A versatile δ -aminolevulinic acid (ALA)-cyclodextrin bimodal conjugate-prodrug for PDT applications with the help of intracellular chemistry

Article abstract of DOI:10.3762/bjoc.10.251

Grafting of aδ-aminolevulinic acid (1) moieties on the narrow periphery of a β-cyclodextrin (β-CD) derivative through hydrolysable bonds was implemented, in order to generate a water-soluble, molecular/drug carrier with the capacity to undergo intracellular transformation into protoporphyrin IX (PpIX), an endogenous powerful photosensitizer for photodynamic therapy (PDT). The water-soluble derivative 2 was prepared by esterifying aδ-azidolevulinic acid with heptakis(6-hydroxyethylamino-6-deoxy)-β- cyclodextrin, with an average degree of substitution, DS = 3. Delivery of water-soluble, colorless 2 to cells resulted in intense red fluorescence registered by confocal microscopy, evidently due to the engagement of the intracellular machinery towards formation of PpIX. Conjugate 2 was further complexed with a fluorescein-labeled model guest molecule which was successfully transported into the cells, thereby demonstrating the bimodal action of the derivative. The present work shows the versatility of CDs in smart applications and constitutes advancement to our previously shown PpIX-β-CD conjugation both in terms of water solubility and lack of aggregation. lack of aggregation.

Full text of DOI:10.3762/bjoc.10.251

A versatile δ-aminolevulinic acid (ΑLA)-cyclodextrin bimodal
conjugate-prodrug for PDT applications with the help of
intracellular chemistry
Chrysie Aggelidou, Theodossis A. Theodossiou*§, Antonio Ricardo Gonçalves,
Mariza Lampropoulou and Konstantina Yannakopoulou*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 2414–2420.
Institute of Advanced Materials, Physicochemical Processes,
Nanotechnology & Microsystems, National Center for Scientific
Research “Demokritos”, Patriarchou Gregoriou & Neapoleos, Aghia
Paraskevi Attikis, 15310 Greece. Tel. +30210 6503796, Fax: +30 210
doi:10.3762/bjoc.10.251
Received: 10 July 2014
Accepted: 25 September 2014
Published: 17 October 2014
6511766
Email:
This article is part of the Thematic Series "Superstructures with
cyclodextrins: Chemistry and applications II".
Theodossis A. Theodossiou* -
Theodossis.Theodossiou@rr-research.no;
Konstantina Yannakopoulou* - dyanna@chem.demokritos.gr
Guest Editor: G. Wenz
*
Corresponding author
© 2014 Aggelidou et al; licensee Beilstein-Institut.
License and terms: see end of document.
§
Current address: Department of Radiation Biology, Institute for
Cancer Research, The Norwegian Radium Hospital, Montebello, 0379
Oslo, Norway
Keywords:
cyclodextrins; PDT; protoporphyrin IX; prodrug; δ-aminolevulinic acid
Abstract
Grafting of δ-aminolevulinic acid (1) moieties on the narrow periphery of a β-cyclodextrin (β-CD) derivative through hydrolysable
bonds was implemented, in order to generate a water-soluble, molecular/drug carrier with the capacity to undergo intracellular
transformation into protoporphyrin IX (PpIX), an endogenous powerful photosensitizer for photodynamic therapy (PDT). The
water-soluble derivative 2 was prepared by esterifying δ-azidolevulinic acid with heptakis(6-hydroxyethylamino-6-deoxy)-β-
cyclodextrin, with an average degree of substitution, DS = 3. Delivery of water-soluble, colorless 2 to cells resulted in intense red
fluorescence registered by confocal microscopy, evidently due to the engagement of the intracellular machinery towards formation
of PpIX. Conjugate 2 was further complexed with a fluorescein-labeled model guest molecule which was successfully transported
into the cells, thereby demonstrating the bimodal action of the derivative. The present work shows the versatility of CDs in smart
applications and constitutes advancement to our previously shown PpIX-β-CD conjugation both in terms of water solubility and
lack of aggregation.
