperature in a magnetic stirrer. EDC/NHS solution was
removed and activated NPs were recovered by ultracentrifugation
at 40,000 rpm for 20 min at 4 ◦C (Sorvall Ultraspeed Centrifuge,
Kendro, USA). We have used NLS-FITC for conjugation to know
the amount of NLS conjugated to NPs. For coupling NLS-FITC, the
EDC activated NPs were suspended in 2 ml of PBS (0.02 M) and
to this 100 ␮l of NLS-FITC solution (1 mg/ml) in 0.02 M PBS was
added drop wise and stirred for another 2 h at room temperature
and the above solution was incubated for overnight at 4 ◦C. Next
day unconjugated NLS-FITC was removed by ultracentrifugation
and the supernatant was collected to calculate the conjugation
efficiency. NLS conjugated NP suspension was lyophilized to get
the powder for further use. The collected supernatant was used
to estimate the amount of NLS-FITC which was not conjugated
to DOX loaded NPs by fluorescence spectrophotometer (Synergy
HT, BioTek® Instruments Inc., Winooski, VT). The average number
of NLS-FITC conjugated to NPs was calculated by subtracting the
amount of NLS-FITC in supernatant from the total amount of NLS-
FITC used in conjugation process. The average number of NLS-FITC
molecules conjugated per one NP was calculated by dividing the
number of NLS molecules bound to NPs by the calculated average
number (n) of NPs using the following equation: (Sahoo et al., 2004)
2.5. Estimation of encapsulation efficiency by fluorescence
spectrophotometer
The encapsulation efficiency (EE) of NPs was evaluated by flu-
orescence spectrophotometer (Synergy HT, BioTek® Instruments
Inc., Winooski, VT). For EE determination, a known amount of freeze
dried NPs was completely dissolved in 12.5% (v/v) methanol in chlo-
roform solution. The solution was sonicated for 1 min in ice bath
and centrifuged for 10 min at 13,800 rpm and 4 ◦C (SIGMA 1-15K,
Germany). The supernatant was used to estimate the drug con-
centration in NPs by a fluorescence spectrophotometer (Synergy
HT, BioTek® Instruments Inc., Winooski, VT) at ꢁex = 485 nm and
ꢁem = 591 nm. A standard plot of DOX was prepared previously in
the same condition to calculate the amount of drug loaded in the
NPs. The encapsulation efficiency was calculated by the following
equation
Mass of drug in nanoparticles
Encapsulation efficiency(%) =
Mass of drug used in formulation
× 100
2.6. Fourier transform infrared (FTIR) spectral studies
To investigate the possible chemical interactions between the
drug and the polymer matrix, FTIR spectra were obtained from
(PerkinElmer Model Spectrum1, USA). For this the samples were
crushed with KBr to get the pellets by applying a pressure of
300 kg/cm2. FTIR spectra of native DOX, DOX loaded NP and void
6m
n =
˘ × D3 × ꢀ
where m is weight of the NP, D is the number based on mean diam-
eter of NP as measured by photon correlation spectroscopy (PCS)
and ꢀ = density of the NP. The above same procedure was followed
to conjugate NLS to NPs (NLS was used instead of NLS-FITC) for
further experiments.
NP were scanned in the range between 4000 and 500 cm−1
.
2.7. Differential scanning calorimetric (DSC) studies
The physical state of DOX encapsulated in NPs was character-
ized using a differential scanning calorimetric (DSC) thermogram
analysis (Shimadzu, DSC-50). Each sample (PLGA, native DOX and
DOX loaded NP) was sealed separately in a standard aluminum
pan, the samples were purged in DSC with pure dry nitrogen
gas set at a flow rate of 10 ml/min, the temperature speed was
set at 10 ◦C/min, and the heat flow was recorded from 0 to
350 ◦C.
2.4. Physico-chemical characterization of NPs
2.4.1. Particle size analysis and zeta potential measurement
Dynamic laser scattering (DLS) was used to measure the hydro-
dynamic diameter (d nm) and laser Doppler Anemometry (LDA)
was used to determine zeta potential (mV). The DLS and LDA
analyses were performed using a Zetasizer Nano ZS (Malvern
Instruments, Malvern, UK). The DLS measurements were done with
a wavelength of 532 nm at 25 ◦C with an angle detection of 90◦. To
determine the particle size and zeta potential, a dilute suspension
of nanoparticles (100 ␮g/ml) (either unconjugated or NLS conju-
gated NP) was prepared in double distilled water and sonicated
on an ice bath for 30 seconds and subjected to particle size and
zeta potential measurement using Zetasizer (Nano ZS, ZEN 3600,
Malvern Instrument, UK). All measurements were performed in
triplicate.
2.8. In vitro release of doxorubicin from NPs
In vitro release kinetics of DOX from DOX loaded NPs was
determined in PBS buffer (0.01 M, pH 7.4, containing 0.1% Tween
80) at 37 ◦C. 10 mg of NPs was dissolved in 3 ml of above buffer.
The NP suspension was equally divided in three tubes contain-
ing 1 ml each. These tubes were kept in shaker at 37 ◦C and
150 rpm (Wadegati Labequip, India). At particular time inter-
vals these tubes were taken out from shaker and centrifuged at
13,800 rpm, 4 ◦C for 10 min (SIGMA 1-15K, Germany). The super-
natants were taken out to estimate the amount of drug released
at that particular time through fluorescence spectrophotome-
ter (Synergy HT, BioTek® Instruments Inc., Winooski, VT). To
the residue same amount of fresh PBS (0.01 M, pH 7.4, contain-
ing 0.1% Tween 80) was added and kept in shaker for further
study.
2.4.2. Transmission electron microscopic studies
NPs were also evaluated for size by a transmission electron
microscope (Philips/FEI Inc, Barcliff, Manor, NY). For this purpose,
a sample of NPs (0.5 mg/ml) was suspended in water and sonicated
for 30 s. One drop of this suspension was placed over a carbon
coated copper TEM grid (150 mesh, Ted PELLA Inc., Rodding, CA)
and negatively stained with 1% uranyl acetate for 10 min and then
allowed to dry. Images were visualized at 120 kV under microscope.

R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
155
2.9. In vitro cellular uptake study
treatment was calculated as a percentage inhibition against the
respective controls.
