Communication pubs.acs.org/bc
Production of Cisplatin-Incorporating Hyaluronan Nanogels via Chelating Ligand−Metal Coordination Seiichi Ohta,† Syota Hiramoto,‡ Yuki Amano,§ Mayu Sato,§ Yukimitsu Suzuki,§ Marie Shinohara,∥ Shigenobu Emoto,¶ Hironori Yamaguchi,¶ Hironori Ishigami,¶ Yasuyuki Sakai,∥ Joji Kitayama,¶ and Taichi Ito*,†,‡,§ †
Center for Disease Biology and Integrative Medicine and ¶Department of Surgical Oncology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ‡ Department of Bioengineering and §Department of Chemical System Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ∥ Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan S Supporting Information *
ABSTRACT: Hyaluronan (HA) is a promising drug carrier for cancer therapy because of its CD44 targeting ability, good biocompatibility, and biodegradability. In this study, cisplatin (CDDP)-incorporating HA nanogels were fabricated through a chelating ligand−metal coordination cross-linking reaction. We conjugated chelating ligands, iminodiacetic acid or malonic acid, to HA and used them as a precursor polymer. By mixing the ligand-conjugated HA with CDDP, cross-linking occurred via coordination of the ligands with the platinum in CDDP, resulting in the spontaneous formation of CDDP-loaded HA nanogels. The nanogels showed pH-responsive release of CDDP, because the stability of the ligand−platinum complex decreases in an acidic environment. Cell viability assays for MKN45P human gastric cancer cells and Met-5A human mesothelial cells revealed that the HA nanogels selectively inhibited the growth of gastric cancer cells. In vivo experiments using a mouse model of peritoneal dissemination of gastric cancer demonstrated that HA nanogels specifically localized in peritoneal nodules after the intraperitoneal administration. Moreover, penetration assays using multicellular tumor spheroids indicated that HA nanogels had a significantly higher ability to penetrate tumors than conventional, linear HA. These results suggest that chelating-ligand conjugated HA nanogels will be useful for targeted cancer therapy.
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Various types of polymer-based nanogels have been synthesized and used as drug carriers to date.4,5,8−10 One of these nanogels, a hyaluronan (HA)-based nanogel, is a promising candidate for a drug carrier targeting tumor tissues. HA is a natural linear polysaccharide that can be found throughout the body and is composed of alternating Dglucuronic acid and N-acetyl-D-glucosamine groups. HA is widely used for biomedical applications because of its good biocompatibility and biodegradability.11−13 One unique feature of HA is that it selectively binds to CD44, which is overexpressed in a wide range of cancer cells and was also recently recognized as a marker for cancer stem cells.14,15 Because of its CD44 selectivity, HA has been examined as a targeted drug carrier for cancer therapy.16−18 Though most studies have used drug-conjugated HA in its linear polymer
anoparticles have been widely used in biomedical applications, such as drug delivery systems and diagnostic imaging. 1,2 Nanogels have recently attracted significant attention as a carrier for drugs and biological molecules (e.g., proteins and DNA), because of their high drug loading capacity, good pharmacokinetic properties, and responsiveness to environmental factors such as pH and ionic strength.3 Nanogels are hydrogels of nanometer size that are composed of a swollen and cross-linked network of polymers. Physical cross-linking, or in other words, self-assembly, of associating polymers is often used for their synthesis.4,5 For example, cholesteryl-groupbearing pullulan spontaneously forms physically cross-linked nanogels through hydrophobic interaction between its cholesteryl groups.4 Chemical cross-linking of polymers, e.g., by Michael-type addition,6 carbodiimide coupling,7 or disulfide bond formation,8 can also be used to synthesize nanogels. In addition to the cross-linking of preformed polymers, polymerization of monomers in a nanoscale environment, such as emulsions, has also been reported to generate nanogels.9 © XXXX American Chemical Society
Received: December 17, 2015 Revised: January 9, 2016
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DOI: 10.1021/acs.bioconjchem.5b00674 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 1. Illustration of nanogel formation from chelating ligand-conjugated HA, induced by ligand−CDDP coordination. The ligand-conjugated HA is cross-linked via bridging of ligands by CDDP or hydrophobic interaction of CDDP-coordinated ligands that lose their hydrophilicity through coordination.
and then dialyzed them. The purified samples were lyophilized for storage, and they can be easily redispersed in aqueous media. Figure 2a shows TEM images of the resultant nanogels.
