Hydrogel Complex In Cancer Therapy - Biomacromolecules

Mar 2, 2009 - The release of the drug in phosphate buffer solution showed an initial burst effect, followed by a near zero-order release phase over th...
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Cisplatin/Hydrogel Complex In Cancer Therapy Mario Casolaro,*,† Renzo Cini,† Barbara Del Bello,‡ Marco Ferrali,‡ and Emilia Maellaro‡ Dipartimento di Scienze e Tecnologie Chimiche e dei Biosistemi, and Dipartimento di Fisiopatologia, Medicina Sperimentale e Sanita` Pubblica, Universita` degli Studi di Siena, Via Aldo Moro 2, I-53100 Siena, Italy Received December 23, 2008; Revised Manuscript Received February 2, 2009

Hydrogels containing R-amino acid residues (L-phenylalanine, L-histidine) were used to complex the chemotherapeutic agent cisplatin. The release of the drug in phosphate buffer solution showed an initial burst effect, followed by a near zero-order release phase over the seven days of reported period. Unlike the nonreleasing pattern of the hydrogel poly(N-acryloyl-L-phenylalanine-co-N-isopropylacrylamide) (CP2), the homopolymer poly(N-acryloyl-L-phenylalanine) (P9) hydrogel showed a released amount of cisplatin loaded from a water/ DMSO mixture that was three times greater than that loaded from simple water. The hydrogel P9 formed with cisplatinum(II) complex species of well-defined stoichiometry; the drug species was released by a chemically controlled process. The Pt(II)/L (L is the monomeric unit of the polymer) stoichiometric molar ratio of 0.5, corresponding to two close carboxylate groups per Pt(II), was found by the viscometric data on the soluble polymer analogue. The platinum species released from cisplatin-loaded (from water) hydrogel retained its cytotoxic activity toward Me665/2/21 human melanoma cell line, in the same manner shown by the native cisplatin. On the contrary, the platinum species released from cisplatin-loaded (from water/DMSO) hydrogel was devoid of any cytotoxic effect.

Introduction Among the antitumor agents, cisplatin [cis-diamminedichloroplatinum(II)] is a commonly used drug in the treatment of a variety of solid tumors, usually administered intravenously. A total of 90% of the cisplatin is bound to plasma proteins in the blood and, thus, does not enter the cells;1 the remaining unbound drug enters the cell as uncharged small molecule.2 Its applicability is limited to a narrow range of tumors, like genitourinary, head and neck, and lung tumors.3 Some tumors have acquired resistance to cisplatin, while others develop resistance after the initial treatment. In addition, this drug causes many severe toxic side effects, including nephrosis, neurosis, myelosis, and hematosis, along with gastrointestinal reactions.4-6 In view of these limitations, the research has been extensively focused on drug delivery systems to achieve the higher concentration of drug in the tumor tissue and the controlled release profile for extended time periods. Most of these systems have been usually designed for intravenous administration on the basis of the EPR (enhanced permeability retention) effect7 and include liposomes,8 microspheres,9-11 and polymeric micelles.12 Recent strategies aimed at overcoming some drawbacks of cisplatin consist in developing platforms for chemotherapy that deliver the drug to the local environment of a solid tumor. Besides the cisplatin nanocapsules, based on lipid formulations, reported by Burger et al.,13 the approach of synthetic microparticles is also of interest.14 The polymeric systems are attractive for their easy tailoring and in vivo tolerance, enhabling the delivery of various bioactive substances after implantation.15,16 The platform developed by Gemeinhart was based on polymeric microparticles14 containing the acrylic acid moiety, with the * To whom correspondence should be addressed. Phone: +39 0577 234388. Fax: +39 0577 234177. E-mail: [email protected]. † Dipartimento di Scienze e Tecnologie Chimiche e dei Biosistemi. ‡ Dipartimento di Fisiopatologia.

