A novel, anisamide-targeted cyclodextrin nanoformulation for siRNA delivery to prostate cancer cells expressing the sigma-1 receptor

Article abstract of DOI:10.1016/j.ijpharm.2015.12.055

Prostate cancer is a leading cause of cancer-related death in men and RNA interference (RNAi) has emerged as a potential therapeutic option. However, the absence of a safe and specific delivery vector remains a major obstacle to the clinical application o

Full text of DOI:10.1016/j.ijpharm.2015.12.055

Accepted Manuscript
Title: A novel, anisamide-targeted cyclodextrin
nanoformulation for siRNA delivery to prostate cancer cells
expressing the sigma-1 receptor
Author: Kathleen A. Fitzgerald Meenakshi Malhotra Matt
Gooding Florence Sallas James C. Evans Raphael Darcy
Caitriona M. O’Driscoll
PII:
S0378-5173(15)30455-5
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.ijpharm.2015.12.055
IJP 15448
To appear in:
International Journal of Pharmaceutics
Received date:
Revised date:
Accepted date:
5-11-2015
18-12-2015
19-12-2015
Please cite this article as: Fitzgerald, Kathleen A., Malhotra, Meenakshi, Gooding,
Matt, Sallas, Florence, Evans, James C., Darcy, Raphael, O’Driscoll, Caitriona M., A
novel, anisamide-targeted cyclodextrin nanoformulation for siRNA delivery to prostate
cancer cells expressing the sigma-1 receptor.International Journal of Pharmaceutics
http://dx.doi.org/10.1016/j.ijpharm.2015.12.055
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Title Page
A novel, anisamide-targeted cyclodextrin nanoformulation for siRNA delivery to prostate
cancer cells expressing the sigma-1 receptor.
Author names and affiliations:
Kathleen A. Fitzgerald#a
Meenakshi Malhotra#a
Matt Goodingb
Florence Sallasb
James C. Evansa
Raphael Darcyb,c
Caitriona M. O’ Driscoll*a
a. Pharmacodelivery Group, School of Pharmacy, University College Cork, Ireland
b. Centre for Synthesis and Chemical Biology, University College Dublin, Ireland
c. School of Chemistry, University College Dublin, Ireland
*Corresponding author:
Prof. Caitriona O’ Driscoll, Cavanagh Pharmacy Building, School of Pharmacy, College
Road, University College Cork, Cork, Ireland.
Tel: +353-21-490-3000.
Fax: +353 21 490 1396
Email: caitriona.odriscoll@ucc.ie (Caitriona M. O’ Driscoll)
# These authors made an equal contribution to this work

Graphical abstract:

Abstract
Prostate cancer is a leading cause of cancer-related death in men and RNA interference
(RNAi) has emerged as a potential therapeutic option. However, the absence of a safe and
specific delivery vector remains a major obstacle to the clinical application of RNAi.
Cyclodextrin derivatives are known to be efficient delivery systems with low toxicity in a
variety of cell types. In this study, a cationic cyclodextrin derivative was synthesized to
complex siRNA. The nanoparticle was then further modified by exploiting the ability of the
β-cyclodextrin cavity to form an inclusion complex with the hydrophobic molecule
adamantane. PEGylated adamantane derivatives were synthesized with and without the
anisamide-targeting ligand on the terminal end of the PEG chain. Anisamide is known to bind
specifically to the sigma receptor which is overexpressed on the surface of prostate cancer
cells. The resulting nanocomplexes were slightly cationic and less than 300 nm in size. They
successfully protected siRNA from serum-induced nuclease degradation and were non-toxic
to prostate cancer cells. In addition, the targeted nanoparticles mediated high levels of siRNA
cellular uptake and corresponding PLK1 gene knockdown in prostate cancer cells in vitro. To
our knowledge, this is the first time that the ability of cyclodextrins to form inclusion
complexes with adamantane derivatives has been exploited for the targeted delivery of
siRNA to prostate cancer cells via the sigma receptor.
Keywords
Gene delivery; Prostate cancer; Anisamide-targeted cyclodextrin; Targeted nanoparticle,
Sigma receptor, Inclusion complex formation.

Abbreviations:
AA: anisamide
Ad-PEG500: adamantane-PEG500
Ad-PEG5000: adamantane-PEG5000
Ad-PEG5000-2xAA: adamantane-PEG5000-2xAnisamide
CD: cyclodextin
MTT: 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PEG: polyethylene glycol
siRNA: small interfering RNA

1
Introduction
Cyclodextrins are cyclic oligosaccharides of 6, 7 or 8 glucose units which are known as α, β
or γ-cyclodextrins respectively (1, 2). They exist in a 3D cone-like conformation with a
hydrophilic outer surface and a hydrophobic inner cavity (3). The differing reactivities of the
glucose hydroxyl groups at positions C-2, C-3 and C-6 and the symmetrical structure of
cyclodextrin allows for high yielding regiospecific chemical modification (4).
Of the different classes of cyclodextrin available, β-cyclodextrin is the most widely studied
(5). Each β-cyclodextrin molecule contains 21 hydroxyl groups that can be easily substituted
to make an unlimited number of cyclodextrin derivatives (5). β-cyclodextrins modified with
amphiphilic, cationic entities have been investigated for delivery of small interfering RNA
(siRNA) and have successfully mediated gene knockdown both in vitro (6, 7) and in vivo (8).
However, cationic polyplexes are prone to aggregation in physiological media and further
modification is necessary prior their application in systemic delivery (9, 10).
In terms of developing an optimised nanoparticle for gene delivery, Kastarelos and Miller
have proposed an ‘ABCD’ formula outlining the key components necessary for in vivo gene
delivery. They suggest that nanoparticles for such applications should consist of (A) a gene
therapy molecule which is condensed by (B) a nanoparticle-forming material. (11). To avoid
in vivo toxicity associated with cationic complexes, further modification with a biological
stabilising group (C) is required (11). The most widely investigated stabilising group in the
formulation of nanoparticles is polyethylene glycol (PEG). PEG chains alter the surface
properties of nanoparticles thus reducing the tendency toward aggregation and in addition
increasing circulation in the blood (12). An ideal delivery system should be active only
against the cells of interest, thus avoiding any unwanted activity in healthy cells (13). It is
widely reported that the incorporation of PEG into a nanoparticle can reduce cellular uptake