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Introduction
Porphyrins have long been used as agents for tumor photodiag- guest molecule for intracellular transport, (ii) positively charged
nosis (PDD) and photodynamic therapy (PDT) because of their secondary and primary amino groups at physiological pH thus
preferential accumulation in cancerous rather than squamous able to interact with negatively charged components of the cell
tissues [1]. δ-Aminolevulinic acid (1, ALA, Scheme 1) is a membranes and achieve cell penetration [11] and (iii) cleavable
δ-amino acid precursor in the cellular biosynthesis of heme [2]. ester bonds expected to release 1 by the action of intracellular
enzymes. Taken together, the strategy would combine prodrug
photosensitizer capability with molecular/drug transport in a
single versatile bimodal system.
Results and Discussion
Synthesis: The suitable β-CD derivative to be linked with ALA
(
1) was heptakis(6-hydroxyethylamino-6-deoxy)-β-CD (4,
Scheme 2), obtained from heptakis(6-bromo-6-deoxy)-β-CD 3
and ethanolamine in high yield [12]. The choice of 4 was based
on the presence of readily accessible primary terminal hydroxy
groups on its narrow side, facilitating the formation of
hydrolysable ester bonds with 1. The resulting conjugate 2
Scheme 1: The structure of δ-aminolevulinic acid (1), a precursor in
cellular biosynthesis of protoporphyrin IX and subsequently heme.
The penultimate step in this biosynthetic cycle is the iron chela- (Scheme 1), expected to be water soluble, comprises seven sec-
tion of protoporphyrin IX (PpIX), a porphyrin photosensitizer ondary and multiple primary (maximum seven) amino groups in
(
PS) which was shown to be very efficient for PDT [3]. Com- the final form. Initial attempts to directly couple compound 1
pound 1 and its more lipophilic esters, methyl and hexyl levuli- with the terminal hydroxy groups in 4 consistently failed due to
nate, have been approved for clinical use as PpIX precursors the very high propensity of 1 to reversibly lactonize or dimerize
against several cancers such as basal cell carcinoma, bladder into dihydropyrazine derivatives (DHPY), which then irre-
cancer, actinic keratosis, etc. [4]. Compound 1 is a polar, versibly aromatize to pyrazines [13] thus resulting in various
zwitterionic compound, with inherently poor cell permeability side products that impede purification. Protection of the
and consequently challenging pharmaceutical formulations [5]. δ-amino group in 1 with the fluorenylmethyloxycarbonyl
We have recently published [6] the covalent conjugation of (Fmoc) group did not eventually lead to coupling products with
PpIX to β-cyclodextrin (β-CD), a water-soluble cyclic oligosac- 4. Furthermore, benzyloxycarbonyl (Ζ) protection of 1 and
charide host capable of carrying hydrophobic molecules, such subsequent successful coupling reaction with 4 gave the desired
as drugs, in its hydrophobic cavity [7]. The cyclodextrins (CDs) Z-protected coupling product which, however, resisted removal
are a notable family of semi-natural carbohydrate molecules of the Z protecting group under a variety of conditions [(i)
approved as pharmaceutical excipients that improve the solu- ΜeOH, Pd/C, H2, (ii) ΜeOH, formic acid, Pd/C, H2, (iii)
bility and bioavailability of drugs through molecular encapsula- ΜeOH, acetic acid, Pd/C, H2, (iv) ΜeOH, trifluoroacetic acid,
tion. We demonstrated [6] the conjugate’s (PpIX-CD) bimodal Pd/C, H2]. A successful alternative was subsequently adopted
action of transporting the chemotherapeutic agent tamoxifen comprising the linking of 4 to δ-azidolevulinic acid (8)
intracellularly to engage its targets while simultaneously acting (Scheme 2). The latter, having the nucleophilicity of the δ-pos-
as a PS. In this sense we created a bimodal system with ition silenced, presented a completely straightforward precursor
adequate water solubility (several μM) able to inflict significant to 1 and proved to be amenable to coupling with 4 under
chemo- and phototoxicity on cancer cells. One additional various reaction conditions. Briefly [14], 8 was prepared from
benefit of tethering β-CD to PpIX, was the reduced aggregation levulinic acid (5) through δ-bromination and concomitant
of the conjugate in aqueous solutions. Formation of aggregates methyl ester formation (compound 6), followed by azidation to
is an adverse yet typical characteristic of porphyrins that 7. Finally, enzymatic cleavage of the ester [15] resulted in liber-
reduces their photodynamic efficiency, quenches their fluores- ation of the carboxylic acid functionality that is required for the
cence and complicates their delivery [8-10]. In the system coupling to the CD (Scheme 2, compound 8, Figure S1, Figure
presented herein, the problems of limited aqueous solubility as S2 in Supporting Information File 1).