latter showing a liable complexing ability toward cisplatinum(II) species. The cisplatin entrapped or complexed in polymeric devices has a reduced systemic toxicity and an increased activity.17 The nontoxic effect of the polymers enables the corresponding hydrogels to be tailored in medical treatment for more efficient routes in the administration of pharmaceutical compounds, especially metal-based drugs. Recently, some hydrogels containing R-amino-acid residues were proposed as new polymeric compounds to get tunable delivery rates of fac(Ru(CO)3)2+-based potential drug18 and Cu(oxicam-H)2,19 as based on the therapeutic requirements and prolonged deliveries to ensure a constant concentration of the drug. The advantages of implantable drug delivery tools may include a precise dose control, a high release efficiency, and a low toxicity and overcomes the disadvantages connected with the conventional methods.20 Following this approach, we wish to report the cisplatincomplexing ability of the novel hydrogels containing Lphenylalanine21 and L-histidine residues22,23 (Chart 1). Unlike the former compound containing only the carboxylate group, the polymer with the histidine moiety shows an ampholytic behavior having a stronger complexing ability toward heavy metal ions. In all cases, copolymers with the Nisopropylacrylamide were also studied to show the influence of the coordinating group density on the cis-platinum(II) complexation. The stoichiometry and Pt(II)-complexing ability of the coordinating groups were shown by the soluble polymer analogues. To test the pharmacological efficacy of the cisplatin released from hydrogels, the cytotoxic properties (in terms of apoptotic response) were evaluated in cultured human melanoma cells, also in comparison with the cell death response afforded by the native cisplatin.

10.1021/bm8014939 CCC: $40.75  2009 American Chemical Society Published on Web 03/02/2009

Cisplatin/Hydrogel Complex In Cancer Therapy Chart 1. Structure of the Monomer Unit of PolyPHE and PolyMHist

Experimental Section Chemicals and Instruments. The chemicals and the solvents were from Fluka and Sigma-Aldrich. They were used without further purification. cis-Diamminedichloroplatinum(II) was purchased from Alfa Aesar. The viscometric measurements were done with an AVS 310 automatic Schott-Gerate viscometer at 25 °C on a dilute PBS solution (pH 7.40).22 A stock solution of cisplatin was stepwise added (400 µL) to 25 mL of a polymer solution containing a weighed amount of the polymer. The measurements of pH were performed with a TitraLab 90 titration system (from Radiometer Analytical).23 The spectrophotometric measurements were carried out with a Specord 210 (Analytikjena) with 10 mm quartz cuvettes. The mass spectrometry (MS) analysis was performed with a VG 70-250S Instrument (Micromass, Manchester) using electron ionization and liquid secondary ion mass spectrometry techniques. Infrared spectra were recorded on a ThermoNicolet 6700 FT-IR spectrometer. Synthesis of the Polymeric Materials. The four cross-linked polymers (hydrogels) named P9, CP2, MH2, and CMH2 used in this study were synthesized as previously reported.21,22 In particular, the two acrylic hydrogels P9 and CP2, containing the carboxyl group, were obtained by a free radical polymerization of the monomers PHE (N-acryloyl-L-phenylalanine) and NIPAAm (N-isopropylacrylamide) and were cross-linked with EBA (N,N′-ethylenebis-acrylamide). While the compound P9 was obtained with only PHE and cross-linked with 9 mol % of EBA, the compound CP2 was a copolymer of PHE with NIPAAm at a NIPAAm/PHE molar ratio of 10 and was cross-linked with 2 mol % of EBA. The two linear polymers were synthesized in a similar way but without the cross-linking agent; while the polyPHE was a homopolymer, the co-2 was a copolymer of PHE with NIPAAm at a NIPAAm/PHE molar ratio of 2. The two hydrogels MH2 and CMH2 were ampholytes obtained by the free radical polymerization of the methacrylate monomer Nmethacryloyl-L-hystidine (MHist) and NIPAAm; besides the MH2 that was obtained with only MHist, the CMH2 was a copolymer with NIPAAm at a NIPAAm/MHist molar ratio of 12. Both the compounds were cross-linked with 2 mol % of EBA. Cisplatin Loading. The loading of cisplatin into the hydrogels was performed in water and in water/DMSO (98.4:1.6, v/v), the two-stock solution being 8.35 and 5.42 mmol/L, respectively. Dry hydrogel samples (40 mg), in the form of small particles, were soaked in 20 mL PBS solution (pH 7.40) for three days. The swollen samples were filtered through the Strainer cell, blotted with a tissue paper, and immersed in the stock cisplatin solution (20 mL) in the dark and at room temperature. Cisplatin complexation to the gel was considered completed after 1 week, when the hydrogel formed tightly compact yellow particles. The complexed samples were washed with

