Riboflavin-Photosensitized Changes in Aqueous Solutions of Alginate

Biomacromolecules 2003, 4, 429-436

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Riboflavin-Photosensitized Changes in Aqueous Solutions of Alginate. Rheological Studies Stefania G. Baldursdo´ ttir,† Anna-Lena Kjøniksen,‡ Jan Karlsen,† Bo Nystro¨m,*,‡ Jaan Roots,‡ and Hanne H. Tønnesen† Department of Pharmaceutics, School of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, N-0316, Oslo, Norway, and Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, N-0315, Oslo, Norway Received November 4, 2002; Revised Manuscript Received January 2, 2003

Interactions between photoexcited riboflavin (RF), promoted by irradiation in the range of 310-800 nm, and alginate have been studied in air equilibrated aqueous solutions with the aid of rheological methods. Light irradiation of RF causes under aerobic conditions fragmentation of alginate and a decrease in the shear viscosity and other rheological parameters of its solutions. The decrease is most pronounced in concentrated polymer solutions. The photochemical degradation of alginate is inhibited in the presence of the quenchers/scavengers D-mannitol, glutathione, potassium iodide, and sodium azide and in excess oxygen. The addition of thiourea to alginate-RF solutions leads to enhanced degradation of the polymer. Significant shear-thinning effects and deviations from the Cox-Merz rule are observed at higher polymer concentrations. Introduction Modified-release pharmaceutical dosage forms have been developed1,2 and studied with the intention to improve the pharmacological activity, diminish toxic effects, and reduce the number of daily administrations to obtain better patient compliance. Many oral pharmaceutical formulations on the market are systems designed for sustained release or prolonged action of the drug. The idea with sustained drug release systems is to give an acceptable therapeutic concentration of the drug at the site of action and keep it constant for the desired duration of the treatment. However, in some cases, the use of oral sustained release formulations may cause a reduction in the therapeutic efficiency1,3 and enhanced side effects, especially for drugs that are subject to large metabolic degradation due to the first-pass effect. Moreover, there are a number of clinical situations where constant rate drug delivery systems are not sufficient.1,2,4 Therefore, it is of great interest to develop drug delivery systems that can be tuned so that drug release coincides with the underlying rhythm of a pathophysiological state. Programmed dosage forms can be characterized as a controlled-release delivery system, and this device is better designed to meet the biological needs than sustained drug release formulations. The inherent feature of pulsed and programmed drug release systems is that they deliver the desired amount of drug at different times over a definite period to correlate with biological needs.2 These systems can be regulated internally or externally. For internally or self-regulating systems the release rate is controlled by a feedback mechanism, based on physiological parameters such as pH, enzymes, competitive binding, or metal concentration de† ‡

Department of Pharmaceutics, School of Pharmacy, University of Oslo. Department of Chemistry, University of Oslo

pendent hydrolysis. In this case the rate control mechanism is independent of external influence. This is in contrast to externally regulated systems where external triggers such as temperature, electricity, magnetism, ultrasound, or light for pulsed delivery are starters for the processes.5-12 Hydrophilic swellable polymer matrices may constitute an interesting system for pulsed delivery induced by light. In an aqueous polymer system in the presence of a photosensitizer and a drug, light irradiation may cause scission of polymer chains and thereby changes in morphological characteristics of the polymer matrix. As a result, the mobility of the polymer segments will change13-16 as well as the drug release rate. A pulsed drug delivery system can be designed by exposure to irradiation under carefully controlled conditions. In the process of developing a system of this type, it is important to gain a fundamental insight into the effect of a photosensitizer on the polymer matrix in vitro. In the present work, the effect of photoexcited riboflavin (RF) on the rheological features of aqueous solutions of the polysaccharide alginate is reported. RF or vitamin B2 is a yellow pigment and an efficient endogenous cellular photosensitizer that is known13,15 to cause scission of polysaccharide chains when exposed to visible light. Alginate is an anionic copolymer from mannuronic acid (M) and guluronic acid (G) and is extracted mainly from the seaweed Laminaria hyperborea in a large-scale industrial production.17 This polysaccharide is a biocompatible polymer that is widely used in the food industry and approved by the European Pharmacopoeia (Ph.Eur) and the Food and Drug Administration (FDA) as an excipient for pharmaceutical products. A schematic illustration of the chemical structures of RF and alginate is displayed in Figure 1. The objective of this investigation is to gain insight into the rheological behavior and photochemical mechanism during the photoinduced degradation of alginate under

