Photoinduced Reactivity of Doxorubicin: Catalysis and Degradation

Article pubs.acs.org/JPCA

Photoinduced Reactivity of Doxorubicin: Catalysis and Degradation Krzysztof Nawara,†,‡ Pawel Krysinski,† and G. J. Blanchard*,‡ †

Department of Chemistry, University of Warsaw, Pasteura 1, Warsaw 02-093 Poland Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States

‡

ABSTRACT: Doxorubicin exhibits unusual photoreactivity in aqueous solutions. Our data show that there are two distinct photoreactive pathways for doxorubicin. One is a two-step process that leads to the formation of 3-methoxysalicylic acid, a stable degradation product. The other pathway is a photoreduction of doxorubicin to form the corresponding dihydroquinone, which undergoes spontaneous oxidation mediated by dissolved oxygen to recover doxorubicin with the formation of hydrogen peroxide. Our data account for the known nonlinear dependence of doxorubicin fluorescence intensity on concentration.

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INTRODUCTION Doxorubicin ((7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4methoxy-8,10-dihydro-7H-tetracene-5,12-dione) has been used for over 50 years to treat a variety of cancers, including breast, esophageal, gastric, and liver carcinomas, Hodgkin’s and nonHodgkin’s lymphomas, osteosarcoma, Kaposi’s sarcoma, softtissue sarcomas, bile-duct carcinoma, and pancreatic and endometrial carcinomas.1−3 The wide ranging anticancer activity of doxorubicin is ascribed to its inhibition of cell replication because of the formation of a stable complex with double-stranded DNA.4 The efficiency of doxorubicin therapy is, however, limited by adverse effects, which include myelosuppression, leukopenia, neutropenia, hand-foot syndrome, risk of developing secondary leukemias, 5 and anthracycline-induced heart failure.2 Doxorubicin (Adriamycin) is a compound that has attracted a great deal of research activity because of its utility as a pharmaceutical and because of its reactivity under a variety of conditions. Doxorubicin is a member of the anthracycline family of antibiotics, which also has characteristically strong anticancer activity. Anthracyclines contain four connected sixmembered rings (Figure 1) three of which are conjugated to form an aromatic chromophore with the fourth ring containing a pendant daunosamine moiety. Doxorubicin in its native form possesses a quinoid ring with the adjacent ring containing pdihydroxy functionality. The hydroxyl and hydroxyacetyl groups on the fourth, nonaromatic ring are thought to play an important role in stabilizing the ternary complex structure that doxorubicin forms with double-stranded DNA.1 Doxorubicin has been studied in a variety of ways, and much information has been learned about its chemical properties. The fluorescence spectroscopic behavior of doxorubicin is atypical in both the time and frequency domains. Fluorescence spectroscopy can, in principle, be a valuable tool for the quantitation of pharmaceutical species, but in the case of © 2012 American Chemical Society

Figure 1. Structure of doxorubicin.

doxorubicin, recent work has shown that there is a linear relationship between fluorescence intensity and solution-phase concentration6 except for higher doxorubicin concentrations, where significant deviation from linearity is seen. This is an important issue for both fundamental and practical reasons. In practical terms, the nonlinear relationship between fluorescence intensity and concentration makes it difficult to quantitate higher concentrations of doxorubicin present either in specific locations or as a function of system conditions.7 For higher doxorubicin concentrations, time-resolved fluorescence lifetime data reveal the presence of multiple exponential decay components indicating that either multiple species are present or unanticipated photochemical events contribute to the observed data. The primary focus of this paper is on resolving the chemical and physical causes of this anomalous behavior. Received: April 4, 2012 Revised: April 11, 2012 Published: April 11, 2012 4330

