P-glycoprotein targeted nanoscale drug carriers

Article abstract of DOI:10.1166/jnn.2013.6084

Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein (P -gp) efflux pump is one of the mostly studied drug carrying processes that shuttle the drugs out of tumor cells. Thus, P -gp inhibitors have attracted a lot of attention as they can stop cancer drugs from being pumped out of target cells with the consumption of ATP. Using quantitive structure activity relationship (QSAR), we have successfully synthesized a series of novel P -gp inhibitors. The obtained dihydropyrroloquinoxalines series were fully characterized and then tested against bacterial and tumor assays with over-expressed P -gps. All compounds were bioactive especially compound 1c that had enhanced antibacterial activity. Furthermore, these compounds were utilized as targeting vectors to direct drug delivery vehicles such as silica nanoparticles (SNPs) to cancerous Hela cells with over expressed P -gps. Cell uptake studies showed a successful accumulation of these decorated SNPs in tumor cells compared to undecorated SNPs. The results obtained show that dihydropyrroloquinoxalines constitute a promising drug candidate for targeting cancers with MDR. Copyright

Full text of DOI:10.1166/jnn.2013.6084

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Journal of
Nanoscience and Nanotechnology
Vol. 13, 1399–1402, 2013
P-Glycoprotein Targeted Nanoscale Drug Carriers
Wengang Li1, Dina Abu Samra2, Jasmeen Merzaban2, and Niveen M. Khashab1ꢀ∗
1Controlled Release and Delivery Lab, Department of Chemical and Life Sciences and Engineering,
King Abdullah University of Science and Technology, Saudi Arabia
2Stem Cell Trafficking Lab, Department of Chemical and Life Sciences and Engineering,
King Abdullah University of Science and Technology, Saudi Arabia
Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop
cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein
(P -gp) efflux pump is one of the mostly studied drug carrying processes that shuttle the drugs
out of tumor cells. Thus, P -gp inhibitors have attracted a lot of attention as they can stop cancer
drugs from being pumped out of target cells with the consumption of ATP. Using quantitive structure
activity relationship (QSAR), we have successfully synthesized a series of novel P -gp inhibitors.
The obtained dihydropyrroloquinoxalines series were fully characterized and then tested against
bacterial and tumor assays with over-expressed P -gps. All compounds were bioactive especially
compound 1c that had enhanced antibacterial activity. Furthermore, these compounds were utilized
as targeting vectors to direct drug delivery vehicles such as silica nanoparticles (SNPs) to cancerous
Hela cells with over expressed P -gps. Cell uptake studies showed a successful accumulation of
these decorated SNPs in tumor cells compared to undecorated SNPs. The results obtained show
that dihydropyrroloquinoxalines constitute a promising drug candidate for targeting cancers with
MDR.
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Keywords: P -Glycoprotein, Inhibitor, SNPs, Drug Delivery, Targeting.
Copyright: American Scientific Publishers
1. INTRODUCTION
coating was used to increase the dispersability of SNPs
in water, which also helps to enhance the biocompati-
bility. Published reports have focused on functionalizing
the surface of SNPs with external mechanical interlocked
molecules for controlled release.11–14 Herein, we report
the preparation of SNPs decorated with synthesized P-gp
inhibitors and their potential to be used inP-gp targeted
drug delivery.
Multidrug resistance (MDR) is a trend whereby tumor
cells exposed to one cytotoxic agent develop cross-
resistance to a range of structurally and functionally unre-
lated compounds.1–3 P-gp belongs to the ATP-binding
cassette (ABC) family of transporters.4 The expression of
P-gp is higher in tumors than normal cells, resulting in
the potential of resistance to some cytotoxic agents before
chemotherapy is initiated.5
Inhibiting P-gp activity is a way of reversing MDR, and
it has been extensively studied for more than two decades.6
In order to overcome the disadvantages of low binding
affinities of P-gp, as well as toxicity from high doses,
people have developed 3 generations of P-gp inhibitors.7ꢀ8
Using QSAR, we have successfully synthesized a series of
novel P-gp inhibitors. The obtained molecules were fully
characterized and subsequently assayed for their ability to
target cancer cells that over-expressed P-gps.