10.1021/bm020117a CCC: $25.00 © 2003 American Chemical Society Published on Web 02/01/2003

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Biomacromolecules, Vol. 4, No. 2, 2003

Figure 1. Chemical structure of riboflavin and the chemical units of alginate (M ) mannuronic acid and G ) guluronic acid).

various conditions. For this purpose, we have carried out rheological measurements on alginate solutions of different concentrations, with and without RF, irradiated in the wavelength range 310-800 nm. The effects of different quenchers and scavengers on the rheological properties of these systems have also been scrutinized. Experimental Section Materials and Solution Preparation. An alginate sample, designated LF 10/60 LS (# 912912), was supplied from FMC Biopolymers, Drammen, Norway. According to the specifications from the manufacturer, this sample has a weightaverage molecular weight of 152 000 and the guluronic acid to mannuronic acid (G/M) ratio is 0.75. The pKa values of G and M have been determined to 3.7 and 3.4, respectively.17 RF, glutathione (GSH), thiourea, D-mannitol, sodium azide (NaN3), and potassium iodide (KI) were supplied by Sigma or Merck and were of analytical grade. These chemicals were used without any further purification. The polymer was dissolved in a phosphate buffer (pH ) 7.4) in the absence or presence of the RF (0.1 mM) photosensitizer and in some cases also with a quencher or a scavenger. Solutions of different polymer concentration were prepared and all samples were stored in the dark at 6 °C prior to measurement. Great care was exercised to ensure that the samples were homogeneous and freshly prepared. All experiments were conducted at 25 °C.

Baldursdo´ ttir et al.

Irradiation Source. The samples were irradiated in a Suntest CPS (Heraeus GmbH, Hanau) apparatus, equipped with a 1.8 kW xenon lamp and a glass filter transmitting irradiation corresponding to exposure behind window glass (wavelength range 310-800 nm). The light intensity was measured to 1.4 × 105 lux and 15 W/m2 in the visible and UV range, respectively, using a lux meter (Hagner EC1 Digital luxmeter) in combination with a UV-filter radiometer (Hagner EC1 UV-A). Both the alginate-RF solution and the corresponding alginate solution without RF (blank) were illuminated. Alginate-buffer solutions served well as blanks, because for nonirradiated samples the small amount of added RF had no effect on the rheological results of the solutions. In most experiments, an irradiation time of 15 min is sufficient for a substantial photochemical degradation of the polymer (see the discussion below). The alginate-RF samples were kept in the dark after irradiation. Absorbance and fluorescence characteristics of the systems were determined by the use of a Shimadzu UV-2101PC scanning spectrometer and a Perkin-Elmer LS50B luminescence spectrometer, respectively. Rheological Experiments. Oscillatory shear and viscosity experiments were conducted in a Paar-Physica MCR 300 rheometer using a cone-and-plate geometry, with a cone angle of 1° and a diameter of 75 mm. The sample was applied on the plate and to prevent dehydration from the solution, the free surface of the sample was always covered with a thin layer of low-viscosity silicone oil (the viscoelastic response of the sample is not affected by this layer). The measuring device is equipped with a temperature unit (Peltier plate) that gives a very good temperature control over an extended time. Some of the measurements were carried out in a Bohlin VOR rheometer system using an ordinary concentric cylinder geometry (C14 cup-and-bob geometry). The values of the strain amplitude were checked to ensure that all oscillatory shear experiments were performed within the linear viscoelastic regime, where the dynamic storage modulus (G′) and loss modulus (G′′) are independent of the strain amplitude. The viscosity measurements were conducted over an extended shear rate range (covering both the linear and nonlinear viscoelastic regimes). The shear rate dependence of the viscosity was usually monitored as a function of increasing shear rate. However, to check possible hysterises effects, the shear-rate dependence of the viscosity for some systems was registered as a function of increasing shear rate (up-ramp curve), and the subsequent decline in shear rate (down-ramp curve) was also probed. No significant hysterises effects were detected under the considered experimental conditions, and the up-ramp curve and the down-ramp curve practically coincided. Results and Discussion Before we present and discuss the rheological results obtained from alginate solutions with and without RF, it may be instructive to give some aspects on the reactions that are caused by photoexcited RF leading to scission of the alginate chains. We should note that the fragmentation