well as that of aggregation are completely bypassed, as multiple
copies of 1 are attached to a β-CD derivative bearing hydroxy- The connection of 8 to 4 proceeded under standard coupling
ethylamino groups in all seven positions of the narrow opening. conditions (DCC, DMAP) [17] affording a water-soluble com-
Τhe resulting conjugate (Scheme 1, 2) will encompass (i) the pound. The 1H NMR spectrum of the product verified the pres-
wide β-CD opening available for inclusion of a hydrophobic ence of peaks arising both from 8 chain and 4 components,
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Beilstein J. Org. Chem. 2014, 10, 2414–2420.
Scheme 2: Synthesis of conjugate 2 i) Br2, Ph3P, DMF, 75–80 °C, 93% according to [16]; ii) 2-hydroxyethylamine, 80 °C, 76%, according to [12];
iii) Βr2, MeOH, rt, 41%; iv) NaN3, THF, 25–30 °C, 90%; v) phosphate buffer, pig liver esterase, pH 8, 25–30 °C, 82%; vi) DCC, DMAP, DMF,
25–30 °C, 53%; vii) H2, Pd/C, 25–30 °C, 95%.
while the relative broadness of the signals indicated incomplete/ (2104 cm−1, (Supporting Information File 1, Figure S5). The so
random substitution, allowing, however, the assignment of obtained conjugate 9 was subsequently reduced under strongly
peaks via the HSQC-edited spectrum (Supporting Information acidic catalytic hydrogenation conditions to 2 in nearly quanti-
File 1, Figure S3). The ratio 8 to 4 was ~3, as indicated by inte- tative yield.
gration of the respective signals and also confirmed by
MALDI–TOF MS measurements (m/z 1853, Supporting Infor- Crucial to the spectroscopic characterization of the product was
mation File 1, Figure S4), whereas the IR spectrum exhibited the 13C NMR spectrum (Figure 1) where the ester and ketone
the characteristic stretching vibrations of the azido group carbonyl signals were observed along with the signals of the
Figure 1: 13C NMR spectrum of 2 (D2O, 125 MHz) with assignment of the peaks.
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Beilstein J. Org. Chem. 2014, 10, 2414–2420.
β-CD moiety. Moreover, in the IR spectrum of 2 (Supporting tical conditions were analyzed with regard to the intensity of red
Information File 1, Figure S5) the azido-group band disap- color with the MATLAB software. 2D analysis and superimpo-
peared, whereas prominent peaks were observed in the carbon- sition of the 1D images along the x-axis (Supporting Informa-
yl region. Finally the mass spectrum confirmed the identity of tion File, Figure S7) revealed that the intensity of red fluores-
the product, which maintained an average molecular weight cence was measurably higher in both images taken from cells
corresponding to ~3 δ-aminolevulinic acid moieties attached. after treatment with 2 than in those treated with 1, irrespective
of the laser intensity applied. Incubation with compound 1 and
Cell studies with 2: The capability of the conjugate to cross use of 100% laser intensity resulted in only small enhancement
cell membranes and to produce PpIX intracellularly was exam- of red fluorescence.