2. EXPERIMENTAL DETAILS
2.1. Synthesis of P-Gp Inhibitors
Dichlorophenylenediamine and isaꢀ tine were dissolved in
acetic acid, and refluxed at 120 C for 1 hour (Fig. 1).
After cooling down, acetic acid was evaporated under vac-
uum. Flash column chromatography was ran to purify
the product. The obtained products were fully character-
ized by 1H NMR, 13C NMR and high resolution mass
spectroscopy.
Silica nanoparticles (SNPs) are attractive in the field
of drug delivery and controlled release due to their high
surface area, availability, as well as the ease of sur-
face functionalization.9ꢀ10 In the recent decade, polymer
2.2. Preparation of SNPs
SNPs were prepared by the Stöbermethod. Ethanol, watꢀer,
andammonia solution (25%) was first thenstirred at 40 C
∗Author to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2013, Vol. 13, No. 2
1533-4880/2013/13/1399/004
doi:10.1166/jnn.2013.6084
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P-Glycoprotein Targeted Nanoscale Drug Carriers
Li et al.
O
Cl
85~90%
Cl
Cl
NH2
NH2
N
N
Cl
Cl
N
N
Acetic acid
O
Reflux, 90%
N
N
H
N
H
Cl
R1
R1 = H, Me, Ac, Boc, Bn
R1
Novel
Yield (%)
M.P. (ꢀC)
MS
1H NMR shifts (400 MHz, CDCl3)
1a
1b
1c
H
N
N
Y
92
88
90
N/A
N/A
288.01
302.02
330.02
N/A
Me
Ac
N/A
223.5–227.4
381.2–384.0
239.5–241.2
8.45–8.44 (d, 1 H, J = 0.9 Hz), 8.43 (s, 1 H),
8.25 (s, 1 H), 7.66–7.62 (m, 1 H), 7.40–7.37 (m, 2 H),
7.34–7.27 (m, 5 H), 5.67 (s, 2 H)
8.40–8.35 (m, 2 H), 8.37 (s, 2 H),
7.76–7.71 (m, 1 H), 7.54–7.50 (m, 1 H),
1.81 (s, 9 H)
1d
1e
Bn
Y
Y
85
86
378.06
388.06
Boc
8.79–8.77 (d, 1 H, J = 8ꢁ4 Hz),
8.38–8.36 (d, 1 H, J = 5ꢁ1 Hz),
8.36 (s, 1 H), 8.25 (s, 1 H), 7.77–7.72 (m, 1 H),
7.56–7.52 (m, 1 H), 3.20 (s, 3 H)
Fig. 1. Synthetic scheme and characterization of the P-gp inhibitors.
for 2 hr followed by addition of tetraethoxylsilane (TEOS).
3. RESULTS AND DISCUSSION
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ꢀ
The mixture was stirred at 40 C overnight. After dry-
IP: 108.160.87.183 On: Sat, 05 Apr 2014 11:17:05
A series of Pgp inhibitors 1a–e (Fig. 1) were success-
ing, SNPs were dispersed in toluene, heated to 95 ꢀC,
with vigorous stirring. And 3-aminopropyltriethoxylsilane
was added drop wise into the mixture. The amino-
functionalized SNPs (ASNPs) were washed and collected
after stirring overnight. The product was characterized by
high resolution TEM.
Copyright: American Scientific Publishers
fully synthesized and characterized by NMR and HRMS.
The products were then assayed for anti-bacterial activ-
ity. Compound1c demonstrated the greatest anti-bacterial
potential. One major challenge when performing the anti-
tumor assays was that none of these compounds was sol-
uble in 5% DMSO solution. To overcome this obstacle,
we decided to prepare the silica nanoparticles (SNPs) and
directly functionalize them with these compounds for the
cell uptake tests.
