Pii: s0142-9612(01)00003-5

Hydrogels for combination delivery of antineoplastic agents Kamal H. Bouhadir , Eben Alsberg , David J. Mooney * Department of Biologic and Materials Sciences, University of Michigan, 3074 H.H. Dow Building, 2300 Hayward Street, Ann Arbor, MI 48109-2136, USA Department of Chemical Engineering, University of Michigan, 3074 H.H. Dow Building, 2300 Hayward Street, Ann Arbor, MI 48109-2136, USA Department of Biomedical Engineering, University of Michigan, 3074 H.H. Dow Building, 2300 Hayward Street, Ann Arbor, MI 48109-2136, USA Received 1 September 2000; accepted 27 December 2000 The systemic delivery of anticancer agents has been widely investigated during the past decade but localized delivery may o!er a safer and more e!ective delivery approach. We have designed and synthesized a novel hydrogel to locally deliver antineoplasticagents, and demonstrate the di!erent types of release that can be achieved from these hydrogels using three model drugs:methotrexate, doxorubicin, and mitoxantrone. Alginate was chemically modi"ed into lowmolecular weight oligomers and cross-linked with a biodegradable spacer (adipic dihydrazide) to form biodegradable hydrogels. The model antineoplastic agents wereloaded into the hydrogel via three di!erent mechanisms. Methotrexate was incorporated within the pores of the hydrogel and wasreleased by di!usion into the surrounding medium. Doxorubicin was covalently attached to the polymer backbone via a hydrolyti-cally labile linker and was released following the chemical hydrolysis of the linker. Mitoxantrone was ionically complexed to thepolymer and was released after the dissociation of this complex. These three release mechanisms could potentially be used to delivera wide selection of antineoplastic agents, based on their chemical structure. This novel delivery system allows for the release of singleor combinations of antineoplastic agents, and may "nd utility in localized antineoplastic agent delivery.
Keywords: Alginate; Controlled release; Biodegradable; Antineoplastic agents; Doxorubicin; Methotrexate; Mitoxantrone reduce the side e!ects associated with the systemic deliv-ery of anticancer agents [3}6]. Several drugs have been A broad spectrum of antineoplastic agents has been found to amplify the anticancer activity of other drugs found to be e!ective in combating di!erent types of [7}10]. This synergistic e!ect can potentially lead to cancer. However, to achieve complete eradication of tu- reduced doses for each drug administered [11}13].
mors, antineoplastic agents are administered systemically Hence, the administration of several drugs simulta- in high doses, and almost all drugs e!ective in killing neously could reduce the side e!ects caused by high doses cancer cells cause damage to other healthy tissues and of single drugs and could prevent the development of organs. This is due to the non-speci"c uptake of these multi-drug resistance (MDR) [14,15].
agents by healthy organs such as the kidney, liver, bone An alternative approach to systemic delivery of anti- marrow, and heart. The adverse side e!ects include neoplastic agents is the localized release from a polymer severe immune suppression, myelosuppression, neph- [16]. We have designed and synthesized a novel hydrogel rotoxicity, and cardiotoxicity [1,2]. During the past dec- to deliver anticancer agents locally. We have oxidized ade, many researchers have investigated the sequential sodium alginate to form lowmolecular weight oligomers and simultaneous delivery of drug combinations to that are cross-linked with adipic dihydrazide to formhydrogels. We hypothesize that a variety of antineoplas-tic agents could be locally delivered, alone or in combina-tion from these hydrogels. The kinetics of drug release * Correspondence address: Department of Chemical Engineering, could potentially be controlled by exploiting three types University of Michigan, 3074 H.H. DowBuilding, 2300 Hayward St., interaction, and covalent coupling via degradable E-mail address: [email protected] (D.J. Mooney).
linkages. We have incorporated three model drugs into 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 0 0 3 - 5 K.H. Bouhadir et al. / Biomaterials 22 (2001) 2625}2633 cross-linked oxidized alginate hydrogels: methotrexate, mixture to reduce any unreacted periodate. The reaction doxorubicin, and mitoxantrone to test this possibility. All was stirred for 0.5 h at ambient temperature, and the three drugs are potent antineoplastic agents that have been extensively utilized in cancer chemotherapy.
