Design of Bifunctional Dendritic 5-Aminolevulinic Acid and Hydroxypyridinone Conjugates for Photodynamic Therapy

Article abstract of DOI:10.1021/acs.bioconjchem.8b00574

Iron chelators have recently attracted interest in the field of photodynamic therapy (PDT) owing to their role in enhancement of intracellular protoporphyrin IX (PpIX) generation induced by 5-aminolevulinic acid (ALA) via the biosynthetic heme cycle. Although ALA is widely used in PDT, cellular uptake of ALA is limited by its hydrophilicity. In order to improve ALA delivery and enhance the PpIX production, several dendrimers incorporating both ALA and 3-hydroxy-4-pyridinone (HPO) were synthesized. The ability of the dendrimers to enter cells and be metabolized to the PpIX photosensitizer was studied in several human cancer cell lines. The dendrimers were found to be significantly more efficient than ALA alone in PpIX production. The higher intracellular PpIX levels showed a clear correlation with enhanced cellular phototoxicity following light exposure. Dendritic derivatives are therefore capable of efficiently delivering both ALA and HPO, which act synergistically to amplify in vitro PpIX levels and enhance PDT efficacy.

Full text of DOI:10.1021/acs.bioconjchem.8b00574

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Article
Design of Bifunctional Dendritic 5-Aminolevulinic Acid and
Hydroxypyridinone Conjugates for Photodynamic Therapy
Tao Zhou, Sinan Battah, Francesca Mazzacuva, Robert C. Hider, Paul S. Dobbin, and Alexander J. MacRobert
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00574 • Publication Date (Web): 24 Sep 2018
Downloaded from http://pubs.acs.org on September 25, 2018
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Design of Bifunctional Dendritic
ꢀAminolevulinic Acid and Hydroxypyridinone
Conjugates for Photodynamic Therapy
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,§,a
§,b,d
c
c
b
Tao Zhou,* Sinan Battah,
Francesca Mazzacuva, Robert C. Hider, Paul Dobbin,
d
Alexander J. MacRobert
a. School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou,
Zhejiang 310035, P. R. China.
b. School of Biological Sciences, University of Essex, Wivenhoe Park CO4 3SQ
c. Division of Pharmaceutical Sciences, King’s College London, 150 Stamford Street,
London SE1 9NH
d. Division of Surgery and Interventional Science, University College London, Charles Bell
House, 43ꢀ45 Foley St, London W1W 7TS
Corresponding author.
*
(T.Z.) Eꢀmail: taozhou@zjgsu.edu.cn
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ABSTRACT
Iron chelators have recently attracted interest in the photodynamic therapy (PDT) field owing
to their role in enhancement of intracellular protoporphyrin IX (PpIX) generation induced by
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5ꢀaminolevulinic acid (ALA) via the biosynthetic haem cycle. Although ALA is widely used
in PDT, cellular uptake of ALA is limited by its hydrophilicity. In order to improve ALA
delivery and enhance the PpIX production, several dendrimers incorporating both ALA and
3ꢀhydroxyꢀ4ꢀpyridinone (HPO) were synthesized. The ability of the dendrimers to enter cells
and be metabolized to the PpIX photosensitizer was studied in several human cancer cell
lines. The dendrimers were found to be significantly more efficient than ALA alone in PpIX
production. The higher intracellular PpIX levels showed a clear correlation with enhanced
cellular phototoxicity following light exposure. Dendritic derivatives are therefore capable of
efficiently delivering both ALA and HPO which act synergistically to amplify in vitro PpIX
levels and enhance PDT efficacy.
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INTRODUCTION
5ꢀAminolevulinic acid (ALA) has been shown to be effective at inducing PpIX after
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ꢀ4
oral, intravenous (i.v.) and topical applications in vivo
.
ALAꢀPDT currently finds vast use
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in dermatology for the treatment of actinic keratosis, squamous cell carcinoma, and Bowen’s
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,6
disease, as well as cutaneous microbial infections such as acne, onychomycosis, and
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verrucae. Promising results have also emerged from studies in gastroenterology and
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urology. PpIX from administered ALA may be used not only to directly treat conditions
such as Barret’s esophagus, inflammatory bowel disease, and bladder cancer by PDT but also
as a diagnostic tool for the visualization of precancerous changes in the mucosae by
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fluorescence spectroscopy.
However, ALA is a hydrophilic molecule and has a limited
ability to penetrate biological barriers, and is less efficient at inducing intracellular porphyrin
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generation compared to esters of ALA.
Several studies have confirmed that a significant
enhancement of cellular uptake can be achieved by esterifying the carboxylic terminal with
17ꢀ21
alkyl or aryl groups, resulting in increased levels of PpIX.
An attractive alternative is to
incorporate ALA into a short peptide which is susceptible to intracellular enzymatic
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activity.
ALAꢀinduced PDT can be modulated in the presence of iron chelators such as
24,25
26,27
EDTA,
and desferioxamine,
the chelators inhibiting ferrochelatase by scavenging the
intracellular labile iron pool. Several studies have also demonstrated enhancement of PpIX
concentration in cells or tissues exposed to a combination of ALA and the membrane
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permeable iron chelator 1,2ꢀdiethylꢀ3ꢀ hydroxypyridinꢀ4ꢀone (CP94).
However, optimal
tissue accumulation and localization remains a clinical problem. One approach adopted to
reduce this problem involves the use of dendrimeric drug carriers which show an appreciable
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efficacy for ALA delivery and subsequent production of PpIX.
