Localized in vivo activation of a photoactivatable doxorubicin prodrug in deep tumor tissue

Article abstract of DOI:10.1111/php.12045

Sparing sensitive healthy tissue from chemotherapy exposure is a critical challenge in the treatment of cancer. The work described here demonstrates the localized in vivo photoactivation of a new chemotherapy prodrug of doxorubicin (DOX). The DOX prodrug (DOX-PCB) was 200 times less toxic than DOX and was designed to release pure DOX when exposed to 365 nm light. This wavelength was chosen because it had good tissue penetration through a 1 cm diameter tumor, but had very low skin penetration, due to melanin absorption, preventing uncontrolled activation from outside sources. The light was delivered specifically to the tumor tissue using a specialized fiber-optic LED system. Pharmacokinetic studies showed that DOX-PCB had an α circulation half-life of 10 min which was comparable to that of DOX at 20 min. DOX-PCB demonstrated resistance to metabolic cleavage ensuring that exposure to 365 nm light was the main mode of in vivo activation. Tissue extractions from tumors exposed to 365 nm light in vivo showed the presence of DOX-PCB as well as activated DOX. The exposed tumors had six times more DOX concentration than nearby unexposed control tumors. This in vivo proof of concept demonstrates the first preferential activation of a photocleavable prodrug in deep tumor tissue.

Full text of DOI:10.1111/php.12045

Photochemistry and Photobiology, 2013, 89: 698–708
Localized In Vivo Activation of a Photoactivatable Doxorubicin Prodrug in
Deep Tumor Tissue
1
2
3
3
3
4
Stuart Ibsen* , Eran Zahavy , Wolf Wrasidlo , Tomoko Hayashi , John Norton , Yongxuan Su ,
5
6
Stephen Adams and Sadik Esener
1
Department of Bioengineering, Moores Cancer Center, University of California San Diego, La Jolla, CA
Department of Infectious Disease, Israel Institute for Biological Research, Ness-Ziona, Israel
Moores Cancer Center, University of California at San Diego, La Jolla, CA
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA
Department of Pharmacology, University of California at San Diego, La Jolla, CA
Department of Nanoengineering, University of California at San Diego, La Jolla, CA
2
3
4
5
6
Received 3 October 2012, accepted 5 January 2013, DOI: 10.1111/php.12045
ABSTRACT
resection (3,4). The 85% positive outcome rate for Wilms tumor
treatment makes managing and reducing the chemotherapy-
related toxicities the main problem of this disease (3). These
toxicities occur because only a small fraction of the injected
chemotherapy dose ever reaches the tumor. The excess drug cir-
culates through healthy tissue resulting in detrimental side effects
Sparing sensitive healthy tissue from chemotherapy exposure
is a critical challenge in the treatment of cancer. The work
described here demonstrates the localized in vivo photoactiva-
tion of a new chemotherapy prodrug of doxorubicin (DOX).
The DOX prodrug (DOX-PCB) was 200 times less toxic than
DOX and was designed to release pure DOX when exposed
to 365 nm light. This wavelength was chosen because it had
good tissue penetration through a 1 cm diameter tumor, but
had very low skin penetration, due to melanin absorption,
preventing uncontrolled activation from outside sources.
The light was delivered specifically to the tumor tissue using
a specialized fiber-optic LED system. Pharmacokinetic studies
showed that DOX-PCB had an a circulation half-life of
(5). The biggest concerns are adverse effects to the gastrointesti-
nal tract (6) and damage to the hematopoietic system causing
suppression of the immune system (6). DOX exposure also
increases the chances of long-term cardiomyopathy and conges-
tive heart failure (2,7,8). These side effects are of particular con-
cern in children (9,10).
Reducing the amount of active DOX that reaches the heart
and healthy tissue has been shown to reduce these side effects as
®
demonstrated with Doxil , a liposomal formulation of DOX
1
0 min which was comparable to that of DOX at 20 min.
(11,12). However, major hurdles remain such as the slow release
DOX-PCB demonstrated resistance to metabolic cleavage
ensuring that exposure to 365 nm light was the main mode
of in vivo activation. Tissue extractions from tumors exposed
to 365 nm light in vivo showed the presence of DOX-PCB as
well as activated DOX. The exposed tumors had six times
more DOX concentration than nearby unexposed control
tumors. This in vivo proof of concept demonstrates the first
preferential activation of a photocleavable prodrug in deep
tumor tissue.
of DOX from the liposomes limiting the maximum levels of
bioavailable drug in the tumor over time (13), and nonspecific
®
accumulation of Doxil in healthy tissue (11,14). Radiofrequen-
cy heating (15) and focused ultrasound heating (16) are being
evaluated as triggers to cause preferential release of pure DOX
from lyso-thermosensitive liposomes specifically in the tumor
tissue. However, the liver and other healthy tissues where these
vehicles also accumulate will receive a dose of pure DOX once
the carriers eventually break down.