(Spectra/Pro membrane, MWCO 3500) against double- Methotrexate is a folic acid antimetabolite inhibitor of distilled water (dd. HO) for three days. The water was dihydrofolate reductase that has been widely used in the changed at least 3 times a day. The solutions were then treatment of neoplastic diseases [17]. Doxorubicin is an concentrated to around 100 ml and freeze dried under antineoplastic agent from the anthracycline antibiotic reduced pressure to yield a white product (6.9 g, 86%). IR family that has been most commonly used to treat solid (KBr pellet, cm\) 3336, 2942, 1730, 1622, 1406, 1321, tumors [18,19]. Mitoxantrone, an anthracenedione, is an intercalating agent that is e!ective in treating varioustumors [18]. Methotrexate, an anionic drug, is used in 2.3. Determination of the degree of oxidation this study as it is expected to rapidly di!use out into thesurrounding medium. We have used doxorubicin as The degree of oxidation of alginate was determined a model drug for chemical-controlled release. Doxo- by measuring the percentage of periodate that was rubicin is expected to be released following the hydrolysis consumed before quenching with ethylene glycol. The of the degradable bond linking it to the gel [20]. In consumption of sodium periodate was determined by addition, we have used mitoxantrone as a model drug for spectrophotometrically measuring the formation of ionic-controlled release. Mitoxantrone is expected to a complex between unreacted periodate anion and form ionic complexes with the carboxylate groups on the thyodene. Brie#y, equal volumes of freshly prepared sugar residues of the polymer backbone.
aqueous solutions of potassium iodide (20% w/v in pH7.0 sodium phosphate bu!er) and thyodene solution(10% w/v in pH 7.0 sodium phosphate bu!er) were mixed as an indicator solution. An Erlenmeyer #ask (100 ml)was covered with aluminum foil and charged with an aqueous solution of alginate (50 ml, 1.0% w/v) and anaqueous solution of sodium periodate (10.1 ml, 0.25 M).
Sodium alginate was purchased from Pronova Bio- The mixture was stirred at room temperature. At di!er- materials (Drammen, Norway). Sodium periodate, adipic ent time intervals, aliquots (0.3 ml) were rapidly removed dihydrazide, ethylene glycol, and anhydrous KBr were and diluted to a volume of 100 ml using dd. HO.
purchased from the Aldrich Chemical Company (Mil- A 0.5 ml aliquot of this solution was immediately mixed waukee, WI) and used as received. Ethanol (95%), with 1.0 ml of the indicator solution in a cuvette. The methanol, and concentrated hydrochloric acid were pur- concentration of the unreacted periodate was measured chased from Fisher Scienti"c Company (Fair Lawn, NJ) spectrophotometrically at 486 nm. This number was then and were used as received. Doxorubicin and mitoxan- subtracted from the original concentration of periodate trone hydrochlorides were purchased from Sigma to yield the amount of periodate that was consumed.
Chemical Company (St. Louis, MO). Methotrexate waspurchased from Fluka Chemical Corporation (Ronkon- 2.4. Size exclusion chromatography (SEC) koma, NY). Phosphate bu!er saline (PBS) and Dul-becco's SEC analysis was performed on a liquid chromato- purchased from Life Technologies (Grand Island, NY).
graph consisting of a SpectraSystem P1000 pump (Ther- Eagle's Minimum Essential Medium (EMEM), fetal bo- mal Separation Products), a Rheodyne 7010 manual vine serum, and MCF-7 breast epithelial cell lines were injector, a dual di!erential viscometer and right-angle purchased from the American Tissue Culture Collection laser light scattering (RALLS) detector (Viscotek T 60, (Manassas, VA). Cell culture plates (96-well) were pur- " 670 nm) and a laser refractometer detector (Viscotek chased from Falcon (Lincoln Park, NJ).