dx.doi.org/10.1021/jp303218r | J. Phys. Chem. A 2012, 116, 4330−4337

The Journal of Physical Chemistry A

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components parallel (0°) and perpendicular (90°) to the vertically polarized excitation pulse using a polarizing cube beam splitter (Newport, extinction ratio ≥ 500:1). The parallel and perpendicular polarized signal components are detected simultaneously using two microchannel plate photomultiplier tubes (MCP-PMT, Hamamatsu R3809U-50) each mounted on a subtractive double monochromator (Spectral Products CM112). The detection electronics (Becker & Hickl SPC-132) resolve the parallel and perpendicular transients separately yielding ca. 30 ps response functions for each detection channel. Data acquisition, detector bias, and collection wavelength are all controlled using an in-house written LabVIEW (National Instruments) program on a PC. Before each measurement, the fresh solution of 200 μM doxorubicin was introduced into the cuvette. The intensity dependence of the emission decay time constants was measured. The light intensity was controlled by neutral optical density filters: ND 0.2 (63%T), 0.4 (40%T), 0.7 (20%T), 1.0 (10%T). Steady-State Absorption, Excitation, and Emission Spectra. Absorbance was measured using a Cary model 300 double beam UV−visible absorption spectrometer with a spectral resolution of 1 nm. Excitation and emission spectra were recorded using a SPEX Fluorolog 3 fluorescence spectrometer equipped with a 450 W Xe arc lamp and single monochromators for both excitation and emission. Laser Degradation of Doxorubicin. An aliquot of 1.5 mL of 200 μM doxorubicin solution was placed in a quartz cuvette equipped with a magnetic stirrer. The sample was irradiated with a laser beam at 483 nm at ca. 100 mW average power for 48 h. Measurement of Doxorubicin Degradation Kinetics. Kinetic studies were performed using the SPEX Fluorolog 3 spectrometer. Photodegradation of doxorubicin was performed using 320 nm excitation (2 nm bandpass). The progress of the reaction was monitored by recording emission at 590 nm (2 nm bandpass) for doxorubicin and at 440 nm (2 nm bandpass) for 3-methoxysalicylic acid. The kinetics of doxorubicin degradation was measured for 35 and 71 μM solutions in 1 mm path length cell. For these measurements, the pH of the solutions was 6.5. Photodegradation of N2-Purged Doxorubicin Solutions. The 40 μM doxorubicin solution was placed in the quartz cuvette equipped with a magnetic stirrer. The sample was purged with N2(g) and was sealed. The sample was irradiated at 300 nm (29 nm bandpass) for 4 h in the fluorometer sample housing. The emission spectra for 320 nm (2 nm bandpass) and 420 nm (2 nm bandpass) excitation were recorded after photodegradation and 20 min after sample exposure to air. The emission spectra of oxygen-mediated doxorubicin recovery were recorded for 466 nm excitation (2 nm bandpass). Chemical Reduction of Doxorubicin. To investigate the spectroscopic properties of reduced doxorubicin, a 40 μM doxorubicin solution was placed in a quartz cuvette and was treated with excess NaBH4. After 0.5 h, the excitation and emission spectra of the solution were recorded in SPEX Fluorolog 3 fluorescence spectrometer. Computational Chemistry. Semiempirical calculations were performed using Hyperchem V8.0 with PM3 parametrization. Mass Spectrometry Analysis. The sample of 100 μM doxorubicin aqueous solution irradiated with 300 nm (29 nm bandpass) for 4 h was subjected to flow injection mass

Degradation is typically accomplished by chemical or enzymatic means. Most doxorubicin degradation products have been characterized following their extraction into organic solvents, and they exhibit prominent fluorescence. While the isolation and characterization of degradation products has been studied extensively and under a variety of conditions,8−12 understanding the photodegradation reactions we report can be addressed most effectively by examining the kinetics of these reactions, and this is the focus of the work we present here. We have extracted information about doxorubicin photodegradation that has not been reported previously. The kinetic approach we have taken has allowed the determination of changes in the reaction mixture over time that would have been difficult or impossible to elucidate by quantitation of the final reaction products. One goal of this work is to investigate the degradation of doxorubicin in an aqueous environment and to focus on a means of degradation that has received limited attention to this point. Doxorubicin is known to photodegrade upon UV excitation, and we are interested in determining the details of this process and its consequences. We have found that in an aqueous environment there are two dominant photoinduced processes with one being the catalytic formation of H2O2. Our data suggest that the mechanism for the catalytic formation of this reactive oxygen species (ROS) involves radical formation.