Currently there is no reliable way for a clinician to limit the
contact of active DOX to a single region within the body. This
research focuses on the development of a system where the clini-
cian can use light to highlight a defined region of tissue encom-
passing the tumor, such as the kidneys for Wilms tumor, creating
a zone where a photocleavable DOX prodrug is locally activated
to a therapeutic state. The light can be delivered with fiber-optic
and light emitting diode (LED) technology. The recent miniaturi-
zation of LEDs to as thin as a human hair (17) allows them to be
temporarily implanted anywhere a biopsy needle, endoscope or
catheter can go. They can also be left in the lesion site after
tumor resection. To respond to the delivered light we have devel-
oped a prodrug of DOX which uses a covalently bound photo-
cleavable linker (18) to render DOX 200 times less toxic to tissue
with the unique ability to restore full therapeutic function when
phototriggered (19). The prodrug, referred to as DOX-PCB, was
INTRODUCTION
Systemically administered chemotherapy is an important treat-
ment option used to reduce tumor mass before surgical removal
(
1,2) and for primary tumors that have not yet metastasized, such
as childhood nonmetastatic Wilms tumor (3). In these cases the
chemotherapy needs to be active only in the affected tissue. For
the treatment of Wilms tumor, doxorubicin (DOX) or a combina-
tion of dactinomycin and vincristine is usually administered
before or after the surgical removal of the tumor. The goal is to
either shrink the tumor to reduce the possibility of surgical com-
plications, or to kill any tumor margins left behind after the
*
Corresponding author email: stuart.ibsen@gmail.com (Stuart Ibsen)
2013 Wiley Periodicals, Inc.
Photochemistry and Photobiology © 2013 The American Society of Photobiology 0031-8655/13
©
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Photochemistry and Photobiology, 2013, 89 699
formed by blocking the free amine of the sugar moiety with a ni-
trophenyl compound conjugated to a short PEG linker and termi-
completely independent of the biochemical or environmental
properties of the tumor allowing the production of free DOX to
be limited to just the highlighted region. The light activation is
also independent of the biochemical variability in tumors
between patients (33) and can be applied to many different types
of solid tumors without requiring prior knowledge of individual
tumor biochemistry. The triggering wavelength of 365 nm light
was chosen because internal tissue has low absorption of
365 nm light (34), including DNA (35). The 365 nm light is also
heavily absorbed by melanin and so does not penetrate signifi-
cantly more than 1 mm through human skin. This ensures the
only significant amount of 365 light present in the body is that
delivered to the tumor region. The localized light delivery for the
in vivo experiments in this work was achieved by using a cus-
tom-designed 365 nm LED/fiber-optic system that physically
penetrated into the middle of the tumor itself. The high-intensity
light could allow for a burst release of DOX inside the tumor tis-
sue allowing the concentration to rise rapidly.
This study demonstrates the feasibility of using 365 nm light
to activate the DOX-PCB prodrug in vivo and cause preferential
accumulation of active DOX in the tumor tissue through three
sets of experiments.
The first set of experiments quantifies 365 nm light penetra-
tion through ex vivo tumor tissue and subsequent DOX-PCB acti-
vation using the custom-designed 365 nm LED/fiber-optic
delivery system.
nated with
a
biotin (PCB) as shown in Fig. 1. The
photocleavable linker was chosen to be resistant to metabolic
degradation preventing nonspecific activation of the prodrug in
healthy tissue, especially the liver (19,20). This limits activation
just to the highlighted tissue region. Systemically administered
prodrugs have been shown to substantially lower toxic effects on
healthy tissue where they are not activated (8,21,22).
The resistance of DOX-PCB to metabolic activation is differ-
ent from traditional prodrug designs which have relied heavily
on inherent chemical and biochemical properties of the tumor to
act as activation triggers. These triggers include environmental
differences between the tumor tissue and normal tissue, such as
hypoxia or low pH (23,24), enzymatic cleavage by enzymes that
are over secreted by the tumor (25–27) and antibody/tumor anti-
gen binding and internalization into the cell where activation is
usually triggered by the low pH of the intracellular endosomal
environment (28). The challenge is that these triggers often have
low contrast between the tumor and healthy tissue (29,30), espe-
cially with the normal enzymatic activity of the liver. Tumor
cells are also not homogenous in their gene expression (31) and
there can be significant differences between tumors from differ-
ent patients (32) making a standardized trigger difficult to
develop. All of these factors make developing a truly tumor-spe-
cific biochemical trigger a challenge.
The illumination of the tumor with the custom LED/fiber-
optic system provides a stark differentiation between the tumor
and the healthy tissue to address these challenges, as the light is
found no where else in the body. The light activation trigger is
The second set of experiments look at the circulation half-life
and elimination of DOX-PCB in mice to determine the proper
time for 365 nm light administration to prevent activation of
DOX-PCB in the vasculature.
The third set of experiments look at the in vivo activation of
DOX-PCB within tumor tissue after systemic administration of
DOX-PCB.
MATERIALS AND METHODS
Materials. Doxorubicin hydrochloride (DOX) was obtained from Qventas
(
Branford, CT) and Sigma (St. Louis, MO). Water soluble photocleavable
biotin-NHS (PCB) was purchased from Ambergen (Watertown, MA).
The components of the synthesis buffer, NaCHO and NaOH, were from
3
EMD (San Diego, CA). High-pressure liquid chromatography (HPLC)
grade acetonitrile and methanol were purchased from Fisher Scientific
(
Fairlawn, NJ). The athymic nu/nu nude mice were purchased from the
Jackson Laboratory (Bar Harbor, ME). The human lung cancer cell-line
A549 was purchased from the American Type Culture Collection
®
(
ATCC) (Manassas, VA). The Captisol was purchased from Ligand
Pharmaceuticals (La Jolla, CA).
Preparation of DOX-PCB. Synthesis of DOX-PCB was preformed as
described in Ibsen et al. (19). The DOX-PCB was then dissolved in
5
0 lL of DMSO and was then purified on a semiprep HPLC system
using a 10–90 ACN:H O 0.05%TFA gradient set over 20 min. The
2
resulting fractions containing the two DOX-PCB isomers were collected
and pooled into the same sample. The sample was then lyophilized and
stored at À20°C wrapped in foil. A study was conducted that found no
difference in biological activity on A549 cells in vitro between the two
isomeric forms (data not shown) so all the studies presented here have
the two isomers pooled together.