Bearing in mind the aforementioned studies and the parameters affecting PpIX levels in
tissues, in principle PpIX can be elevated in three different ways: chelation of iron in
intracellular compartments, thereby inhibiting ferrochelatase; derivatization of one or both
termini of ALA, thereby enabling membrane permeation; and attachment of ALA to
macromolecular assemblies, in particular dendrimers. In a previous study, it was
demonstrated that ALAꢀHPO conjugates via ester or amido linkages effectively improve the
intracellular PpIX production when compared to the use of ALA alone and combination of
37ꢀ39
ALA and HPO.
Thus, we designed dendritic carriers simultaneously incorporating both
ALA and ironꢀchelating agents (HPOs). Dendrimers have been shown to rapidly enter cells
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by triggering endocytotic processes
and as such, dendrimers are predicted not to use the
same uptake pathway as ALA and simple ALA prodrugs. Dendritic carriers were selected for
this study as the synthesis of such molecules can be controlled with respect to the size and
loading of both ALA and HPOs. The amino groups of ALA molecules will be protonated
under in vivo conditions, and the presence of the HPO hydroxyl groups, further confers good
water solubility of such dendrimers. Dendrimers can be constructed in a stepwise manner
4
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yielding structurally and topologically defined, monodisperse unimolecular entities. The
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convergent growth strategy adopted for the synthesis of ALAꢀHPOꢀcontaining dendrimers in
this study has permitted the dendrimer to be assembled in segments. The dendrons were
attached to a dipodent core unit in the final step of dendrimer construction. We have
investigated the properties of first generation dendritic carriers containing three residues of
ALA and either one or three residues of HPO. The structure of the dendrimer was designed to
enable ALA and the HPO moieties to be attached by controlled synthesis via ester linkages to
a multipodent aromatic core.
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RESULTS AND DISCUSSION
Chemistry. Five first generation dendrimers containing three residues of ALA and with
either one residue of HPO (10a and 25a) or three residues of HPO (20a, 29a and 30a) were
synthesized (Chart 1). The loading of controlled amounts of both ALA and HPO into a
wellꢀdesigned carrier was rendered possible by using standard ester and amide bond forming
reactions and selective protection strategies. The synthetic sequence involved: (a)
esterification of the three hydroxyl or the three carboxy groups of tripodal molecules; (b)
construction of a core molecule with attached βꢀalanine spacer groups; (c) successive amide
bond formation to give dendrons and then dendrimers; (d) removal of the N and O protecting
groups.
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Chart 1
The choice of HPO in this approach was based on ClogP values of a range of
Nꢀ(ωꢀhydroxyalkyl)ꢀHPOs, a compound with appreciable hydrophobicity being necessary to
45
inhibit ferrochelatase. As a result of this analysis, we selected a 3ꢀhydroxypyridinone, with
either hexanoic or hexanol substituents at position 1, for the branching building blocks of
dendrimers 25a, 29a, and 30a.
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The stability of the dendrimers containing the CbzꢀALA dendron was a challenging
issue because subsequent to catalytic hydrogenation of the Cbz protecting group, there was
considerable decomposition of the resulting dendrimer, particularly when the hydrogenation
step exceeded 30 min. We therefore employed phenylalanine and methionine as protection
groups, which can be cleaved by cytoplasmic enzymes. These two amino acids were selected
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on the basis of their efficacy when conjugated to ALA.
Synthesis of dendrimer 10a. Firstly, the dendron containing 3 ALA moieties (8) was
synthesized (Scheme 1).
Commercially available nitromethanetrispropanol (2) was
triacetylated to produce 3, which was then reduced by hydrogenation to generate amine 4.
The free amino group of 4 was coupled to NꢀBocꢀβꢀalanine in the presence of
N,N′ꢀdicyclohexylcarbodiimide (DCC) and 1ꢀhydroxybenzotriazole (HOBt) to afford 5. The
purpose of introducing a small spacer (βꢀalanine) to aminomethanetrispropanol was to reduce
steric hindrance in subsequent conjugation reactions. Alkaline hydrolysis of 5 afforded 6.
CbzꢀALA was coupled to the branching unit 6 by formation of an ester bond using
DCC/DMAP reagents. The crude product was purified by flash chromatography on silica gel
to provide triꢀester 7, together with monoꢀ and diꢀesters. Cleavage of the Boc protected group
1
13
provided dendron 8. The structure of dendron 8 was confirmed by H and C NMR and
LCꢀMS/MS mass spectrometry. Dendron 8 was coupled to 9 using the standard amide
coupling reagents DCC and HOBt to provide 10 in a reasonable yield (48%). Removal of the
protecting groups was accomplished using hydrogen (40 psi) and 10% Pd/C in presence of
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benzyl chloride to isolate the corresponding dendrimer 10a as the tetraꢀhydrochloride. Some
decomposition occurred during the hydrogenation step, necessitating further HPLC
purification.