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PBS solution (50 mL) and dried to a constant weight. Four replicates were performed, each loading cisplatin from water and water/DMSO mixture. In Vitro Cisplatin Release. A weighed amount (10-30 mg) of dry cisplatin-loaded hydrogel contained in a Strainer cell was suspended in 40 mL of PBS (pH 7.40) solution. The temperature was maintained at 25 °C and the solution was slowly stirred with magnetic bar. At intervals, aliquots (400 µL) of the dissolution medium were sampled and immediately replaced with fresh PBS. The amount of released Pt(II) was monitored by the colorimetric o-phenylenediamine method.24 Replicates were performed on different quantities and different samples of cisplatin-loaded hydrogels. Cell Culture Conditions and Treatments. The human melanoma cell line Me665/2/21 derived from a cutaneous metastasis was used in this study. The cells were cultured in RPMI 1640 medium (Sigma, Milan, Italy), supplemented with 10% heat-inactivated fetal calf serum (FCS; Invitrogen, Milan, Italy), 2 mM L-glutamine (Sigma), and 50 mg/L gentamycin, at 37 °C, under 95:5% CO2 in a humidified atmosphere. The cells were routinely harvested by phosphate-buffered saline (PBS)-EDTA (0.2 g/L) and reseeded before reaching the confluence. For experiments, the cells were seeded at 1.5 × 106 cells on 78 cm2 plastic dishes (Sarstedt, Newton, NC) overnight, whereupon the medium was changed. The cells were treated for 72 h with native cisplatin (Sigma, 1 µg/mL) or with cisplatin-loaded P9 hydrogel, in which cisplatin was loaded in water (H-H) or in water/DMSO (H-H/ D; 30 µg/mL, each). Cytotoxicity Assay. At the end of the experiments, the floating cells were harvested separately from the still adhering cells, which were also harvested after a gentle detachment by PBS-EDTA. Small aliquots (2 µL) of both the adherent and the floating cell suspensions were counted in a Bu¨rker chamber. The apoptotic response was evaluated as percentage of floating cells on total cells of each sample; as we previously described in the same experimental model,25,26 such a percentage strictly correlates with the rate of apoptotic cell death, as measured by biochemical and morphological parameters. Moreover, from each sample (adherent + floating cells) 100 µL of cell suspension were lysed and used for the fluorogenic assay of caspase-3/-7 activity, as previously described,25,27 to further confirm the apoptotic mode of cell death.

Results and Discussion Coordination Properties of Polymers. The coordination properties by the functional groups from the polymers toward cisplatin were investigated in PBS buffer at physiological pH (7.40). Viscometric titrations of dilute polymer solutions with cisplatin were carried out to investigate the stoichiometry of complex species. Figure 1 shows the viscometric data of the polyPHE and its copolymer co-2 at different cisplatin/L molar ratio (L is the monomer unit of the polymer containing the carboxyl group). Each plotted data point of the viscometric titration curve was obtained in the equilibrium conditions that were reached after five days from addition of the titrant owing to the inertness of Pt-ligand bonds.28 Contrary to the copolymer co-2, the polyPHE regularly decreased its coil dimension until a cisplatin/L molar ratio close to 0.5 was obtained. This indicates that each metal center was coordinated to two monomer units of the polymer. The stoichiometry confirms what is usually reported for complex formation of Pt(II) with carboxylate anions.16,28 On the other hand, any shielding of these groups with, for instance, NIPAAm, leads to a lack in the coordination ability. The reduced viscosity did not change significantly in case each titration point was recorded after a longer time. The lower amount of randomly distributed carboxylate groups lacks the ability of the copolymer to anchor the platinum species in