The Stöber method was successfully used to prepare
ASNPs with a diameter around 120 nm (Fig. 2). Sub-
sequently we used Ullmann coupling to crosslink the
inhibitors directly to ASNPs. As illustrated in Figure 3,
the inhibitor (1c) has an excitation wavelength at around
2.3. Functionalization of SNPs
Ullmanncoupling reaction was usedfor the functional-
ization of silica nanoparticles. ASNPs were first dis-
persed in dimethyl sulfoxide (DMSO) followed by
the addition of functionalized Pgp inhibitors, N,N-
dimethylethylenediamine, K2CO3, and CuI. The mixture
was heated at 80 ꢀC for 20 hr. The product was then cooled
down to room temperature and washed 5 times with N,N-
dimethylformide (DMF). The particles were dried at 80 ꢀC
under vacuum.
2.4. Cellular Test
HeLa cells were chosen as a model cell line for these
targeting assays using the inhibitor decorated SNPs. HeLa
cells express higher P-gps population than normal cells.15
Confocal microscopic images were taken after incubating
HeLa cells for 24 with either the decorated SNPs or ASNP.
ꢂ-actin (cytoplasmic structures) and DAPI (nuclear stain)
were used to stain the cells.
Fig. 2. TEM of the silica nanoparticles synthesized.
J. Nanosci. Nanotechnol. 13, 1399–1402, 2013
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Li et al.
P-Glycoprotein Targeted Nanoscale Drug Carriers
NH2
Cl
N
N
Cl
Cl
N
N
DMEDA, K2CO3
N
R
N
R
CuI, DMSO, 80ºC, 20hr
R = H, Me, Bn, Ac, Boc
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Fig. 3.
Ullmann coupling of inhibitors IoPn:to1t0he8.s1ur6fa0c.e8o7f.1A8S3NPOs n(a:bSovae)t;, U0V5-vAispsrp2ec0tr1um4 1of1S:1N7P:s0, 5P-gp inhibitors and SNPs decorated with
inhibitors in DMF (below).
Copyright: American Scientific Publishers
363 nm and 400 nm, while for SNPs, the peak lies below
300 nm. When they were re-dispersed in DMF, there was
an obvious UV-vis absorption peak at 363 nm, which is
also the character peak for the inhibitor, indicating that
the coupling reaction was successful. However, for 1d
(R1 = Bn) and 1e (R1 = Boc), there was no peak at
363 nm. It is possible that steric hinderance and hydropho-
bic character of the side groups push the molecules away
from the hydrophilic ASNPs and thus dramatically slow-
ing down or even stopping the reaction.
We used the three decorated SNPs (1a–c) and ANPS
as a control, in cellular assays. Confocal studies using
an excitation wavelength of 405 nm was used for the
analysis of the SNPs with HeLa cells. It is apparent
from Figure 4 thatthe emission of the inhibitor deco-
rated SNPs almost overlapped with the HeLa cell struc-
tures outlined with beta-actin and DAPI staining, implying
that the P-gp-inhibitor decorated SNPs were targeted
to the P-gp expressing HeLa cells whereas the ASNPs
were not.
Fig. 4. Confocal microscope image of Dapi stained HeLa cells with decorated SNPs (1b) with HeLa: (A) Fluorescence images of the decorated SNPs
at 405 nm; (B) Bright field images of HeLa cells; (C) Overlap of A and B.
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P-Glycoprotein Targeted Nanoscale Drug Carriers
Li et al.
4. CONCLUSIONS
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We have successfully synthesized a series of compounds
with a high potential of inhibiting P-gp efflux pumps. Ull-
mann coupling was used to get three of the compounds
crosslinked to amino group decorated silica nanoparticles.
After performing the in-vitro testing with HeLa cell line,
we found that the decorated SNPs preferentially accumu-
lated in tumor cells compared to undecorated SNPs that
were not taken up by the cells. Thus, targeting P-gp can be
a new method for improving the operation of drug delivery
vehiclesand increasing the retention of drug in diseased
cells versus healthy ones.
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Acknowledgments: We thank King Abdullah Univer-
sity of Science and Technology (KAUST) for the generous
support.
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Received: 12 September 2011. Accepted: 30 November 2011.
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J. Nanosci. Nanotechnol. 13, 1399–1402, 2013