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O
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O
OH
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a
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c
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b
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OH
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H
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Cbz
NH
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OH
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e
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O
d
R
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Boc
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N
OH
OH
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H
N
O
N
H
H
H
O
H
O
O
O
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NH
7: R = Boc
f
Cbz
8: R = H
R'
NH
O
O
O
RO
h
O
O
O
R'
O
N
N
N
O
O
N
H
H
O
H
BnO
O
O
O
N
OH
O
NH
R'
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g
10: R = Bn, R' = Cbz
10a: R = H, R' = H
i
O
O
OBn
1
Scheme 1. Reagents and conditions: (a) Acetic anhydride, pyridine, rt, 18h; (b) Raney Ni,
MeOH, H , 30psi; (c) DCC, HOBT, DCM/DMF (3:1), rt, argon, 24h; (d) 2M NaOH/MeOH
1:1), 10 °C, 30 min; (e) CbzꢀALA, DCC, DMAP, DCM/DMF (3:1), rt, argon, 24h; (f)
2
(
2
1%TFA in DCM, 30 min; (g) 8ꢀamino octanoic acid, EtOH/H O (1:1), 2M NaOH, reflux 18h;
(
h) DCC, HOBT, Bmim, DCM/ DMF (3:1), rt, Argon, 24h; (i) Pd/C, H /40 psi, BnCl,
2
MeOH/EtAc (1:4), rt, 30 min.
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Synthesis of dendrimer 20a. Synthesis of dendrimer 20a is outlined in Scheme 2. The
core for this dendrimer was the dicarboxylic aromatic acid 12. The synthesis of 12 utilized
phthalolyl chloride, providing 11 in good yield by reaction with the ethyl ester of βꢀalanine.
Alkaline hydrolysis of 11 afforded core 12. Coupling of Fmocꢀβꢀalanine and aminomethane
tris (3ꢀtertꢀbutoxycarbonyl propionate) (13) in the presence of HOBt and DCC provided 14 in
excellent yield (92%). Triꢀacid 15 was obtained in quantitative yield by the treatment of 14
with formic acid. 3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ1ꢀ(6ꢀhydroxyhexyl)pyridinꢀ4(1H)ꢀone (16H) was
coupled to triꢀacid 15 to provide triꢀester 17H in the presence of DCC and DMAP in
reasonable yield (42%). Cleavage of Fmoc in 17H was achieved by treatment with piperidine
to yield dendron 18H. Coupling of 18H to the diꢀcarboxy functionalized core 12 gave 19H in
a moderate yield (56%). 19H was then coupled with the ALA dendron 8 to provide the
protected dendrimer 20, which on subjection to hydrogenation, yielded dendrimer 20a. As
with 10a, there was some degradation during the hydrogenation step. After preparative HPLC,
the dendrimer was isolated as the hexaꢀhydrochloride.
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O
OBn
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OBu-t
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c
e
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14: R = Bu-t
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17H: R = Fmoc
18H: R = H
O
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5: R = H
BnO
f
OBn
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g
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OH
H
N
HO
N
H
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H
H
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H
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OH
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O
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O
O
O
b
O
O
O
O
O
O
EtO
N
H
N
OEt
H
19H
8
N
BnO
11
a
O
O
O
Cl
Cl
h
OR
N
R'
NH
O
O
O
O
O
O
O
O
RO
O
R'
H
H
H
N
H
N
H
H
O
O
N
N
N
N
N
N
H
O
O
O
O
O
O
O
O
O
O
O
O
NH
R'
N
20: R = Bn, R' = Cbz
20a: R = H, R' = H
RO
i
O
Scheme 2. Reagents and conditions: (a) βꢀalanine ethyl ester hydrochloride, DCM/DMF (5:1),
o
Et
3
N, 0 C then rt; (b) 2M NaOH/MeOH (1:1), 10°C, 30 min, 1M HCl neutralized to pH 6; (c)
Fmocꢀβꢀalanine, DCC, HOBT, DCM/DMF (3:1), rt, Argon, 24h; (d) HCOOH, rt, 24h; (e)
DCC, DMAP, DCM/DMF (3:1), rt, Argon, 24h; (f) Piperidine, DMF, rt, 10 min; (g) DCC,
o
o
HOBT, DMF, Bmim, Argon, 0 C 1h then rt 24h; (h) DCC, HOBT, DMF, Bmim, Argon, 0 C
1
h, then rt 24h; (i) Pd/C, H
2
/40 psi, BnCl, MeOH/EtAc (1:4), rt, 30 min.
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Synthesis of dendrimer 25a. NꢀCbzꢀLꢀphenylalanine was coupled to ALA methyl ester
in the presence of DCC, HOBt and 1ꢀbutylꢀ3ꢀmethylimidazolium bromide (Bmim) to provide
46
2
1 in high yield (>95%). 21 was hydrolyzed in alkaline solution to give 22P. 22P was
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
coupled to 6 in the presence of DCC and DMAP to provide the triꢀbranched ALA amino acid
moiety 23, which when treated with TFA cleaved Boc, yielding dendron 24. Dendron 24 was
conjugated to HPO 9, providing the protected dendrimer 25, which on hydrogenation gave
dendrimer 25a (Scheme 3).