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Figure 2. Release pattern of platinum(II) from the hydrogel P9 [loaded with cisplatin in water (H-H) and in water/DMSO (H-H/D) solutions] into PBS buffer (40 mL, pH 7.40 and 25 °C). The different amounts of loaded-hydrogels: blue diamonds, 10.8 mg; pink squares, 12.7 mg; yellow triangles, 10.3 mg; open squares, 22.3 mg.

Figure 1. Reduced viscosity (dL/g) in relation to the platinum(II)/L molar ratio (L is the ionic PHE monomer unit). The cisplatin solution was added to a polymer solution (0.0436 mmol polyPHE and 0.0458 mmol co-2) in PBS buffer (pH 7.40) and 25 °C.

a planar geometry. These data may be helpful in developing microparticulates as a tailoring platform to release cisplatin in tumor chemotherapy. Cisplatin Loading and Release. The cisplatin was loaded into the hydrogels following two methods. First, a water solution of cisplatin was allowed to react for more than one week with the gel previously swollen at pH 7.40. In the second method, a water/dimethylsulfoxide (98.4:1.6, v/v) was used as described in Gemeinhart’s work.14 Subsequently, both the procedures for cisplatin loading consisted of the same steps. The white hydrogels in the swollen state in PBS buffer (pH 7.40) appeared prevalently yellowish and in a more tightly compact form upon complexation with the cisplatin. In all cases, a yellow to grayish powder of hard consistency was obtained in the dry form and easily manipulated. Among the hydrogels investigated, the homopolymeric compounds P9 and the MH2 showed a loading capacity corresponding to a ligand/Pt molar ratios of 2. The total amount of bounded Pt(II) was evaluated by considering the molar ratio 1:2 metal/ionic group stoichiometry. The results for the P9 and the MH2 were quite in agreement with the weights of the dry samples obtained before and after the loading process. Typically, 39.1 mg of dry P9 was weighed and loaded with cisplatin in water/DMSO mixture; the dry weight of the loaded P9 resulted in 59 mg. Similarly, the MH2 showed 71 mg of the dry loaded gel from 44 mg of the native gel in the dry state. By considering the amount of cross-links, both loaded hydrogels have the same wt % of platinum(II) complexed species that satisfy the evaluated stoichiometric molar ratio. The release of platinum(II)-complex species was monitored via the colorimetric assay with o-PDA on samples collected at increasing time from the buffer solution in which a loaded gel was soaked. Figure 2 shows the release profile for Pt(II) (mg) from dry gel P9 (g). A good reproducibility of data was obtained even different amounts of different loaded-hydrogel samples were tested. The amount of released platinum(II)-complex species from the same hydrogel P9 strongly depends on the initial cisplatin