ined. MCF7 cells were incubated with the colorless conjugate
solution for 4 h, to allow conversion of prodrug 1 into PpIX These qualitative results show that the conjugate 2 worked
[
5,18]. Indeed, upon confocal microscopy imaging of the much more efficiently than 1 alone, either due to the multi-
treated cells (λex = 568 nm), intense red fluorescence was plicity of 1 attached and/or due to more efficient cell penetra-
observed (Figure 2), demonstrating that either (i) cell internal- tion. What can be, however, deduced from the images in
ization was accompanied by esterolysis and release of 1 which Figure 2 and Figure S7 is that although the 1-treated cells were
was subsequently converted into PpIX, or (ii) one molecule of 2 imaged at more than 3-times the laser power used for the
with five molecules of 1 produced PpIX-(mono)CD or (iii) two 2-treated cells, the latter exhibited a much more intense PpIX
molecules of 2 interacted with two molecules 1 to form PpIX- fluorescence. This indicates that it is not the 3×δ-aminole-
(
bis)CD. The first of these three scenarios is the most probable vulinic acid molecular multiplicity of 2 that is solely respon-
given the abundance of intracellular esterases, which would sible for the PpIX fluorescence enhancement, but either an
engage into activity as with the approved and marketed augmented cell entry or the inhibition of ferrochelatase-
lipophilic esters of 1 [5], however, the other two possibilities catalyzed iron insertion to form non-fluorescent iron-complexed
cannot be excluded. The red fluorescence of cells incubated heme [19] (possibly due to the existence of PpIX-CD moieties).
with 2, evidently originating from PpIX formation, was more These suggestions as well as quantitation of subcellular
intense in comparison with the flurorescence from cells treated processes (hydrolysis rate of 1, PpIX concentration) could only
with the same concentration of 1·HCl. Specifically, images be confirmed with specific mechanistic biological studies,
taken from cells that were treated with either 1 or 2 under iden- which lie outside the scope of the present work.
Figure 2: Representative confocal microscopy images of MCF7 cells incubated with 1 mM 2 (a, b and c), and 1 mM 1 (d, e, f), (λex = 568 nm,
λem > 585 nm). The laser intensity used for the acquisition of images a, b, c was 30% of that used for images d, e and f.
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Beilstein J. Org. Chem. 2014, 10, 2414–2420.
We further incubated MCF7 cells with 2 pre-complexed with the system was exemplified by the intracellular formation of
water-insoluble fluorescein isothiocyanate-labeled 1-adamantyl- PpIX using “intracellular chemical synthesis”. At the same time
amine (Figure 3). The characteristic confocal microscopy 2 was able to carry inside the cytoplasm a model guest mole-
images revealed red fluorescence upon excitation at λex = cule. The possibility that the intracellular biosynthetic
5
68 nm, as well as green fluorescence upon excitation at 488 machinery may have produced some PpIX-CD species cannot
nm. Following superimposition of the two fluorescence chan- be ruled out. Overall, successful δ-aminolevulinic acid intracel-
nels it could be concluded that PpIX production and CD guest lular delivery with complete aqueous solubility, PpIX produc-
intracellular localization, largely coincided within the cell cyto- tion and transport of a model guest were achieved.