(14). In order to ensure targeted delivery and enhance uptake into disease cells a distal
biological-targeting or cell penetrating ligand (D) must also be attached to the nanoparticle
(11).
A number of formulation strategies to incorporate a PEG shielding layer following siRNA
complexation with a cationic CD have previously been reported. Such methods include a ‘co-
formulation’ approach using two different amphiphilic cyclodextrins (one cationic and one
with a PEG chain) (10, 14, 15) and a ‘post-insertion’ method using an amphiphilic cationic
cyclodextrin and a PEGylated DSPE chain (16). In addition, a prostate cancer cell-targeted
cyclodextrin derivative was successfully prepared with a cationic group on the primary face
(to complex siRNA) and a biologically targeted-PEG500 chain conjugated directly onto the
secondary face (17).
An alternative strategy for developing PEGylated cyclodextrins exploits the non-polar inner
cavity of β-cyclodextrin which permits the formation of ‘inclusion complexes’ with
hydrophobic guest molecules. This strategy has been exploited in gene delivery where a β-
cyclodextrin was attached to a cationic polymer (to bind siRNA) and a targeting ligand
(Adamantane-PEG-Transferrin) was then incorporated via formation of an inclusion complex
into the CD cavity (18). Adamantane is a highly hydrophobic molecule which demonstrates
strong binding to β-cyclodextrin with an association constant in the order 104-105 M-1 (19).
This strategy led to the first targeted delivery of siRNA in humans for the treatment of solid
tumors in 2008 (18). The sole function of adamantane in these formulations is to facilitate
inclusion complex formation as a means of functionalising cyclodextrins with different
moieties, which are chemically conjugated to adamantane.
Prostate cancer is a leading cause of cancer-related death in men (20) and treatment using
siRNA presents great potential (17, 21, 22). Our group previously synthesized an anisamide-

targeted cyclodextrin vector which facilitated receptor-mediated uptake in prostate cancer
cells which overexpress the sigma receptor (17). In the current study, a cationic CD
(Dilysine-CD, Figure 1 (a)) was synthesized and used to complex siRNA. This complex was
modified by exploiting inclusion complex formation with three different PEGylated
adamantane derivatives: Adamantane-PEG500 (Ad-PEG500, Figure 1 (b)) and Adamantane-
PEG5000 (Ad-PEG5000, Figure 1 (c)) to mask the positive charge, and an Adamantane-
PEG5000-anisamide derivative (Ad-PEG5000-2xAA, Figure 1 (d)) to ensure cell-specific
uptake. A schematic outlining the nanoparticle structure is given in Figure 2. The
physicochemical properties of the nanoparticles were assessed and the ability of the CD.Ad-
PEG5000-2xAA nanoformulation to specifically target the sigma receptor and elicit
therapeutic gene silencing was quantified.

2
Materials and Methods
2.1 Reagents
All reagents were purchased from Sigma unless otherwise stated. Penicillin-Streptomycin
(Gibco) was purchased from Biosciences (Dublin, Ireland). PLK1 siRNA (sense strand
sequence
5’-AGAmUCACCCmUCCUmUAAAmUAUU-3’)
was
procured
from
Genepharma (Shanghai, China), where “m” represents 2’ O methylated nucleotide. Negative
control non-silencing siRNA (sense strand sequence 5’-UUC UCC GAA CGU GUC ACG
UdT dT-3’) was purchased from Sigma (Wicklow, Ireland). A similar sequence modified
with 6FAM on the 3’ end of the sense strand was used for fluorescent experiments (Sigma,
Wicklow, Ireland).
Reactions were monitored by TLC on precoated aluminium plates of silica gel (Merck 60F254
0.25 mm). Carbohydrates were visualised by dipping in 5 % sulfuric acid by volume in
ethanol and charring. Amines were visualised by dipping in ninhydrin solution (1 g in 30 ml
of ethanol) and charring. Other compounds were visualised under a UV lamp (254 nm).
Column chromatography was performed on silica gel (Davosil LC60A, 40-63 µm).
1H NMR and 13C NMR were recorded at 25 °C with Varian spectrometers at 400 and 100, or
500 and 125 MHz. Electrospray ionisation mass spectra (ESI-MS) were recorded on a
Quattro Micro LC/MS/MS system (Waters Corporation, USA).
MALDI spectra were
recorded on a MALDI Q-Tof Premier mass spectrometer (Waters Corporation, USA) using a
matrix of either trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile
(DCTB) or α-cyano-4-hydroxycinnamic acid (CHCA).

2.2 Synthesis of Dilysine Cyclodextrin (D)
2.2.1 Synthesis of N,N’,N’’-tri-Boc-di-l-lysine (A)
Dilysine dihydrochloride (100 mg, 0.28 mmol) and diisopropylethylamine (372 mg, 500 µL,
2.88 mmol) were dissolved in dimethylformamide (10 ml) and water (5 ml) and stirred for 15
min at 0 °C. Di-tert-butyl dicarbonate (314 mg, 1.44 mmol) was added and the mixture
warmed to room temperature and stirred for 17 h, concentrated, and purified by column
chromatography over silica gel (DCM/MeOH: 9/1) to yield the product A (115 mg, 72%).
1H NMR (400 MHz, CDCl3) δ 7.05 (s, 1H), 5.47 (s, 2H), 4.85 (s, 2H), 4.52 (s, 2H),
4.12 (s, 2H), 3.06 (t, J = 11.9 Hz, 4H), 1.99 – 1.54 (m, 5H), 1.38 (s, 31H).
13C NMR (101 MHz, CDCl3) δ 172.37 (s), 162.75 (s), 156.23 (s), 79.97 – 79.77 (m),
79.10 (s), 54.26 (s), 40.18 (s), 36.55 (s), 32.04 (s), 31.49 (s), 29.52 (s), 28.34 (d, J = 9.9 Hz),
22.45 (s).
MS (ESI+-TOF) m/z: [M + Na]+ calcd for C27H50N4O9Na 597.3475; found 597.3477.
2.2.2 Synthesis of N,N’,N’’-tri-Boc-di-l-lysine hydroxysuccinimide ester (B)
A (102 mg, 0.18 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (38 mg, 0.2
mmol) were dissolved in anhydrous dichloromethane (10 ml) and stirred for 30 min at room
temperature. N-hydroxysuccinimide (23 mg, 0.2 mmol) was added and the mixture was
stirred for 17 h at room temperature under a nitrogen atmosphere. The reaction was diluted
with DCM (25 ml), washed with water (3 x 20 ml), dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by column chromatography over silica gel
(DCM/MeOH: 9.5/0.5) to yield the product B (52 mg, 43%).