solution used for the loading process. The cisplatin solution containing DMSO showed an amount of releasing drug that is more than three times greater with respect to the cisplatin dissolved in water. In both cases, however, the cumulative in vitro release show to have a biphasic pattern; the first initial release was ascribed to the burst effect that vanishes within a few hours; the remaining longer pattern show a linear near zeroorder release. In the case of the H-H/D groups, the cumulative release was also higher as shown by the greater slope of the curve. The different behavior may be ascribed to the original platinum(II)-complex structure present in the mother solution used for the loading process. Unlike the cisplatin solution (from water), both mother (from DMSO) and sample cisplatin solution released from P9 showed the presence of a DMSO molecule linked to Pt(II) because of the replacement of a chloride leaving group.29 The electrospray MS spectra confirmed the composition and the structure of the cisplatin-DMSO adduct; among others, a prevailing peak at m/z ) 343 was consistent with the Pt(NH3)2ClDMSO species.30 This leads to the hypothesis that once linked the DMSO molecule remains complexed to the cisplatin, and only the chlorine plays a role in the coordination ability with the carboxyl groups of the hydrogel. In this case, the cisplatin remains more weakly complexed to the carboxylate group and then its release becomes faster. The presence of a DMSO molecule linked to the P9-loaded cisplatin was supported by the FT-IR spectrum, as it showed two strong SdO stretching bands at 1028 cm-1 and 1124 cm-1.29 The typical FT-IR spectra of P9 loaded with cisplatin in water is shown in Figure 3, along with the hydrogel P9 and the native cisplatin. A comparison of the spectra shows the disappearance of the broadband at 1593 cm-1, assigned to the COO- group that is present in the free P9 (green line) and absent in the P9 loaded with cisplatin, as well as the native cisplatin. Slight shifts, in opposite directions, of the frequencies appeared at 1646 cm-1 and 1547 cm-1, assigned at the CdO amide I and N-H amide II frequencies, respectively. The content of carboxyl groups also plays a major role on the amount of loaded and released platinum(II)-complex species from the hydrogels. Contrary to the P9, the hydrogel CP2 does not reveal a releasing profile. Figure 4 shows the cumulative in vitro release of Pt(II)-complex species from the CP2 loaded with cisplatin solution containing DMSO.

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Figure 3. FT-IR spectra of the native cisplatin (blue line) and of the hydrogel P9 in the free (green line) and cisplatin-loaded (red line) form.

Figure 4. Release pattern of platinum(II) from the hydrogel CP2 and P9 (loaded with cisplatin in water/DMSO solution) in PBS buffer pH 7.40.

After the burst phase in the first few hours, a flatter pattern was recorded for more than one week. This result confirms the absence or the lower Pt(II)-complexing ability of the copolymeric (CP2) microparticulates in comparison to the homopolymeric one (P9), as also reported above for the viscometric titrations. Besides the carboxyl hydrogels, the amphoteric compounds containing histidine residues showed an opposite pattern. In Figure 5 is reported the release profile comparison of the MH2 and CMH2 hydrogels. In this case, the complexing ability of the imidazole strongly suppresses the release of Pt(II) species from MH2 even though a slight release trend is observed with the CMH2. In this system the amount of Pt(II) released was much lower with respect to the P9 or CP2 systems because of the presence of the stronger coordinating imidazole sp2-nitrogen group. Moreover, the randomly distributed histidine residues in the CMH2 carrier allow a weaker coordination to the Pt(II)-species, thus favoring a greater release from the hydrogel. It is noteworthy that, besides the tightly complexed hydrogels P9 and MH2, only in the hydrogels containing the NIPAAm units (CP2 and CMH2) was observed a very small swelling phenomenon during the release of the drug. The release from P9 and CMH2 produced only a small percentage of Pt(II)species in the downstream solution even for prolonged times, whereas the hydrogels remained in the tightly compact state.

Figure 5. Release pattern of Pt(II) from the hydrogels MH2 and CMH2 (loaded with cisplatin in water/DMSO solution) in PBS buffer pH 7.40.