plasm (Figure 3). This indicates that possibly some of the
levulinic acid ester moieties in 2 not have undergone enzymatic Experimental
cleavage or hydrolysis but instead have proceeded as a conju- Ester of δ-azidolevulinic acid with heptakis(6-hydroxyethyl-
gate through the heme biosynthesis, resulting in the biosyn- amino-6-deoxy)-β-cyclodextrin (9). Heptakis(6-hydroxyethyl-
thesis of PpIX-CD species. This hypothesis, however, can only amino-6-deoxy)-β-cyclodextrin (4, 62 mg, 0.043 mmol) was
be confirmed by using 1-CD species, bearing no cleavable dissolved in dimethylformamide (DMF, 2 mL), cooled to 0 °C
tethers to the cyclodextrin core. Moreover, is well known that and stirred for 15 min. Then, 4-dimethylaminopyridine (DMAP,
β-CDs affect cells by interacting with their membrane compo- 3.7 mg, 0.0302 mmol) and Ν,Ν΄-dicyclohexylcarbodiimide
nents, namely depleting and enriching cholesterol in lipid raft (DCC, 68.6 mg, 0.332 mmol) were added, followed by
and non-raft regions as well as removing and redistributing δ-azidolevulinic acid (8, 52.2 mg, 0.332 mmol), which had
phospholipids [20]. Thus, the transport of an adamantyl-substi- previously been stirred in DMF (1 mL) for 6 h in the presence
tuted guest that blocks the β-CD cavity is anticipated to alter the of a KOH pellet. The mixture was stirred at rt for 48 h under
above host–membrane interactions. In this respect, the primary argon. Then it was centrifuged to remove insoluble side prod-
side enrichment of 1-CD with amino groups is expected to play uct dicyclohexylurea and the supernatant was collected and
a major role in the interaction with the anionic parts of phos- condensed to dryness. The solid residue was dissolved in a
pholipids, rather than the β-CD cavity and its encapsulation small volume of water and dialyzed from a benzoylated cellu-
ability of hydrophobic membrane components.
lose membrane (MWCO ~2000) for 24 h. The solvent of the
dialyzed solution was evaporated and the residue was
lyophilized, to afford an off-white solid with degree of substitu-
Conclusion
The present work demonstrates the synthesis and characteriza- tion ~3, (53%). 1H ΝΜR (500 ΜHz, D2O, 298 K) δ 5.16 (brs,
tion of the bimodal conjugate prodrug 2, bearing ~3 δ-aminole- 7H, CD-H1), 4.28 (s, 5H, H5), 3.96 (brs, 14H, CD-H3, CD-H5),
vulinic acid moieties per β-CD macrocycle. The dual nature of 3.78 (brs, 14H, CD-H8), 3.67 (brs, 14H, CD-H2, CD-H4),
Figure 3: Representative confocal fluorescence microscopy images of MCF7 cells after incubation with a solution of 2–fluorescein isothiocyanate-
labeled 1-adamantylamine (1 mM/0.2 mM) PBS: (a, d) red fluorescence (λex = 568 nm, λem > 585 nm); (b, e) green fluorescence (λex = 488 nm, λem =
522 nm); (c, f) overlay of images a, b and of d, e: yellow color indicates guest co-localization with PpIX core; (g) blank experiment of fluorescein iso-
thiocyanate-labeled 1-adamantylamine (0.2 mM in PBS, λex = 488 nm, λem = 522 nm ).
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Beilstein J. Org. Chem. 2014, 10, 2414–2420.