1H NMR (400 MHz, CDCl3) δ 6.98 (s, 1H), 4.98 – 4.48 (m, 2H), 4.08 (s, 1H), 3.20 –
2.95 (m, 2H), 2.86 – 2.74 (m, 2H), 2.10 – 1.10 (m, 21H).
13C NMR (101 MHz, CDCl3) δ 172.19, 168.59, 167.71, 156.12, 80.02, 79.05, 50.18,
39.85, 31.58, 29.53, 29.12, 28.41, 28.27, 25.55, 22.41.
2.2.3 Synthesis of Heptakis-[2-aminoethylthio]-β-cyclodextrin trifluoroacetate
(C)
2-(Boc-amino) ethanethiol (605 µL, 3.58 mmol) was dissolved in anhydrous DMF (20 ml)
and sodium hydride (82 mg, 3.41 mmol) was added. The mixture was heated to 60 °C and
stirred under nitrogen for 2 h. The mixture was then cooled to room temperature and
heptakis-6-bromo-6-deoxy-β-cyclodextrin (250 mg, 0.16 mmol) was added in anhydrous
DMF (5 ml). The mixture was heated to 60 °C and stirred under nitrogen overnight. The
solvent was removed in vacuo and the residue washed several times with cyclohexane and
water. The product was dissolved in DCM/TFA (1:1, 10 ml) and stirred for 6 h under
nitrogen. The mixture was concentrated and the product precipitated from diethyl ether. The
product C was purified on Sephadex LH20 (MeOH: 100%) (159 mg, 42%).
1H NMR (400 MHz, DMSO-d6) δ 7.97 (s, 3H), 5.91 (m, 2H), 4.87 (s, 1H), 3.81-3.22
(m, 10H), 3.07-2.89 (m, 2H), 2.89-2.65 (m, 2H).
13C NMR (101 MHz, DMSO-d6) δ 102.4, 84.8, 72.9, 72.5, 72.2, 65.2, 33.1, 30.3.
MS (MALDI-TOF) m/z: [M + Na]+ 1575.6176
2.2.4 Synthesis of Heptakis-6-[dilysine-ethylthio]-β-cyclodextrin (D)
C (155 mg, 0.023 mmol) and DIPEA (112 µL, 0.64 mmol) were dissolved in anhydrous
DMF (5 ml) and stirred for 15 min. N,N’,N’’-tri-Boc-di-L-lysine hydroxysuccinimide ester
(151 mg, 0.23 mmol) was added and the solution was stirred at room temperature overnight.

The mixture was concentrated in vacuo and purified on Sephadex LH20 (MeOH: 100%).
The compound was dissolved in TFA/DCM (1/1, 10 ml) and stirred for 6 h. The product D
was precipitated and washed several times with diethyl ether (141 mg, 100%).
1H NMR (400 MHz, CD3OD) δ 5.00 (m, overlaps with D2O, H1), 4.37-4.25 (m, 1H,
Lysα), 4.07-3.73 (m, 2H, H3, H5), 3.70-3.31 (m, 4H, H2, H4, CH2NH), 3.02-2.69 (m, 4H,
Lysε, SCH2), 2.45-2.33 (m, 1H, NH), 2.02-1.61 (m, 4H, Lysβ, Lysδ), 1.59-1.37 (m, 2H,
Lysγ).
13C NMR (101 MHz, CD3OD) δ 172.2, 168.7, 159.9 (q), 102.24 (m), 78.1, 77.8, 77.5,
72.8, 53.6, 52.5, 39.3-38.5 (m), 33.0, 32.4, 31.3, 30.5, 28.1-26.2 (m), 22.5.
MS (MALDI-TOF) m/z: [M + Na]+ 3365.8286.
2.3 Synthesis of Adamantane Derivatives
2.3.1 Ad-PEG500 and Ad-PEG5000
1 g of PEG monomethyl ester (PEG5000: 0.2 mmol; PEG500: 2.1 mmol) was dissolved in
anhydrous DCM (PEG5000: 25 ml; PEG500: 150 ml) and cooled to 0 °C. Adamantane
carbonyl chloride (5 eq), DIPEA (10 eq) and DMAP (1% cat.) were added and the mixture
warmed to room temperature and stirred overnight under nitrogen. The mixture was
transferred to a separating funnel and washed with 5 % citric acid (50 ml), saturated NaHCO3
(50 ml), and brine (50 ml). The organic layer was dried over MgSO4, filtered and
concentrated in vacuo. Ad-PEG500 was purified by column chromatography over silica gel
(DCM/MeOH: 9/1) to yield the product E as a yellow oil (1.1 g, 83%). Ad-PEG5000 was
dissolved in a minimum amount of DCM and precipitated from diethyl ether to yield the
product F as a white solid (805 mg, 78 %).

Ad-PEG500 (E):
1H NMR (400 MHz, CDCl3) δ 4.24 – 4.15 (m, 2H), 3.75 – 3.59 (m, 30H), 3.59 – 3.51
(m, 2H), 3.38 (s, 2H), 2.09 – 1.95 (m, 5H), 1.95 – 1.80 (m, 8H), 1.80 – 1.63 (m, 9H).
13C NMR (101 MHz, CDCl3) δ 177.55, 71.84, 70.41, 69.14, 63.22, 58.92, 40.60,
39.37, 38.71, 36.85, 36.41, 28.42, 27.85.
MS (ESI-TOF) m/z: [M + Na]+ 657.63.
Ad-PEG5000 (F):
1H-NMR (400 MHz, CDCl3) δ 4.22 – 4.18 (m, 2H), 3.84 – 3.79 (m, 2H), 3.64 (s,
271H), 3.57 – 3.53 (m, 2H), 3.49 – 3.44 (m, 2H), 3.38 (s, 2H), 2.05 – 1.97 (m, 6H), 1.92 –
1.87 (m, 4H), 1.77 – 1.65 (m, 6H).
13C-NMR (101 MHz, CDCl3) δ 71.9, 70.6, 63.2, 38.8, 36.5, 27.9.
MALDI-MS: Mavg = 5193.0122 g mol-1
2.3.2 Ad-PEG5000-2xAA (L)
2.3.2.1 Synthesis of Fmoc- Lys(Boc)-anisamide (G)
To a solution of Fmoc-Lys(Boc)-COOH (1.5 eq, 1 g) in dry DCM (50ml) was added
EDC.HCl (1.6 eq, 436 mg), TEA (1.6eq, 317 µl) and anisidine (175 mg). The reaction
mixture was stirred at room temperature overnight then diluted with DCM and extracted. The
organic phase was washed with 5 % citric acidaq and brine then dried (MgSO4) and
evaporated. The residue was purified on silica gel column chromatography (From DCM 100
% to DCM 99:MeOH 01) to afford compound G (495 mg, 61 %).