form, some recent studies have shown that using HA in nanogel form further improves its antitumor activity.19−23 However, methods for the fabrication of HA nanogels are, as yet, not completely established, thereby constraining their use. For example, although most reported work used self-assembly of hydrophobic moiety-conjugated HA to form HA nanogels,19,20,24 such a design allows only hydrophobic drugs to be incorporated. Nanosized hydrophilic drug-HA complexes have been fabricated by simply mixing HA with cisplatin (CDDP), which is a platinum-containing anticancer drug, but their size was comparatively large, greater than 100 nm, and the release rate of CDDP was relatively fast.21,22 Therefore, novel designs of HA-based nanogels still require exploration. Herein, we used chelating ligand−metal coordination to fabricate drug-incorporating HA nanogels. Chelating ligands, such as iminodiacetic acid (IDA), are known to form stable coordinate bonds with metal ions, including platinum contained in certain kinds of anticancer drugs, such as CDDP. In addition, the resultant metal−ligand complex dissociates in acidic environments through a decrease in affinity,25 which is suitable for pH-responsive drug release. In this study, we conjugated metal chelating ligands to the HA backbone, as shown in Figure 1. By mixing the chelating ligandconjugated HA with CDDP, they were cross-linked via ligand− platinum coordination, resulting in the spontaneous formation of CDDP-loaded HA nanogels that responded to pH changes. Combined with the original characteristics of HA described above, this CDDP-loaded HA nanogel is expected to comprise an efficient drug carrier for antitumor therapy. IDA and malonic acid (MA) were chosen as chelating agents in this study. They were conjugated to HA using carbodiimide chemistry (Supporting Information Figure S1). The conjugation was confirmed by observing methylene protons of conjugated IDA (2.92 ppm) or methine proton of conjugated MA (2.89 ppm) in 1H NMR spectra (Supporting Information Figure S2). The degrees of substitution of IDA-conjugated HA (HA-IDA) and MA-conjugated HA (HA-MA) calculated from the 1H NMR spectra were 35% and 36%, respectively. When a 1:1 complex is formed with CDDP, the conjugated IDA is expected to form an eight-membered chelate ring, whereas a six-membered chelate ring is expected in the case of MA (Supporting Information Figure S3). To fabricate CDDP-loaded HA nanogels, we mixed HA-IDA or HA-MA with CDDP, heated the mixtures at 95 °C for 1 h,
Figure 2. (a) TEM images of HA-IDA and HA-MA nanogels. The scale bar is 100 nm. (b) Hydrodynamic size distribution of HA-IDA and HA-MA nanogels in PBS measured by DLS. (c) Release profile of CDDP form HA-IDA and HA-MA nanogels at different pH values (pH 7.5 and 4.5). PBS was used as a solvent and its pH was changed by addition of HCl at 37 °C. Data are averages ± standard deviations (n = 4).
HA-IDA and HA-MA nanogels of spherical shape were successfully obtained. Bridging of chelating ligands by CDDP or hydrophobic association of CDDP-coordinated chelating ligands that lose their hydrophilicity through coordination is a possible explanation for these nanogel formations (Figure 1). The sizes of HA-IDA and HA-MA nanogels analyzed by TEM images were 10.5 ± 2.8 and 7.9 ± 2.3 nm, respectively. The hydrodynamic sizes of HA-IDA and HA-MA nanogels were also measured by dynamic light scattering (DLS) (Figure 2b), resulting in average diameters of 21 and 9.7 nm, respectively. These values were larger than the sizes obtained by TEM B
DOI: 10.1021/acs.bioconjchem.5b00674 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry images because the nanogels swelled in aqueous media. By comparison of HA-IDA with HA-MA, both size measurements showed that HA-MA nanogels were smaller than HA-IDA nanogels. In addition, the amount of loaded CDDP differed between HA-IDA and HA-MA, 16.8 wt % for HA-IDA and 28.3 wt % for HA-MA; whereas it was 11.4 wt % for unmodified HACDDP nanogel prepared in the same way as HA-IDA/MA nanogels. We believe that these differences reflect the chelate stability of the ligands. Due to the high affinity of IDA and MA with platinum, the CDDP loading efficiency of the ligandconjugated HA nanogels was higher than that of conventional unmodified HA-CDDP nanogels. In addition, when comparing between the two chelating ligands, because a six-membered chelate ring is generally more stable than an eight-membered ring,26,27 the stability of the MA-platinum complex is concluded to be higher than that of the IDA-platinum complex. This higher chelate stability would lead to binding of a larger amount of CDDP to HA-MA than to HA-IDA, giving a higher loading efficiency for CDDP. Consequently, cross-linking would become denser in HA-MA, resulting in the smaller size of the formed nanogels. The CDDP release profiles from HA-IDA and HA-MA nanogels at different pH values are shown in Figure 2c. At neutral pH (pH 