Cellular uptake of native DOX and nanoparticular formulation
(NLS conjugated and unconjugated NPs) was studied with little
modification (Sahoo et al., 2002). For this, 24-well plates (Corning,
NY, USA) were seeded with the MCF-7 cells at 50,000 per well and
the cells were allowed to attach for 24 h. The medium in the wells
was replaced with 1 ml of freshly prepared NP suspension (NLS
conjugated or unconjugated NPs) (200 ␮g of NPs/well). Similar con-
centration of native DOX in solution was added and the plates were
incubated for 2 h. Cells were washed three times with PBS (0.1 M,
pH 7.4) to remove native DOX and NPs which were not internalized
in to the cells and then incubated with 0.1 ml of 1 X cell lysis reagent
(Sigma–Aldrich, St. Louis, MO, USA) for 20 min at 37 ◦C. 5 ␮l of cell
lysate was used for cell protein determination using micro BCA pro-
tein assay (Pierce, Rockford, IL, USA) and the remaining portion was
lyophilized. DOX from the lyophilized NPs was extracted by dis-
solving each sample in 1 ml of 12.5% (v/v) methanol in chloroform
solution. The samples were centrifuged at 14,000 rpm for 10 min
in a microcentrifuge tube to remove cell debris. The supernatant
was analyzed for DOX using fluorescence spectrophotometer (Syn-
ergy HT, BioTek® Instruments Inc., Winooski, VT). A standard plot
with different concentration of NPs was constructed simultane-
ously under similar conditions to determine the amount of NPs
in cell lysate. The data was normalized to per milligram cell pro-
tein.
2.12. Intracellular uptake and nucleus-cytoplasm distribution of
DOX
After intracellular uptake of native DOX and NP formulations
(NLS conjugated or unconjugated NPs), the amount of DOX in
nucleus and cytoplasm was determined by separating the nucleus
and cytoplasm as mentioned below. Nuclei were separated from
cytoplasm by dissolving the plasma membrane and subsequent
centrifugation steps. Briefly, cells were stained with Hoechst 33342
and then suspended in 500 ␮l of ice cold buffer (HEPES, pH 7.9,
KCL, EDTA, DTT and IGEPAL) to dissolve the plasma membrane.
The cytoplasm was collected by centrifugation at 4 ◦C, 13,800 rpm
for 3 min (Sigma–Aldrich, St. Louis, MO, USA). The cell pellet was
dispersed in 150 ␮l of buffer (HEPES, pH 7.9, NaCl, EDTA and Glyc-
erol) and the above suspension was shaken in a rocker (Labnet
S 2030-RC-220, Labnet Inc., Woodbridge, NJ, USA) for 2 h at 4 ◦C.
Subsequently, centrifugation at 4 ◦C, 13,800 rpm for 5 min (SIGMA
1-15K, Germany) was carried out to obtain the nuclei from the
supernatant. Thus, the cytoplasm and nuclei were collected sep-
arately. The obtained nuclear yield was evaluated by comparing
the fluorescent absorbance (ꢁex = 355 nm and ꢁem = 450 nm) of the
collected nuclear suspension with that of the intact cell suspension
after Hoechst staining.
To determine the amount of DOX present in nucleus or in cyto-
plasm, 1 × 106 cells/5 ml were seeded in T-25 flask (Corning, NY,
USA) and incubated overnight for better attachment. The cells were
exposed to 5 ␮M/ml of free DOX or DOX loaded NPs (NLS conju-
gated or unconjugated NPs) in culture medium and incubated at
37 ◦C for 2 and 6 h. At predetermined time, the cells were washed
three times with ice-cold PBS (0.1 M, pH 7.4) to terminate the
uptake and remove the free DOX or DOX–NPs (NLS conjugated or
unconjugated NPs) which were not internalized into the cells. The
cells were collected by a short trypsin treatment. Cytoplasm and
nuclei were collected separately, as describes above. 5 ␮l of cell
lysate was taken for cell protein estimation using micro BCA pro-
tein assay (Pierce, Rockford, IL, USA). The concentration of DOX in
cytoplasm or nucleus was determined by the fluorescence mea-
surement. Cellular DOX levels were expressed in microgram DOX
per milligram protein.
2.10. Intracellular localization by confocal laser microscopy
Intracellular localization of native DOX and nanoparticular for-
mulations (conjugated and unconjugated NPs) was studied in
confocal microscopy with slight modification(Jain et al., 2005).
MCF-7 cells were seeded in Bioptechs plates (Bioptechs, Butler, PA)
at 50,000 cells/plate in 1 ml of complete RPMI medium 24 h prior to
the experiment. To study the intracellular retention of drug, cells
were treated with 5 ␮M/ml of either drug in solution, drug-loaded
NPs or NLS conjugated drug-loaded NPs and were incubated for
2, 24, 48 and 120 h. Then cells were washed three times with PBS
(0.1 M, pH 7.4) to remove the untrapped DOX. After some time the
nuclei of cells were stained with DAPI by incubating the cells in dye
for 15 min and washed with PBS twice before being imaged under a
confocal microscope (Leica TCS SPS, CLSM system) at ꢁex = 488 nm
and a long pass filter of 515 nm for detecting the emission light. The
images were processed using Leica Application Suite software.
2.13. Cell cycle analysis and determination of apoptotic cells by
flow cytometry
2.11. Cytotoxicity assay
Cell cycle analysis and apoptosis study of native DOX and NP
formulations (NLS conjugated or unconjugated NPs) were done
with slight modification (Noh et al., 2004). 5 × 105 cells/5 ml were
seeded in T-25 flask (Corning, NY, USA) and incubated overnight for
better attachment. The cells were exposed to a particular concen-
tration (0.2 ␮M/ml) of drug in solution, drug-loaded NPs and NLS
conjugated drug-loaded NPs in RPMI medium and incubated for
48 h. Void nanoparticles-treated cells and untreated cells were used
as respective controls. Then the cells were collected by trypsiniza-
tion, washed with PBS (0.1 M, pH 7.4) twice and resuspended in
hypotonic propidium iodide solution (10 ␮g of propidium iodide,
10 ␮g of RNase A, and 0.5% Tween 20 in 1 ml of PBS) for 1 hour
at room temperature and kept in dark at 4 ◦C before analysis. Cell
cycle distribution was determined by analyzing 10,000 cells using
FACScan flow cytometer and Cell Quest software Caliber (Becton-
Dickinson, Sanjose, CA). The percentage of apoptotic cells was
determined by the subG1 peak in the DNA histogram as reported
earlier (Noh et al., 2004).
A comparison of cytotoxicity of NP formulations (NLS con-
jugated or unconjugated NPs) and native DOX was performed
on the test cells (MCF-7) with an in vitro proliferation assay
using MTT (Jain et al., 2005). Briefly, 5000 cells per well were
plated in 96-well plates (Corning, NY, USA) and incubated for
24 h to allow the cells to attach. Then the cells were exposed
to different concentrations of DOX either as solution or encap-
sulated in NPs (NLS conjugated or unconjugated NPs) with void
NPs (without drug) or medium serving as respective controls.