1H NMR (500 MHz, CDCl3) δ (ppm): 8.22 (s, 1H, NH), 7.76 (d, J = 7.5 Hz, 2H, CH,
Ph), 7.59– 7.27 (m, CH, Fmoc), 6.84 (d, J = 8.9 Hz, 2H, CH, Ph), 5.64 (s, 1H, NH), 4.65 (s,
1H, NH), 4.44 (m, 2H, CH2, Fmoc), 4.29 (m, 1H, CH, Fmoc), 4.21 (t, J = 6.9 Hz, 1H, CH,
Lys), 3.79 (s, 3H, OMe), 3.16 – 3.08 (m, 2H, CH2, Lys), 1.97 and 1.71 (s, 1H each, CH2,
Lys), 1.54 (m, 2H, CH2, Lys), 1.44 (s, 10H, t-Bu; CH2 Lys).
2.3.2.2 Synthesis of Fmoc-Lys-Di-anisamide (I)
A solution of G (380mg) in dioxane (20ml) was cooled to 0 ºC. A solution of HCl 4N in
dioxane (10 ml) was added and the reaction mixture was stirred at 0 ºC for 2h then at room
temperature for another 2 h. The solvents were evaporated to dryness and the residue
dissolved in a minimum of MeOH. The free amino intermediate (H) was precipitated by
addition of Et2O. The precipitate was filtered and washed with Et2O and used without further
purification (268 mg, 79 %).To a solution of the crude amine H (265 mg) in dry DCM (100
ml) was added EDC.HCl (1.7 eq, 170 mg) followed by TEA (10 eq, 725 µL) and anisic acid
(1.5 eq, 119 mg). The reaction mixture was stirred at room temperature overnight then diluted
with DCM and extracted. The organic phase was washed with 5 % citric acidaq, water and
brine then dried (MgSO4) and evaporated. The residue was purified on silica gel column
chromatography (DCM 97:MeOH 03) to afford compound I (147mg, 47%).
1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.27 (t, 3H, 3 x NH), 7.85 (d, J = 7.6 Hz,
2H, CH, Ph), 7.77 (d, J = 8.5 Hz, 2H, CH, Ph), 7.71– 7.27 (m, CH, Fmoc), 6.92 (d, J = 8.3
Hz, 2H, CH, Ph), 6.84 (d, J = 8.6 Hz, 2H, CH, Ph), 4.25-4.03 (m, 4H, CH2 Fmoc; CH
Fmoc; CH Lys), 3.76 and 3.68 (2x s, 2x3H, 2xOMe), 3.21 (m, 2H, CH2, Lys), 1.65 (m, 2H,
CH2, Lys), 1.50 (m, 2H, CH2, Lys), 1.34 (m, 2H, CH2 Lys).

13C NMR (125 MHz, DMSO-d6) δ (ppm): 169.81 , 156.55 , 143.69 , 141.29 , 127.74 ,
127.09, 125.01 , 121.82 , 119.97 , 114.12 , 67.18 , 55.47 , 47.15 , 39.64 , 31.82 , 29.53 , 28.43
, 22.50.
2.3.2.3 Fmoc-PEG5000-Lys (Anisamide)2 (K)
To a solution of I (147mg) in DMF (5 ml) was added piperidine (500 µL). The reaction
mixture was stirred at room temperature for 2 h. DMF was evaporated under reduced
pressure. The residue was purified on silica gel column chromatography (DCM 97:MeOH
03:Et3N:0.35) to afford compound J (92 mg, Q.Y.). The crude amine was used without
further purification.
To a solution of Fmoc-PEG5000-OSu (294mg) in dry DCM (10ml) was added amine J (4 eq,
92 mg) and DIPEA (7 eq, 73 µL). The reaction mixture was stirred at room temperature for 3
days then diluted with DCM and extracted. The organic phase was washed with saturated
ammonium chloride and brine then dried (MgSO4) and evaporated. The residue was taken up
in a minimum amount of DCM and precipitated by addition of Et2O. The precipitate was
filtered and washed with Et2O then loaded on a LH-20 gel filtration chromatography (MeOH
100 %) to afford K (224 mg, 71 %).
1H NMR (400 MHz, CDCl3) δ (ppm): 8.67 (t, 3H, 3 x NH), 7.77 – 7.26 (m, CH,
Fmoc; CH, Ph CH, Ph), 6.87 (d, J = 8.8 Hz, 2H, CH, Ph), 6.80 (d, J = 9 Hz, 2H, CH,
Ph), 6.57 (t, NH), 4.50-4.45 (m, 1H, CH Lys), 4.38 (d, J = 6.9 Hz, 2H, CH2 Fmoc,), 4.21-
4.18 (m, 1H, CH Fmoc,), 3.82 (s, 3H, OMe), 3.80-3.36 (m, 196H, PEG; OMe; CH2, Lys),
2.36-1.41 (m, 6H, CH2, Lys; CH2, Lys; CH2 Lys).
13C NMR (100 MHz, CDCl3) δ 175.28, 174.07, 162.02, 156.19, 149.70, 149.42,
143.97, 128.81, 127.60, 126.98, 121.59, 119.90, 113.97, 113.59, 97.40, 70.54, 55.41, 54.41,
53.60, 47.25, 38.77, 32.96, 28.83, 22.51.

2.3.2.4 Ad-PEG5000-Lys (Anisamide)2 (L)
To a solution of K (210mg) in DMF (5ml) was added piperidine (1ml). The reaction mixture
was stirred at room temperature for 2 h. DMF was evaporated under pressure. The residue
was taken up in a min of DCM and precipitated by addition of Et2O. The precipitate was
filtered and washed with Et2O then loaded on a LH-20 gel filtration chromatography (MeOH
100 %) to afford the free amino intermediate which was used without further purification
(137 mg, 68 %). A solution of this crude amine (137 mg) in dry DCM (10 ml) was cooled to
0 ºC. Adamantane carbonyl chloride (5 eq, 27 mg) and DIPEA (10 eq, 47 µL) were added.
The reaction mixture was stirred at room temperature overnight then the solvent was
evaporated under reduced pressure. The residue was loaded on a LH-20 gel filtration
chromatography (MeOH 100 %) to afford compound L (113 mg, 80 %).
1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H, NH), 7.76 (d, J = 8.8 Hz, 2H, CH, Ph),
7.43 (d, J = 9 Hz, 2H, CH, Ph), 6.87 (d, J = 9 Hz, 2H, CH, Ph), 6.80 (d, J = 9 Hz, 2H, CH,
Ph), 4.47 (m, 1H, CH Lys), 3.81-3.40 (m, 176H, PEG; OMe; CH2, Lys), 2.35-1.41 (m,
11H, CH2, Lys; CH2, Lys; CH2 Lys; CH and CH2 ADAM).
13C NMR (100 MHz, CDCl3) δ 174.05, 169.31, 162.01, 156.18, 128.81, 121.58,
113.96, 113.58, 70.53, 55.34, 39.16, 38.78, 36.51, 32.94, 30.19, 28.82, 28.11, 25.47, 22.59.
2.4 Cell Culture
DU145 cells (human prostate cancer cell line, kindly donated by the Conway Research
Institute, University College Dublin, Dublin, Ireland) and PC3 cells (human prostate cancer
cell line, European Collection of Cell Cultures (ECACC), UK) were maintained in RPMI-
1640 medium, supplemented with 10 % fetal bovine serum (FBS), 2 mM L-Glutamine, 50
units/ml penicillin and 50 µg/ml streptomycin. VCaP cells (human prostate cancer cell line,