Riboflavin-Photosensitized Changes

Figure 2. Absorption spectra of riboflavin (0.1 mM) in the presence of alginate (2 wt %) and with added thiourea (0.4 mM) at different times of irradiation.

of alginate discussed below is due to photoexcited RF because the reaction does not take place in the dark or in the absence of RF. Time evolution of the absorption spectrum (in the wavelength range of about 275-800 nm) of RF (0.1 mM) in aqueous alginate (2 wt %) solution under aerobic conditions is shown in Figure 2a. In the wavelength range above 300 nm, the spectrum of nonirradiated RF consists of two structureless peaks centered at 446 and 375 nm, while after approximately 10 min exposure to light most of the RF has been consumed and converted into other products. In the presence of thiourea (0.4 mM), we can see (Figure 2b) that the degradation of RF is retarded (it takes about 30 min for RF to be decomposed). Thiourea has previously been reported18 to photostabilize solutions containing RF. In photochemical degradation of RF under neutral aqueous conditions, lumichrome (LC) is probably a dominant product.16,19 When aqueous solutions are exposed to irradiation in the range of 310-800 nm in the presence of an efficient photosensitizer such as RF under aerobic conditions, RF absorbs energy and reacts, predominantly via the triplet excited state (3RF*), with other molecules such as substrates with easily abstractable hydrogen or molecular oxygen, generating reactive species. However, it has been suggested20,21 that in some cases the reaction of the radical cation RF•+ with substrates is even more important than direct attack by 3RF*. It is frequently argued22-25 that a triplet sensitizer can react via two general pathways, commonly referred to as type I and type II mechanisms. Type I photosensitized oxidation involves the formation of radical species (including reactive oxygen species such as superoxide ion (O2•-) and hydroxyl radical (OH•)) due to hydrogen or electron abstraction by interaction of the triplet sensitizer with other molecules. In the type II process, energy is transferred from a triplet sensitizer to O2 with the formation of singlet oxygen

Biomacromolecules, Vol. 4, No. 2, 2003 431

Figure 3. Simplified schematic representation of some possible pathways for reactions leading to free radical-induced degradation of alginate. RF ) riboflavin and LC ) lumichrome.

(1O2) that subsequently reacts with a substrate. Both type I and II processes can take place simultaneously. The distribution between the processes depends on the experimental conditions, and one of the processes may be dominant in a specific system. In addition, RF can also directly oxidize a suitable substrate by electron abstraction in the absence of molecular oxygen.21,26,27 The final photochemical degradation of the polymer is frequently attributed28-31 to the oxidative cleavage of glycosidic bonds. On the basis of the findings discussed above, a simplified schematic representation of some possible pathways for reactions leading to alginate degradation due to the freeradical-induced scission of the glycosidic linkage is depicted in Figure 3. The two pathways that are sketched involve abstraction of the H atom from alginate to yield a C-centered radical on alginate and electron transfer from the carboxyl group and the subsequent formation of a C-centered radical, respectively. However, we should bear in mind that secondary reactions might generate species (e.g., OH• and O2•-), which may be involved in the photosensitized degradation of alginate. Independent of the reaction pathway, the final step is the production of radicals on alginate that can cause scission of glycosidic linkages. Effects of Quenchers and Scavengers on Alginate Degradation. Figure 4 shows the effect of shear rate on the measured viscosity for nonirradiated and irradiated (30 min) alginate solutions (2 wt %) and alginate (2 wt %)-RF (0.1 mM) mixtures in the absence and presence of thiourea and different quenchers or scavengers. The shear-rate dependence of the viscosity for the nonirradiated samples is virtually not influenced by the presence of the different additives, suggesting that the rheological properties of a nonirradiated alginate solution are essentially unaffected by the addition