LR40, "670 nm), the detectors being connected in par-allel. The mobile phase consisted of aqueous sodium nitrate (0.1 M) and was periodically degassed with anon-line degasser. The mobile phase was delivered at am- A 1 l Erlenmeyer #ask was wrapped with aluminum bient temperature with a nominal #owrate of 0.7 ml/min.
foil and charged with sodium alginate (8.0 g). Double- The separations were carried out on two TSK GMPW6* distilled water (800 ml) was added, and the mixture was (TosoHaas, 7.8;300 mm) mix bed columns. Polymers stirred until the solid dissolved. An aqueous solution of were dissolved in mobile phase solvent at a concentration sodium periodate (0.25 M, 162 ml) was added and the of 1}3 mg/ml by mechanical stirring for a minimum of 6 h reaction was stirred for 24 h at room temperature.
until completely hydrated. A 100 l injection volume was Ethylene glycol (2.3 ml) was then added to the reaction used for all analyses. The chromatograms were analyzed K.H. Bouhadir et al. / Biomaterials 22 (2001) 2625}2633 using the TriSEC 3.0 GPC software (Viscotek). A di!er- ance of the drug at 327.5 nm (methotrexate), 480 nm ential index of refraction (dn/dc) of 0.154 ml/g was used spectra were collected on a Perkin Elmer Lambda 12UV/VIS spectrophotometer.
2.8. In vitro drug release from cross-linked oxidized Hydrogels were formed at various concentrations of oxidized alginate, adipic dihydrazide and calcium chlor-ide in 24-well plates. The contents of each well were Sterile tubes were charged with aqueous solutions of mixed and allowed to gel for 1 h at ambient temperature adipic dihydrazide (150 l, 0.5 M), calcium chloride (20 l, on a mechanical shaker. The hydrogels were immersed in 1.0 M), and DMEM (20 l). Solutions of methotrexate, de-ionized water and incubated at 373C for 24 h to reach doxorubicin hydrochloride, or mitoxantrone (10 l, the equilibrium swelling condition. The hydrogels were 25 mg/ml in DMSO) were then added to the above aque- transferred to 2 ml vials and weighed (wet weight). The ous solutions, and the mixtures were mixed for 15 min.
gels were then frozen, lyophilized, and the dried samples An aqueous solution of oxidized alginate (300 l, were weighed (dry weight). The swelling ratio was de"ned 10% w/w) was then added, and the contents of the tubes as the ratio of (wet weight ! dry weight)/(dry weight).
were mixed thoroughly and allowed to gel for 1 h. Aque- Infrared spectra were recorded as percent transmittance ous DMEM solutions (5 ml) containing penicillin and using a Nicolet 5DX FTIR spectrophotometer and streptomycin were added to each tube, and the tubes a Hewlett Packard 7470A plotter. Samples were pressed were incubated at 373C. The medium was removed as KBr pellets using a hydraulic press (Carver, Inc.). IR periodically and replaced with fresh DMEM, and the (KBr pellet, cm\) 3554, 3472, 3414, 3236, 1660, 1622, released drug was quanti"ed as described previously.
A total of 0.25 mg of each drug was always loaded per gelsample. Release data are reported as a percentage of this 20% w/w) and adipic dihydrazide (125 l, 0.5 M) contain- ing calcium chloride (80 mM) were mixed in 15 ml conicaltubes (in quadruplicates) and allowed to gel for 5 h.
Aqueous solutions of sodium alginate were oxidized in Solutions of Dulbecco's Modi"ed Eagles Medium the dark using sodium periodate at room temperature (DMEM, 10 ml) were added, and the tubes were incu- following a modi"ed procedure reported previously bated at 373C. The medium was replaced with fresh [23}25]. The amount of sodium periodate used in these medium on a weekly basis. Four tubes were removed reactions was varied to form alginates with di!erent every week, and the medium was decanted. The gels were degrees of oxidation. FTIR analysis of the oxidized prod- frozen, lyophilized, and the dry solid was weighed.
uct revealed a newpeak at 1730 cm\ corresponding forthe vibrational symmetric stretching vibration of the 2.7. In vitro drug release from alginate hydrogels aldehyde groups. The degree of oxidation was deter-mined indirectly by measuring the percentage of sodium An aqueous alginate solution was prepared by dissolv- periodate that was consumed in each reaction. Sodium ing sodium alginate (2 g), sodium chloride (0.8 g), and periodate was almost quantitatively consumed in all con- sodium hexametaphosphate (0.4 g) in 96.8 ml of dd. HO.