Sigma, Wicklow, Ireland) were maintained in DMEM and supplemented with 10 % FBS, 50
units/ml penicillin and 50 µg/ml streptomycin. VCaP cells were cultured and seeded in Poly-
D-Lysine (1x) coated tissue culture flasks and wells. All cells were cultured in a humidified,
5 % CO2 atmosphere tissue culture incubator at 37 °C.
2.5 Isothermal Calorimetry
A MicroCalTM PEAQ-ITC microcalorimeter (S/N MAL1119955, Malvern, United Kingdom)
was used to investigate the ability of Adamantane-PEG5000 and Adamantane-PEG5000-
2xAnisamide to form inclusion complexes with Dilysine-CD. The analysis was performed
using the MicroCalTM PEAQ-ITC analysis software. The corresponding samples for each
experiment were loaded into the sample cell and the titrant was loaded into the syringe, using
280 µL and 40 µL respectively. The reference cell was loaded with Milli-Q water. After a 60-
second delay, a typical experiment involved a series of 19 injections of 2 µL of the titrant
solution, plus an initial injection of 0.4 µL and a spacing of typically 120 seconds between
injections. The data point from the initial injection was discarded before the data analysis.
The sample cell was stirred at 750 rpm throughout the experiment and the experimental
temperature was maintained at 25 °C. The feedback/gain mode was set to high. Consecutive
titrations were combined using the freeware software CONCAT. The area under each peak of
the resulting heat profile was integrated, normalized by the concentrations, and plotted
against the molar ratio of ligand to protein using MicroCal PEAQ-ITC Analysis Software.
The resulting binding isotherms were fitted by nonlinear regression using a one set of sites
model. The stoichiometry of the interaction (n), the equilibrium dissociation constant (KD),
and the change in enthalpy (ΔH) were obtained during the fitting of all titration data. Gibbs
free energy change (ΔG) was calculated from the following equation ΔG=-RT lnKa and the
entropy (ΔS) from ΔG = ΔH – TΔS.

2.6 Preparation of Complexes with siRNA
Dilysine-CD and adamantane derivatives were soluble in aqueous solutions and were
dissolved at a concentration of 1 µg/µl in ddH2O. These aqueous solutions were stored at -20
°C until use and were thawed and sonicated at room temperature for 30 min, before forming
nanocomplexes with siRNA. The Dilysine-CD.siRNA nanocomplexes were formed at a fixed
mass ratio (mr) of 10. Specifically, 30 µg of Dilysine-CD and 3 µg of siRNA were separately
dissolved in ddH2O to give a final volume of 50 µl each. The siRNA (50 µl) was then added
to the Dilysine-CD solution (50 µl) and vortexed at a mild speed for 10 seconds, to give a
final volume of 100 µl and was left to stabilize at room temperature 30 min. Required
amounts of adamantane-PEG derivatives were prepared (to form untargeted and targeted
nanoparticles) in separate Eppendorf vials to which Dilysine-CD.siRNA complexes were
added. Specifically, for untargeted nanoparticles, Ad-PEG-500 and Ad-PEG-5000 were used
in the molar ratio of 0.5:0.5 to 1 mole Dilysine-CD. For targeted nanoparticles Ad-PEG500,
Ad-PEG5000 and Ad-PEG5000-2xAA were used in the molar ratio of 0.5:0.25:0.25 to 1
mole Dilysine-CD. The Dilysine-CD.siRNA complexes containing adamantane derivatives
were then kept at 900 rpm at 25 °C for 30 mins in a thermomixer after which the
nanoparticles were brought to 1 ml of total volume with ddH2O. The nanoparticles were kept
at room temperature for 30 min to stabilize, before performing any further analysis.
2.7 Size, Charge and Aggregation
Particle size and zeta potential were measured in RNase-free water using a Malvern Nano-ZS
(Malvern Instrument, UK) with negative control non-silencing siRNA. Complexes were
prepared as detailed above. For size and charge measurement, each sample contained 3 µg
siRNA (at Dilysine-CD.siRNA mass ratio 10) in 1 ml RNase-free water. Nanoparticle
stability in high salt solution was assessed by preparing complexes to a final volume of 1 ml
in OptiMEM Reduced Serum Media (Gibco) (50 %) as previously employed (14).

Complexes were incubated at 37 ºC and particle size was measured at 1 h, 4 h and 24 h. Each
sample was measured in triplicate.
2.8 Transmission Electron Microscopy
The morphology of the CD.siRNA complexes was visualised using transmission electron
microscopy (TEM). Complexes were prepared as detailed above using negative control non-
silencing siRNA. 15 µl of the CD.siRNA complex was mixed with 15 µl 2 % (w/w) uranyl
acetate and applied to 400 mesh carbon-film copper grids (Agar Scientific) for a couple of
minutes. Images were taken using a JEOL 2000 FXII transmission electron microscope.
2.9 Binding and Release Gel
The ability of the cyclodextrins to bind and release siRNA was assessed using gel
electrophoresis. For the binding study, complexes were prepared as detailed above and mixed
with 1x loading buffer. Samples were loaded onto a 1 % agarose gel containing SafeViewTM
(NBS Biologicals Ltd, England) (6 µl/100 ml) (14). For the release study, complexes were
incubated with 5 µl Heparin (1,000 U/ml) for 60 minutes and loaded onto the gel. Each well
contained 0.3 µg siRNA. Electrophoresis was carried out at 90 V for 45 minutes using a Tris-
borate-EDTA buffer. Naked siRNA was used as a control.
2.10 Serum Stability
Serum stability was evaluated by incubating the formulations with 50 % FBS for various time
periods (30 min, 1 h, 2 h, 4 h, 8 h and 24 h) and stored at -20 °C until gel electrophoresis was
performed. Each time point contained 0.35 µg of siRNA for all nanoparticle formulations. To
inactivate the serum, samples were incubated in a bath incubator at 80 °C for 5 min, followed
by addition of Heparin (10 µl at 1000 U/ml) and kept at room temperature for an hour. All the
samples were mixed with a 1x Blue Juice Gel loading buffer (Invitrogen, U.S.A) and were
added to the wells of a 1.5 % (w/v) agarose (Sigma, MO, USA) solution, prepared with 1x