The medium was changed on every alternate day and no further
dose of drug was added. Cell viability was determined on 5th day
following drug treatment using the 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) based colorimetric assay.
10 ␮l of the MTT solution (5 mg/ml) was added and incubated
at 37 ◦C for another 3 h, and then the media was replaced with
100 ␮l of DMSO to dissolve the formazan crystals. The absorbance
was measured at 540 nm using a microplate reader (Synergy
HT, BioTek® Instruments Inc., Winooski, VT). The effect of each

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R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
Fig. 1. Schematic representation of conjugation of NLS to DOX loaded NPs by EDC/NHS method to form NLS conjugated DOX loaded NPs.
2.14. Cell culture
MCF-7 cells were purchased from American Type Culture Collec-
tion (ATCC, Manassas, VA) and were grown in RPMI 1640 (Himedia
Laboratories Pvt. Ltd., Mumbai, India) supplemented with 10% fetal
bovine serum and 100 ␮g/ml penicillin G and 100 ␮g/ml strepto-
mycin (Himedia Laboratories Pvt. Ltd., Mumbai, India) at 37 ◦C in a
humidified and 5% CO2 atmosphere (Hera Cell, Thermo Scientific,
Waltham, MA).
2.15. Statistical analysis
Statistical analyses were performed using a Student’s t test. The
differences were considered significant for p values of <0.05.
3. Results and discussion
3.1. Physico-chemical characterization of NPs
Doxorubicin loaded NPs were prepared by single emulsion
method. NLS was covalently conjugated to the carboxylic group
of PLGA nanoparticles by EDC/NHS activation method (Kocbek
et al., 2007). EDC forms an active ester intermediate by reacting
with carboxylic group of PLGA NPs. This active intermediate forms
amide bond by reacting with amine group of NLS peptide. Thus
NLS was successfully conjugated onto the surface of DOX loaded
NPs as schematically represented in Fig. 1. The amount of NLS
conjugated to DOX loaded NPs was estimated by fluorescence spec-
trophotometer. It was found that approximately 6 ␮g of NLS was
conjugated per mg of NP, which represents approximately 433 NLS
molecules per NP. The size of the NPs was around 226 3.1 nm
(Fig. 2a) as measured by photon correlation spectroscopy (PCS)
with a low polydispersity index of 0.15, suggesting a uniform par-
ticle size distribution. On measuring the size of conjugated NPs
it was observed that there was a negligible increase in size of NPs
(226–234 nm) after conjugation of NLS to NP surface (Table 1). Such
observations were also reported by (Nam et al., 2002). They have
found that conjugation of Tat peptides on surface of PLGA NPs leads
to increase in particle size. The reason for this may be the different
chemical compositions of NLS conjugated NPs which might have
resulted in different internal morphology of the nanoparticles as a
result the size may increases slightly (Zhang and Eisenberg, 1995).
The zeta potential of the NLS conjugated NPs was comparatively
less than unconjugated NPs (−9.93 2.3 mV vs −5.56 2.8 mV)
(Table 1). The decrease in zeta potential is likely due to the con-
Fig. 2. Particle size and shape analysis. (a) Nanoparticle size distribution measured
by photon correlation spectroscopy (data as mean SEM, n = 6). (b) Transmission
electron microscopy of DOX loaded NPs (c) Scanning electron micrograph of DOX
loaded NPs.
tribution of cationic amino acid residues present in NLS peptide.
The mean particle size of NPs as observed with TEM was around
100 nm (Fig. 2b), which is less than the particle size measured by
photon correlation spectroscopy. Prabha et al. have reported that
the particle size measured by TEM is different from size measured
by PCS because it measures hydrodynamic diameter and hydra-
tion of the surface associated PVA probably contributes towards
increasing the hydrodynamic diameter of the NPs (Prabha et al.,
Table 1
Physico-chemical characteristics of nanoparticle formulation.
Formulation
Sizea
Zeta potentialb
PDIc
DOX–NP
DOX–NP–NLS
226 3.1
234 4.3
−9.93 2.2
−5.48 2.5
0.15
0.11
a
Size in nm as measured by photon correlation spectroscopy.
Zetapotential in mV measured by zetasizer.
Polydispersity index measured by photon correlation spectroscopy.
b
c

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157
2002). This may be the reason for getting bigger sized NPs when
measured by PCS than TEM. We have studied the surface morphol-
ogy of drug-loaded NPs by SEM. The particles were spherical with
a smooth exterior as evident from SEM results (Fig. 2c). The encap-
sulation efficiency of NPs was around 46% (i.e., 46% of the drug
added in formulation was entrapped in NPs) estimated by fluo-
rescent spectrophotometry. After conjugation the encapsulation
efficiency slightly decreased to ∼44%. The reason for this slight
decrease in encapsulation efficiency of NLS conjugated NPs may
be due to release of some surface adsorbed drug from NPs during
washing steps in conjugation process.
3.2. Fourier transform infrared (FTIR) spectral studies
FTIR results confirmed the chemical stability of DOX in nanopar-
ticles. Fig. 3 shows the FTIR spectra of native DOX, DOX loaded
NPs and void NPs. Native DOX showed characteristic bands due
to presence of different functional groups. A band appearing at
3436 cm−1 is due to O-H/N-H stretching vibrations, while those
observed at 2954 cm−1 is due to the C-H stretching vibration. Bands
at 1673 and 1583 cm−1 are due to primary amide (N–H) bending
and aromatic N–H bending vibrations, respectively. Carbonyl (C O)
stretching vibrations are seen at 1634 cm−1, while the bands at
1448 and 1384 cm−1 are due to –CH2 bending and C–H bending
vibrations respectively. The bands at 1276 and 1176 cm−1 belong
to C–N stretching vibrations. The bands occurring in void NPs are
almost similar to the bands in DOX loaded NPs in addition with
some extra bands due to DOX. Mundagri et al. have encapsulated
doxycycline in PLGA:PCL microspheres and have shown that in
microspheres some of the characteristic bands of native doxycy-
cline are also found by FTIR that indicates the chemical stability of
drug inside the formulation (Mundargi et al., 2007). In our exper-
iment the distinctive bands of native DOX are appearing at 3436
and 1673 cm−1 that are also present in DOX loaded NPs indicating
the chemical stability of DOX in our nanoparticle formulation.
Fig. 4. Differential scanning calorimetry thermogram curves of (a) PLGA, (b) native
DOX and (c) DOX loaded NP.