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HN Cbz
R'
O
NH
O
O
O
O
O
a
c
R'
O
H
3
N
O
Cbz
H
O
O
O
H
O
N
OR
N
N
R
Cl
O
N
N
O
N
Cbz
H
H
R'
O
O
O
H
H
O
O
HN
2
1: R = Me, R' = PhCH2
6: R = Me, R' = MeSCH CH
2
NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
R'
b
2
Cbz
2
3: R = Boc, R' = PhCH2
2
2P: R = H, R' = PhCH2
22M: R = H, R' = MeSCH CH
2
7: R = Boc, R' = MeSCH CH
2
2
2
2
d
2
4: R = H, R' = PhCH2
28: R = H, R' = MeSCH
Ph
R'
HN
2
CH
2
e
9
NH O
O
O
Ph
O
H
N
R'
N
H
O
H
H
O
N
N
N
O
OR
O
O
O
O
O
O
H
Ph
g
19C
N
O
2
5: R = Bn, R' = Cbz
5a: R = H, R' = H
R' N
H
O
f
2
RO
O
O
S
N
N
O
N
HNR'
O
O
H
O
O
O
O
g
RO
S
19H
H
H
H
H
H
H
O
N
N
O
N
N
N
N
N
N
O
H
HN
O
R'
O
O
O
O
O
O
O
O
O
O
H
HN
R'
RO
O
O
N
29: R = Bn, R' = Cbz
9a: R = H, R' = H
O
O
S
h
2
OR
N
O
S
O
N
O
HNR'
O
O
H
O
O
O
RO
O
O
O
S
N
H
H
H
H
N
H
H
O
N
N
N
N
N
N
O
H
HN
O
O
R'
R'
O
O
O
O
O
O
O
O
O
H HN
N
O
3
0: R = Bn, R' = Cbz
0a: R = H, R' = H
N
O
S
RO
h
O
3
Scheme 3. Reagents and conditions: (a) NꢀCbzꢀLꢀphenyl alanine or NꢀCbzꢀLꢀMethionine,
DCC, HOBt, Bmim, THF, DIPEA, rt, Argon, 24h; (b) 2M NaOH/MeOH (1:1), 4 °C, 30 min.;
(c) 6, DCC, DMAP, DCM/DMF (3:1), rt, Argon, 48h; (d) DCM/HCOOH (10:1), 18h; (e) 9,
DCC, HOBt, DCM/DMF (3:1), rt, Ar, 24h; (f) Pd/C, H /20psi, EtOAc/MeOH (4:1), 2 min;
2
(g) DCC, HOBt, Bmim, DCM/THF (1:1), rt, Argon, 48h; (h) Pd black, H
MeOH (7N) and liquid ammonia, 2h.
2
, ammonia in
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Synthesis of dendrimer 29a and 30a. The synthetic route for dendron 19C is presented in
Scheme 4. 6ꢀ(3ꢀ(Benzyloxy)ꢀ2ꢀethylꢀ4ꢀoxopyridinꢀ1(4H)ꢀyl)hexanoic acid was coupled to
the triꢀol 6 to provide the triꢀester 17C, which yielded dendron 18C after removal of Boc.
Coupling of 18C to the diꢀcarboxyl functionalized core 12 gave 19C in a yield of 62%.
Dendron 28, which contains three CbzꢀMet functionalized ALA branches and one free amino
group, was then synthesized in a similar method to that of 24 (Scheme 3). Coupling of
dendron 28 with 19H and 19C via amide bond formation in the presence of DCC, HOBt and
Bmim provided the protected dendrimers 29 and 30, respectively. Hydrogenation of 29 and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
30 yielded dendrimers 29a and 30a (Scheme 3). Cleavage of Nꢀbenzyloxycarbonyl and
Oꢀbenzyloxy groups of compounds 29 and 30 was found to be more difficult than with
compounds 10, 20 and 25, as both 29 and 30, contain the sulphur group of methionine which
47
poisons the catalyst. Thus for these hydrogenation procedures methanolic ammonia and Pd
4
8
black was adopted, the dendrimers being isolated as formate salts.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
O
O
N
OBn
N
O
OBn
O
OBn
O
O
O
O
O
O
b
N
O
a
Boc
N
O
H N
N
O
2
N
N
H
O
H
OBn
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
OBn
OH
O
O
O
O
Boc
O
OH
OH
OH
O
N
O
N
H
N
N
O
H
OBn
OBn
1
6C
6
17C
18C
BnO
O
N
N
O
c
O
BnO
O
H
H
H
H
O
N
O
N
N
N
N
OH
O
O
O
O
O
HO
N
H
N
OH
O
O
O
O
H
O
BnO
O
1
2
O
19C
Scheme 4. Reagents and conditions: (a) DCC, DMAP, DCM/DMF (3:1), rt, Argon, 48h; (b)
o
DCM/HCOOH (10:1), rt, 18h; (c) DCC, HOBT, DMF, Bmim, Argon, 0 C 1h then rt 24h.
Fluorescence Pharmacokinetics. Several processes control the enhancement of
dendrimerꢀinduced PpIX generation: (a) the enhanced rate of drug uptake; (b) the rate of
1
3ꢀ15
enzymatic conversion of dendrimers to active principles;
(c) the enhanced retention of
dendrimers at the application site and (d) the accessibility of the HPO moiety to
4
9,50
ferrochelatase.
In this study we compared intracellular PpIX fluorescence levels induced
by the dendrimer conjugates compared to ALA as a function of incubation time and
concentration in a range of cell lines. MCFꢀ7 cells (human breast adenocarcinoma) and its
doxorubicinꢀresistant subline, MCFꢀ7R, were selected since Feuerstein et al have shown that
51
the resistant subline exhibits lower ferrochelatase expression. In addition, the human
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9
epithelial carcinoma KB cell line of cervical origin was selected, which is relevant to the
clinical applications of ALAꢀPDT.