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Figure 4. Viscosity as a function of shear rate for nonirradiated (a) and irradiated (30 min) (b) alginate (2 wt %) and for alginate (2 wt %)-riboflavin (0.1 mM) solutions without and with thiourea (0.4 mM) or in the presence of different quenchers/scavengers (GSH, 0.01 M; NaN3, 0.01 M; and KI, 0.01 M).

of small amounts of these cosolutes. At this polymer concentration (located in the semidilute regime; see the discussion below) shear thinning is observed, which probably can be ascribed to disruption of entanglements and other intermolecular interactions. The largest degradation effect on the polymer is observed in the presence of RF in combination


with thiourea (see Figure 4b), while the presence of scavengers and quenchers inhibit the RF-mediated photochemical degradation of alginate. A more detailed analysis of these effects is given below. The effect of irradiation time on the normalized viscosity (ηn ≡ ηt/η0, where the subscripts denote the time of irradiation) for an alginate solution (2 wt %) and alginate (2 wt %)-RF (0.1 mM) solutions in the presence of different quenchers or scavengers is displayed in Figure 5. In the lower panel, effects of the different additives on ηn after 30 min irradiation are shown. By recording absorption and fluorescence spectra (data not shown) for alginate-RF solutions in the presence of the different quenchers/scavengers no sign of interactions (no spectral shift) between any of these compounds was detected. The common feature of all the systems containing RF is that the main part of the photoinduced cleavage of the chains has occurred during the first 10 min of irradiation. In the case of alginate without any cosolutes, ηn is constant and independent of the irradiation time. In the presence of additives, except for thiourea, the photochemical degradation of alginate is retarded as compared with the pure alginate-RF system. The more pronounced decrease of ηn upon addition of the electron donor thiourea (0.4 mM) to the alginate-RF system indicates enhanced cleavage of the alginate chains. The conjecture is that thiourea will slow the rate of photodegradation of RF to LC, and the result is that more radicals are generated per time unit, which may promote the scission of the glycosidic bonds in alginate. The agents D-mannitol (0.01 M) and GSH (0.01 M) are both known to scavenge OH• and other free radicals. There is a competition between different reactions, namely hydrogen abstraction from a scavenging/quenching agent or from polymer fragments. Because of the higher mobility of the low molecular weight agents as compared to the polymer species, it is likely that reactions between RF and agent molecules predominate. Since the abstraction of hydrogen is more favorable from GSH than from

Figure 5. (a) Time evolution of the normalized viscosity at a fixed shear rate for irradiated alginate (AL) (2 wt %) and for alginate (2 wt %)riboflavin (0.1 mM) solutions without and with thiourea (0.4 mM) or in the presence of different quenchers/scavengers (D-mannitol, 0.01 M; GSH, 0.01 M; NaN3, 0.01 M; and KI, 0.01 M). (b) Effects of thiourea and various quenchers/scavengers on the normalized viscosity of alginate (2 wt %)-riboflavin solutions after irradiation time of 30 min (see text for more details).