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MATERIALS AND METHODS Chemicals. Ampliflu Red (>98% purity), horseradish peroxidase (HRP, type VI-A), dimethyl sulfoxide (>99.5% purity), and 2-hydroxy-3-methoxybenzoic acid (97% purity) were purchased from Sigma-Aldrich. Doxorubicin hydrochloride was purchased from Selleck Chemicals (>99% purity), and sodium borohydride was purchased from Spectrum Chemicals (>98% purity). All chemicals were used as received. For aqueous solution preparation, Milli-Q water was used. The N2(g) used was boil-off from in-house N2(l). The concentration of aqueous solutions of doxorubicin, horseradish peroxidase, and resorufin solutions was determined spectrophotometrically using molar absorptivity constants ε480 = 1.15 × 104 M−1 cm−1 (doxorubicin),13 ε403 = 1.02 × 105 M−1 cm−1 (HRP),14 and ε571 = 6.97 × 104 M−1 cm−1 (resorufin).15 The solution of Ampliflu Red was prepared by mixing excess Ampliflu Red reagent and horseradish peroxidase (6 μM) in 2:1 water:DMSO. Time-Correlated Single-Photon-Counting (TCSPC) Spectrometer. Fluorescence lifetime and anisotropy decay data were acquired using a time-correlated single-photoncounting (TCPSC) instrument that has been described previously,16 and we recap its salient features here. The source laser is a CW passively mode-locked, diode-pumped Nd:YVO4 laser (Spectra Physics Vanguard) that produces 2.5 W average power at 355 nm and at 532 nm, at 80 MHz repetition rate, with 13 ps fwhm pulses at both wavelengths. The Nd:YVO4 laser pumps a cavity-dumped dye laser (Coherent 702-2), which operates in the range of 430−850 nm producing 5 ps pulses. The repetition rate of the dye laser is adjustable between 80 MHz and 80 kHz through cavity-dumping electronics (Gooch & Housego). The laser output is linearly polarized with a polarization extinction ratio of ca. 100:1. The excitation pulse at 483 nm from the dye laser is divided with one portion directed to a reference photodiode (Becker & Hickl PHD-400N), and the other portion sent to the sample. Emission at 590 nm is collected using a 40× reflecting microscope objective (Ealing). The collected emission is separated into polarization 4331



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spectrometry analysis. The sample was ionized by electrospray ionization. For comparison, the mass spectrum of pure 3methoxysalicylic acid solution was also collected.

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RESULTS AND DISCUSSION The focus of this work is on understanding the optical spectroscopic properties of doxorubicin. As noted above, this compound exhibits anomalous fluorescence behavior, and in many instances, such behavior is indicative of relatively complex photochemical or photophysical processes operating in the molecule. Htun has reported that doxorubicin exhibits a biexponential fluorescence decay in both protic and polar aprotic solvents.17 Our data indicate that the fractional contribution of the second fluorescence decay component depends on excitation intensity (Table 1). Fluorescence Table 1. Effect of Light Intensity on Fluorescence Lifetimea

Figure 2. Emission spectra of an aqueous doxorubicin solution after laser degradation. Excitation wavelengths are as indicated: 320, 350, 400, 466 nm. The absence of emission between 550 and 650 nm for 466 nm excitation indicates complete degradation of doxorubicin.