3
65 nm LED/fiber-optic delivery system. A custom designed 365 nm
LED/fiber-optic delivery system was developed in collaboration with
Prizmatix Ltd. that coupled a fiber optic to a 365 nm LED. The end of
the fiber was encased inside a beveled metal tube that was the equivalent
of a 22 gauge needle to allow for tumor penetration as shown in Fig. 2.
Tumor preparation. A549 cells were expanded in 25 mL flasks. They
were trypsinized and washed with cold PBS. They were then resuspended
Figure 1. Chemical structure of DOX-PCB with the three products
8
À1
in cold PBS at a concentration of 1 9 10 cells mL . 1.2 mL of the
cell suspension was added to 1.2 mL of matrigel and kept on ice.
resulting from photocleavage including pure DOX. The molecular weight
À1
of DOX-PCB is 1221.44 g mol
.

7
00 Stuart Ibsen et al.
Fiber Optic
Embedded
B
C
LED
2
2 Gauge
A
Housing
Needle
Fiber
Optic
Power
Supply
Cable
Figure 2. The 365 nm LED/fiber-optic delivery system. (A) The power supply and the LED housing are shown with the fiber-optic cable. As 365 nm
light is not directly visible, the end of the cable was placed on a piece of white paper to show the blue autofluorescence of the paper caused by the
365 nm light emitted from the end of the fiber. (B) An expanded view of the fiber-optic embedded in a 22 gauge needle without the 365 nm light. (C)
This expanded view shows the 365 nm light coming from the end of the needle causing secondary autofluorescence of the paper which is visible as the
blue light.
2
00 lL of the mixture was injected in the left and right flanks of five
lyophilized DOX-PCB powder was dissolved in 500 lL of methanol and
dried in a speedvac. The dried DOX-PCB was then dissolved in 50 lL
of DMSO. This was also dried down in a speedvac but only until it was
female athymic nu/nu nude mice. One of the two tumors in the same
mouse would serve as a control and would not receive a 365 nm light
dose. The tumors were allowed to grow for 1 month until they reached
the size of 1 cm . Mice were maintained under standard conditions in the
University of California, San Diego Animal Facility. All animal protocols
received prior approval by the institutional review board.
3
in an almost dry tacky state. 10 lL of a NaHCO /NaOH buffer at pH 9
3
was added. The entire mixture was then dissolved in 500 lL of methanol
and put in a small agate mortar. The mortar was placed under low
vacuum for at least 5 h to evaporate the methanol. The 10 lL of a
Ex vivo 365 nm light penetration of tumor tissue. A Newport 1830-C
Optical Power Meter with a Newport 818-UV detector head and a New-
port 883-UV OD3 filter was used to measure the intensity of light com-
ing from the 365 nm LED/fiber-optic system turned to max power.
Measurements were taken 10 mm directly in front, from the right side
and from behind the fiber end. To measure tumor tissue penetration and
scattering of the light, the system needle was inserted into the middle of
a 9 9 6 9 5 mm tumor prepared in the manner stated above that was
collected from a mouse never injected with DOX-PCB or DOX. The
measurements were taken again from a distance of 10 mm from the
tumor surface.
3
NaHCO /NaOH buffer helped to change the pH as the methanol evapo®-
rated ionizing the DOX-PCB to assist in the formation of the Captisol
complex.
After the methanol was evaporated from the mortar, 80 mg of Capti-
®
sol brand cyclodextrin dry powder was added and mechanically ground
with the dried DOX-PCB with the small pestle until a fine purple colored
powder was formed. 200 lL of sterile saline was added and then solution
bath sonicated for 10 min. The maximum concentration achieved of the
®
DOX-PCB in the Captisol solution was 2 mM. The samples were centri-
fuged at 17 500 g for 5 min to eliminate any precipitated DOX-PCB.
The concentration of DOX-PCB in the supernatant was then measured
using absorbance on a NanoDrop ND-1000 spectrophotometer (Thermo
Scientific, Wilmington, DE, USA).
DOX-PCB activation in ex vivo tumor tissue. An ex vivo experiment
was run to establish the activatability of DOX-PCB in an actual tumor
sample prepared in the manner stated above that was collected from a
mouse never injected with DOX-PCB or DOX. The tumor was cut into
three sections of 30 mg each. The first sample was injected with 10 lL
DMSO containing 224 ng of DOX-PCB and 20 ng of DOX to ensure
that a small amount of DOX would be detectable. The other two samples
were injected with 10 lL DMSO containing just 224 ng of DOX-PCB.
One of these samples was exposed to 30 min of 365 nm light using the
LED/fiber-optic system described above. The tissue was allowed to
absorb the DMSO for 15 min giving the DMSO enough time to penetrate
deep into the sample carrying the dissolved compounds with it. The
tissue integrity was not compromised by the DMSO.
DOX-PCB blood circulation half-life and urine elimination. Two
®
female nude mice were injected with 200 lL of the DOX-PCB/Captisol
solution. Blood samples were collected at 5, 15, 30, 60 min and at 24 h.
The blood was centrifuged and 15 lL of serum was collected. Urine was
collected when naturally possible from the mouse out to 24 h.
Serum extraction method. 30 lL of acetonitrile (ACN) was added to
the collected 15 lL blood serum samples from each time point. The
samples were bath sonicated for 10 min. The sample was centrifuged at
17 500 g for 10 min and the supernatant collected. 30 lL of the
supernatant was used for LC/mass analysis.
Urine extraction method. ACN was added to the urine sample (1:1)
(vol:vol). The sample was then bath sonicated for 10 min. The sample
was centrifuged at 17 500 g for 10 min. The supernatant was taken and
dried in a speedvac. The precipitant was redissolved in 50 lL of ACN
and sonicated. The sample was centrifuged again and the supernatant
removed for analysis.