7.5), release of CDDP plateaued around 2 days and almost 40% of loaded CDDP was retained in the HA nanogels after 1 week (33% in HA-IDA nanogels and 41% in HA-MA nanogels). The residual amount of CDDP in HA-MA nanogels was larger than that in HA-IDA nanogels, because of the higher stability of the MA-platinum complex. On the other hand, in the case of unmodified HA-CDDP nanogels, almost 90% of loaded CDDP was released in 48 h (Supporting Information Figure S5), due to the lower stability of the complex. Moreover, the release of CDDP from HA-IDA and HA-MA nanogels was accelerated in an acidic environment (pH 4.5), which can be attributed to a decrease in the stability of the ligand−platinum coordination. Within 24 h, 90% of the loaded CDDP was released from HA-IDA nanogels, while 75% was released from HA-MA nanogels. These results indicate that HA-IDA and HA-MA nanogels can be used as a pH-responsive drug carrier. After they are internalized by cells via endocytosis, they are expected to selectively release CDDP in response to an endosomal pH decrease (usually from 7.5 to ca. 5.0). In addition, it should be noted that the release of CDDP observed here was accompanied by a size increase in the HA nanogels (Supporting Information Figure S6). This is consistent with our explanation that CDDP works as a “cross-linking agent” during nanogel formation. The potential of HA-IDA and HA-MA nanogels as a drug carrier against gastric cancer was examined by using a human gastric cancer cell line MKN45P, which expresses high levels of CD44. MKN45P cells were incubated with fluorescein-5thiosemicarbazide (FTSC)-labeled HA-IDA or HA-MA nanogels at 37 °C for 4 h, washed with PBS, and then observed by confocal laser scanning microscope (CLSM). Results are shown in Figure 3a. Fluorescence of HA-IDA and HA-MA nanogels was clearly detected in MKN45P cells after incubation. Colocalization with Lyso-tracker Red, which selectively labels late endosomes/lysosomes, indicated that HA-IDA and HA-MA nanogels were accumulated in late endosomes/lysosomes. These results suggest that HA-IDA and HA-MA nanogels were internalized by the gastric cancer cells via endocytosis and then transported to endosomes/lysosomes.
Figure 3. (a) Confocal microscopic images of MKN45P cells after incubation with HA-IDA and HA-MA nanogels. The scale bar is 10 μm. (b) Cell viability assay for MKN45P and MeT-5Acells exposed to HA-IDA nanogels, HA-MA nanogels, and free CDDP (control). Data are averages ± standard deviations (n = 8).
Anticancer activity of HA-IDA and HA-MA nanogels against MKN45P cells was examined by the MTT assay (Figure 3b). Met-5A human mesothelial cells were also used as normal control cells. In the case of MKN45P gastric cancer cells, the cytotoxic effects of HA-IDA and HA-MA nanogels were comparable to or even higher than that of the control CDDP group. In contrast, in the case of Met-5A mesothelial cells, the cytotoxicities of HA-IDA and HA-MA nanogels were lower than that of the control CDDP group. Cell viabilities after 24 h incubation with HA-IDA nanogel, HA-MA nanogel, and CDDP at a concentration of 0.5 mg/mL CDDP were 73.5%, 94.4%, and 37.1%, respectively. These results suggest that HA-IDA and HA-MA nanogels selectively inhibit the growth of gastric cancer cells. We believe that this selectivity can be attributed to the CD44 targeting ability of HA-IDA and HA-MA nanogels. Compared with MKN45P, Met-5A expresses a lower level of CD44, about 5 times lower in mean fluorescent intensity of flow cytometric analysis, though it is not negative (Supporting Information Figure S7). Therefore, HA-IDA and HA-MA nanogels would be internalized more prominently via CD44mediated endocytosis by MKN45P than Met-5A, resulting in selective cytotoxicity. On the other hand, however, according to Figure 2c, free CDDP is released especially in first 24 h and it could also contribute to the cytotoxic activity. Thus, the mechanism of the observed antitumor effect is not simple and it will be further examined in our future study. Although a more detailed examination is still needed to clarify the reason for this selectivity, our results indicate the utility of HA-IDA and HAMA nanogels as a targeted CDDP carrier against gastric cancer. The utility of chelating ligand-conjugated HA nanogels was further examined in vivo using a mouse model of peritoneal dissemination of gastric cancer. BALB/c nude mice were inoculated intraperitoneally with MKN45P cells and fed for 2 C
DOI: 10.1021/acs.bioconjchem.5b00674 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 4. (a) Fluorescent images of peritoneal dissemination of MKN 45P cells in mice after the administration of FTSC-modified HA-IDA linear polymer and nanogels. Arrows indicate peritoneal nodules that are labeled by HA nanogels. (b) Distribution of HA-IDA linear polymer and nanogels in the central sections of the multicellular tumor spheroids, observed by CLSM. The scale bar is 50 μm.
