Photocaged permeability: A new strategy for controlled drug release

Article abstract of DOI:10.1039/c2cc30819c

Light is used to release a drug from a cell impermeable small molecule, uncloaking its cytotoxic effect on cancer cells.

Full text of DOI:10.1039/c2cc30819c

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COMMUNICATION
Photocaged permeability: a new strategy for controlled drug releasew
M. Michael Dcona,ab Deboleena Mitra,ab Rachel W. Goehe,bc David A. Gewirtz,bc
Deborah A. Lebmanbd and Matthew C. T. Hartman*ab
Received 5th February 2012, Accepted 7th March 2012
DOI: 10.1039/c2cc30819c
Light is used to release a drug from a cell impermeable small
molecule, uncloaking its cytotoxic effect on cancer cells.
EDANS (Fig. 2), a small fluorophore, chosen because it contains
a sulfonic acid moiety known to hinder cellular entry.22 To
connect Dox to EDANS we utilized a light-cleavable nitroveratryl23
linker. The nitroveratryl moiety has been used previously as a
photocaging group for a wide variety of biomolecules.24
Off-target toxicity plagues conventional cancer chemotherapy. One
strategy to enhance selectivity of anti-cancer drugs involves un-
masking the cytotoxicity of a molecule in the vicinity of the tumor.1
This type of activation can be mediated by enzymes,2,3 changes in
pH,4 or exogenous factors such as temperature5 or light.6–8
Light is an ideal external stimulus since it provides a broad
range of adjustable parameters9 that can be optimized for
biological compliance. Several approaches that use light for
biomolecular activation have been reported.10–15 One established
method to enable selectivity of drug action using light is photo-
dynamic therapy (PDT). In PDT,16,17 light activation of a
photosensitizer generates cytotoxic singlet oxygen killing only
illuminated cells. PDT is currently used in several types of
malignancies16 including skin, lung, esophageal, bladder, head
and neck, and prostate cancer. A related strategy, photochemical
internalization,18,19 also uses photosensitizers. In this case, the
photosensitizers are used to release macromolecular cytotoxins
from endosomes, enabling their entry into the cytosol. However,
these approaches suffer from disadvantages,20 including unpre-
dictable drug uptake rates, the limited diffusion and lifetime of
1O2, and the requirement for moderate levels of O2 which may
not always be available in the tumor environment.
Fig. 1 Photocaged permeability strategy for drug delivery.
The synthesis (Scheme 1) began with commercially available
nitroveratryl carboxylic acid (1). The N-hydroxy succinimide
ester was prepared followed by coupling with propargylamine
to give amide (2). This compound was converted to the
p-nitrophenyl carbonate which was then treated with doxo-
rubicin3 to generate photocaged doxorubicin carbamate (3).
EDANS was coupled with azido benzoic acid (5) and was
finally attached to the photocage using click chemistry to
generate the final Dox-EDANS conjugate (6).
In this communication, we report a new light-targeted drug
delivery system, which operates independently of the creation
of 1O2. The basis of the system is the attachment of a cell
impermeable small molecule to a drug via a linker that can be
removed in presence of light, allowing cellular entry (Fig. 1).
We call this new strategy photocaged permeability (PCP).21
More specifically, we report the controlled release of the anti-
cancer drug, doxorubicin (Dox) (Fig. 2), upon illumination.
To prevent entry of Dox in the dark we attached Dox to
Fig. 2 (a) The anticancer drug doxorubicin. (b) Cell impermeable
EDANS.
a Department of Chemistry, Virginia Commonwealth University
(VCU), 1001 West Main Street, P. O. Box 842006, Richmond,
VA 23284, USA. E-mail: mchartman@vcu.edu
The photolytic release of doxorubicin from the drug conjugate
was analyzed using HPLC. The drug conjugate was dissolved in
PBS buffer and was exposed to UV light at 365 nm (9.0 mW/cm2).
Aliquots of the reaction mixture were collected at various times
and were analyzed on RP-HPLC. During the course of the
reaction, the peaks corresponding to the Dox-EDANS conjugate
disappeared with concomitant increase in the intensity of a peak
with the same retention time as Dox (See ESI Fig. S3w), confirm-
ing time-depended drug release in the presence of light.
b Massey Cancer Center, Virginia Commonwealth University,
401 College Street, Richmond, VA 23298, USA
c Department of Pharmacology and Toxicology, VCU, Richmond,
VA 23298, USA
d Department of Microbiology and Immunology, VCU, Richmond,
VA 23298, USA
w Electronic supplementary information (ESI) available: Details of
synthesis and characterization of the photocaged drug conjugate,
photolytic drug release studies, cytotoxicity studies, confocal and flow
cytometry data. See DOI: 10.1039/c2cc30819c
c