®
DOX-PCB solubilization with Captisol cyclodextrin. DOX-PCB
required a solubilization agent for in vivo injection because it displayed
low inherent water solubility. DOX-PCB was observed to be soluble in
cell culture media if first dissolved in DMSO and injected straight into
the media. This apparent solubility was most likely due to DOX-PCB
adsorption to proteins found in the media itself because the same injec-
tion of DMSO/DOX-PCB into water resulted in instant precipitation. For
in vivo injection the concentration of DOX-PCB in the injectant needed
to be 2 mM which made it necessary to use the well-characterized and
DOX-PCB activation in vivo. The experimental design for the in vivo
®
activation is shown in Fig. 3. The 200 lL of DOX-PCB/Captisol was
injected slowly into the tail vein of two mice. The mice were anesthetized
with 120 lL of ketamine 4 min later which was enough to keep the
mouse anesthetized for the 30 min duration of the 365 nm light adminis-
tration. Ten minutes after the DOX-PCB injection the 365 nm LED was
inserted into the middle of the tumor and turned to full power delivering
®
nontoxic cyclodextrin solubilizing agent Captisol . This kept the DOX-
PCB soluble in saline at a sufficient concentration without the use of
organic solvents to make it safe for injection.
Solubilization of DOX-PCB with the cyclodextrin required the forma-
tion of a DOX-PCB solvent complex and possible salt. 1 mg of the
À2
240 lW cm . Only the tumors on the right side of the mice were
exposed. The 365 nm light was administered for a total of 30 min. Blood

Photochemistry and Photobiology, 2013, 89 701
The high degree of DOX sensitivity was necessary due to the poorly vas-
cularized nature of the A549 tumors where it was expected that small
amounts of drug would accumulate.
RESULTS
Ex vivo 365 nm light penetration of tumor tissue
As discussed in the introduction, the photocleavable group of
DOX-PCB was activated by 365 nm light which has minimal
absorption by protein and DNA, but is highly adsorbed by mela-
nin resulting in low skin penetration. Such characteristics enable
Mouse
with 2
implanted
tumors
Systemic
injection of
photo-
Localized light Release of active
delivery to
therapeutics in
tumor through tumor cells and
activatable
the custom
microvasculature
designed to quickly
penetrate tumor
cells
prodrug, very fiber-
3
65 nm light penetration into soft tissues once delivered across
little effect on optic/LED
the skin. The attenuation spectra for the tumor tissue was mea-
sured on a NanoDrop 1000 Spectrophotometer using a 4.9 mg
tissue sample and is shown in Fig. 4A. The attenuation is 0.38 at
365 nm over a 0.2 mm path length. Not all of this attenuation is
due to absorption. The 365 nm light is highly scattered in tissue
all tissue
system.
Exposure set
to cover
entire tumor
area
(
37) which helps to create a uniform coverage of the tumor area
and contributes to the attenuation value. To establish effective
65 nm exposure through tumor tissue, including the tumor
Figure 3. Schematic of photoactivatable prodrug delivery scheme and in
vivo experimental setup.
3
periphery, ex vivo tumor tissue samples were studied. The ex vivo
tumors were exposed from the center and the resulting intensity
of the light that penetrated through the surface of the tumor
tissue was measured at different angles.
The intensity of the light coming from just the 365 nm LED/
fiber-optic itself, without tumor tissue, was measured at 240 lW
from 10 mm directly in front of the fiber-optic end. The 365 nm
light was undetectable from 10 mm above the fiber-optic end
and 10 mm from directly behind.
was drawn right after the 365 nm light administration (40 min after injec-
tion) to obtain the circulating concentration. The mice were then sacrificed
and the tumors and other internal organs collected for analysis.
DOX in vivo control. A control mouse was injected with a 2mM dose
®
of DOX dissolved in saline with 80 mg of Captisol to compare with
DOX-PCB. The DOX mouse was treated the same as the DOX-PCB
mice being given 120 lL of ketamine 4 min after tail vein injection.
However, no 365 nm light was administered to the mouse. Blood was
drawn at 30 min after injection to obtain the circulating concentration.
The mouse was then sacrificed and the tumors and other internal organs
collected for analysis. The tumor extraction from the DOX control mouse
The same power settings were maintained and the fiber optic
was inserted into the middle of a tumor sample. The 365 nm
light was heavily scattered in the tissue causing it to exit the
tumor tissue at all angles. A visual demonstration of the experi-
ment is shown in Fig. 4B. The 365 nm light exiting the tumor
surface is shown by autofluorescence on a piece of paper held
behind the tumor. The measured intensity of the 365 nm light
(
data not shown) showed that DOX was present in the tumor when
injected systemically confirming that this xenograft tumor model allowed
drug accumulation within the tumor.
Tissue extraction protocol and LC-MS\MS method. Both the tumor
tissue extraction protocol and the LC-MS\MS method used here to quan-
tify the DOX and DOX-PCB content are fully detailed in the literature
(
36). These procedures were chosen because they were shown to have a
À2
À2
detection limit of 7.8 pg for DOX from tumor tissue extraction samples.
Achieving this low limit of detection was impart due to salt precipitations
of the DNA to denature the double helix and release intercalated DOX
with subsequent precipitation of the salt for LC-MS\MS compatibility.
was 2 lW cm from 10 mm directly in front, 2.9 lW cm
À2
from 10 mm directly above and 1.4 lW cm
from 10 mm
directly behind the tumor sample. The measured intensity above
A
B
Autofluorescence from paper background
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Fiber Optic
3
00 350 400 450 500 550 600 650 700 750 800
Autofluorescent
Tumor Sample
Wavelength nm
10mm
Figure 4. 365 nm light scattering and penetration through an excised tumor sample. (A) Attenuation spectra for the tumor tissue over a 0.2 mm path
length. Attenuation is 0.38 at 365 nm which is contributed to by scattering as well as absorption. (B) The fiber optic is inserted into the center of a
9
9 6 9 5 mm tumor sample. The tumor glows light blue from 365 nm light-induced autofluorescence. A piece of white paper is placed behind the
tumor and glows blue from the autofluorescence caused by the 365 nm light that exits the tumor sample.