gastric cancer cells. Compared with unmodified HA-CDDP nanogels, use of the chelating ligands enabled increased CDDP loading, suppressed burst release of CDDP and pH responsiveness. To the best of our knowledge, this is the first study that has used chelating ligand−metal coordination for nanogel formation. Our methodology should not be limited to the specific pairings of HA with IDA or MA, but is expected to be applicable to other polymers and ligands. In addition, because the safety of both HA and chelating ligands has been widely recognized, future translation of the chelating ligandconjugated HA nanogels into the clinic is expected to be promising. One important observation in the HA nanogels reported here was that their CDDP loading, release properties, and consequential anticancer activities depended on the type of chelating ligands used. These results suggest that we could control the properties of nanogels by choosing a suitable combination of chelating ligand and metal ions. On the other hand, to achieve such control, improved understanding of the interactions of conjugated ligands with CDDP is needed. For example, although we are supposing two possible mechanisms for nanogel formation, i.e., CDDP bridging and hydrophobic interactions, details of the cross-linking structure has not yet been well clarified. The coordination state of CDDP inside the nanogels must also be explored, because various kinds of coordination state could be possible, such as monodentate coordination to unmodified carboxyl groups in HA or bridging of those unmodified carboxyl groups with the chelating ligand, in addition to the simple bidentate coordination structure shown in Supporting Information Figure S3. By clarifying these points, we expect to be able to further improve the properties of chelating ligand-conjugated HA nanogels, including more suppression of CDDP burst release and control of degradation kinetics in biological media.
weeks to create the peritoneal dissemination model. HA-IDA nanogel was chosen as a representative nanogel formulation and its in vivo behavior was compared to that of HA-IDA linear polymer. FTSC-labeled HA-IDA linear polymer and nanogels were intraperitoneally injected into the mice and their distribution was observed after 6 h using a fluorescent imaging system (Figure 4a). HA-IDA nanogels specifically accumulated in peritoneal nodules, while no specific localization was observed in HA-IDA linear polymer. These results suggested the superior targeting ability of HA nanogels over that of their linear analogue. To further examine the origin of this improved targeting property, multicellular spheroids were employed as a model tumor tissue. Multicellular spheroids of MKN74 human gastric cancer cells were cultured on a polydimethylsiloxane (PDMS) microwell recently developed by us,28 whose high oxygen permeability allows us to grow large-sized multicellular spheroids without losing activity. FTSC-labeled HA-IDA with linear polymer and nanogel forms were exposed to the multicellular tumor spheroids, and their penetration behaviors were observed by CLSM (Figure 4b). The results showed that linear HA-IDA polymers were not able to penetrate the multicellular tumor spheroid effectively, so the florescent signal was detected only from the peripheral region of the spheroid. On the other hand, because of their smaller size, HA-IDA nanogels penetrated deeply into the spheroid and thus the fluorescent signal was uniformly observed over the entire spheroid. A similar enhanced penetration behavior was observed with HA-MA and unmodified HA nanogels (Supporting Information Figure S9). These results suggest that HA-IDA and HA-MA nanogels have significantly higher tumor penetration ability than their linear analogue, which is an important quality for nanocarriers to achieve high antitumor effects.29 This tumor penetration ability, as well as the CD44 targeting property, would be the reason for the observed specific localization of HA nanogels into peritoneal nodules in vivo. In the medical field, chelating ligands have been used to remove toxic heavy metals from the body, a treatment known as chelation therapy. A variety of chelating agents, such as dimercaptosuccinic acid and deferoxamine, are now approved by the US Food and Drug Administration. In this study, by using their strong coordination to metals as a cross-linking reaction, we fabricated CDDP-incorporating HA nanogels that can deeply penetrate into tumor tissues and selectively target
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00674. Experimental details and supporting figures (PDF) D
DOI: 10.1021/acs.bioconjchem.5b00674 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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AUTHOR INFORMATION
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[email protected]. Tel: +81-3-5841-1696. Fax: +81-3-5841-1697. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Kikkoman Biochemifa Co. for supplying hyaluronic acid. We also thank K. Hashimoto for his help in the optimization of dialysis procedure. This work was supported by a Health Labour Sciences Research Grant from the Ministry of Health, Labour and Welfare of Japan, and a Grant-in-Aid for Young Scientists (B) (No. 26820356) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. S.O. thanks the Japan Society for the Promotion of Science (JSPS) for a Research Fellowship (PD, No. 5621). We also thank the Center for NanoBio Integration, The University of Tokyo, Japan, for providing access to the CLSM.
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DOI: 10.1021/acs.bioconjchem.5b00674 Bioconjugate Chem. XXXX, XXX, XXX−XXX