We initially compared PpIX fluorescence levels induced using a fixed concentration at
various incubation times. The intracellular PpIX fluorescence levels following administration
to various dendrimers using a concentration of 100 ꢁM increased with time (2ꢀ 24 hr) as
shown in Figure 1A and B for the KB cell line. Higher levels were observed for all the
dendrimers compared to ALA, with the most striking enhancement apparent at shorter
incubation times (2ꢀ4 hr) where ALA induced very low PpIX levels. The dendrimer which
lacked a chelating agent (triꢀALA, Figure 1) was found to be slightly less effective at shorter
incubation times than dendrimer 10a which contains one iron chelating HPO moiety.
Dendrimer 20a which also contains three ALA residues like 10a but three HPO chelating
moieties was more effective for PpIX generation than 10a at all the timeꢀpoints. These data
are consistent with a synergistic enhancement in PpIX generation resulting from the presence
of the HPO moieties. Inhibition of ferrochelatase activity by the HPO chelator results in the
inhibition of PpIX conversion to haem thereby leading to enhanced intracellular PpIX
accumulation, as found in previous studies using coꢀadministration of HPOs and the
administration of single conjugates of HPO with ALA where higher PpIX levels were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7,39
observed in the presence of HPO conjugates compared to ALA alone.
In a further
modification to the dendrimer structure, attachment of a phenylalanine residue at the
Nꢀterminus of the ALA (dendrimer 25a) enhanced the PpIX levels measured at shorter times
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compared to 20a. Attachment of methionyl residues (dendrimers 29a and 30a) further
enhanced PpIX levels and generated approximately 12ꢀ15 fold of PpIX fluorescence than the
equivalent of ALA within 24 h. These results are consistent with a previous study
demonstrating enhancement of PpIX generation for single conjugates of ALA with amino
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
23
acid residues.
90
80
70
60
50
40
30
20
10
0
ALA
tri-ALA
A
1
2
2
0a
0a
5a
2h
4h
6h
24h
ALA-HPO Dendrimer Incubation Time
ALA
B
8
6
4
0
2
3
9a
0a
0
0
20
0
2
h
4h
6h
24h
Dendritic ALA-HPOs Incubation Time
Figure 1. Kinetics of PpIX fluorescence in KB cell line. A timeꢀcourse of fluorescence
intensities produced after incubation with 100 ꢁM of dendritic ALAꢀHPO. (A) Comparison
of 10a, 20a and 25a with triꢀALA and ALA; (B) comparison of 29a and 30a with ALA.
In the next part of the study, a range of concentrations of each compound was
investigated for a fixed incubation timeꢀpoint of 4 hours, which is a typical exposure time
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employed clinically for ALA administration. The magnitude of PpIX generation induced by
exposure to ALAꢀcontaining dendrimers in the KB and MCFꢀ7 cell lines was found to exhibit
a dose dependent response. All dendrimers were found to be more effective than ALA at all
concentrations investigated in KB (Figure 2A) and MCFꢀ7 (Figure 2 B). Clearly the
protection of the ALA moieties with methionine has an advantage, at lower concentrations
the effect being apparently irrespective of whether there is a HPO with either a
ωꢀhydroxyalkyl side chain (29a) or a ωꢀcarboxylalkyl side chain (30a). We note that previous
studies on single conjugates of amino acids with ALA also showed that methione conjugation
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
23
was effective at improving PpIX efficiency in certain cell lines. The high efficacy of all the
dendrimers was evident in a comparative study of three cell lines, KB, MCFꢀ7 and MCF 7R
(Figure 3). The dendrimer efficacies (at 50 ꢁM) for each cell line were found to be
comparable, which is consistent with Figure 2 where PpIX levels tended towards a common
plateau at higher concentrations.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
0
ALA
A
1
2
0a
0a
45
4
3
3
2
0
5
0
5
25a
9a
30a
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20
1
1
5
0
5
0
0
20
40
60
80
100
Concentration of Dendritic ALA-HPOs (µM)
4
5
0
5
0
5
ALA
B
1
2
0a
0a
4
3
3
2
25a
2
3
9a
0a
20
15
1
0
5
0
0
20
40
60
80
100
Concentration of Dendritic ALA-HPOs (µM)
Figure 2. Concentration dependence profile of PpIX generation induced by ALA and the
dendritic compounds. Fluorescence measurements were performed after incubation with
variable concentration (20ꢀ100 ꢁM) of ALA and dendritic ALAꢀHPO for 4 h. (A) in KB cell
line; (B) in MCFꢀ7.
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50
40
30
KB
MCF-7
MCF-7R
2
1
0
0
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ALA
10a
20a
25a
29a
30a
Compound (50 µM)
Figure 3. Comparison of PpIX production in three cell (KB, MCFꢀ7 and MCFꢀ7R) lines after
incubation for 4 h with 50 ꢁM of dendritic ALAꢀHPO (10a, 20a, 25a, 29a and 30a) versus
ALA.
In an attempt to investigate the ability of dendrimers to act as a prolonged source of
ALA and chelator, 30a was incubated with KB cells for various time periods (15ꢀ60 min) and
the PpIX fluorescence intensity was measured after 4h (Figure 4A). Exposure for 45 and 60
min led to approximately 50% of the fluorescence being observed when the dendrimers were
incubated with cells for the entire period. A similar set of data was observed when the study
was extended to 24h (Figure 4B). This is in complete contrast to incubation with ALA at a
fiveꢀfold higher concentration so that the ALA dose to the cells was comparable to the
dendrimers containing multiple ALA moieties and shows that the dendrimers are capable of
generating ALA and presumably HPO chelators over a prolonged time period. The
experiment was repeated with MCFꢀ7 and MCFꢀ7R which gave similar results but with
slightly lower fluorescence intensities than KB cell line (Figure S1 in Supporting
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6
Information). These data are consistent with our previous studies of second generation ALA
35
dendrimers, where we also observed sustained release of ALA over prolonged periods.