(Fig. 4). Different compounds show their particular characteristic
endothermic peaks in DSC. The endothermic peak of PLGA polymer
was found approximately at ∼45 ◦C due to glass transition tempera-
ture (Tg) of PLGA and there is no shifting of PLGA peak in DOX loaded
NPs. The endothermic peak of native DOX was found approximately
at 235 ◦C. This characteristic peak was not observed in DOX loaded
nanoparticles (Fig. 3) which indicates that the drug encapsulated
in NPs is in the amorphous or disordered- crystalline phase or
in solid-state solubilized form in the polymer matrix, resulting in
molecular dispersion of drug inside the NPs. Similar type of results
has been reported by (Sahoo and Labhasetwar, 2005). They have
entrapped paclitaxel inside PLGA NPs and in the final formulation
no peak of paclitaxel was observed. Thus it can be concluded that
DOX in NPs is in amorphous or disordered crystalline phase which
inhibited crystal growth thereby leading to enhanced stability of
formulation.
3.3. Differential scanning calorimetric (DSC) studies
3.4. In vitro release of doxorubicin from NPs
The stability of nanoparticles depends on the state of drug in
formulation. Controlling crystallization is one of the key issues
in formulating successful and stable nanoparticulate formulations
because decrease in crystalline fractions and increase in amor-
phous fractions of drug enhances the saturation solubility of drug,
increasing shelf life and stability thereby preventing Ostwald’s
ripening phenomenon (Kalaria et al., 2009). DSC study shows the
nature of the drug encapsulated in the nanoparticles. This analysis
was performed on native PLGA, native DOX and DOX loaded NPs
The in vitro release behavior of DOX loaded nanoparticles is
summarized in the cumulative percentage release shown in Fig. 5.
The NPs demonstrated a sustained release of the encapsulated drug,
with ∼63% cumulative drug release in 60 days. It was observed
that NPs give a comparatively faster release (∼9% in 1 day) fol-
Fig. 5. In vitro release of DOX from PLGA nanoparticles in PBS (0.01 M, pH 7.4)
(values reported are mean SD; n = 3).
Fig. 3. FTIR spectra of (a) native DOX, (b) DOX loaded NPs and (c) void NPs.

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R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
lowed by a slow release (∼63% in 60 days) of the drug from the
polymeric matrix. Tewes et al. have prepared DOX loaded PLGA
NPs by single emulsion method and reported that approximately
6.5% of DOX was released from the NPs in 24 h (Tewes et al., 2007).
They have also found an initial fast release followed by a sustained
release of drug from the NPs. The reason for this may be that ini-
tially the drug deposited at the NP interface is released at a faster
rate via diffusion through the water channels created in the NPs.
The slower and sustained release of the drug at later stages can
be attributed to the diffusion/erosion of polymeric matrix which
releases the encapsulated drug (Sahoo et al., 2004).
the nucleus(Jarver and Langel, 2004; Vasir and Labhasetwar, 2007).
Thus it is believed that NLS conjugated NPs may enter the cells by
some other way besides receptor mediated endocytosis. As a result
NLS conjugated DOX loaded NPs were capable of showing higher
cellular uptake than native DOX and unconjugated NPs (Futaki et
al., 2002; Lochmann et al., 2004; Schirmbeck et al., 2003). After
entering the cells, NLS peptide mediates binding of the karyophilic
protein to importin ␣. This complex formation triggers binding to
importin ␤ and the ternary complex is then carried through the
nuclear pore with the help of the GTPase Ran (Zanta et al., 1999).
In our study, we believed that the NLS conjugated NPs may enter
the nucleus by the above-mentioned way. In addition to this, these
NPs may also bind to the NPC (as NLS has binding affinity to NPC)
where the NPs may give a sustained release of the drug which will
later diffuse into the nucleus resulting in greater efficiency of NLS
conjugated DOX loaded NPs. The proposed mode of entry of all the
three formulations into the cells and the mechanism of transport
of NLS conjugated NPs into the nucleus through nuclear membrane
is depicted schematically in Fig. 7.
3.5. In vitro cellular uptake study
Targeting effect of NLS conjugated NPs was evaluated by study-
ing the cellular uptake of native DOX, NP formulations (NLS
conjugated or unconjugated NPs) for 2 h in breast cancer MCF-7 cell
line. Fig. 6 shows that NP formulation (NLS conjugated or uncon-
jugated NPs) have higher cellular uptake than native DOX. NLS
conjugated NPs showed 6-times and 2.5-times more uptake than
native DOX and unconjugated NPs respectively. Previous studies
have shown that the native drug enters the cells by simple dif-
fusion (Sahoo and Labhasetwar, 2005). In diffusion process after
a particular concentration, the drug reaches a saturation point,
hence further diffusion of native drug is prevented leading to lower
uptake by the cells (Sahoo and Labhasetwar, 2005). Besides, DOX
has also been found to be a substrate of P-glycoproteins (P-gp)
which are present in cancer cells leading to multi-drug resistance
(MDR) effect of the cancer cells, so the native drug may efflux out
of the cells through P-gp efflux pump (Kubota et al., 2001; Mi and
Lou, 2007). This may be another reason for lower uptake of native
drug by the cells. In case of NPs the mode of entry into the cells is
through endocytosis (Qaddoumi et al., 2003). The process of endo-
cytosis helps the NPs in escaping the MDR effect of cancer cells
leading to the entry of NPs in more amounts as compared to native
drug (Panyam and Labhasetwar, 2003a,b). It has also been reported
that drug-loaded PLGA NPs prevent endo-lysosomal degradation
of drug inside the cells (Panyam et al., 2002) thereby increasing
the half-ife of encapsulated drug. However, it has been reported
that NLS sequences are able to cross cell membranes to reach the
nucleus within few minutes. These positively charged peptides are
able to bypass the cell membrane and deliver the drugs directly to
3.6. Intracellular localization by confocal laser microscopy
Confocal microscopy of cells treated with native DOX and
NP formulations (NLS conjugated and unconjugated NPs) for 2,
24, 48, and 120 h showed that the drug was localized more in
nucleus in case of NLS conjugated NPs than in unconjugated NPs
and drug in solution (Fig. 8). Clear distinction between nucleus
and cytoplasm due to DAPI staining is also observed by confo-
cal microscopy. Confocal results illustrate that more amount of
native DOX is present inside the nucleus during the first 24 h of
incubation but with increasing incubation time the fluorescence
intensity decreases. The reason for this may be attributed to the
saturation of drug entry through diffusion process. It is known
that native DOX has its site of action inside the nucleus. So, it
first crosses the cell membrane by simple diffusion and then dif-
fuses into nucleus, but gradually the process of diffusion reaches a
saturation point after which further entry of native DOX is pre-
vented. Therefore a decrease in fluorescence intensity is clearly
evident in cells treated with native DOX with time. As we have
discussed in cellular uptake study that NP formulations showed
more uptake than native drug, the sustained release of encap-
sulated drug from NPs lead to a steady increase in fluorescence
intensity with increasing incubation. However, enhanced uptake
of NLS conjugated NPs by MCF-7 cells resulted in greater accumu-
lation of released drug in nucleus thereby increasing fluorescence
intensity to maximum level. Tkachenko et al. have prepared differ-
ent peptide-modified gold particle complexes and have evaluated
the cellular trajectory of these particles using different cell lines.