Figure 6. Frequency dependencies of the dynamic moduli for irradiated (15 min) alginate and alginate-riboflavin (0.1 mM) solutions of different polymer concentration. D-mannitol, it is not a surprise that GSH is more effective to prevent chain break in the alginate network than Dmannitol. By adding NaN3 (0.01 M) (quencher of triplet state) to the system with GSH, we observe that the RF-mediated photodegradation of alginate is further arrested. Most of the degradation process is blocked when the alginate-RF solution is exposed to an excess of O2 (by bubbling O2 through the solution for several minutes prior to the experiments) or when KI (0.01 M) is added to the solution. The reason is probably that both agents are efficient 3RF* quenchers. This finding supports the hypothesis16,26,32,33 that the photosensitized degradation of alginate and similar polymers in water under visible light irradiation and aerobic conditions predominantly proceeds through the triplet state of the sensitizer. Effect of Polymer Concentration on Degradation. In this section our attention will be focused on the rheological features of solutions of various alginate concentration, with or without RF (0.1 mM), that have been irradiated for 15 min. In dilute polymer solutions the molecules act as individual moieties and the intermolecular interactions are suppressed. As the concentration increases, the intermolecular interactions become important, and at a certain concentration, the overlap concentration c*, a transition to the semidilute regime occurs and a transient network is formed. Dobrynin et al.34 have suggested criteria of the relative viscosity for the determination of the overlap concentration (η = 2ηs for c*, where ηs is the solvent viscosity) and the entanglement concentration ce (η ≈ 50ηs for ce). For the present system, c* is estimated to be 0.14 wt % and ce is 0.9 and 1.25 wt % for the irradiated (15 min) alginate and alginate-RF solutions, respectively. This value of c* is fairly close to that (0.16 wt %) estimated from c* ) 1/[η], where [η] is the intrinsic viscosity. In dilute solution, the cleavage of chains will occur within the individual molecules, while scission of chains in the semidilute solution can be regarded as fragmentation of the apparent polymer network. The surmise is that the latter effect has a greater impact on the rheological properties.

Figure 7. Log-log plots of G′′ vs G′ for irradiated (15 min) alginate (a) and alginate-riboflavin (0.1 mM) (b) solutions of the polymer concentrations indicated. The values of the power-law exponent R (see text) at different conditions are listed in the inserts.

Typical illustrations of the frequency dependencies of G′ and G′′ for irradiated alginate and alginate-RF samples of the concentrations indicated are depicted in Figure 6. We note that, for both types of sample, G′′ is higher than G′ throughout the frequency domain studied at all concentrations, which is a characteristic feature of systems exhibiting a typical viscous behavior. In general, the values of the dynamic moduli are higher for the alginate samples than for the corresponding alginate-RF mixtures. This effect is

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Figure 8. (a) Frequency dependencies of the complex viscosity for irradiated (15 min) alginate and alginate-riboflavin (0.1 mM) solutions of different polymer concentration. (b) Shear rate dependencies of the viscosity for irradiated (15 min) alginate and alginate-riboflavin (0.1 mM) solutions of different polymer concentration.

associated with the photochemical degradation of the alginate-RF mixtures. In the analysis of viscoelastic features in the linear regime, the data are frequently portrayed35,36 in a Cole-Cole plot of G′′ against G′. If the simple Maxwell model applies, the data should be in the form of a semicircle. However, for systems that exhibit a more complex behavior, with a distribution of relaxation times, it has been reported for different polymer systems37-40 that if the loss modulus is plotted as a function of the storage modulus, a linear relation in a log-log plot is obtained. The value of the power-law exponent R (G′′∝ (G′′)R) is equal to 0.5 for a single Maxwell element. Hence this type of plot can be employed to characterize deviations from the Maxwell model.41 Details of this procedure have been discussed elsewhere.38 Figure 7 shows log-log plots of G′′ vs G′ for illuminated alginate and alginate-RF solutions of different concentration. The data have been shifted vertically by a factor b (see the insert in Figure 7a) to avoid overlap. The introduction of the exponent R should only be considered as a first attempt to characterize the viscoelastic properties and to reveal deviations from the simple Maxwellian behavior. It was observed for all systems that straight lines could represent the data over the studied frequency domain (the values of R at all conditions are given in the insets of Figure 7b). The values of R are ca. 0.8 for concentrations where the chains become entangled and somewhat lower for the lowest concentrations and there is no significant difference between the corresponding values of R for alginate and alginate-RF