30
30a (60µmol/L)
A
0
1
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3
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8
9
0
ALA (300 µmol/L)
20
10
0
15 min
30 min
45 min
60 min
Dendritic ALA-HPO Exposure Time
30
30a (60µmol/L)
B
ALA (300 µmol/L)
20
10
0
15 min
30 min
45 min
60 min
Dendritic ALA-HPO Exposure Time
Figure 4. PpIX production in KB cells treated with 300 ꢁM of ALA or 60 ꢁM ALAꢀHPO
dendrimer 30a. Cells were exposed to the compounds for times up to 60 min and then washed
to remove the excess compounds. Fluorescence reading were recorded at two later
timeꢀpoints to compare porphyrin levels. (A) fluorescence readings taken after 4 h from the
time point at which cells were first exposed to the compound; (B) fluorescence readings were
taken after 24 h.
Photodynamic studies. Following illumination of cells incubated with dendrimers
containing both ALA and chelators, efficient phototoxicity was observed at the lowest
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concentrations investigated (2 ꢁM) as shown in Figure 5 where cell viability is plotted versus
concentration. In contrast negligible phototoxicity was observed using ALA. The calculated
LC50 values are given in Table 1, with the exception of ALA where we can only set a lower
limit since estimation of an LD50 value would have required a much higher concentration
range. The LD50 values for all the dendritic compounds in both cell lines were calculated to
be between 2ꢀ10 ꢁM. From Figures 5A, 5B and Table 1, the relative phototoxicities of 10a
and 20a, which contains three times more HPO moieties than 10a, show that 20a is more
phototoxic in each cell line which is consistent with the greater propensity for PpIX
generation by 20a (Figure 2). The same correlation applies to the other compounds studied
and the trend in efficiency for PpIX generation from Figure 2 versus the phototoxicity data
shows a clear correlation between higher PpIX generation efficacy and higher phototoxicity,
with compounds 29a and 30a being the most phototoxic. Moreover higher phototoxicities
and correspondingly lower LD50 values were observed for the KB cell line than MCFꢀ7 cell
line, which is consistent with higher PpIX generation efficiency in the KB cell line, as is
evident in Figure 2 for the lower concentrations, which is the relevant range since the
phototoxicity studies were conducted with a relatively low concentration of 10 ꢁM. The
1
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9
0
‘dark’ toxicity of the dendritic ALAꢀHPO was also assessed (i.e. their cytotoxicity in the
absence of irradiation) and there was negligible toxicity present even at effective ALA levels
of 100 ꢁM after 24h (Figure S2 in Supporting Information).
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ALA
10a
20a
25a
29a
30a
20
0
0
2
4
6
8
10
ALA and ALA-HPO dendrimers conceentration (µM)
1
1
20
00
80
B
6
0
ALA
1
2
2
2
0a
0a
5a
9a
40
2
0
30a
0
0
2
4
6
8
10
ALA and ALA-HPO dendrimers Concentration (µM)
Figure 5. Phototoxicity dependence on concentration after incubation with ALA and
dendritic ALAꢀHPO at a range of concentrations assessed by MTT assay: (A) dependence in
KB cells; (B) dependence in MCFꢀ7 cells. Cells were incubated with the compounds for 4 h
2
and illuminated (2.5 J/cm ).
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Table 1. LD50 of ALA and ALAꢀHPO dendrimers phototoxicity in KB and MCFꢀ7 cell lines
2
(
4 h incubation with the compounds then irradiation/light exposure (2.5 J/cm ) and further 24
h incubation before MTT tests).
LD50
KB (ꢁM)
>100
MCFꢀ7 (ꢁM)
>100
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
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3
4
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0
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0
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9
0
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0
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2
3
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9
0
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2
3
4
5
6
7
8
9
0
ALA
10a
20a
25a
29a
30a
6.9
3.8
6.1
2.8
2.1
±
±
±
±
±
0.9
0.8
2.3
1.7
1.1
10.2
±
2.1
5.6
8.8
5.1
4.6
±
1.2
±
±
±
3.2
1.9
2.2
Notes: LD values were calculated by using the exponential trendline, then finding the
5
0
correspondent concentration of each compound that caused 50% of cell death/survival.
Mechanistic investigation of dendritic ALA-HPO. We also considered the mechanism
of cellular uptake. Whereas small drugs are generally taken up via passive diffusion across
the cell membrane, larger drug carriers such as dendrimers are taken up via active transport
5
2
35
endocytic mechanisms. In our previous study of second generation ALA dendrimers, we
found that uptake occurred via an endocytic mechanism (macropinocytosis). In order to
provide an insight into utilized endocytic pathways, the cellular uptake mechanism was
investigated by measuring PpIX fluorescence induced during incubation of dendritic
ALAꢀHPO following preꢀtreatment with two endocytosis inhibitors: 5ꢀ(NꢀethylꢀNꢀisopropyl)
53
54
amirolide (EIPA) and colchicine (Colch). Figure 6 shows that insignificant effects were
observed for all investigated compounds with concentration of 50 ꢁM employing EIPA or
colchicine. PpIX generated by ALA was too weak to assess but it is not likely to be affected
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with EIPA or colchicine on preꢀtreatment. These results on first generation ALA dendrimers
35
contrast with our previous study, where preꢀtreatment with one of these inhibitors (EIPA)
significantly reduced PpIX generation.