7
02 Stuart Ibsen et al.
the tumor was higher than directly in front due to the asymmetry
of tissue distribution around the end of the fiber optic. When
integrated over the surface of a 15 mm radius sphere the total
amount of 365 nm light exiting the tumor is estimated to be
DOX-PCB + DOX Spike No UV
A
100
DOX
EPI
i) 50
5
9.3 lW. Using uncaging rates already measured for DOX-PCB
À1
À1
at 0.0031% lW min (19) the 59.3 lW would be sufficient
intensity to uncage DOX-PCB at rate of 0.19%/min at the
periphery of the 9 mm long tumor. After the 30 min exposure
an estimated 5.7% of the DOX-PCB would be converted into
DOX at the tumor periphery. The intensity of 365 nm light can
be increased four times by directly inserting the LED into the
tumor tissue, due to loss in the LED fiber-optic coupling.
Directly inserting the LED’s will be explored in future experi-
ments. This shows that the 365 nm light has significant penetra-
tion through the tumor tissue to activate DOX-PCB.
0
9
9
10
11
12
13
14
14
1
00
DOX-PCB
Isomers
ii) 50
0
10
11
12
13
DOX-PCB Spike No UV
100
B
EPI
i) 50
The antitumor effect of just the 365 nm light itself is expected
to be minimal based on observations of in vitro cancer cell via-
0
bility (19). A549 human lung cancer cells were exposed to
9
9
10
11
12
13
14
14
À2
1
.8 mW cm of 330–380 nm band pass filtered light from a
100
mercury arc lamp with no observed change in viability out to
0 min of exposure.
DOX-PCB
Isomers
2
ii) 50
0
DOX-PCB activation in ex vivo tumor tissue
10
11
12
13
The results of the ex vivo tumor exposure experiment are shown
in Fig. 5. Figure 5A shows the tissue extraction LC/MS/MS
trace for a tumor sample spiked with DOX, epirubicin (EPI) as
an internal standard and DOX-PCB. Epirubicin is an isomeric
form of DOX and can be separated from DOX on HPLC.
Figure 5Ai shows the LC/MS/MS trace from a tumor spiked with
DOX and EPI. Figure 5Aii shows the two DOX-PCB isomer
peaks. Figure 5B shows the LC/MS/MS trace for the tumor
sample spiked with just DOX-PCB and EPI. Trace 4Bi shows
the presence of only EPI and trace 4Bii shows the presence of
DOX-PCB. Figure 5C shows the trace for the tumor sample
spiked with just DOX-PCB and EPI with subsequent exposure
to 30 min of 365 nm light. As can be observed in trace 4Ci, the
EPI peak is present along with a restored DOX peak. Moreover,
the DOX-PCB peaks (trace 4Cii) have been dramatically
reduced in magnitude. The disappearance of the DOX-PCB peak
with the appearance of the DOX peak demonstrates that DOX-
PCB can be converted into actual DOX within tissue using the
DOX-PCB Spike 30 min UV
C
100
i) 50
0
DOX
EPI
9
10
11
12
13
14
1
00
DOX-PCB
Isomers
ii) 50
0
9
10
11
12
13
14
Time in min
Figure 5. LC/MS/MS traces of an ex vivo tumor tissue extraction. The
tumor tissue samples were spiked with: (A) DOX and the internal stan-
dard EPI (i), DOX-PCB (ii); (B) EPI (i), DOX-PCB (ii); (C) EPI and
DOX-PCB and then exposed to 30 min of 365 nm light from the LED
system. As can be seen the DOX peak is restored (i) whereas the DOX-
PCB isomer peaks are greatly reduced in intensity (ii). Additional peaks
appear along with the DOX-PCB peaks because the DOX-PCB concen-
tration has been reduced to the point where it is no longer the dominate
compound in the sample.
3
65 nm light.
In vivo activation of DOX-PCB in tumor tissue
After it was shown successfully that active DOX was formed
upon local exposure of DOX-PCB in cell lines (19) and in
ex vivo tumor tissue samples, a set of experiments were con-
ducted to determine the in vivo accumulation of DOX-PCB in
tumor tissue and the photocleavage of that DOX-PCB to active
DOX. The steps in achieving this were to first solubilize DOX-
DOX content. EPI was spiked into these tumor samples during
the extraction process to serve as an internal standard.
®
DOX-PCB solubilization with Captisol cyclodextrin
®
®
PCB using Captisol cyclodextrin to achieve high concentrations
The solubilization of DOX-PCB with Captisol cyclodextrin was
in saline for injection. The circulation half-life of the solubilized
DOX-PCB was measured as well as urine content to look for
elimination. The circulation half-life was used to determine the
best time to start 365 nm exposure of the tumor tissue to prevent
significant activation of DOX-PCB in the tumor vasculature.
Reducing free DOX production in the vasculature should reduce
the possibility of free DOX being swept into systemic circula-
tion. The tumor samples were then analyzed for DOX-PCB and
successful and resulted in the desired concentration of 1.2 mg of
DOX-PCB in 200 lL of saline. The three dimensional structure
of the b-cyclodextrin molecule (38) has a hydrophobic internal
®
barrel. Captisol is a slightly modified version of b-cyclodextrin
where a sulfobutylether is used to separate a sodium sulfonate salt
from the hydrophobic cavity to improve water solubility. The
structure of DOX interacting with the hydrophobic barrel through
the conjugated ring structure has been proposed (38) and should

Photochemistry and Photobiology, 2013, 89 703
1
2000000
0000000
bined to meet the tissue mass requirement and to represent one
extreme end of the tumor. Sections 3 and 6 were likewise com-
bined to represent the other extreme end of the tumor. Sections 2
and 5 met the mass requirements individually and represented
two halves of the central portion of the tumor closest to the nee-
dle insertion site. The tumors from Mouse 1 were bigger and
more vascularized than the tumors from Mouse 2.