Figure 9. Polymer concentration dependencies of the complex viscosity (a) (at the indicated frequency) and the zero-shear viscosity (b) for irradiated (15 min) alginate and alginate-riboflavin (0.1 mM) solutions. The inset plots show a better representation of the differences at low concentrations.

solutions. These results reveal deviations from the Maxwell model and indicate that the relaxation process is not controlled by a single mode, but a rather broad distribution of relaxation modes. This type of behavior is not rare but is frequently observed for cross-linked and/or entangled polymer systems.37-40 Figure 8a illustrates the frequency dependence of the complex viscosity (η*) for irradiated alginate and alginateRF solutions of various concentrations. Above ce the negative frequency dependence of η* becomes gradually more marked as the concentration increases (growth of entanglements). The behavior of η* is similar for the alginate and alginateRF systems, but at higher polymer concentrations the values of η* are perceivably lower for the alginate-RF solutions. In Figure 8b, an analogous illustration of the shear rate dependence of the viscosity is shown for the same solutions. The features are similar as for the complex viscosity, and we observe a progressively stronger shear-thinning effect with increasing polymer concentration and lower values for the alginate-RF solutions. The difference in viscosity behavior between alginate and alginate-RF solutions can be traced to the RF-induced photochemical degradation of the polymer. The concentration dependencies of η* (at an angular frequency of 7.6 rad/s) and the zero-shear viscosity (η0) for irradiated alginate and alginate-RF solutions are depicted in Figure 9. The results from the complex viscosity and the zero-shear viscosity are very similar and reveal a difference in η* or η0 between the two systems that gradually is strengthened as the polymer concentration is increased. The



Conclusions

Figure 10. Shear viscosity compared to the magnitude of the complex viscosity for irradiated (15 min) alginate (a) and alginateriboflavin (b) solutions of different polymer concentration.

difference is discernible even at low concentrations (see the insets in Figure 9), but the most conspicuous feature is the much lover values of η* or η0 for the alginate-RF system at the highest concentrations. This may indicate that the number of effective entanglement couplings has been reduced due to the RF-mediated photochemical scission of polymer chains. For many solutions of nonassociating polymers, the shear viscosity as a function of shear rate is virtually identical to the complex viscosity as a function of frequency, an empirical finding known as the Cox-Merz rule.42 Deviations from this rule are usually observed for more complex polymer systems, such as solutions of hydrophobically modified polymers.39,43-48 Shear viscosity and complex viscosity properties of irradiated solutions of alginate and alginate-RF mixtures of various polymer concentrations are shown in Figure 10 as a function of shear rate or frequency. The most pronounced deviations from the CoxMerz superposition rule are observed for the most entangled solutions. In this case, the complex viscosity is lower than the steady shear viscosity over a wide shear rate/frequency domain. This type of behavior has been reported for many associating polymer systems that display shear-thinning effects. Simulation studies of associating polymer systems have revealed that the cross-link dissociation rate49 increases with increasing shear rate and shear forces may induce dramatic structural reorganizations50 of the network. It is possible that in strongly entangled solutions, the topological constraints are strengthened when moderate shear rates are imposed on the system, while oscillatory shear does not have this effect.