I(t) = A1·exp(−t/τ1) + A2·exp(−t/τ2) neutral density filter 0.2 0.4 0.7 1

content % 26 74 39 61 58 42 100

τi (ps)

Ai 772 2228 962 1512 1497 1069 2326

± ± ± ± ± ± ±

28 20 45 34 44 31 47

1026 508 1025 578 1024 571 971

± ± ± ± ± ± ±

To gain insight into the photodegradation of doxorubicin, we monitored the emission intensity of a doxorubicin solution as a function of irradiation time. The photodegradation of doxorubicin exposed to 320 nm light is characterized by three distinct regions (Figure 3a): an apparent first-order decay

5 8 4 16 2 23 3

a

For all measurements, we recorded single exponential anisotropy decays with R(0) = 0.23 ± 0.01 τOR = 329 ± 24 ps.

lifetime and anisotropy decay data were measured for doxorubicin in aqueous solution as a function of excitation intensity. For low excitation light intensity, we observe a single exponential fluorescence lifetime decay and, as the excitation intensity is increased, a second decay component is seen with its fractional contribution increasing proportionally. The time constant of the second component is faster than that of the first (doxorubicin) by a factor of ca. 2.5. These findings suggest that doxorubicin undergoes a photochemical reaction to form some product that is spectrally overlapped with the emission spectrum of doxorubicin. Despite the presence of two fluorescence lifetime components, we obtained a single exponential anisotropy decay functionality with the same time constant to within the experimental uncertainty for all excitation intensities. This finding indicates that both doxorubicin and its photoproduct have similar hydrodynamic volumes, and the lifetime data cannot be accounted for by dimer or excimer formation. The reaction product is not sufficiently stable to produce an emission spectrum that can be resolved from that of doxorubicin. The absorbance spectrum (not shown) of a solution after exhaustive photodegradation of doxorubicin by laser irradiation does not reveal any bands that are obviously the result of photoproduct formation, but the emission spectrum exhibits a variety of new bands with the dominant spectral feature depending on the excitation wavelength (Figure 2). The fact that several fluorescent products are formed indicates that there are either multiple independent degradation pathways or a sequential degradation pathway with multiple steps. In either case, these data are not consistent with the direct formation of a single, stable photoproduct.

Figure 3. (a) Kinetics of doxorubicin degradation monitored via emission intensity at 590 nm. The top trace is for a 71 μM aqueous doxorubicin solution, and the bottom trace is for a 35 μM solution. (b) Comparison of the time-resolved emission profiles of 590 nm degradation (top) and 440 nm growth (bottom) for a 71 μM doxorubicin solution.

(ca. 0.5 h), followed by a plateau with an intensity and duration that depends on solution concentration and irradiation intensity, and at long times a slow decay. This photodegradation profile cannot be described in the context of simple first order kinetics. The most prominent and revealing feature in these data is the presence of a plateau region. Such a feature is not consistent with the decomposition of a single species coupled directly to the formation of another. For this functional form to exist, there must be at least one intermediate 4332



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High resolution mass spectral data show mass-to-charge ratio (m/z) to be 167.034 for both 3-methoxysalicylic acid and the photodegradation product. The only mechanism by which this photoproduct can be formed is cleavage of the anthraquinone ring structure. To understand the functional form of the data presented in Figure 3a, we have constructed a kinetic model (Figure 5). In

species. The intermediate species can arise either from the formation of an emissive doxorubicin decomposition product with an emission profile that overlaps that of doxorubicin closely or by the autoinhibition of doxorubicin photodegradation by the decomposition products. In either scenario, an additional fluorescent species should be detectable, and changes in the fluorescence spectrum of the doxorubicin solution as a function of irradiation time indicate the formation of a product with a broad emission feature centered near 440 nm (Figure 4a).

Figure 5. Kinetic model to account for the data shown in Figure 3. The reactions are assumed to be irreversible and to proceed according to the model indicated. The profile of compound C produces a mirrorlike image of the sum A and B. A and B both emit at the same wavelength. Products C and D do not exhibit spectral overlap with A.