DOX-PCB was found in all sections of the tumor tissue in
both the exposed and unexposed tumors for both mouse 1 and 2.
As an example, the LC/MS/MS data from the Mouse 1 tumors
are shown in Fig. 7C–F and Fig. 8C–F. For both Mouse 1 and
1
8
6
4
2
000000
000000
000000
000000
0
0
10
20
30
40
50
60
70
Time in min
2
, DOX appeared in all four sections in roughly equal amounts
Figure 6. The serum concentration of DOX-PCB as a function of time
as measured by LC/MS. No DOX was detected at any time point.
of the 365 nm light-exposed tumor as shown in Fig. 7C–F. A
trace amount of DOX was found in two sections of the unex-
posed tumor as shown in Fig. 8C and f covering the left side of
the tumor in Mouse 1. Mouse 2 showed a trace amount of DOX
in only one side of the tumor as well.
To summarize the data shown in Fig. 7, each of the four
graphs were adjusted to account for small elution time differ-
ences from run to run and then added together and normalized
as shown in Fig. 9A. This gives the relative amplitude of DOX
to the EPI internal standard for the entire 365 nm light-exposed
tumor. The same was done for the four graphs in Fig. 8 and the
summed results are shown in Fig. 9B. The intensity of the EPI
internal standard is the same in Fig. 9A,B allowing both to be
directly compared. The 365 nm light-exposed tumor had a higher
amount of DOX than the unexposed tumor due to the conversion
of DOX-PCB into DOX. This trend was the same for the second
mouse as well.
be similar to how DOX-PCB interacts with the cyclodextrin.
Interaction with the hydrophobic barrel of the cyclodextrin pre-
vents interaction between the hydrophobic conjugated ring struc-
ture of DOX and the hydrophobic nitrophenyl compound
avoiding aggregation in saline solution.
DOX-PCB circulation half-life and urine concentration
DOX-PCB circulation in vivo was studied by analyzing serum
samples collected at different time points from mice injected with
®
the DOX-PCB/Captisol complex. DOX-PCB was found to have
an alpha phase circulation half-life of 10 min (Fig. 6), which is
comparable to that of DOX at around 20 min (39). No DOX
was found in the blood at any time point.
No DOX-PCB or DOX was found in the urine at any of the
collected time points out to 24 h (data not shown). The absence
of DOX in the serum and urine indicated that there is no meta-
bolic or self-conversion of DOX-PCB to DOX. This is consistent
with earlier findings that DOX-PCB was resistant to metabolic
activation when incubated with human liver microsomes (19).
This establishes exposure to 365 nm light as the major mode of
activation.
No DOX was found in the serum of the mouse after the
30 min of 365 nm light exposure in the tumor as shown in
Fig. 10.
Preferential DOX accumulation in the 365 nm light-exposed
tumor
The same amount of EPI internal standard was spiked into each
extraction sample to evaluate and quantitize DOX formation
within the tumors. This allowed a ratio to be calculated between
the area under the curve of the LC/MS/MS traces for DOX and
EPI. This ratio allowed for direct comparisons to be made
between different samples to estimate relative DOX amounts in
each. This allowed for direct comparison between the 365 nm
light-exposed and unexposed tumors as well as between the two
mice. When the mass of the entire tumor was accounted for,
there was six times more DOX/gram of tissue in the tumor
DOX-PCB activation in vivo
Two tumors were collected from each DOX-PCB injected
mouse. Only one of the two tumors was exposed to the 365 nm
light with the other serving as a control. The tumors collected
from the mice were sectioned into six pieces as shown in
Fig. 7A,B. The tumors were not spherical but rather oblong in
shape as shown schematically edge on in Fig. 7A. The line down
the center shows where the tumor was cut in half. The two
halves were subsequently cut into six pieces as shown in
Fig. 7B. The mass of the different pieces from both Mouse 1
and 2 is shown in Table 1. This dissection scheme was designed
to fulfill two requirements. The first was to obtain tissue samples
that had individual masses between 30 and 60 mg. It was found
that this mass range worked best for the extraction protocol.
Tissue samples larger than 60 mg showed incomplete freeze
fracturing reducing the extraction efficiency. Tissue samples
above 30 mg ensured that sufficient DOX would be present to
yield a clear signal. The second requirement was to understand
the DOX distribution throughout the entire tumor. The LED
fiber-optic needle was inserted into the center of the tumor so
the tumor was first dissected down the center through where the
needle end had been. The two end sections 1 and 4 were com-
Table 1. Mass in grams of the different tumor sections for both the
3
65 nm light-exposed and unexposed tumors.