They have studied the intracellular localization of these particles
through video-enhanced color differential interference contrast
microscopy and showed enhanced nuclear localization of NLS con-
jugated gold NPs in different cell lines (Tkachenko et al., 2004).
Thus NLS conjugated NPs probably act as an intracellular depot
and sustained the drug release slowly for longer time in the
nucleus.
3.7. Cytotoxicity assay
To investigate the therapeutic efficiency of the formulations,
MCF 7 cells were treated with native DOX, NP formulations (NLS
conjugated or unconjugated NPs) at different concentrations for
5 days, and cell proliferation was measured by a standard MTT
colorimetric assay (Fig. 9). It was found that with increasing DOX
concentration, the cell viability of cancer cells decreased (or equiv-
alently, the cell mortality increased). Conjugation of NLS peptide
Fig. 6. Cellular uptake of native DOX (DOX), unconjugated NPs (DOX–NP) and NLS
conjugated NPs (DOX–NP–NLS) in MCF-7 cells. A suspension of NPs (200 ␮g/ml) and
similar concentration of drug in solution was incubated with MCF-7 cells (5 × 104
cells) in 24 well plates for 2 h, cells were washed, and DOX levels in cells treated with
above three samples were determined by fluorescence spectrophotometer. Data as
mean S.E.M. (n = 6). *p < 0.05, DOX–NP vs. DOX. **p < 0.005, DOX–NP–NLS vs. DOX.

R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
159
Fig. 7. Schematic representation showing quick cellular entry and endo-lysosomal escape of NLS conjugated NPs (I), cellular internalization of unconjugated NPs by endo-
cytosis, endo-lysosomal escape of NPs and release of drug in cytosol (II), diffusion of native DOX through plasma membrane (III), efflux of native drug by P- glycoprotein
present in the plasma membrane due to MDR effect in cancer cells (IV). After endolysomal escape of NLS conjugated NPs, these formulations enter the nucleus either by
active transport of importin bounded NLS conjugated NP complex through NPC (V) or by binding of NLS conjugated NPs to NPC leading to sustained release of drug in its
close proximity (VI). This enhanced accumulation of DOX inside the nucleus by NLS conjugated NPs leads to greater therapeutic efficiency of such nuclear targeting drugs.
to nanoparticle surface elevated the cytotoxic effect of drug which
is evident from the IC50 values. NLS conjugated NPs exhibited a
lower IC50 value of 2.3 ␮M/ml than drug in solution and unconju-
gated NPs where the observed IC50 values were 17.6 and 7.9 ␮M/ml
respectively. This could be explained on the basis of the differences
in the cellular uptake results. Cellular uptake of NLS conjugated
NPs was 6 times and 2.5 times higher than native DOX and uncon-
jugated NPs respectively. As the antiproliferative effect of drug is
very well correlated with the duration of its intracellular retention,
a small fraction of free drug that is available in the cytoplasmic
compartment (due to initial diffusion of drug molecules) is respon-
sible for eliciting the antiproliferative effect of native drug in MCF-7
cells (Acharya et al., 2009). However, cytotoxicity of NLS conju-
gated NPs was improved sharply due to better accumulation of
drug at its site of action (nucleus) due to targeted delivery. It is also
noteworthy that unconjugated NPs demonstrated a lower antipro-
liferative activity than their NLS conjugated counterparts but they
had a better efficacy than drug in solution. Higher uptake of NPs
by endocytosis is responsible for educing such a response in MCF-7
cells.
3.8. Intracellular uptake and nucleus-cytoplasm distribution of
DOX
To support the fact that NLS conjugated NPs have enhanced
antiproliferative activity due to direct targeting of DOX to the
nucleus, the intracellular concentration and distribution of DOX

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R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
Fig. 8. Time course study of intracellular retention of florescent DOX in MCF-7 cells by confocal microscopy. Cells were treated with native drug (DOX), unconjugated NPs
(DOX–NPs) and NLS conjugated NPs (DOX–NP–NLS) (dose = 5 ␮M) in the growth medium and incubated for 2, 24, 48 and 120 h. The fluorescence intensity increases in case
of cells treated with DOX–NPs and DOX–NP–NLS while a decrease in fluorescence intensity was observed in case of native DOX with increase in incubation time.
were measured after the separation of cytoplasm and nucleus in
different time intervals. The obtained nuclear yield was about 71.1%
which was evaluated by comparing the fluorescent absorbance of
the collected nuclear suspension with that of the intact cell sus-
pension after Hoechst staining. Fig. 10a and b depict that in cells
treated with NLS conjugated NPs the concentration of DOX in nuclei
increased gradually with time, while a decrease in DOX concentra-
tion in cytoplasm was observed with passage of time. Cells treated
with native DOX had nearly equal concentration of the drug in cyto-
plasm and nucleus during initial hours of incubation (2 h). However
with time more and more amount of DOX gets accumulated in cyto-
plasm while DOX concentration in nucleus remains steady even
after 6 h. It indicates that native DOX is not further transported
into the nucleus and is retained in the cytoplasm. From Fig. 10b
the above fact is starkly evident where after 6 h of incubation the
concentration of native DOX in cytoplasm of MCF-7 cells increased
by 2.6 and 2.2 times more than cells treated with NLS conjugated
NPs and unconjugated NPs respectively. You et al. have reported
that the transport of DOX–HCl is quite different from free DOX in
different cell lines (You et al., 2008). They found that after removal
of HCl molecule from DOX–HCl, the obtained DOX base was always
present in the cytoplasm. The reason for this is still unclear, how-
ever this can also be considered as another reason for retention of
native DOX in cytoplasm. In case of unconjugated and NLS conju-
gated NPs more amount of DOX was concentrated in nucleus rather
than in cytoplasm the value being higher in the later. Initially, cells
treated with NLS conjugated NPs had 3.1 and 1.7 times more DOX
in nucleus than cells treated with native DOX and unconjugated
NPs respectively. With time the concentration of DOX increased in
nucleus corroborating our view that surface modification of PLGA
NPs with NLS peptide resulted in better delivery of drug to its
site of action (Fig. 10a). As we have discussed that in case of NLS
conjugated NPs the cellular uptake is higher than native DOX and
unconjugated NPs, so increased amount of DOX is taken by the cells

R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
161
in case of NLS conjugated NPs. As the drug is having its action inside
the nucleus after cellular uptake more amount of DOX is targeted to
nucleus in case of NLS conjugated NPs than native DOX and uncon-
jugated NPs. This resulted in an enhanced antiproliferative activity
of NLS conjugated NPs over unconjugated NPs and native DOX.