Tris borate EDTA (TBE) buffer. 6 µl of SafeView™ (NBS Biologicals Ltd., England) was
added to the 100 ml of 1 % (w/v) agarose-TBE solution. The electrophoresis was carried out
at 90 V for 30 min in TBE buffer. The bands were visualised by UV using DNR Bioimaging
Systems MiniBis Pro and Gel capture US B2 software.
2.11 Cellular Uptake
DU145 cells (1x104 cells/well), VCaP cells (5x104 cells/well) and PC3 cells (1x104
cells/well) were seeded in complete growth media 24 hours prior to transfection in 96-well
plates. CD.siRNA complexes were prepared as detailed previously using FAM-labelled
siRNA (Sigma). After 24 hours, 50 µL of the CD.siRNA complex (prepared in ddH2O) was
added to the cells with 150 µl serum-free and antibiotic-free transfection media. The cells
were then incubated at 37 °C in 5 % CO2 for 24 h, after which the quantitative measurement
of transfection was performed by measuring fluorescence intensity using a Perkin Elmer
Victor2 1420 fluorescent plate reader (excitation 485 nm, emission 535 nm). Samples were
normalised to untreated control by subtracting the background fluorescence of untransfected
cells.
2.12 MTT
The MTT assay was used to assess the cell viability after transfection with the complexes as
previously employed (14, 16). DU145 cells (2x104 cells/well), VCaP cells (5x104 cells/well)
and PC3 cells (2x104 cells/well) were seeded 24 h prior to transfection in 96-well plates.
CD.siRNA complexes were prepared as previously described using non-silencing siRNA
(Sigma). Transfection was carried out using 50 nM siRNA in antibiotic-free media for 24 h.
After this time period the media was removed and replaced with 100 µl serum-free media and
20 µl MTT reagent (5 mg/ml solution in PBS). Cells were incubated for 4 h at 37 ºC after
which 100 µl DMSO was added to each well. Absorbance was measured at 570 nm using a

UV plate reader. The results are express as the percentage cell viability relative to untreated
controls.
2.13 Competitive Uptake
DU145 cells (2x104 cells/well), VCaP cells (5x104 cells/well) and PC3 cells (2x104
cells/well) were seeded in 96-well plates in antibiotic-free medium 24 h prior to transfection.
After 24 h and prior to transfection, cells were pre-incubated with 100 µM Ad-PEG5000-
2xAA in serum-free and antibiotic-free medium for 1 h. CD.siRNA complexes (prepared
with FAM-labelled non-silencing siRNA) were then added to the media and co-incubated
with the free Ad-PEG5000-2xAA for a further 5 hours in serum-free and antibiotic-free
medium (37 °C with 5 % CO2) at a final siRNA concentration of 50 nM. Cell uptake was
done by measuring fluorescence intensity using a Perkin Elmer Victor2 1420 fluorescent
plate reader (excitation 485 nm, emission 535 nm). Samples were normalised to untreated
control by subtracting the background fluorescence of untransfected cells.
2.14 Gene Knockdown
Complexes were prepared as detailed above. DU145 cells (5 x104 cells/well), VCaP cells
(1.5x105 cells/well) and PC3 cells (5x104 cells/well) were seeded 24 h prior to transfection in
24-well plates. Complexes were added to the cells in serum-free and antibiotic-free media to
give a final concentration of 100 nM siRNA. After 24 h the media was changed to serum-
containing medium. 48 h after the initial transfection experiment, cells were lysed open and
RNA was extracted using GenEluteTM Mammalian Total RNA Miniprep Kit (Sigma) as per
manufacturer’s instructions. A NanoDrop ND-1000 UV-vis Spectrophotometer was used to
quantify the concentration of RNA in the samples. First-strand complementary DNA (cDNA)
was generated from the total RNA using a High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems). Gene expression was assessed by real-time qPCR using the Applied
Biosystem’s Real Time PCR System (Model 7300). Cycling conditions were as follows: 10

min (min) at 95 °C, 40 cycles of [15 s at 95 °C; 1 min at 60 °C]. Assays were performed
using appropriate primer sets for PLK1 (catalogue number Hs00153444_m1) and β-actin
(catalogue number Hs01060665_g1) (Taqman®, Applied Biosystems). The quantitative level
of each PLK1 mRNA was measured as a fluorescent signal corrected according to the signal
for β-actin mRNA. The 2-delta Ct method was used to quantify the relative changes in
mRNA (23).
2.15 Statistics
GraphPad Prism (San Diego, CA, USA, version 5.0) was used for statistical calculations.
One-way analysis of variance (ANOVA) was used to compare multiple groups followed by
Bonferroni’s post hoc test except in Figure 12 where a two-tailed unpaired student t-test was
used to compare PLK1 siRNA knockdown with its non-silencing counterpart.

3
Results/Discussion
3.1 Synthesis
A cationic cyclodextrin modified with a dilysine group on the primary face (Figure 1 (a)).
was successfully synthesized ( Figure 3 (a)). The β-cyclodextrin was modified to include two
lysine amino acids on the primary face as it was anticipated that the presence of two cationic
residues would lead to enhanced siRNA complexation efficiency. In addition, β-cyclodextrin
is known to form inclusion complexes with lipophilic guest molecules (2). One such
molecule is adamantane, a highly hydrophobic entity with a diameter that fits well into the β-
cyclodextrin inner cavity resulting in a high binding constant (24). Such inclusion complex
formation has previously been exploited for the targeted delivery of gene therapy agents to
cancer cells (18). In a similar fashion, the CD.siRNA nanoparticle was modified utilising the
inclusion complex formation strategy with three different PEGylated adamantane derivatives;
Ad-PEG500 and Ad-PEG5000 (Figure 1 (b) and (c)) and Ad-PEG5000-2xAA (Figure 1
(d)). The Ad-PEG derivatives were chosen to mask the cationic charge. The Ad-PEG5000-
2xAA molecule (synthesis described in Figure 4 (b)) was prepared with two anisamide-
targeting moieties on the terminal end of the PEG chain. Anisamide was selected as the
targeting ligand as it is known to increase cellular uptake via binding to the sigma receptor on
prostate cancer cells (17, 25). Two anisamide ligands were used to further enhance clustering
on the cell surface and subsequent receptor-mediated uptake.
3.2 Physicochemical Characterisation
3.2.1 ITC
To confirm the ability of the Ad-PEG derivatives to form inclusion complexes with the
Dilysine-CD, isothermal calorimetry (ITC) was used (26, 27). This technique is based on the
change in heat when two molecules interact and can be employed to determine the