In fact, after 8 days, the released amount of Pt(II) was evaluated to be 12 wt % and 30 wt %, from the P9 loaded with cisplatin in water and in water/DMSO mixture, respectively. Moreover, the hydrogel P9 showed larger swellings when loaded from water/DMSO than from pure water; this phenomenon was only observed but not measured. The results above-reported were analyzed to understand the drug release from hydrogels. The analysis was performed on the basis of the empirical equation proposed by Peppas et al.:31 Mt/M∞ ) ktn (where Mt and M∞ is the amount of drug released up to any time t and the final amount of drug; k is a structural/ geometric constant and n is a release exponent related to the intimate mechanism of release) as a means of identification of the transport mechanism of the drug through a polymer slab. The power law is easy to use and can be applied to most diffusion-controlled release systems, even if it is too simple to offer a robust prediction for complicated release phenomena. The advantage of this approach is that it yields a convenient measure of the constancy of release rate in the value of n. It also provides a quick way to check the quality of data, as values of n < 0.5 or n > 1 for slabs indicate that another process in addition to diffusion occurs. In Table 1 is reported the values for k and n as obtained from the best fitting of our experimental curves. The low n values lead to exclude significant swelling and simple diffusion controlled delivery mechanism. The complexed Pt(II)-species obey to two correlated processes within the

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Table 1. Comparison of the Release Exponent (n) and Structural Constant (k) Values in the Empirical Peppas’s Model: Mt/M∞ ) ktn system

n

k

P9 (water/DMSO) P9 (water)

0.16 0.21

0.14 0.04

Table 2. Me665/2/21 Melanoma Cells (1.5 × 106) were Treated for 72 h with Cisplatin (1 µg/mL or 3.3 µM) or with Cisplatin-Loaded Hydrogels (30 µg/mL or 45 µM of Pt Loaded from Water)a sample control cisplatin hydrogel-cisplatin (in water) hydrogel-cisplatin (in water/DMSO)

total cell number (×10-6)

% detached/total cells

12.04 ( 0.68 3.02 ( 0.44 3.00 ( 0.38

ND 80.8 ( 1.2 77.0 ( 4.9

11.22 ( 0.44

ND

a The percentages of detached cells on total cells in control (untreated) and in hydrogel-cisplatin (in water/DMSO) samples were minor, ranging from 2 to 5%. Results are given as mean ( SE from 3-5 experiments.

delivery matrix: first, a chemically controlled event related to the breakage of coordinate Pt-gel bonds; second, a diffusioncontrolled step. The species released are then subjected to a concentration-dependent diffusion. The stepwise binding cleavage and diffusion of the drug from the inner to the external interface of the hydrogel reach a broad range of delivery timescales. In the present investigation it is clear that the drug release rate is determined by the dissociation of Pt-gel coordinate bonds. Cytotoxicity of Cisplatin-Loaded Hydrogels. In vitro experiments were performed on Me665/2/21 melanoma cells to compare the cytotoxic effect of native cisplatin with the cytotoxic effect of cisplatin released from the P9 hydrogel loaded in different solvents. The concentration chosen for native cisplatin was 1 µg/mL (corresponding to 0.67 µg/mL or 3.3 µM of Pt), which is close to the plasma concentration found in patients treated with cisplatin for solid tumors.32 In the same experiments, cells were also treated with 30 µg/mL of P9 hydrogels (containing 8.7 µg/mL or 45 µM of Pt loaded from water); with the hydrogel containing cisplatin loaded from water, which is slower in terms of cisplatin release, melanoma cells received a dose of platinum(II) (0.51 µg/mL or 2.6 µM, released within the first 3 h, see Figure 2) comparable to that of native cisplatin. As shown in Table 2 and Figure 6, the cisplatin released from the cisplatin-loaded (from water) hydrogel, similarly to the native cisplatin, induced a remarkable apoptotic cell death, as evaluated by cell loss, cell detachment from the monolayer, and increased activity of caspase-3/-7, the latter being a well-known hallmark of apoptosis. On the contrary, no effect on cell growth and viability was obtained by the same hydrogel loaded with cisplatin from a water/DMSO mixture, although it releases a higher amount of cisplatinum(II) species (more than three times, as showed in Figure 2). In the latter case, the loss of activity of the cisplatin released from the hydrogel may be likely attributed to its exclusion into cells because of the charged platinum(II)-complex species anchoring sterically hindered DMSO molecules.14 Alternatively, the presence of the DMSO molecule in the cisplatin complex species could suppress the intercalation between the two DNA strands.30 Solvolysis reactions of cisplatin in DMSO might be expected to form primarily monofunctional DNA adduct.29 It is worth that different platinum(II) species are released from the same hydrogel P9; unlike the cisplatinum(II) species released from loaded P9 in water, the adduct