ditions except when 100 percentage equivalents was used A 50 ml conical tube was charged with 5 g of the above (Table 1). In this case, only 69% of the sodium periodate solution. A solution of the drug (165 l, 25 mg/ml in was consumed after 24 h. This is consistent with earlier DMSO) was then added and mixed thoroughly. Aqueous papers reporting that the aldehyde groups of the oxidized calcium sulfate slurry (200 l, 158 mg/ml) was added, uronate react with neighboring alcohol groups to form mixed thoroughly, and the mixture was cast between hemiacetals [23]. The formation of hemiacetals protects parallel glass plates separated with glass spacers (2 mm in the alcohol groups of neighboring uronates from further thickness). The gels were allowed to set for 8 h. The glass plates were separated, and the disks were punched outwith a 12.7 mm hole puncher (McMaster-Carr). The hy- 3.1. Molecular weight distribution of oxidized alginate drogel disks were placed in scintillation vials (2 disks ineach vial). Aqueous PBS (pH 7.4) was added, and the The molecular weight distribution of oxidized alginate vials were incubated at 373C. The medium was replaced was analyzed by aqueous gel permeation chromatogra- periodically, and the amount of drug that was released phy. The weight-average molecular weight of the in the medium was quanti"ed by measuring the absorb- starting alginate was 394 kDa. Alginate oxidized with "ve K.H. Bouhadir et al. / Biomaterials 22 (2001) 2625}2633 Table 1The experimental degree of oxidation of cross-linked oxidized alginatehydrogels. Reactions were run at a concentration of 0.8% w/w alginatein the dark at room temperature for 24 h Table 2Molecular weight distributions of alginate and oxidized alginates Represents the percentage equivalents of sodium periodate that was initially added to the reaction mixture (based on the uronate groups).
F(x) is the weight fraction of the polymer that have a molecular equivalents of sodium periodates formed a polymer witha weight-average molecular weight of 198 kDa (Table 2).
The weight-average molecular weight then decreased asthe percentage equivalents of periodate was increased toreach 29 kDa with 100% equivalents of sodium peri-odate. As a result, the intrinsic viscosity of the polymers Fig. 1. Synthesis and cross-linking of oxidized alginate: (a) sodium decreased as periodate concentration was increased.
Only 12.5% weight fraction of the original unmodi"edalginate has a molecular weight below 80 kDa. This num-ber increased to reach a value of 96% for alginate thatwas oxidized with 100 equivalents of sodium periodate The degree of swelling of cross-linked oxidized alginate hydrogels was analyzed after the hydrogels reached the equilibration swelling in dd. HO. The swelling ratio of these hydrogels varied signi"cantly depending on the Hydrogels were subsequently formed by the reaction concentrations of both the ionic and the covalent of adipic dihydrazide and the oxidized alginates. The cross-linkers. The swelling ratio of hydrogels made with hydrazide group reacts with the aldehyde groups in oxi- oxidized alginate (100 equivalents periodate) and cross- dized alginate to form hydrazone bonds (Fig. 1). The linked at 150 mM adipic dihydrazide was 29.9$1.2 in dd.
hydrogels were washed with water and soaked in dd.
HO (Table 3). The swelling ratio then decreased with HO for 24 h to release the unreacted adipic dihydrazide.
increasing concentrations of calcium to reach a minimum The gels were frozen and lyophilized. FTIR spectroscopic of 11.7$0.3 at 40 mM calcium ions. A similar trend was analysis of the discs indicated the disappearance of the observed when the concentration of covalent cross-links peak at 1730 cm\ and the appearance of another peak was increased. The swelling ratio was 20.1$1.2 at 40 mM at 1660 cm\ that corresponds to the stretching vibra- calcium ions at 50 mM adipic dihydrazide, and decreased tion of the carbonyl in the hydrazide group.