this model, we assume that photochemical reactions are mediated by free radicals, which participate in irreversible reactions. If the photoinduced reaction is a free-radical reaction, direct back-conversion to doxorubicin is not possible. To explain the functional form of these data (Figure 3a), doxorubicin (designated A in Figure 5) must undergo photodecomposition to (at least) two distinct products. One of them, which we designate as B in this model, exhibits emission spectral overlap with doxorubicin. B will undergo subsequent reactions to form product C, which we identify as 3-methoxysalicylic acid. C is expected in this model (Figure 5) to produce a mirrorlike image to the degradation kinetics of A (Figure 3b). The photodegradation studies presented to this point were performed with solutions that had been exposed to ambient air. To determine whether or not O2 played a role in doxorubicin photodegradation, we purged a doxorubicin solution with N2 prior to irradiation. The resulting photodegradation of doxorubicin in the absence of O2 revealed the formation of an intermediate product which we assign as B (Figure 6a). This photoproduct exhibits two emission bands at 462 and 489 nm, and we identify it as B because of its spectral overlap with

Figure 4. (a) Normalized emission spectra of 3-methoxysalicylic acid (dashed line) and one photodegradation product (solid line). (b) Electrospray ionization (ESI) mass spectrum of the photodegradation product after 4 h irradiation with 300 nm. High resolution mass spectrometry confirmed molecular formula of 3-methoxysalicylic acid (C8H7O4, 167.034). (c) ESI mass spectrum of 3-methoxysalicylic acid (167.034).

The formation kinetics of the 440 nm feature mirrors the 590 nm decay kinetics indicating that the 440 nm band is associated with the photoproduct (Figure 3b). Ramu et al. identified that for the riboflavin-mediated photodegradation of doxorubicin one product is 3-methoxysalicylic acid.11 We present the normalized emission spectrum of 3-methoxysalicylic acid in Figure 4a in comparison to the emission spectrum of our photoproduct. The emission spectra in conjunction with the ESI mass spectra of the photoproduct (Figure 4b) and 3methoxysalicylic acid (Figure 4c) provide a sufficiently good match to identify the photoproduct as 3-methoxysalicylic acid. 4333



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Figure 7. (a) Normalized excitation and emission spectra after reduction of doxorubicin by NaBH4. (b) Normalized emission spectra of chemically reduced doxorubicin (dotted line) and one photodegradation product (solid line).

Figure 6. (a) Formation of intermediate product B. Bands with maxima at 560 and 590 nm are characteristic of doxorubicin emission. The additional peaks at 462 and 489 nm belong to product B. Product B undergoes further reaction upon exposure to air. The solid line emission spectrum was measured for a N2 purged sample. The dotted line emission spectrum was measured shortly after exposure to air. (b) Emission spectra of 3-methoxysalicylic acid and doxorubicin. The solid line is emission spectrum measured under N2 purging conditions. The dotted line is the emission spectrum measured 20 min after exposure of the solution to air.

doxorubicin. Upon exposure to the photodegraded solution to air, the concentration of the photoproduct B decreases and, simultaneously, the concentration of 3-methoxysalicylic acid increases (Figure 6b). We cannot isolate B or identify its structure unambiguously because it is unstable and reacts rapidly with O2 to form 3-methoxysalicylic acid. For our kinetic model to reflect the observed experimental data accurately, another decomposition pathway is required. We designate the product of this pathway as D (Figure 5). D is formed directly from doxorubicin (A), and its emission spectrum does not overlap that of doxorubicin. One possible photochemical reaction is photoreduction analogous to known reactions for anthraquinones18 and α-hydroxyanthraquinones.19 To evaluate whether or not a reduction product of doxorubicin contributes to our observed spectra, we have reduced doxorubicin chemically using NaBH4 (Figure 7). The excitation spectrum we report for the reduced compound is similar to the absorbance spectrum of the quinizarin semiquinone radical.20 This spectral feature is also similar to the triplet state absorbance spectrum of daunomycin.21 A comparison of normalized emission spectra of photodegraded doxorubicin with chemically reduced doxorubicin indicates that the same compound is formed by both reduction routes (Figure 7). We thus assign the photoproduct formed under N2 purging conditions to the reduced form of doxorubicin, an assignment consistent with the known photochemistry of other anthracyclines. This reduced photoproduct oxidizes spontaneously in solutions exposed to air to produce doxorubicin (Figure 8).19 There is a general agreement that under the appropriate conditions (e.g., the enzymatic action in tissues) the chemistry of anthracyclines lends itself to the generation of reactive free