0
1
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0
30
25
20
15
10
5
0
Without Inhibitor
With Colch.
With EIPA
ALA
10a
20a
25a
Dendritic ALA-HPOs
Figure 6. Effect of inhibitors on cellular uptake mechanism of ALA and dendritic
ALAꢀHPO. PpIX fluorescence produced by 50 ꢁM ALA or dendritic ALAꢀHPO 10a, 20a,
and 25a incubated for 4 h in MCFꢀ7R cells. Cells were preꢀtreated with or without colchicine
(100 ꢁM) or EIPA (100 ꢁM) inhibitors for 1 h before prodrugs incubation.
However in support of the present study, we have also investigated a dendrimer
55
containing six ALA residues, albeit in a macrophage cell line instead of cancer cells, where
we found that EIPA elicited little effect on porphyrin generation. In that study we used
another assay for endocytic uptake by examining the effect of incubation at a lower
temperature (18C), which inhibits transport between endosomes and lysosomes, and observed
a reduction in porphyrin generation by a factor of two. The first generation dendrimers
studies in this work have a similar size therefore active endocytic uptake may therefore be
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important but further work including the temperature dependence is needed since both
passive and active mechanisms may be relevant.
In another mechanistic study on dendrimer uptake the uptake mechanisms were reported
1
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1
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0
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56
to be dependent on the dendrimer generation (ie size). The in vitro cytotoxic and
intracellular oxidative stress responses to exposure to poly(propylene imine) dendritic
compounds of increasing generation (G0–G4) identified the threshold between the active
endocytic uptake of the larger poly(propylene imine) generations G3–G4, and passive uptake
of the smaller G0 and G1 dendrimers. The G2 dendrimers exhibited intermediate behaviour
with a contribution of both active and passive uptake.
CONCLUSIONS
Although ALA is a useful agent for PDT studies, it is important to improve its conversion
efficiency into protoporphyrin IX in order to optimize the phototherapeutic response. The
synergistic combination of iron chelating agents with ALA offers a means to improve
ALAꢀPDT efficiency by increasing cellular PpIX accumulation. A series of ALAꢀHPO
dendrimers with an aromatic core with suitable spacers have been synthesized utilizing
convergent methodology to enable coꢀadministration of the ALA and HPO to cells. By this
methodology the number of ALA molecules, the size, and the molecular weight of the
dendrimers were precisely controlled. The concept of using dendrimers as carriers for the
delivery of ALA and HPO or other synergistic agents to tumour cells is a novel approach to
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ALAꢀPDT. The ALAꢀHPO dendrimers demonstrated higher efficacies for PpIX generation
than ALA and phototoxicities in two human cancer cell lines compared to ALA. In a further
modification, conjugation of methionine at the Nꢀterminus of the ALA moieties in the
dendrimers amplified PpIX generation. In future the use of other cleavable substituents at the
0
1
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0
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0
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7
Nꢀterminus could be investigated. In conclusion ALAꢀHPO dendrimers provide a promising
means for enhancing the efficacy of ALAꢀPDT.
EXPERIMENTAL SECTION
1
13
General. H and C Nuclear Magnetic Resonance spectra were recorded on Bruker
Avance 400 or Bruker Avance 500 spectrometer. Chemical shifts are quoted in ppm
measured downfield relative to TMS. Ultravioletꢀvisible spectra were recorded on a
Unicam UV2 spectrometer (PerkinꢀElmer, Beaconsfield, UK) in dichloromethane solution.
Electrospray ionization (ESI) mass spectrometer (Quattro Premier XE, Micromass
Technologies) coupled to High Performance Liquid Chromatograpy (HPLC, Waters/AcQuity
Ultrapreformance LC, Waters, Manchester, UK). Analytical thin layer chromatography was
carried out using aluminiumꢀbacked, silicaꢀcoated plates (VWR, Germany) which were
visualised using ultraviolet light (254nm). Column chromatography was carried out using
f
Alfa Aesar silica gel (220ꢀ440 mesh flash grade. R values are quoted using the same solvent
system as used for column chromatography unless otherwise stated. HPLC was performed on
a 5 C18, 250×2.1 mm column attached to a Agilent instrument (Agilent, Life Sciences &
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Chemical Analysis Group, UK). All the chemicals were purchased from Aldrich, fisher and
Alfa Aesar and used without further purification unless otherwise stated; where stated,
solvents were purified according to standard laboratory procedures. HPLC purification of the
final compounds performed on both semiꢀpreparative Agilent systems 1100 (Life Sciences &
Chemical Analysis Group UK). The semiꢀpreparative system consisted of G1367A
QuatPump and collector 1330B compartment, the column was Hypersil ODS C18 HPLC
(Thermo Scientific, UK). The method is described under the synthetic sections for the final
compounds.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The purity of the final compounds was analysed with the Agilent analytical systems
1100 (Life Sciences & Chemical Analysis Group UK). The analytical system consisted of a
G1311A QuatPump fitted with an internal vacuum degasser, a WPSꢀ300SL autosampler
equipped H3BDSC10ꢀH column compartment. The separations were performed on a Gemini
5ꢁ C18 H3BDSC10ꢀH column, 100 mm × 2.1 mm (Phenomenex, UK), equipped with a
Security Guard C18 4 mm × 2.1mm, guard column (Phenomenex, UK), at 35 ± 0.1 °C. The
mobile phase consisted of 0.05% formic acid in 98% water (solvent A) and 0.05% formic
acid in 98% methanol (solvent B). A gradient elution for the mobile phases A and B was
carried out in 10 min. Isocratic run was carried out with 0% B for 4 min then gradient from
0ꢀ100% up to 8 min then reconditioning the column to 0% solvent B. The flow rate was
0.2 mL/min and the elution was monitored at the wavelengths (268 nm and 285 nm). The
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6
purity of the products was between 90ꢀ95% for the final compounds 10a, 20a, 25a, 29a and
30a.