to 11.7$0.3 at 40 mM as the concentration of adipic K.H. Bouhadir et al. / Biomaterials 22 (2001) 2625}2633 Table 3Swelling ratio of cross-linked oxidized alginate hydrogels as a functionof the concentrations of the ionic and covalent cross-linkers. Hydrogelswere formed at 6% w/w oxidized alginate in dd. HO Fig. 2. Percentage weight loss of cross-linked oxidized alginate hydro- gels as a function of time. Hydrogels were formed at (ⅷ) 100 mM and (*) 150 mM adipic dihydrazide and 40 mM CaCl prepared with 10% w/w oxidized alginates (69% oxidized) in dd. H dihydrazide increased to 150 mM (Table 3). The swelling Methotrexate was quantitatively released within 2 days ratio slightly increased as the adipic dihydrazide con- of incubation from hydrogels formed with 50 mM adipic centration was further increased. This latter result is dihydrazide. The overall release time of methotrexate consistent with past studies which indicated a decreased increased to 3 days at 75 mM adipic dihydrazide and cross-link density, and increased content of dangling 7 days at 150 mM adipic dihydrazide. However, 75% of cross-linkers, above 150 mM adipic dihydrazide [25].
the loaded drug was released initially at a constant rate of37.5% per day in all conditions. Drug release from hy- drogels formed with concentrations of adipic dihydrazideabove 150 mM exhibited similar release kinetics (not Alginate hydrogels degrade in an uncontrolled manner following the release of calcium ions into the surrounding Doxorubicin was covalently incorporated into the hy- medium. To evaluate whether the degradation of cross- drogel by reacting it with excess adipic dihydrazide as linked oxidized alginates can be controlled, gels were reported previously for daunomycin [20]. The ketone formed with 10% w/w oxidized alginates (oxidized with group on the C13 position of doxorubicin reacts with the 100 equivalents of periodate) and cross-linked with hydrazide group to form the doxorubicin}adipoyl hy- adipic dihydrazide and/or calcium. The percentage drazide conjugate (Fig. 3). Upon mixing with oxidized weight loss of these gels was determined following incu- alginate, the free hydrazide group on this conjugate re- bation in medium (Fig. 2). Hydrogels formed at 100 mM acts with the pendant aldehyde group on the backbone adipic dihydrazide degraded after 3 weeks of incubation of oxidized alginate to form a labile hydrazone bond at a rate of 5% per day. Hydrogels cross-linked at (Fig. 3). The release of doxorubicin from cross-linked 150 mM adipic dihydrazide and 40 mM calcium chloride oxidized alginate was dependent on the concentration of degraded at a lower rate of 2.5% per day (Fig. 2). Only the covalent cross-linker (Fig. 4b). Doxorubicin was 40% of the gel weight dissolved after 15 weeks. Therefore, quantitatively released within 2 days from all hydrogels hydrogels may be formed with this approach that de- formed at 50 mM adipic dihydrazide. At 75 mM adipic grade in time frames from 3 weeks to more than 4 months dihydrazide, doxorubicin was released after 3 days. In- by simply varying the number of covalent and ionic creasing the concentration of adipic dihydrazide to 100 mM prolonged the total release of doxorubicin to6 days of incubation, and at a higher concentration of 150 mM adipic dihydrazide, only 20% of doxorubicin wasreleased after 22 days of incubation at 373C.
The three model drugs were "rst separately incorpor- Mitoxantrone was expected to form ionic complexes ated in cross-linked oxidized alginate hydrogels. Metho- with the hydrogels (Fig. 3), and would thus be released trexate (Fig. 3) was not expected to interact ionically or only as the gels degraded. The release of mitoxantrone covalently with the hydrogel. The release pro"le of from cross-linked oxidized alginate hydrogels was methotrexate was not signi"cantly dependent on the con- in#uenced by the concentration of adipic dihydrazide centration of the covalent cross-linker (Fig. 4a), as ex- (Fig. 4c). The entire loaded drug was released after only pected at the high degrees of swelling in these hydrogels.
2 days of incubation from hydrogels formed with 50 mM K.H. Bouhadir et al. / Biomaterials 22 (2001) 2625}2633 Fig. 3. Proposed mechanism for drug incorporation. Doxorubicin is chemically linked to oxidized alginate with a hydrazone bond. Mitoxantroneforms an ionic complex with the carboxylate groups.
adipic dihydrazide. Hydrogels formed at 75 and 100 mM of incubation as expected for an ionically interacting adipic dihydrazide released the entire drug after 3 and 6 days, respectively. However, hydrogels formed at To test the utility of oxidized alginate hydrogels in 150 mM adipic dihydrazide released only 7% of the delivering combinations of drugs, cross-linked oxidized loaded drug after 21 days of incubation.