Figure 8. Oxygen-mediated doxorubicin regeneration after reduction. (a) After reduction with NaBH4. Time-resolved emission intensity at 590 nm (530 nm excitation). The initial emission intensity at t = 0 was 3.15 × 106 counts. (b) Frequency domain spectra at several locations along the signal recovery showing the recovery to be associated with doxorubicin.

radicals and oxidative stress.22−25 We assert that the photoreduction of doxorubicin results in the formation of the corresponding semiquinone radical. Spontaneous regeneration of doxorubicin is thus expected to proceed with the formation of hydrogen peroxide,26,27 which is in agreement with our experimental findings (Figure 9). 4334



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of the energy balance of the overall reaction. The reaction sequence consistent with our data is shown in Scheme 1. For this reaction sequence to operate, the addition of ca. 3.9 eV of optical energy is required. The initial step, the photoreduction of doxorubicin to the dihydroquinone, is accompanied by the oxidation of water 2H 2O → 4H+ + 4e− + O2

Ered 0 = 1.23 V vs NHE

and the auto-oxidation of the dihydroquinone is accompanied by the reduction of O2 O2 + 2H+ + 2e− → H 2O2

Ered 0 = 0.68 V vs NHE

These oxidation and reduction half reactions provide some insight into the operation of the overall reaction. Specifically, for the photoreduction of doxorubicin to proceed, the energy provided by a photon exciting the doxorubicin S2 ← S0 transition is required for efficient photoreduction (i.e., to overcome the activation energy). When photoexcited to the S1 state, the peroxide forming auto-oxidation reaction proceeds much less efficiently. It is not possible to determine more closely the reduction potential for photoexcited doxorubicin because we do not know whether the photoreaction proceeds directly from the singlet manifold or from the triplet manifold, which would require intersystem crossing. We do know that photoexcitation of doxorubicin accesses a state with a standard reduction potential greater than 1.23 V versus normal hydrogen electrode (NHE). We also know that oxidation of the dihydroquinone to doxorubicin has a standard reduction potential in excess of 0.68 V. At a minimum, for both the photoreduction and auto-oxidation reactions to operate, the standard reduction potential of photoexcited doxorubicin must be at least 0.55 V higher in energy than the standard reduction potential of the dihydroquinone. This energy difference is related to the net free energy of the overall reaction but does not address the activation barrier for the photoreduction reaction. A word is in order regarding the role of protons in this catalytic process because their presence or absence may affect the reaction pathway. The photodegradation studies we have performed were at a pH of 6.5 ([H+] = 3 × 10−7 M), and the doxorubicin concentrations used were 35 μM or greater giving

Figure 9. Absorbance spectrum of doxorubicin after photoreduction (dotted line). The solid line spectrum shows the absorbance of doxorubicin (after oxygen-mediated recovery) and formation of resorufin (peak at 572 nm), an indicator of hydrogen peroxide.

In the context of our kinetic model (Figure 5), the photoproduct D should not be spectrally overlapped with doxorubicin. While there is slight spectral overlap experimentally, its contribution is exceedingly small for excitation at 320 nm, and on this basis, we believe the contribution of D to the decay kinetics seen at 590 nm (Figure 3a) is negligible. It is also important to consider that we have not included reversibility, per se, in the kinetic model. Because the A ↔ D process operates in such a way that A is an intermediate species in the kinetic pathway, the inclusion of reversibility in this model will not affect the functional form of the model predictions. As a practical matter, the rate of doxorubicin recovery is slow relative to photoreduction for the UV photon flux we apply rendering the A ↔ D process a reaction that can be described effectively as A → D. The photoreduction of doxorubicin, followed by autooxidation back to the starting compound with the formation of H2O2, can be understood in terms of thermodynamics expressed in the context of the relevant electrochemical reactions. This approach is not in conflict with a free-radical mechanism of auto-oxidation, and it allows for an examination