The effects of photoexcited RF on the shear viscosity and other rheological quantities of aqueous solutions of alginate at pH ) 7.4 have been reported in this work. Illumination of RF in alginate solutions, under aerobic conditions, causes disruption of the polymer network and a change in the rheological parameters; e.g., the shear viscosity decreases. This effect is more pronounced at higher polymer concentrations. The main results can be summarized in the following way: (1) The RF-sensitized photochemical degradation of alginate is inhibited in the presence of quenchers/scavengers, while the degradation of the polymer is further enhanced if the electron donor thiourea is added to the alginate-RF solution. (2) From a rheological point of view, cleavage of polymer chains has a larger impact on the rheological response in strongly entangled polymer solutions than in dilute solutions. (3) The frequency dependences of the dynamic moduli for the solutions, recorded in the terminal zone of the mechanical spectrum, cannot be described by a single Maxwell element which suggests that there is not a single relaxation time that controls the time scale, but a distribution of relaxation times. (4) Significant shear-thinning effects and deviations from the Cox-Merz rule are observed in entangled polymer solutions. The deviations from CoxMerz rule are often observed in associating systems that display shear thinning, and are probably due to shear induced structural reorganizations.50 References and Notes (1) Kost, J.; Langer, R. AdV. Drug DeliVery ReV. 2001, 46, 125. (2) Vyas, S. P.; Sood, A.; Venugopalan, P.; Mysore, N. Pharmazie 1997, 52, 815. (3) Pozzi, F.; Furlani, P.; Gazzaniga, A.; Davis, S. S.; Wilding, I. R. J. Controlled Release 1994, 31, 99. (4) Freichel, O. L.; Lippold, B. C. Int. J. Pharm. 2001, 216, 165. (5) Gupta, V. K.; Beckert, T. E.; Price, J. C. Int. J. Pharm. 2001, 213, 83. (6) Khan, M. Z. I.; Prebeg, Z.; Kurjakovic, N. J. Controlled Release 1999, 58, 215. (7) Ashford, M.; Fell, J. T. J. Drug Targeting 1994, 2, 241. (8) Jeong, B.; Bae, Y. H.; Kim, S. W. J. Controlled Release 2000, 63, 155. (9) Scherlund, M.; Brodin, A.; Malmsten, M. J. Colloid Interface Sci. 2000, 229, 365. (10) Saslawski, O.; Weingarten, C.; Benoit, J. P.; Couvreur, P. Life Sci. 1988, 42, 1521. (11) Marin, A.; Muniruzzaman, M.; Rapoport, N. J. Controlled Release 2001, 75, 69. (12) Vanhillegersberg, R.; Kort, W. J.; Wilson, J. H. P. Drugs 1994, 48, 510. (13) Yui, N.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 26, 141. (14) Rabek, J. F. Photodegradation of Polymers, Physical Characteristics and Applications; Springer-Verlag: Berlin, 1996. (15) Frati, E.; Khatib, A. M.; Front, P.; Panasyuk, A.; Aprile, F.; Mitrovic, D. R. Free Radical Biol. Med. 1997, 22, 1139. (16) Heelis, P. F. Chem. Soc. ReV. 1982, 11, 15. (17) Smidsrød, O.; Draget, K. I. Carbohydr. Eur. 1996, 14, 6. (18) Asker, A. F.; Habib, M. J. Drug DeV. Ind. Pharm. 1990, 16, 149. (19) Cairns, W. L.; Metzler, D. E. J. Am. Chem. Soc. 1971, 93, 2772. (20) Heelis, P. F.; Parsons, B. J.; Phillips, G. O.; Swallow, A. J. J. Phys. Chem. 1986, 90, 6833. (21) Lu, C.-Y.; Wang, W.-F.; Lin, W.-Z.; Han, Z.-H.; Yao, S.-D.; Lin, N.-Y. J. Photochem. Photobiol. B.: Biol. 1999, 52, 111. (22) Foote, C. S. Science 1968, 162, 963. (23) Laustriat, G. Biochimie 1986, 68, 771. (24) Kanofsky, J. R. Chem. Biol. Interact. 1989, 70, 1. (25) Huber, R. A. Eur. J. Biochem. 1990, 187, 283.

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BM020117A

Riboflavin-Photosensitized Changes in Aqueous Solutions of Alginate

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