alginate hydrogels have been loaded with all three drugs Methotrexate, doxorubicin, and mitoxantrone have simultaneously. Methotrexate was quantitatively re- also been separately incorporated into calcium cross- leased after 9 days of incubation (Fig. 6). Doxorubicin linked alginate hydrogels, and their release monitored was released slowly at a rate of 1.7% per day for 13 days spectrophotometrically as a control. Methotrexate was followed by a rapid release of 19.5% per day during the quantitatively released after 8.5 h of incubation at 373C last 4 days (Fig. 6). Mitoxantrone, on the other hand, was as expected. Doxorubicin was released over a 3.5 day not signi"cantly released during the initial 10 days. How- time period (Fig. 5). The release of mitoxantrone ever, a rapid release of 24% per day was observed during from alginate hydrogels was negligible over the "rst week K.H. Bouhadir et al. / Biomaterials 22 (2001) 2625}2633 Fig. 5. The cumulative release of (ⅷ) methotrexate and (᭿) doxorubicinfrom alginate hydrogels. Hydrogels were formed with 2% w/w alginatein dd. HO and cross-linked with calcium sulfate.
Fig. 6. The release of (ⅷ) methotrexate, (᭿) doxorubicin, and (᭡)mitoxantrone from cross-linked oxidized alginate hydrogels loadedwith all three drugs. Hydrogels were formed at 100 mM adipic dihyd-razide and 6% w/w oxidized alginates (69% oxidized) in dd. HO.
utilized the reactive aldehyde groups to cross-link thesepolymers and form hydrogels. These polymers could besynthesized in a relatively short period of time and could Fig. 4. The cumulative release of (a) methotrexate, (b) doxorubicin, and be formed with a wide range of molecular weights. The (c) mitoxantrone from cross-linked oxidized alginate hydrogels. Hydro- weight fraction of the product that had a molecular gels were formed at (ⅷ) 50 mM adipic dihydrazide, (*) 75 mM adipicdihydrazide and (᭿) 100 m weight below 80 kDa could also be readily controlled.
M adipic dihydrazide and (᭡) 150 mM adipic dihydrazide. All hydrogels were prepared with 6% w/w oxidized al- This is very attractive for biomedical applications of alginate derivatives since polymers with molecularweights lower than 80 kDa are expected to be clearedfrom the body in a similar manner to lowmolecular We have separately incorporated three model drugs We have synthesized a novel hydrogel derived from into cross-linked oxidized alginate hydrogels: metho- alginate for the single or simultaneous delivery of anti- trexate, doxorubicin, and mitoxantrone to demonstrate neoplastic agents. We have previously demonstrated the that various drug}hydrogel interactions can be exploited synthesis and cross-linking of poly(aldehyde guluronate), to control the kinetics of drug release. Methotrexate was PAG to form hydrogels [24]. However, we were limited quantitatively released over a 2 days period from hydro- with that polymer by the low molecular weight of PAG gels formed at 50 mM adipic dihydrazide, and the release (6 kDa), and the synthesis required several labor intensive was extended up to 7 days from hydrogels formed at puri"cation steps. In the present study, we oxidize so- higher concentration of adipic dihydrazide. This rapid dium alginate directly, bypassing the hydrolysis step, and release is expected for a drug with minimal hydrogel K.H. Bouhadir et al. / Biomaterials 22 (2001) 2625}2633 interaction. The higher concentrations of adipic dihyd- The simultaneous delivery of a combination of thera- razide led to a lower swelling ratio, and this likely caused peutical active agents has recently been shown to be the slower release. These "ndings are consistent with bene"cial in combating cancer and HIV infection. How- many past reports of the rapid release of non-coupled or ever, it might even be more attractive to deliver each drug non-interacting drugs from alginate hydrogels [27].
with a di!erent release pro"le. We have incorporated all To demonstrate the utility of these hydrogels to deliver the three drugs simultaneously into cross-linked oxidized drugs via a chemical-controlled release mechanism, we alginate hydrogels and have observed three di!erent re- have incorporated doxorubicin by covalent coupling to lease pro"les (Fig. 6). Methotrexate was completely re- the alginate backbone. Doxorubicin was released from leased after 9 days of incubation. Both doxorubicin and days to weeks depending on the concentration of adipic mitoxantrone were completely released following the dihydrazide used (Fig. 5b). This clearly indicates that degradation of the hydrogel after 17 days of incubation.