Scheme 1. Reaction Sequence for Photocatalytic Reaction of Doxorubicin in Aqueous Solution to Form H2O2a

The first reaction is photoinduced reduction and the second reaction is spontaneous oxidation of the photoproduct. These reactions are consistent with energetic considerations but do not address the mechanistic pathway. a

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the two states. Direct excitation of the S1 manifold gives rise to much less efficient generation of the photoproduct implying that the ∼8000 cm−1 (1 eV) of energy that is dissipated because of rapid relaxation from the S2 to the S1 states is sufficient to overcome the activation barrier for the reaction if the reaction proceeds from a singlet state. If the reaction proceeds from the triplet manifold, there are several possible reaction pathways that depend on the states from which intersystem crossing (ISC) occurs. If ISC proceeds from the S2 state to the T3 state, there is ca. 4000 cm−1 (0.5 eV) energy difference between them that will be dissipated nonradiatively. Presuming relaxation within the triplet manifold is rapid, relaxation from the T3 state to the T1 state yields ca. 13 000 cm−1 (1.6 eV) of excess energy dissipated nonradiatively. ISC from the S1 state to the T2 state is calculated to be almost isoenergetic, yielding ∼0 eV excess energy, and subsequent relaxation to the T1 state provides ∼9000 cm−1 (1.1 eV) excess energy. Because of the near degeneracy of the S1 and T2 states, it is tempting to assume that ISC occurs between these two states. As noted above, however, direct excitation of the S1 state does not give rise to efficient photoproduct generation indicating that T2 to T1 relaxation (1.1 eV) is not sufficient to overcome the activation barrier. Taken collectively, these calculations provide limits on the height of the activation barrier for the photoreduction of doxorubicin. The probability of the reaction proceeding from the singlet versus triplet manifold favors the triplet on the basis of lifetime grounds alone. If S1 is the reactive state, then S2 to S1 relaxation gives 8000 cm−1 of excess (thermal) energy which is enough to cross the activation barrier. Direct S1 excitation provides essentially no excess energy, and the reaction should proceed at a very low rate. If the reaction proceeds from the T1 as the reactive state, then S1 → T2 → T1 relaxation yields ∼9000 cm−1 of excess energy while S2 → T3 → T1 or S2 → S1 → T2 → T1 relaxation yields ∼17 000 cm−1 of excess energy. If 17 000 cm−1 of excess energy gives rise to efficient doxorubicin photoreduction while 9000 cm−1 of excess energy does not, the activation barrier for the photoreduction reaction must lie between these values (i.e., between 1.1 and 2.1 eV), and electrochemical data places a lower bound on this value of 1.23 eV (vide supra). Neither the electrochemical considerations nor the spectroscopic energy dependence data speak directly to the mechanism of the reactions. For similar reactions, it has been established that the mechanism is free radical in nature and that the initial photoreduction reaction is thought to proceed from the triplet manifold for anthraquinones.18

rise to a stoichiometric excess of doxorubicin that is 100:1 or more. This is potentially significant because for this proton concentration, the doxorubicin chromophore will not experience protonation to a significant extent consistent with our observation of the known doxorubicin absorbance spectrum and prominent fluorescence in all cases. With this ansatz, we consider the calculated singlet and triplet energy levels of doxorubicin and the roles they may play in determining the reaction pathway. We presume that the photoreduction is an activated process, and we recognize that the electrochemical information is related to the thermodynamics of the reactions. We have used Hyperchem to calculate the Jablonski diagram (Figure 10) for

Figure 10. Jablonski diagram for doxorubicin computed using Hyperchem 8.0 semiempirical calculations using the PM3 parametrization.