3.9. Cell cycle analysis and determination of apoptotic cells by
flow cytometry
The decreased cell growth induced by native DOX and nanopar-
ticulate formulation (NLS conjugated and unconjugated NPs) can be
attributed to decreased cell cycle progression or increased apopto-
sis. After 48 h of treatment with 0.2 ␮M/ml concentration of DOX,
the percentage of cells in G2/M phase was higher in case of cells
treated with NLS conjugated NPs (with a concurrent lower propor-
tion of cells in the proliferative S phase) than cells treated with the
native DOX in solution and unconjugated NPs (Table 2). However,
the unconjugated NPs had a relatively lower percentage of cells
in the S phase than the cells treated with drug in solution. Void
NPs had no effect on cell cycle distribution confirming its nontoxic
nature. Doxorubicin is known to have a variety of pharmacological
actions, including its effect on genes controlling the cell cycle and
induction of pro-apoptotic genes (Di et al., 2009; Li et al., 2007; Liu
et al., 2008; Wang et al., 2004). Primarily, DOX inhibits cell growth
and proliferation by its effect on the cell-cycle arrest in the G2 phase
of cell cycle (Yamashita et al., 2007). In our studies it was found that
treatment of DOX resulted in a greater proportion of cells in the
G2/M phase, compared to the proportion of cells left untreated or
treated with void nanoparticles. A higher proportion of cells were
in G2/M arrest phase in the NLS conjugated NPs treatment groups
than in the group treated with the drug in solution and unconju-
gated NPs. Thus, by conjugating NLS to DOX loaded NPs, excess of
DOX can be targeted to its site of action, i.e. nucleus, to prolong
the availability of drug for a longer period of time resulting in its
greater antiproliferative activity. The hypodiploid peak (sub G1)
appearing before the G1 phase on histogram is called the apoptotic
peak (Liu et al., 2003), which indicates reduced DNA content in
apoptotic cells in flow cytometry analysis. Table 2 shows that cells
incubated with NLS conjugated NPs resulted in 9.4 1.3% apopto-
sis, indicating that NLS conjugated NPs induced similar apoptotic
progression in MCF-7 cells as native DOX (8.3 1.0%) and the bio-
logical function of DOX was retained even after conjugation of NLS
to the NPs. 7.3 0.7% of cells were in apoptotic stage after treatment
with unconjugated NPs. It was observed that native DOX and NLS
conjugated NPs exhibited nearly similar percentage of cell death
by apoptosis. In case of native DOX treated cells the availability of
entire dose of native drug all at once leads to enhanced rate of apop-
tosis (Panyam and Labhasetwar, 2003a,b). However, unconjugated
NPs were retained in the cytoplasm, with a lower dose of released
drug entering the nucleus leading to less apoptosis. Direct targeting
of DOX to nucleus by NLS conjugated NPs resulted in slight increase
of apoptotic MCF-7 cells. Importantly, little apoptosis or cell death
Fig. 9. Dose dependent cytotoxicity of DOX in MCF-7 cells. Different concentration
of native DOX (ꢀ), DOX loaded NPs (᭹) and NLS conjugated NPs (ꢁ) was added to
wells with NPs (without drug) or medium acting as respective controls. The medium
was changed at 2 days and then on every alternate day with no further addition of
drug. The extent of growth inhibition was measured on 5 days using MTT assay. The
percentage of survival was determined by standardizing the absorbance of controls
to 100% (n = 6). Data as mean S.E.M.
Table 2
Cell cycle analysis by flow cytometry.
Treatment
Sub G1a
G1b
Sc
G2/Md
Control cells
Void NP
Native DOX
DOX–NP
1.7 0.9
1.9 0.7
8.3 1.0
7.3 0.7
9.4 1.3
67.8 1.2
65.5 1.6
37.3 1.1
27.4 1.8
14.7 1.6
18.7 0.6
20 0.9
13.7 0.9
12.7 1.2
15.7 1.5
11.8 2.1
12.6 1.1
40.7 1.3
52.6 1.1
60.2 0.9
Fig. 10. Intracellular distribution of doxorubicin in MCF-7 cells treated with native
drug (DOX), unconjugated NPs (DOX–NP) and NLS conjugated NPs (DOX–NP–NLS)
with different time of incubation (2 and 6 h). In all cases, cells were treated with a
particular drug concentration (5 ␮M). (a) Accumulation of doxorubicin in nucleus
after 2 h (white column) and 6 h (black column) treatment. Data as mean S.E.M.
(n = 6). *p < 0.05, 6 h vs. 2 h. (b) Accumulation of doxorubicin in cytoplasm after 2 h
(white column) and 6 h (black column) treatment. Data as mean S.E.M. (n = 6).
**p < 0.005, 6 h vs. 2 h for native DOX and *p < 0.05, 6 h vs. 2 h for DOX–NP and
DOX–NP–NLS.
DOX–NP–NLS
a
Number of cells undergoing apoptosis (sub G1) estimated by flow cytometry
Number of cells present in G1 phase of cell cycle estimated by flow cytometry.
Number of cells present in S phase estimated by flow cytometry.
b
c
d
Number of cells present in G2/M phase estimated by flow cytometry

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R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
was observed in controls that had MCF-7 cells grown in media alone
(1.7 0.9%) and with void treated cells (1.9 0.7%). DOX is an ener-
getic chemotherapeutic agent used in medical oncology. It triggers
apoptosis through numerous mechanisms. It induces DNA damage
by inhibiting the action of topoisomerase II and also intercalates
between the DNA base pairs leading to DNA breakage. In breast
cancer, dysregulated apoptotic pathways include down-regulated
death receptor pathway function, p53 mutations and abnormal Bcl-
2 pathway function (Li et al., 2007). DOX may help in triggering
the biological regulators like p53, Bcl-2 family proteins, caspases
and DNA fragmentation factor resulting in cell death by apopto-
sis (Wang et al., 2004). Therefore, activation of specific apoptotic
mechanisms could be an effective method in treating breast cancer.
Kocbek, P., Obermajer, N., Cegnar, M., Kos, J., Kristl, J., 2007. Targeting cancer cells
using plga nanoparticles surface modified with monoclonal antibody. J. Control.
Release 120, 18–26.