Synthetic Procedures.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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0
1
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0
Tris(3ꢀacetoxypropyl) nitromethane (3). Tris(3ꢀhydroxypropyl) nitromethane (2) (10g,
42mmol) was dissolved in pyridine (50mL). Acetic anhydride was added (25mL) to the
mixture and stirred at room temperature for 18h. The reaction mixture was quenched with
water and stirred for 30 min. The solvents were evaporated and the residue was dissolved in
DCM and washed with sodium carbonate then water. The solvents were evaporated under
vacuum to yield product as an oil (14.2g, 95% yield). No further purification was required.
1
3
H NMR (CDCl , 400MHz) δ: 1.53 (m, 6H, 3CH
2
), 1.97 (m, 6H, 3CH
2
), 2.02 (s, 9H,
1
3
3
COCH
62 ([M+H] ), 384 ([M+Na] ).
Tris(3ꢀacetoxypropyl) aminomethane (4). Compound 3 (10 g, 27.7 mmol) was dissolved
3 2
), 4.03 (m, 6H, 3CH ); C NMR δ: 20.9, 24.0, 32.4, 63.6, 93.3, 171.0. ESIꢀMS: m/z
+
+
3
in ethanol. Raney nickel was added and the mixture was stirred at room temperature for 24 h.
The catalyst was filtered off and the solvent was evaporated in vacuum to give an oily
1
product (8.4 g, 94% yield). H NMR (CDCl , 400MHz) δ: 1.36 (m, 6H, 3CH ), 1.57 (m, 6H,
3
2
13
3CH
2
), 2.00 (s, 9H, 3COCH
3
2
), 4.04 (m, 6H, 3CH ). C NMR δ: 21.8, 22.9, 36.0, 52.9, 64.6,
+
+
171.2. ESIꢀMS: m/z 332 ([M+H] ), 354 ([M+Na] ).
ꢀ(3ꢀAcetoxypropyl)ꢀ4ꢀ(3ꢀ((tertꢀbutoxycarbonyl)amino)propanamido)heptaneꢀ1,7ꢀdiyl
diacetate (5). To a solution of compound 4 (10 g, 30.2 mmol) in THF (100 mL) was added
4
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Bocꢀβꢀalanine (6.8 g, 36 mmol), DCC (6.8 g, 33 mmol), HOBt (4.5, 33 mmol), and Bmim
(6.6 g, 30 mmol). The mixture was stirred under argon at room temperature for 24 h. The
precipitate was filtered off and solvent was evaporated in vacuum to yield an oily residue.
The residue was dissolved in DCM and washed with dilute hydrochloric acid, saturated
sodium hydrogen carbonate, and water successively. The solvent was dried over anhydrous
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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0
1
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7
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0
1
2
3
4
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9
0
sodium sulfate, evaporated in vacuum then subjected to column chromatography on silica gel
1
using ethyl acetate/methanol (9:1) (12.4 g, 82% yield). H NMR (CDCl
3
, 400MHz) δ: 1.29
), 2.19 (m, 2H,
CH ), 3.07 (m, 2H, CH ), 4.03 (m, 6H, 3CH ); C NMR δ: 21.5, 22.9, 23.4, 27.0, 28.8, 35.9,
(
2 3 2 3
m, 6H, 3CH ), 1.36 (s, 9H, 3CH ), 1.60 (m, 6H, 3CH ), 2.01 (s, 9H, 3COCH
1
3
2
2
2
+
+
5
3.4, 63.0, 155.9, 171.2, 172.8. ESIꢀMS: m/z 503 ([M+H] ), 525 ([M+Na] ).
Tertꢀbutyl (3ꢀ((1,7ꢀdihydroxyꢀ4ꢀ(3ꢀhydroxypropyl)heptanꢀ4ꢀyl)amino)ꢀ3ꢀoxopropyl)
carbamate (6). Compound 5 (5 g, 9.96 mmol) was dissolved in methanol (50 mL) in iceꢀbath
at 0 ꢀ 4 °C. Sodium hydroxide solution (2M, 100mL) was added, the mixture was stirred for
15 min. The alkaline hydrolysis was confirmed with TLC in ethyl acetate and methanol (9:1).
®
The solution was neutralized with Amberlite IR120 to pH 7 and the solvents were
1
evaporated to give a yellow paste. (3.46 g, 92% yield). H NMR (CDCl , 400MHz) δ: 1.29
3
(
m, 6H, 3CH
2
), 1.36 (s, 9H, 3CH
3
2 2
), 1.55 (m, 6H, 3CH ), 2.21 (m, 2H, CH ), 3.07 (m, 2H,
1
3
CH
2
), 3.33 (m, 6H, 3CH
2
); C NMR δ: 26.0, 28.8, 39.5, 36.0, 37.3, 57.8, 61.9, 78.1, 156.0,
+
+
1
70.2. ESIꢀMS: m/z 377 ([M+H] ), 399 ([M+Na] ).
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