stoichiometry of the interaction (N) as well as the dissociation constant (KD) (27). In addition,
it gives details on the heat released or taken in during the reaction (the enthalpy change, ΔH)
and allows the entropy (ΔS) and Gibbs free energy (ΔG) to be calculated (27).
Isothermal calorimetry was used in this study to investigate the thermodynamics of the
interaction between the Dilysine-CD and the Ad-PEG5000 derivatives (PEG5000 or
PEG5000-2xAA). Representative ITC data plots are shown in Figure 5 and the
thermodynamic properties of the interaction are listed in Table 1.
It is reported that each β-cyclodextrin molecule is capable of interacting with a single
adamantane molecule yielding a maximum N value of 1 (28). As shown in Table 1 the N
values for Ad-PEG5000 and Ad-PEG5000-2xAA are 0.587 and 0.783 respectively indicating
that the cyclodextrin cavities are not fully saturated. This is most likely due to steric
hindrance caused by the long PEG5000 chains on the adamantane molecule as previously
reported (28). In addition, the ΔH for both PEG chains is negative, indicating that energy is
released upon binding due to the favourable interaction between the β-cyclodextrin cavity and
the hydrophobic adamantane derivative (28). This is further supported by the unfavourable
entropic contribution (i.e. positive -TΔS). The Ad-PEG5000 and Ad-PEG5000-2xAA
derivatives form complexes with KD values of 12.8 and 89.8 µM respectively. These are
consistent with those previously reported for the interaction between alkylated cyclodextrin
and mannose-adamantane conjugates (KD 13.9-76.9 µM) (26).
The lower KD value for the Ad-PEG5000 derivative indicates that it forms a tighter inclusion
complex with the Dilysine-CD relative to the Ad-PEG5000-2xAA derivative. However, the
results presented confirm that the PEGylated adamantane derivatives are capable of forming
inclusion complexes with the Dilysine-CD.

3.2.2 The Influence of PEGylation on Size and Charge-Preliminary Screening of
Formulations
The change in particle size and zeta potential of the cyclodextrin nanoparticle was
investigated by varying the amounts of PEGylated adamantane derivatives in the formulation.
A mixture of adamantane derivatives was added to give a total cyclodextrin:adamantane
molar ratio of 1:1. After optimisation experiments, the Dilysine-CD was complexed with
siRNA at mass ratio of 10. A mixture of two Ad-PEG lengths was chosen following an
optimisation experiment investigating stability in high salt solution (Supplementary Figure
1). These results indicated that the PEG500 chain length did not prevent salt-induced
aggregation to the same extent as a mixture of both PEG lengths, i.e Ad-PEG500 and Ad-
PEG5000-2xAA (Supplementary Figure 1). Hence, a combination of the two PEG lengths
was selected for use in the formulation. The final optimised targeted nanoparticle, in terms of
size and charge, contained a mixture of the three adamantane derivatives (1:0.5:0.25:0.25
Dilysine-CD:Ad-PEG500:Ad-PEG5000:Ad-PEG5000-2xAA).
Correspondingly
the
untargeted nanoparticle did not contain any anisamide-targeted PEG (1:0.5:0.5 Dilysine-
CD:Ad-PEG500:Ad-PEG5000). The composition of these nanoparticles as well as their size
and charge properties are summarised in Table 2.
As shown in Table 2 the unPEGylated Dilysine-CD formulation had the lowest particle size
(115.3 nm) and the highest surface charge (+ 27.8 mV). Both the targeted and untargeted
formulations (which contained a mixture of PEG500 and PEG5000 adamantane derivatives)
had a larger nanoparticle diameter (288.9 and 283.2 nm respectively) relative to the Dilysine-
CD. This is in agreement with previous reports whereby the addition of a PEG group to a
cyclodextrin gene delivery vector resulted in increased nanoparticle diameter (10, 29).
Furthermore, the addition of PEG to the nanoparticle successfully reduced the surface charge
from + 27.8 mV for the Dilysine-CD to ~10-14 mV for both the targeted and untargeted

nanoparticles. In addition, all formulations demonstrated polydispersity values (PDI) less
than 0.4 (Table 2). Thus the targeted formulation demonstrates favourable pharmaceutical
properties for use in gene delivery.
3.2.3 Aggregation in Physiological Media
To facilitate in vivo application, stability in biological fluid is critical. Depending on the
surface properties, nanoparticles may interact with electrolytes, proteins and cellular surfaces
in the blood leading to reduced efficacy (30). In addition, aggregation in physiological fluids
in vivo can result in life-threatening conditions (12). As previously outlined, PEG is
frequently included in drug delivery formulations to shield the surface of nanoparticles
against undesirable interactions with the bioenvironment (30).
The stability of the three cyclodextrin-based formulations in a physiologically-relevant
environment was assessed by measuring the increase in nanoparticle size following
incubation in a high salt OptiMEM solution at 37 ºC over a 24 h period (Figure 6) (6, 10, 14,
16). As previously reported for other cationic cyclodextrins (9, 10, 14-16), the highly cationic
unPEGylated Dilysine-CD aggregated in high salt solution. In contrast, the combination of
PEGylated adamantane derivatives used in both the targeted and untargeted formulations
prevented aggregation up to 24 h (Figure 6), thus supporting the incorporation of PEG500
and PEG5000 into the formulation.
3.2.4 Morphology of the Complexes
The morphology of the cyclodextrin complexes (unPEGylated Dilysine-CD, targeted and
untargeted) was analysed by Transmission Electron Microscopy (TEM) (14, 17, 28) (Figure
7 (a)). The unPEGylated Dilysine-CD formed spherical nanoparticles when complexed with
siRNA. On addition of Ad-PEG (as in the case of the both targeted and untargeted
formulations) a noticeable increase in nanoparticle size was detected, further corroborating