Kubota, T., Furukawa, T., Tanino, H., Suto, A., Otan, Y., Watanabe, M., Ikeda, T.,
Kitajima, M., 2001. Resistant mechanisms of anthracyclines—pirarubicin might
partly break through the p-glycoprotein-mediated drug-resistance of human
breast cancer tissues. Breast Cancer 8, 333–338.
Lee, S.J., Jeong, J.R., Shin, S.C., Huh, Y.M., Song, H.T., Suh, J.S., Chang, Y.H., Jeon,
B.S., Kim, J.D., 2005. Intracellular translocation of superparamagnetic iron oxide
nanoparticles encapsulated with peptide-conjugated poly (D,
glycolide). J. Appl. Phys. 97, 10Q913-1-3.
L lactide-co-
Li, S., Zhou, Y., Wang, R., Zhang, H., Dong, Y., Ip, C., 2007. Selenium sensitizes
mcf-7 breast cancer cells to doxorubicin-induced apoptosis through modula-
tion of phospho-akt and its downstream substrates. Mol. Cancer Ther. 6, 1031–
1038.
Liu, T.J., Yeh, Y.C., Ting, C.T., Lee, W.L., Wang, L.C., Lee, H.W., Wang, K.Y., Lai, H.C.,
Lai, H.C., 2008. Ginkgo biloba extract 761 reduces doxorubicin-induced apop-
totic damage in rat hearts and neonatal cardiomyocytes. Cardiovasc. Res. 80,
227–235.
4. Conclusion
Liu, Z.S., Tang, S.L., Ai, Z.L., 2003. Effects of hydroxyapatite nanoparticles on prolifer-
ation and apoptosis of human hepatoma bel-7402 cells. World J. Gastroenterol.
9, 1968–1971.
Targeted nuclear delivery of therapeutic agents could improve
the treatment of different cancers. Accessing the nucleus inside
an intact cell is a difficult task because of presence of different
membrane barriers. In this study we have prepared NLS conjugated
drug-loaded NPs to target the anticancer drug doxorubicin to the
nucleus. This study emphasizes that the concentration of DOX in
the nuclei could be increased via transport by NLS conjugated NPs
leading to increased cytotoxicity of these formulation due to accu-
mulation of drug in its intracellular action site i.e. nucleus more
efficiently. Thus, NLS conjugated DOX loaded NPs showed high
antiproliferative activity than native DOX and unconjugated NPs
by delivering the drug directly to its site of action. Thus this formu-
lation can act as a promising candidate for effective therapy of any
antitumor drugs with nucleus as its site of action.
Lochmann, D., Jauk, E., Zimmer, A., 2004. Drug delivery of oligonucleotides by pep-
tides. Eur. J. Pharm. Biopharm. 58, 237–251.
Mi, Y., Lou, L., 2007. Zd6474 reverses multidrug resistance by directly inhibiting the
function of p-glycoprotein. Br. J. Cancer 97, 934–940.
Mundargi, R.C., Srirangarajan, S., Agnihotri, S.A., Patil, S.A., Ravindra, S., Setty,
S.B., Aminabhavi, T.M., 2007. Development and evaluation of novel biodegrad-
able microspheres based on poly(d,l-lactide-co-glycolide) and poly(epsilon-
caprolactone) for controlled delivery of doxycycline in the treatment of human
periodontal pocket: In vitro and in vivo studies. J. Control. Release 119, 59–
68.
Nam, Y.S., Park, J.Y., Han, S.H., Chang, I.S., 2002. Intracellular drug delivery using
poly(d,l-lactide-co-glycolide) nanoparticles derivatized with a peptide from a
transcriptional activator protein of hiv-1. Biotechnol. Lett. 24, 2093–2098.
Noh, W.C., Mondesire, W.H., Peng, J., Jian, W., Zhang, H., Dong, J., Mills, G.B., Hung,
M.C., Meric-Bernstam, F., 2004. Determinants of rapamycin sensitivity in breast
cancer cells. Clin Cancer Res. 10, 1013–1023.
Paine, P.L., Moore, L.C., Horowitz, S.B., 1975. Nuclear envelope permeability. Nature
254, 109–114.
Pante, N., Kann, M., 2002. Nuclear pore complex is able to transport macromolecules
with diameters of about 39 nm. Mol. Biol. Cell 13, 425–434.
Acknowledgements
Panyam, J., Labhasetwar, V., 2003a. Biodegradable nanoparticles for drug and gene
delivery to cells and tissue. Adv. Drug Deliv. Rev. 55, 329–347.
Panyam, J., Labhasetwar, V., 2003b. Sustained cytoplasmic delivery of drugs with
intracellular receptors using biodegradable nanoparticles. Mol. Pharm. 1, 77–84.
Panyam, J., Zhou, W.Z., Prabha, S., Sahoo, S.K., Labhasetwar, V., 2002. Rapid endo-
lysosomal escape of poly(d,l-lactide-co-glycolide) nanoparticles: implications
for drug and gene delivery. FASEB J. 16, 1217–1226.
SKS would like to thank the Department of Biotech-
nology, Government of India for providing the grants nos.
BT/04(SBIRI)/48/2006-PID and BT/PR7968/MED/14/1206/2006.
We thank Dr. S. Devdas, Scientist, Institute of Life Sciences for his
help in Flow Cytometry Experiments.
Parveen, S., Sahoo, S.K., 2008. Polymeric nanoparticles for cancer therapy. J. Drug
Target. 16, 108–123.
Pouton, C.W., Wagstaff, K.M., Roth, D.M., Moseley, G.W., Jans, D.A., 2007. Targeted
delivery to the nucleus. Adv. Drug Deliv. Rev. 59, 698–717.
References
Prabha, S., Zhou, W.Z., Panyam, J., Labhasetwar, V., 2002. Size-dependency of
nanoparticle-mediated gene transfection: Studies with fractionated nanopar-
ticles. Int. J. Pharm. 244, 105–115.
Qaddoumi, M.G., Gukasyan, H.J., Davda, J., Labhasetwar, V., Kim, K.J., Lee, V.H.L., 2003.
Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival
epithelial cells: role in plga nanoparticle endocytosis. Mol. Vision 9, 559–568.
Sahoo, S.K., Labhasetwar, V., 2003. Nanotech approaches to drug delivery and imag-
ing. Drug Discov. Today 8, 1112–1120.
Acharya, S., Dilnawaz, F., Sahoo, S.K., 2009. Targeted epidermal growth factor
receptor nanoparticle bioconjugates for breast cancer therapy. Biomaterials 30,
5737–5750.
Adcock, I.M., Ito, K., 2000. Molecular mechanisms of corticosteroid actions. Monaldi
Arch Chest. Dis. 55, 256–266.