doxorubicin optimized structurally using the PM3 parametrization. The calculated transition energies are not expected to be in quantitative agreement with experimental results owing to the parametrizations used in these calculations. The calculation of each energy level is, however, performed using the same parametrization and is thus subject to the same systematic errors. Consideration of energy differences between calculated levels should thus prove useful in understanding the activation barrier for the initial photoreduction reaction. It is possible, in principle, for the photoreduction reaction to proceed from either the singlet manifold or the triplet manifold for this system. On the basis of the relative lifetimes of an excitation in the singlet manifold (⟨τ⟩ ∼ 1.5 ns, Table 1) compared to that in the triplet manifold (τT ∼ 1.7 μs),21 we believe that the photoreduction reaction is more likely to proceed in the triplet manifold. The energy levels shown in Figure 10 provide some insight into the possible reaction pathway. If the reaction proceeds in the singlet manifold, excitation to the S2 state provides the excess energy to the reactant doxorubicin. The S2 state remains populated for 10 ps or less following excitation with rapid relaxation to the S1 state. Because the interaction of doxorubicin with water produces the photoreduced species, we believe that the picosecond S2 lifetime is too short for the reaction to proceed from this state directly in high yield. The reaction is much more likely to proceed from the S1 state on the basis of the relative lifetimes of

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CONCLUSIONS The fluorescence dynamics of doxorubicin have been shown to be anomalous in several respects. The data we report here indicate that doxorubicin undergoes several reactions, including a photoreduction to form a dihydroquinone in aqueous solution upon exposure to light. The efficiency of the photoreduction process exhibits a wavelength dependence with excitation to the S2 manifold producing photoproduct more rapidly than excitation to the S1 manifold. The limited electrochemical information relevant to this process in conjunction with semiempirical calculations allows the determination of the activation barrier for the initial photoreduction to be between 9000 cm−1 and 17 000 cm−1. The dihydroquinone undergoes spontaneous reoxidation to recover doxorubicin with the formation of H2O2. These findings are 4336



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(18) Tickle, K.; Wilkinson, F. J. Chem. Soc., Faraday Trans. 1965, 61, 1981−1990. (19) Dibrova, V.; Klimenko, V.; Nurmukhametov, R.; Shigorin, D. J. Appl. Spectrosc. 1990, 53, 242−247. (20) Mukherjee, T. Proc. Indian Natl. Sci. Acad. 2000, 66, 239−265. (21) Andreoni, A.; Land, E. J.; Malatesta, V.; McLean, A. J.; Truscott, T. G. Biochim. Biophys. Acta 1989, 990, 190−197. (22) Gewirtz, D. A. Biochem. Pharmacol. 1999, 57, 727−741. (23) Berthiaume, J. M.; Wallace, K. B. Cell Biol. Toxicol. 2007, 23, 15−25. (24) Keizer, H. G.; Pinedo, H. M.; Schuurhuis, G. J.; Joenje, H. Pharmacol. Ther. 1990, 47, 219−231. (25) Yee, S. B.; Pritsos, C. A. Arch. Biochem. Biophys. 1997, 347, 235− 241. (26) Davies, K. J.; Doroshow, J. H. J. Biol. Chem. 1986, 261, 3060− 3067. (27) Doroshow, J. H.; Davies, K. J. J. Biol. Chem. 1986, 261, 3068− 3074.

consistent with anthraquinone chemistry that leads to the catalytic production of hydrogen peroxide, and the irradiation of doxorubicin gives rise to the same reaction sequence. In parallel, doxorubicin undergoes irreversible photodegradation. This reaction proceeds with generation of an unstable intermediate and its further oxidation to 3-methoxysalicylic acid. Understanding the mechanism of photochemical degradation and photocatalytic generation of H2O2 advances our understanding of doxorubicin and, potentially, some of its known toxic effects. With this understanding in place, the next step in this work will be to establish the yields of these reactions and the mass balance for the system, which will be undertaken in the near future.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +011 517 355 9715 x224. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant CHE 0808677 from the National Science Foundation. K.N. gratefully acknowledges support of the Foundation of Polish Science MPD Program cofinanced by the European Regional Development Fund. We are grateful to the MSU Mass Spectrometry Facility for their assistance in the acquisition of the MS data.

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