Mouse 1
Mouse 2
Tumor No 365 nm 30 min 365 nm No 365 nm 30 min 365 nm
section
light
light
light
light
1
2
3
4
5
6
0.0255
0.0405
0.0267
0.0334
0.0534
0.0258
0.2053
0.0496
0.0825
0.0426
0.0382
0.0833
0.0547
0.3509
0.0247
0.0237
0.0254
0.0262
0.0286
0.0131
0.1417
0.0163
0.0354
0.0101
0.0125
0.0104
0.0217
0.1064
Total

7
04 Stuart Ibsen et al.
A
B
1
4
2
3
6
1
4
5
C
Tumor Sections 1&4
D
Tumor Section 2
1
00
100
DOX
EPI
DOX
EPI
5
0
0
50
0
9
9.5
10
10.5
11
9
9.5
10
10.5
11
1
00
100
50
0
DOX-PCB
Isomers
DOX-PCB
Isomers
5
0
0
1
2
13
14
15
12
13
14
15
E
Tumor Sections 3&6
F
Tumor Sections 5
1
00
100
50
0
DOX
DOX
EPI
EPI
5
0
0
9
9.5
10
10.5
11
9
9.5
10
10.5
11
1
00
1
00
50
0
DOX-PCB
Isomers
DOX-PCB
Isomers
5
0
0
1
2
13
14
15
12
13
14
15
Time in min
Figure 7. Tumor sectioning procedure and the LC/MS/MS data showing the presence of DOX-PCB, EPI and DOX in the in vivo tumor tissue exposed
to 30 min of 365 nm light for Mouse 1. (A) Edge-on view of the tumor in schematic representation showing how the tumor was sectioned in half. (B)
Open view of the halved tumor showing the numbered sections. (C) LC/MS/MS traces of the tumor tissue extraction for combined pieces 1 and 4 show-
ing both DOX and DOX-PCB presence. (D) LC/MS/MS traces for piece 2 showing both DOX and DOX-PCB presence. (E) LC/MS/MS traces of the
tumor tissue extraction for combined pieces 3 and 6 showing both DOX and DOX-PCB presence. (F) LC/MS/MS traces for piece 5 showing both DOX
and DOX-PCB presence.
exposed to 365 nm light than the unexposed control tumor.
Mouse 1 had 6.1 times more DOX in the 365 nm light-exposed
DISCUSSION
tumor. In this case, the 365 nm light-exposed tumor was bigger
than the unexposed tumor and had better vascularization of the
outer margins. Mouse 2 had a ratio of 6.0 times more DOX in
the 365 nm light-exposed tumor. In this case the 365 nm light-
exposed tumor was smaller and was less vascularized than the
control tumor. There appears to be little migration effect of the
DOX into systemic circulation, even when the tumors were more
vascularized.
In this work we have demonstrated the activation of the new
DOX-PCB photocleavable prodrug inside of both ex vivo and in
vivo tumor tissue. The ex vivo tissue penetration of the 365 nm
light was sufficient through a 9 mm tumor sample to uncage
DOX-PCB at an estimated rate of 0.19% per min at the periph-
ery of the tumor. This rate can be increased at least four times
by using more powerful LED light sources and directly inserting
the LED itself into the tumor tissue. These results demonstrate

Photochemistry and Photobiology, 2013, 89 705
A
B
1
4
2
3
6
1
4
5
C
Tumor Sections 1&4
D
Tumor Section 2
EPI
1
00
1
00
DOX
EPI
50
50
0
0
9
9.5
10
10.5
11
9
9.5
10
10.5
11
15
1
00
100
50
0
DOX-PCB
Isomers
DOX-PCB
Isomers
5
0
0
1
2
13
14
15
12
13
14
E
Tumor Sections 3&6
EPI
F
Tumor Sections 5
EPI
1
00
100
DOX
50
50
0
0
9
9.5
10
10.5
11
9
9.5
10
10.5
11
1
00
100
50
0
DOX-PCB
Isomers
DOX-PCB
Isomers
5
0
0
1
2
13
14
15
12
13
14
15
Time in min
Figure 8. Tumor sectioning procedure and the LC/MS/MS data showing the presence of DOX-PCB, EPI and a trace amount of DOX in the in vivo
tumor tissue not exposed to 365 nm light for Mouse 1. (A) Edge-on view of the tumor in schematic representation showing how the tumor was sec-
tioned in half. (B) Open view of the halved tumor showing the numbered sections. (C) LC/MS/MS traces of the tumor tissue extraction for combined
pieces 1 and 4 showing DOX-PCB presence and a trace amount of DOX. (D) LC/MS/MS traces for piece 2 showing only DOX-PCB presence. (E) LC/
MS/MS traces of the tumor tissue extraction for combined pieces 3 and 6 showing only DOX-PCB presence. (F) LC/MS/MS traces for piece 5 showing
DOX-PCB presence and a trace amount of DOX.
that tissue does not inhibit DOX-PCB activation and that the
65 nm light has significant penetration through several millime-
ters of tissue.
The 10 min a phase circulation half-life of DOX-PCB was
comparable with the 20 min half-life of DOX showing that
DOX-PCB was not immediately removed by the liver. The lack
of DOX in the serum or the urine showed that DOX-PCB was
resistant to normal metabolic processes. This also indicates that
any DOX produced in the body would most likely be a result of
the light activation pathway.
measurable quantities. This is significant because the tumor
tissue was poorly vascularized reducing the efficiency of any
compound to accumulate. The absence of DOX in the serum
showed that DOX generated by the 365 nm light did not get
immediately swept into circulation and was able to penetrate into
the local tumor tissue, or was released within the cells them-
selves. This makes it unlikely that a significant systemic expo-
sure would occur from DOX activated within the tumor.