Figure 6. Me665/2/21 melanoma cells (1.5 × 106) were treated for 72 h with cisplatin (1 µg/mL or 3.3 µM) or with cisplatin-loaded hydrogels (30 µg/mL or 45 µM of Pt loaded from water). Caspase-3/ -7 activity is expressed as ∆ arbitrary units of fluorescence (AUFs)/ min/mg protein. Results are given as mean ( SE from 3-5 experiments. Control ) untreated cells; Cis ) native cisplatin; H-H ) cisplatin-loaded (in water) hydrogel; H-H/D ) cisplatin-loaded (in water/DMSO) hydrogel.

containing a DMSO molecule has reduced ability to bind doublestranded DNA and, therefore, has potentially reduced toxicity as showed by the work of Windebank.30 Thus, the affinity of Pt(II) for sulfur donor ligands makes DMSO unsuitable for use in biological studies of the mechanism of action of platinum antitumor drugs and that the results of biologically related experiments employing mixture of solvents containing this molecule must be strongly discouraged.

Conclusions With increasing efforts devoted to controlled drug release, the applications of charged polymers will continue to grow in the future.16 Proper network design can tune the drug release rates and modulate tissue regeneration. The investigated hydrogel containing the carboxyl groups may show a potential application as a platform for chemotherapy due also to its intrinsic nontoxic property. The chemically controlled cisplatin release was favored by the weak metal-based drug linkage. Such a controlled release is also improved with materials composed by homopolymeric networks where cisplatin is loaded from pure water. The well-defined metal-ligand stoichiometry and the ability to show a chemotherapeutic effect similar to the native cisplatin render these systems to be tailored for a broad range of tumors with a desired maximum concentration in the blood stream. The apoptotic cell death due to the released cisplatinum(II) species was remarkable and close to that afforded by native cisplatin; this occurred only when the drug was loaded from pure water, while cis-platinum(II) species containing DMSO molecules were devoid of any cytotoxic effect, as also reported in different experimental systems.14,30 The use of DMSO to dissolve the cisplatin in biological studies is therefore strongly discouraged. Acknowledgment. The research was supported by a grant of Siena University as a PAR project (2005). Thanks are due to Prof. G. Giorgi for MS spectrometry analysis.

References and Notes (1) DeConti, R. C.; Toftness, B. R.; Lange, R. C.; Creasey, W. A. Cancer Res. 1973, 33, 1310–1315. (2) Reedijk, J.; Teuben, J. M. In Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug; Lippert, B., Ed.; Wiley-VCH: Zurich, Switzerland, 1999; p 339. (3) Kelland, L.; Farrel, N. Platinum Based Drug in Cancer Therapy. Humana Press: Totowa, NJ, 2000.

Cisplatin/Hydrogel Complex In Cancer Therapy (4) (5) (6) (7) (8) (9) (10) (11)

(12)

(13) (14) (15) (16) (17) (18)