doxorubicin is not merely interacting ionically with the However, over 20% of doxorubicin was slowly released hydrogel but is covalently linked to the polymer back- during the "rst 13 days whereas mitoxantrone was not bone [20]. Doxorubicin is released following the hy- signi"cantly released during that time. This demonstrates drolysis of the hydrazone bond connecting it to the clearly that we can indeed load a variety of drugs and hydrogel. We can potentially release a variety of drugs release them simultaneously over a wide range of time that contain an aldehyde, a ketone, or a hydrazide group frame. Another approach to achieve delivery of multiple in a similar manner. A detailed analysis of this mecha- drugs simultaneously or in sequence is to deliver each nism of drug release from these types of polymers has from a di!erent polymer, and either mix the formulations been provided in a previous publication [20].
prior to delivery or introduce each separately. However, To con"rm that doxorubicin was covalently linked to the data in this paper demonstrate a single hydrogel may oxidized alginate hydrogels, we used calcium cross-lin- be used to deliver multiple drugs either simultaneously or ked alginate hydrogels as a control. The release of sequentially. This may simplify multi-drug delivery, and doxorubicin is expected to followa di!usion-controlled biomaterial development and regulatory approval.
release in a similar manner to methotrexate in these gels,due to the lack of potential for covalent coupling.
Doxorubicin was released from these gels over 3.5 days (Fig. 5), which was somewhat longer than expected. Thissuggests that doxorubicin, a positively charged molecule, We have designed and synthesized novel hydrogels may be interacting ionically with the hydrogel, and this derived from alginate to simultaneously deliver a variety interaction is slowing down the release of doxorubicin.
of drugs. We can control the degradation pro"le of the However, doxorubicin was released over a longer time hydrogel from days to months and the release of model from oxidized alginate hydrogels (formed at 150 mM antineoplastic agents over a similarly wide range of time adipic dihydrazide) indicating a chemical attachment be- frames. Three di!erent release mechanisms: di!usion- tween the drug and the polymer backbone [20].
controlled, covalent bond degradation, and ionic dis- We have also incorporated and released mitoxantrone sociation-controlled mechanisms, can be utilized in this from oxidized alginate hydrogels to determine if ionic system to control the kinetics of drug release. This novel interactions can be used to control drug release. The delivery system could be potentially used for the control- release of mitoxantrone was coupled with the degrada- led delivery of a variety of anticancer compounds sequen- tion of the hydrogels, as expected for this mechanism.
tially or simultaneously, and in a localized manner.
Mitoxantrone was completely released from 2 to 6 daysfrom hydrogels cross-linked with 50}100 mM adipicdihydrazide (Fig. 4). However, less than 10% of the drug was released after 22 days from hydrogels formed at highconcentrations of adipic dihydrazide (150 mM) suggesting The authors would like to thank Gavin Sy for his help that mitoxantrone is forming ionic junctions with the in this project. The authors would also like to thank carboxylate groups on the polymer backbone similar to Reprogenesis for "nancial support of this research. The divalent cations. Mitoxantrone was not signi"cantly re- research was also supported in part by a grant from leased from calcium cross-linked alginate hydrogels dur- the John and Suzanne Munn Endowed Research Fund of ing the initial 4 weeks of incubation (data not shown).
the University of Michigan Comprehensive Cancer These gels are not expected to degrade in vitro. This supports our prediction that mitoxantrone is forming ionic junctions between the carboxylate groups on the polymer backbone (Fig. 3). Ionic interactionsbetween alginate and many drugs are possible, and have [1] Lowenthal RM, Eaton K. Toxicity of chemotherapy. Oncol Clin K.H. Bouhadir et al. / Biomaterials 22 (2001) 2625}2633 [2] Klein-Szanto AJ. Carcinogenic e!ects of chemotherapeutic com- damaging agents in a human lung-cancer cell line. Int J Cancer pounds. Prog Clin Biol Res 1992;374:167}74.
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