The small amount of DOX found on one side of the unex-
posed control tumor was surprising given that DOX-PCB was
resistant to metabolic activation and no DOX was found in circu-
lation. This means that the DOX was most likely formed by
3
The preliminary in vivo activation data show that DOX-PCB
made it to the tumor tissue where it was converted into DOX in

7
06 Stuart Ibsen et al.
A
B
this level of tumor localization can be a challenge for the highly
penetrating red or near IR light wavelengths used to activate
photodynamic therapy compounds (PDT) (40). These PDT com-
pounds produce free radicals creating a cytotoxic amount of
indiscriminate cellular damage to both tumor and healthy tissue
3
0 min of 365 nm Light
No 365 nm Light
1
00
100
EPI
EPI
5
0
50
0
DOX
DOX
(41). The therapeutic mechanism of DOX on the other hand has
0
9
9.5
10
10.5
11
9
9.5
10
10.5
11
a level of tumor specificity. DOX is well known to preferentially
accumulate in the DNA causing damage and preventing cellular
replication (19) and thereby having the greatest effect on rapidly
dividing cells (42,43). The advancing tumor margins are in
direct contact with healthy tissue and are a major target of
chemotherapy treatment (44). It is desirable to have the light
exposure extend just one or two centimeters beyond the tumor
margin to make sure these regions get a significant dose.
DOX-PCB activation in the healthy tissue of these margins
would have a much lower toxic effect than the activation of
PDT compounds (41). The efficiency of the photoactivation
needs to be considered as well. DOX-PCB activation is more
efficient at wavelengths below 365 nm, but tissue penetration at
these wavelengths is negligible, preventing uncontrolled release
from occurring. The efficiency of 365 nm light activation can be
compensated for by simply increasing the exposure time in the
tumor region.
1
00
100
50
0
DOX-PCB
Isomers
DOX-PCB
Isomers
5
0
0
1
2
13
14
15
12
13
14
15
Time in min
Figure 9. Summary of the data shown in Figs. 7 and 8. (A) The four
individual tumor section graphs from Fig. 7 were adjusted for elution
time differences and added together to give the total DOX content of the
3
65 nm light-exposed tumor. The intensity of the DOX peak is scaled by
the EPI internal standard peak. (B) The four graphs in Fig. 8 were ana-
lyzed in the same manner to give the DOX content of the tumor not
exposed to the 365 nm light scaled to the EPI internal standard. The EPI
internal standard peaks have the same intensity in both frames A and B
allowing the DOX content of the two tumors to be directly compared.
More DOX was seen in the 365 nm light-exposed tumor.
The photoactivation of prodrug inside the tumor cells and
tumor microvasculature is different from an intratumoral injec-
tion of pure DOX. When injecting drug directly into the tumor
tissue most of the injectant is forced into the interstitial space
between tumor cells and is quickly swept into the lymph system
1
00
A
(45). This limits the distance that the drug can diffuse away from
EPI
the needle. Although the center of the tumor might get a thera-
peutic dose, the limited diffusion makes it difficult to achieve a
therapeutic concentration of drug at the advancing tumor margins
where it is needed most (46). The prodrug, however, is delivered
to the tumor through the vasculature and so reaches every part of
the tumor that has an active blood supply, including the well-
vascularized tumor margins. The tumor tissue scatters the
5
0
0
9
9.5
10
10.5
11
100
B
3
65 nm light delivered to the tumor center to achieve prodrug
activation throughout the whole region including the margins. As
the activated DOX was quickly taken up by nearby cells it did
not have a chance to enter systemic circulation, keeping the drug
in the tumor tissue.
DOX-PCB
Isomers
5
0
0
1
2
13
14
15
Another challenge with intratumoral injection is that the tumor
tissue tends to be denser than healthy tissue, like muscle, requir-
ing a higher injection pressure. During intratumoral injection the
drug can simply follow the path of least resistance and reflux
straight back up the needle track. This spreads the highly con-
centrated drug to healthy tissues along the way causing necrosis
and pain (47,48). As the light activation method does not require
an injection pressure, there is no driving force to send the acti-
vated drug up the fiber-optic track. During these experiments no
reflux was observed from the fiber-optic insertion site of the
tumor.
The pharmacokinetics, lack of metabolic activation and local-
ized in vivo light activation of DOX-PCB as described here dem-
onstrate the desirable qualities of this compound. The 200 times
reduction in cytotoxicity of DOX-PCB should allow larger doses
to be administered than possible with pure DOX. This increases
the amount that could reach even poorly vascularized tumors.
The localized release of fully toxic DOX from DOX-PCB can
help create significant tumor response and should reduce side
effects.
Time in min
Figure 10. LC/MS/MS trace of the serum extraction preformed on the
serum collected after 30 min of light exposure in Mouse 1. No DOX was
found indicating that the activated DOX was not immediately swept into
circulation in measurable quantities, but primarily stayed within the tissue
where it was activated.
exposure to 365 nm light that leaked out of the exposed tumor.
The nude mice used were albino and lacked melanin in the skin
which allowed the 365 nm light to leave the exposed tumor and
expose the facing half of the control tumor just 10 mm away.
Without this leaked 365 nm light, the ratio of activated DOX
between the exposed and unexposed tumors would have been
higher than the six times shown in this work.
In the human application, sensitive organs such as the heart
can be considerably further away than a few centimeters from
the tumor making 365 nm light exposure negligible. Achieving

Photochemistry and Photobiology, 2013, 89 707
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CONCLUSIONS
1
®
The DOX-PCB prodrug, solubilized with Captisol cyclodextrin,
was found to have a circulation half-life of 10 min which was
sufficient time to reach even poorly vascularized tumor tissue.
The 365 nm light, administered with the LED/fiber-optic system,
was shown to penetrate the tumor tissue to sufficiently release
measurable amounts of pure DOX from DOX-PCB at the tumor
periphery and within the tumor tissue. Tumor tissue exposed to
the light had six times more DOX than tumor tissue from the
same animal that did not have direct light exposure, demonstrat-
ing preferential tumor tissue retention. No DOX was found in
the serum sample collected after the 30 min light exposure indi-
cating that activated DOX was not immediately swept into sys-
temic circulation, but rather stayed within the tissue where it was
activated. This demonstrates a proof of concept for the approach
of using photoactivation as a trigger to achieve localized prodrug
activation in deep tumors.
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