Bisht, S., Maitra, A., 2009. Dextran-doxorubicin/chitosan nanoparticles for solid
tumor therapy. Adv. Rev. 1, 415–425.
Brana, M.F., Cacho, M., Gradillas, A., de Pascual-Teresa, B., Ramos, A., 2001. Interca-
lators as anticancer drugs. Curr. Pharm. Des. 7, 1745–1780.
Sahoo, S.K., Labhasetwar, V., 2005. Enhanced antiproliferative activity of
transferrin-conjugated paclitaxel-loaded nanoparticles is mediated via sus-
tained intracellular drug retention. Mol. Pharm. 2, 373–383.
Sahoo, S.K., Ma, W., Labhasetwar, V., 2004. Efficacy of transferrin-conjugated
paclitaxel-loaded nanoparticles in a murine model of prostate cancer. Int. J.
Cancer 112, 335–340.
Sahoo, S.K., Panyam, J., Prabha, S., Labhasetwar, V., 2002. Residual polyvinyl alco-
hol associated with poly (d,l-lactide-co-glycolide) nanoparticles affects their
physical properties and cellular uptake. J. Control. Release 82, 105–114.
Sahoo, S.K., Parveen, S., Panda, J.J., 2007. The present and future of nanotechnology
in human health care. Nanomedicine 3, 20–31.
Schirmbeck, R., Riedl, P., Zurbriggen, R., Akira, S., Reimann, J., 2003. Antigenic epi-
topes fused to cationic peptide bound to oligonucleotides facilitate toll-like
receptor 9-dependent, but cd4+ t cell help-independent, priming of cd8+ t cells.
J. Immunol. 171, 5198–5207.
Sokolovskaya, A.A., Zobotina, T.N., Blokhin, D.Y., Kadagidze, Z.G., Baryshnikov, A.Y.,
2001. Comparitive analysis of apoptosis induced by various anticancer drugs in
jurkat cells. Exp. Oncol. 23, 46–50.
Tan, M.L., Choong, P.F., Dass, C.R., 2009. Review: doxorubicin delivery systems based
on chitosan for cancer therapy. J. Pharm. Pharmacol. 61, 131–142.
Tewes, F., Munnier, E., Antoon, B., Ngaboni Okassa, L., Cohen-Jonathan, S., Marchais,
H., Douziech-Eyrolles, L., Souce, M., Dubois, P., Chourpa, I., 2007. Comparative
study of doxorubicin-loaded poly(lactide-co-glycolide) nanoparticles prepared
by single and double emulsion methods. Eur. J. Pharm. Biopharm. 66, 488–492.
Cheng, F.Y., Wang, S.P., Su, C.H., Tsai, T.L., Wu, P.C., Shieh, D.B., Chen, J.H., Hsieh,
P.C., Yeh, C.S., 2008. Stabilizer-free poly(lactide-co-glycolide) nanoparticles for
multimodal biomedical probes. Biomaterials 29, 2104–2112.
Das, M., Mohanty, C., Sahoo, S.K., 2009. Ligand-based targeted therapy for cancer
tissue. Expert Opin. Drug Deliv. 6, 285–304.
Di, X., Shiu, R.P., Newsham, I.F., Gewirtz, D.A., 2009. Apoptosis, autophagy, acceler-
ated senescence and reactive oxygen in the response of human breast tumor
cells to adriamycin. Biochem. Pharmacol. 77, 1139–1150.
Futaki, S., Nakase, I., Suzuki, T., Youjun, Z., Sugiura, Y., 2002. Translocation of
branched-chain arginine peptides through cell membranes: flexibility in the
spatial disposition of positive charges in membrane-permeable peptides. Bio-
chemistry 41, 7925–7930.
Harashima, H., Shinohara, Y., Kiwada, H., 2001. Intracellular control of gene traffick-
ing using liposomes as drug carriers. Eur. J. Pharm. Sci. 13, 85–89.
Jain, T.K., Morales, M.A., Sahoo, S.K., Leslie-Pelecky, D.L., Labhasetwar, V., 2005. Iron
oxide nanoparticles for sustained delivery of anticancer agents. Mol. Pharm. 2,
194–205.
Jarver, P., Langel, U., 2004. The use of cell-penetrating peptides as a tool for gene
regulation. Drug Discov. Today 9, 395–402.
Kalaria, D.R., Sharma, G., Beniwal, V., Ravi Kumar, M.N., 2009. Design of biodegrad-
able nanoparticles for oral delivery of doxorubicin: in vivo pharmacokinetics
and toxicity studies in rats. Pharm. Res. 26, 492–501.

R. Misra, S.K. Sahoo / European Journal of Pharmaceutical Sciences 39 (2010) 152–163
163
Tkachenko, A.G., Xie, H., Liu, Y., Coleman, D., Ryan, J., Glomm, W.R., 2004. Cellular tra-
jectories of peptide-modified gold particle complexes: Comparision of nuclear
localization signals and peptide transduction domains. Bioconjug. Chem. 15,
482–490.
Vasir, J.K., Labhasetwar, V., 2007. Biodegradable nanoparticles for cytosolic delivery
of therapeutics. Adv. Drug Deliv. Rev. 59, 718–728.
inhibitor (nanoliposomal topotecan) and a topoisomerase ii inhibitor (pegylated
liposomal doxorubicin) in intracranial brain tumor xenografts. Neuro Oncol. 9,
20–28.
You, J., Hu, F.Q., Du, Y.Z., Yuan, H., 2008. Improved cytotoxicity of doxorubicin by
enhancing its nuclear delivery mediated via nanosized micelles. Nanotechnol-
ogy 19, 1–8.
Wang, S., Konorev, E.A., Kotamraju, S., Joseph, J., Kalivendi, S., Kalyanaraman, B.,
2004. Doxorubicin induces apoptosis in normal and tumor cells via distinctly
different mechanisms. Intermediacy of h(2)o(2)- and p53-dependent pathways.
J. Biol. Chem. 279, 25535–25543.
Yamashita, Y., Krauze, M.T., Kawaguchi, T., Noble, C.O., Drummond, D.C., Park, J.W.,
Bankiewicz, K.S., 2007. Convection-enhanced delivery of a topoisomerase I
Zanta, M.A., Belguise-Valladier, P., Behr, J.P., 1999. Gene delivery: a single nuclear
localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc.
Natl. Acad. Sci. U.S.A 96., 91–96.
Zhang, L., Eisenberg, A., 1995. Multiple morphologies of “Crew-cut” aggregates of
polystyrene-b-poly(acrylic acid) block copolymers. Science 268, 1728–1731.