the results obtained by dynamic light scattering (Table 2). In addition a distinctive ‘shielding’
PEG layer is visible in both targeted and untargeted formulations as previously reported (14).
3.2.5 Ability of Complexes to Bind and Release siRNA
The degree of binding will influence protection and release of siRNA after cellular uptake.
Thus the binding and release properties of the cyclodextrin-based delivery vectors were
assessed. As observed in Figure 7 (b), the unPEGylated Dilysine-CD formulation at mass
ratio 10 was able to both bind and release siRNA. The addition of Ad-PEG to the formulation
(both targeted and untargeted) did not interfere with either siRNA binding or release.
3.2.6 Serum Stability
To ensure efficacy after systemic administration in vivo siRNA must be protected from
degradation by nuclease enzymes (31). Hence the ability of nanoparticles to protect siRNA
from nuclease digestion was investigated after incubation in 50 % serum for up to 24 hours
and analysed by gel electrophoresis as previously employed (7, 17, 31, 32). Naked siRNA
was partially degraded after 8 h incubation in 50 % serum and was fully degraded after 24 h.
In contrast, an siRNA band was still visible in cyclodextrin samples for up to 24 h indicating
enhanced protection against serum nucleases (Figure 8).
3.3 Uptake of Dilysine-CD.siRNA Formulations in Prostate Cancer Cells
The physicochemical characterisation data of the Dilysine-CD.siRNA nanoparticles
identified the optimum prototype nanoparticle with potential for targeted delivery to prostate
cancer cells. The uptake of fluorescent siRNA over 24 hours in three different sigma-receptor
positive prostate cancer cells lines (DU145, VCaP and PC3 cells) was assessed using targeted
and untargeted nanoparticles. The cationic unPEGylated Dilysine-CD was included for
comparative purposes.

As demonstrated in Figure 9, the targeted formulation had significantly higher uptake
relative to the untargeted formulation in all three prostate cell lines tested (P<0.001), giving a
strong indication that this nanoparticle was taken into the cells via receptor-mediated
endocytosis. Thus, the targeted formulation was further investigated for cell toxicity.
3.4 MTT
A major hurdle that must be overcome in the area of gene therapeutics is biocompatibility of
delivery vectors to avoid cellular toxicity (33). The MTT assay is a widely used method of
assessing eukaryotic cell viability by measuring the reduction of 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide by cellular mitochondrial reductase enzymes (34). It is
frequently used to assess the toxicity of gene therapy delivery vectors and is a good measure
of cell metabolism, proliferation and viability (33). Hence the MTT assay was used to assess
prostate cancer cell viability 24 hours post-transfection in serum-containing media as
previously reported (8, 10, 14, 16). The results for the three different prostate cancer cell lines
(DU145, VCaP and PC3 cells) are shown in Figure 10. Cell viability was maintained at >80
% following transfection with each of the cyclodextrin-based formulations (Dilysine-CD,
targeted and untargeted).
3.5 Competitive Uptake Inhibition
To investigate if uptake into prostate cancer cells occurs specifically via the sigma receptor,
cells were pre-incubated with excess free Ad-PEG5000-2xAA. The specificity of the
anisamide ligand for the sigma receptor has been extensively studied (17, 25, 35, 36). Such
procedures have been previously employed for competitive uptake experiments (28). Results
from the competitive uptake experiment in the three different prostate cancer cells lines
(DU145 cells, VCaP cells and PC3 cells) are shown in Figure 11.

As shown in Figure 11 (a), in the absence of excess free anisamide the targeted formulation
had significantly higher uptake in DU145 cells relative to the untargeted formulation 5 hours
after transfection (P<0.05). However when excess free anisamide was co-incubated with the
formulation, the uptake of the targeted nanoparticle was reduced to the same level as the
untargeted nanoparticle. In contrast the uptake of the untargeted nanoparticle was unaffected
by the absence or presence of free anisamide. This indicates that, in DU145 cells, the targeted
nanoparticle is taken up via sigma receptor-mediated endocytosis whereas untargeted
nanoparticles are not (Figure 11 (a)).
A similar result was found in VCaP cells (Figure 11 (b)) where the targeted nanoparticle had
significantly higher uptake than the untargeted in the absence of excess free Ad-PEG5000-
2xAA (P<0.001). However, following the competitive study, uptake of both nanoparticles
were equal, demonstrating that uptake of the targeted nanoparticle in VCaP cells is via the
sigma receptor.
The results obtained in PC3 cells showed no difference in uptake between the targeted and
untargeted nanoparticles in the absence of excess free Ad-PEG5000-2xAA under these
experimental conditions. In contrast, the targeted nanoparticle had significantly higher uptake
relative to the untargeted nanoparticle at 24 h as seen in Figure 9. This effect was not
observed at 5 h (Figure 11) and suggests that a longer period of transfection (24 h versus 5 h)
is required to observe a significantly higher level of uptake of the targeted nanoparticle in
PC3 cells. Following co-incubation with excess free Ad-PEG5000-2xAA the uptake of both
targeted and untargeted nanoparticles was equally reduced (Figure 11 (c)). The reduced
cellular uptake of the untargeted nanoparticle was surprising and suggests that the excess free
Ad-PEG5000-2xAA ligand may have caused steric hindrance and inhibited non-specific
uptake pathways in PC3 cells. The inconclusive results to try to demonstrate receptor-
mediated uptake of the targeted nanoparticle may be rationalised by either of two

explanations. Firstly, the conditions for the competitive uptake experiment were optimised in
DU145 cells. It is possible that further optimisation may be required to prove receptor-
mediated uptake into PC3 cells. Secondly, PC3 cells have lower sigma receptor expression
than both DU145 and VCaP cells (manuscript in preparation). Hence it is possible that
receptor-mediated uptake of the targeted nanoparticle via binding to the sigma receptor is not
a dominant mechanism for cellular uptake in PC3 cells. However, as previously shown, the
uptake of the targeted nanoparticle 24 h post-transfection was significantly higher than the
untargeted (Figure 9 (c)) and hence it was included for comparative purposes in the gene
knockdown experiment to see if the higher level of uptake would correspond to higher
knockdown of the PLK1 gene (Figure 12 (c)).
It is recognised that, despite the absence of the anisamide-targeting moiety, the untargeted
nanoparticle underwent high levels of non-specific cellular uptake. This non-specific uptake
pathway is also likely to be present for the targeted nanoparticle whereby uptake is further
enhanced by the anisamide targeting ligand. However, as the sigma receptor is overexpressed
in prostate cancer cells, the presence of the targeting ligand should enhance accumulation of
the nanoparticle at its intended site of action relative to the untargeted nanoparticle in vivo.
3.6 PLK1 Gene Knockdown
Polo-like kinase 1 (PLK1) is a serine-threonine-protein kinase enzyme whose overexpression
has been linked with a variety of cancers, including prostate cancer where it is associated
with a higher tumor grade (37). It plays a key role in cellular mitosis and high levels are
associated with increased cell proliferation (38). Recently, targeting the PLK1 gene has been
identified as a potential therapy for prostate cancer (38-40). With this in mind, the PLK1 gene
was selected for mRNA knockdown with our targeted cyclodextrin-based nanoparticle.