Garattini, S.; La Vecchia, C. Eur. J. Cancer 2001, 37, 128–147. Lebwohl, D.; Canetta, R. Eur. J. Cancer 1998, 34, 1522–1534. Ferguson, L. R.; Pearson, A. E. Mutat. Res. 1996, 355, 1–12. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Controlled Release 2000, 65, 271–284. Sasaki, H.; Niimi, S.; Akiyama, M.; Tanaka, T.; Hazato, A.; Kurozumi, S.; Fukusima, S.; Fusushima, S. Cancer Res. 1999, 59, 3919–3922. Araki, H.; Tani, T.; Kodama, M. Artif. Organs 1999, 23, 161–168. Itoi, K.; Tabata, Y.; Ike, O.; Shimizu, Y.; Kuwabara, M.; Kyo, M.; Hyon, S. H.; Ikada, Y. J. Controlled Release 1996, 42, 175–184. Tokuda, K.; Natsugoe, S.; Shimada, M.; Kumanohoso, T.; Baba, M.; Takao, S.; Nakamura, K.; Yamada, K.; Yosizawa, H.; Hatate, Y.; Aikou, T. Int. J. Cancer 1998, 76, 709–712. Mizumura, Y.; Matsumura, Y.; Hamaguchi, T.; Nishiyama, N.; Kataoka, K.; Kawaguchi, T.; Hrushesky, W. J.; Moriyasu, F.; Kakizoe, T. Jpn. J. Cancer Res. 2001, 92, 328–336. Burger, K. N. J.; Staffhorst, R. W. H. M.; De Vijlder, H. C.; Velinova, M. J.; Bomans, P. H.; Frederik, P. M.; De Kruijff, B. Nat. Med. 2002, 8, 81–84. Yan, X.; Gemeinhart, R. A. J. Controlled Release 2005, 106, 198– 208. Kopecek, J.; Kopeckova, P.; Minko, T.; Lu, Z.-R.; Peterson, C. M. J. Controlled Release 2001, 74, 147–158. Komane, L. L.; Mukaya, E. H.; Neuse, E. W.; van Rensburg, C. E. J. J. Inorg. Organomet. Polym. 2008, 18, 111–123. Yapp, D. T. T.; Lloyd, D. K.; Zhu, J.; Lehnert, S. M. AntiCancer Drugs 1998, 9, 791–796. Cini, R.; Defazio, S.; Tamasi, G.; Casolaro, M.; Messori, L.; Casini, A.; Morpurgo, M.; Hursthouse, M. Inorg. Chem. 2007, 46, 79–92.

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(19) Tamasi, G.; Serinelli, F.; Consumi, M.; Magnani, A.; Casolaro, M.; Cini, R. J. Inorg. Biochem. 2008, 102, 1862–1873. (20) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Chem. ReV. 1999, 99, 3181–3198. (21) Casolaro, M.; Paccagnini, E.; Mendichi, R.; Ito, Y. Macromolecules 2005, 38, 2460–2468. (22) Casolaro, M.; Ito, Y.; Ishii, T.; Bottari, S.; Samperi, F.; Mendichi, R. eXPRESS Polym. Lett. 2008, 2, 165–183. (23) Casolaro, M.; Bottari, S.; Ito, Y. Biomacromolecules 2006, 7, 1439– 1448. (24) Golla, E. D.; Ayres, G. H. Talanta 1973, 20, 199–210. (25) Del Bello, B.; Valentini, M. A.; Mangiavacchi, P.; Comporti, M.; Maellaro, E. Exp. Cell Res. 2004, 293, 302–310. (26) Del Bello, B.; Moretti, D.; Gamberucci, A.; Maellaro, E. Oncogene 2007, 26, 2717–2726. (27) Brockhaus, F.; Brune, B. Exp. Cell. Res. 1998, 238, 33–41. (28) Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug; Lippert, B., Ed.; Wiley-VCH: Zurich, Switzerland, 1999. (29) Sundquist, W. I.; Ahmed, K. J.; Hollis, L. S.; Lippard, S. I. Inorg. Chem. 1987, 26, 1524–1528. (30) Fisher, S. J.; Benson, L. M.; Fauq, A.; Naylor, S.; Windebank, A. J. NeuroToxicology 2008, 29, 444–452. (31) Peppas, A. N.; Korsmeyer, R. W. In Hydrogels in Medicine and Pharmacy; Peppas, N. A., Ed.; CRC Press: Boca Raton, FL, 1987; p 109. (32) Sileni, V. C.; Fosser, V.; Maggian, P.; Padula, E.; Beltrame, M.; Nicolini, M.; Arslan, P. Cancer Chemother. Pharmacol. 1992, 30, 221–225.

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