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Fig. 3 (A). The flow cytometry histogram displays the relative fluorescence intensity of untreated controls in the dark (blue) or light (red) or
JH-EsoAd1 cells treated with EDANS-Dox in the dark (orange) or light (green). (B) Quantification of Dox fluorescence for the indicated treatment
conditions. Data are representative of two independent experiments, N = 9. (C) Representative confocal images of JHEsoAd1 cells treated with
EDANS-Dox in the dark or light. The red channel shows Dox fluorescence, the gray channel is a DIC image, and the overlay represents the cellular
localization of Dox fluorescence.
With permeability enhancement established, we proceeded
to investigate the extent to which the release of Dox with light
would lead to enhanced cellular toxicity. Indeed, increased
illumination lead to decreased survival as measured by an
MTT assay (Fig. 4). Survival of cells treated with EDANS-Dox
in the dark was equivalent to controls with no drug added.
Moreover, treatment with light alone at the same dose was not
cytotoxic (See ESI, Fig. S5w).
Scheme 1 Synthesis of photocaged-cell impermeable drug conjugate.
Reagents and conditions: (a) NHS, EDC-HCl; (b) propargylamine, Et3N;
(c) bis p-nitrophenylcarbonate, Et3N; (d) doxorubicin-HCl, Et3N; (e)
Fig. 4 Light-dependent cytotoxicity of Dox-EDANS. Cells were treated
NHS, EDC-HCl; (f) EDANS, EtN(iPr)2; (g) CuSO4Á5H2O, sodium
with 10 mM of Dox-EDANS with (solid diamonds) or without (open
squares) light for the specified times. After 2 h, the cells were washed
with fresh media and allowed to grow for 72 h. The fraction of surviving
cells was evaluated using the absorbance of the formazan product of
MTT reduction. Error bars denote one standard deviation from the
mean.
ascorbate, tris-(benzyltriazolylmethyl)amine, DMSO : water (1 : 1).
We then investigated whether the attachment of EDANS
to Dox via the veratryl linker would enable drug delivery.
JH-EsoAd1 cells, a Barrett’s esophagus associated adenocarci-
noma cell line,25 were incubated with Dox-EDANS in the dark
or with illumination. Cell permeability was measured with flow
cytometry; upon illumination a significant enhancement of
cellular Dox fluorescence was observed (Fig. 3A–B). This enhance-
ment was mirrored in confocal studies with the same cell line
(Fig. 3C)—only light-treated cells show significant Dox fluores-
cence in the nucleus, where it is known to accumulate.26
Finally, we sought to compare the concentration dependence
for our method vs. Dox alone. We first measured the IC50
of Dox alone with the JH-EsoAd1 cells and found it to be
1.0 Æ 0.4 mM (see ESI Fig. S6w). As expected by PCP,
EDANS-Dox was not toxic to the cells in the dark at the
highest value tested (16 mM). However, illumination of
c
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EDANS-Dox (365 nm, 9.0 mW/cm2) leads to cytotoxicity with
an IC50 of 1.6 Æ 1.0 mM (Fig. 5), comparable to that of Dox
alone. Based on our FACS study (Fig. 3), we deduce that this
enhanced cytotoxicity is caused by efficient light-stimulated
release of free Dox from the impermeable EDANS.
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Fig. 5 Concentration-dependent, light-stimulated cytotoxicity. Cells
were treated with EDANS-Dox at various concentrations in the dark
(open squares) or with UV light for 20 min (black diamonds). After 2 h,
the cells were washed with fresh media and allowed to grow for 72 h.
The fraction of surviving cells relative to no drug controls was
evaluated using the absorbance of the formazan product of MTT
reduction. Error bars denote one standard deviation from the mean.
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permeability is blocked by a cell impermeable small molecule.
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In conclusion, we have developed a new and efficient
strategy for drug release based on photocaged permeability
(PCP). In this first report, we have focused on applying PCP to
the light-stimulated delivery of Dox into esophageal adeno-
carcinoma cells. In principle, the PCP approach could be
applied to any small, cell permeable molecule that has a free
amine, hydroxyl, or carboxylic acid group for attachment of
the veratryl-EDANS molecule. Further experiments will focus
on use of other light-scissile linkers that can operate at longer
wavelengths that are able to penetrate farther into tissues, as
well as the use of other molecules that block permeability.
The authors acknowledge financial support from the
American Cancer Society (IRG-73-001-34) and the Massey
Cancer Center. Flow cytometry and confocal microscopy were
supported in part by grants P30CA16059 and 5P30NS047463.
We thank Dr M. Bertino (VCU) and M. Foussekis (VCU)
for help in measuring light intensity, Dr J. Eshleman (Johns
Hopkins) for the JH-EsoAd1 cells, and Dr A. Cropp (VCU)
for helpful comments.
Notes and references
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4755–4757 4757