3
.36–2.77 (m, 28H, CD-H6, CD-H6΄, CD-H7), 2.72 (t, J = 7.0 with 1 mM of 1 or 2 or alternatively 2 (1 mM)/fluorescein iso-
Hz, 5H, H2), 2.47 (t, J = 7.0 Hz, 5H, H3), 2.13–1.06 (m, 1,3- thiocyanate-labeled 1-adamantylamine (0.2 mM) for 4 h. After-
dicycloxylurea byproduct) ppm; 13C NMR (62 ΜHz, CDCl3, wards the cells were washed with PBS and mounted on micro-
2
8
5
3
98 K) δ 207.4 (C4), 183.1–173.4 (C1), 101.7 (br, CD-C1), scope slides for confocal fluorescence live-cell imaging
2.91 (br, CD-C4), 71.3, 72.0, 69.9 (CD-C3, CD-C5, CD-C2), performed as described previously [21]. Intracellular fluores-
8.7 (CD-C8), 57.2 (C5), 50.29, (CD-C7), 48.70 (CD-C6,6΄), cein isothiocyanate-labeled 1-adamantylamine was excited
1.1 (C3), 27.0 (C2), 36.0–22.7 (1,3-dicycloxylurea byproduct) using the 488 nm line of an argon–krypton ion laser while the
ppm; IR ν: 2104.2 (vs) cm−1; MS (MALDI–TOF) m/z: 1853.7 PpIX fluorescence was excited with the 568 nm line of the same
(
(
100%, [M + Η]+); Exact mass calculated for C71H120N16O41 laser. Fluorescein isothiocyanate-labeled 1-adamantylamine
9): 1852.78.
fluorescence was collected through a band-pass filter centered
at 522 (± 35) nm, while PpIX fluorescence was collected after a
Ester of δ-aminolevulinic acid with heptakis(6-hydroxy- long-pass filter at ≥585 nm. In a control experiment cells were
ethylamino-6-deoxy)-β-cyclodextrin (2). To a solution of 9 incubated for 4 h with 0.2 mM ADA-FITC in PBS.
(
20 mg, 0.0112 mmol) in ethanol (1 mL), CF3COOH (250 μL)
was added dropwise followed by addition of Pd/C (7.5 mg,
.0071 mmol, 0.06 equiv). The mixture was hydrogenated with
Supporting Information
0
Η2 gas under stirring at rt for 48 h. Subsequently the catalyst
was removed by repeated centrifugation cycles and the solution
was evaporated to dryness, the residue re-dissolved in water and
dialyzed as above. The resulting solution was evaporated and
the residue lyophilized to obtain the desired solid product
Supporting Information File 1
Full experimental procedures and detailed analytical data
for the synthesis of all precursor molecules of Scheme 1;
additional NMR, IR and mass spectral data.
[http://www.beilstein-journals.org/bjoc/content/
(
95%). 1H ΝΜR (500 MHz, D2O, 298 K) δ 5.18 (brs, 7H,
supplementary/1860-5397-10-251-S1.pdf]
CD-H1), 4.13 (s, 5H, H5), 3.98 (brs, 14H, CD-H3, CD-H5),
3
3
5
.89 (brs, 14H, CD-H8), 3.78–3.57 (m, 14H, CD-H2, CD-H4),
.30 (brs, 28H, CD-H6, CD-H6΄, CD-H7), 2.92 (t, J = 6.5 Hz, Acknowledgements
H, H2), 2.73 (t, J = 6.5 Hz, 5H, H3) ppm; 13C NMR (62 MHz, Funding by the Marie Curie Initial Training Networks (FP7-
CDCl3, 298 K) δ 204.5 (C4), 175.2 (C1), 101.7 (CD-C1), 82.3 People-ITN-2008, Project No 237962 "CYCLON") is grate-
(
(
br, CD-C4), 71.8, 67.6 (CD-C3, CD-C5, CD-C2), 59.1, 56.5 fully acknowledged. Istituto di Ricerche Chimiche e
CD-C8, CD-C8), 52.5, 49.9 (CD-C7, CD-C7), 48.11 (CD- Biochimiche "G. Ronzoni" Milano, Italy, is also thanked for the
C6,6΄), 34.4 (C3), 27.1 (C2) ppm; MS (MALDI–TOF) m/z: MALDI–TOF measurements. We also thank A. Salapatas of our
685.2 (100%, [M + Νa]+), 1798.18 (89%, [M’ + Na]+); exact Institute for the analysis of images in Figure S7.
1
mass calculated for C66H119N9O39 ((1)2-CD = M): 1661.76 and
for C71H126N10O41 ((1)3-CD = M’): 1774.808.
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