- Email: [email protected]
Anticancer drug-based multifunctional nanogels through self-assembly of dextran–curcumin conjugates toward cancer theranostics
Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Anticancer drug-based multifunctional nanogels through self-assembly of dextran–curcumin conjugates toward cancer theranostics Koji Nagahama ⇑, Yoshinori Sano, Takayuki Kumano Department of Nanobiochemistry, Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-Minamimachi, Kobe 650-0047, Japan
a r t i c l e
i n f o
Article history: Received 13 February 2015 Revised 15 April 2015 Accepted 18 April 2015 Available online xxxx Keywords: Curcumin Self-assembly Amphiphilic polymer Nanogel Theranostics
a b s t r a c t Curcumin (CCM) has been received much attention in cancer theranostics because CCM exhibits both anticancer activity and strong fluorescence available for bio-imaging. However, CCM has never been utilized in clinical mainly due to its extremely low water solubility and its low cellular uptake into cancer cells. We fabricated novel CCM-based biodegradable nanoparticles through self-assembly of amphiphilic dextran–CCM conjugates. Significantly high CCM loading contents in the nanoparticles and the high water solubility were achieved. Importantly, the dextran–CCMs nanoparticles were effectively delivered into HeLa cells and exhibited strong fluorescence available for live-cell imaging, although the nanoparticles were not delivered into normal cells. Thus, the dextran–CCMs nanoparticles could be a promising for creation of novel CCM-based cancer theranostics with high efficacy. Ó 2015 Elsevier Ltd. All rights reserved.
Polymer-based nanoparticles are large and fast growing fields, and such nanoparticles have been applied as delivery carriers for therapeutic agents including anticancer drugs and bio-imaging agents, especially for cancer therapy and diagnostics.1,2 Recently, a new field combining both therapy and diagnostics, called theranostics, has been generated for cancer treatment.3 One of the main therapeutic systems in cancer theranostics is imaging-guided drug delivery. The therapeutic system usually employs polymer nanoparticles for their achievement. Polymer nanoparticles for cancer theranostics must be both nano depot and nano carriers for therapeutic and diagnostic agents.4 Common imaging components utilized for cancer theranostics include photoluminescent mostly fluorescent materials, while common therapeutic components are anticancer drugs.2 Nanoparticles consisting of amphiphilic copolymers, such as micelles, polymersomes, and nanogels with hydrophobic domains, are commonly used in cancer theranostics.5 In these cases, both fluorescent materials and anticancer drugs should be loaded in hydrophobic domains of nanoparticles with enough high amounts. However, there are inherent difficulties in achieving enough high drugs and imaging materials loading per nanoparticle because of the limited capacity of hydrophobic domains in nanoparticles for loading.5
⇑ Corresponding author. Tel.: +81 78 303 1328; fax: +81 78 303 1495. E-mail address: [email protected] (K. Nagahama).
Recently, curcumin (CCM), naturally-occurring hydrophobic molecule derived from turmeric, has been received much attention in cancer theranostics because CCM induces apoptosis to various kinds of cancer cells6,7 with a safe manner to healthy cells and exhibits strong fluorescence as biocompatible probes available for bio-imaging.8,9 Thus, CCM can be applied to our concept described above as anticancer drug, imaging material, as well as building block to create nanoparticle for cancer theranostics. However, CCM has never been utilized in clinical mainly due to two problems. The first problem is poor aqueous solubility. CCM is hydrophobic molecule, and thus the maximum water solubility is about 30 nm, whereas CCM induces apoptosis to cancer cells with an IC50 10–75 lM.10 The second problem is low cellular uptake. CCM deeply inserts into cell membrane, anchored by hydrogen bonding to the phosphate group and hydrophobic interaction with fatty acyl group of lipid, and thus only a few parts of CCM translocate into cytoplasm and nucleus whereat it works.11 Thus, intracellular delivery of intact CCM to cancer cells is big issue to achieve clinical application of CCM in cancer theranostics. Herein, we fabricated novel CCM-based polymer nanoparticles by self-assembly approach. Importantly, the obtained CCM-based nanoparticles exhibited not only cell penetrating property in cancer cell-selective manner, but also strong fluorescence in cancer cells enough for visualizing. Therefore, we describe the synthesis of dextran–CCM conjugates, fabrication of their self-assembled nanogels, its fluorescence properties, and the cell penetrating properties. This study is the first example in theranostics studies
http://dx.doi.org/10.1016/j.bmcl.2015.04.062 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Nagahama, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.062
2
K. Nagahama et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
to overcome water-insoluble problems of useful anticancer and fluorescence agents through utilization of polymeric self-assembly. Ulbrich et al. has reported that hydrophilic polymer-hydrophobic drugs conjugates self-assembled into nanoparticles in aqueous media via hydrophobic interactions among the drug molecules.12 Inspired by this paper, we synthesized amphiphilic polymers composed of hydrophobic CCMs and hydrophilic dextran to fabricate CCM-based nanoparticles. Dextran–CCMs conjugates were synthesized through coupling reaction of activated dextran and CCMs (Fig. 1). Dextran–CCMs conjugates are expected to self-assemble into nanoparticles through hydrophobic interactions between CCM side-chains attached to the dextran in aqueous environment (Fig. 1). In this molecular systems, hydrophobic/hydrophilic balance of amphiphilic dextran–CCMs conjugates would affect the water solubility and their self-assembly behavior to form nanoparticles. Therefore, we synthesized a series of dextran–CCMs conjugates with different CCM contents by varying feed molar ratios of CCM to dextran in the synthesis process. Purification of the obtained products were carried out by precipitation technique using excess amount of methanol as poor solvent for the obtained dextran–CCMs conjugates and as good solvent for uncoupled CCM, CDI, and DMAP. Fig. S1 (Supplemental data) shows gel permeation chromatography (GPC) profiles of the dextran–CCMs conjugates. The dextran–CCMs conjugate each shows unimodal peak with a reasonably narrow molecular weight distribution, indicating the complete removal of uncoupled CCM and the reaction byproducts from the obtained dextran–CCMs conjugates. The conjugation of CCMs to dextran was also confirmed by 1H NMR measured in DMSO-d6 as good solvent for dextran–CCMs conjugates (Fig. S2, Supplemental data). UV–vis spectra of the dextran–CCMs conjugates measured in DMSO were shown in Fig. 2a. We used the absorbance at 434 nm to determine the average number of CCM side-chains conjugated to dextran. The characteristics of the
obtained dextran–CCMs conjugates were summarized in Table 1. We used the abbreviations for dextran–CCMs conjugates as DCx (x means numbers of CCM per dextran molecule). DC3, DC16, and DC30 were synthesized and these polymers were used following experiments. It has been reported that amphiphilic polymers composed of polysaccharides main-chain and hydrophobic side-chains, such as cholesterol-conjugated pullulan, form hydrogel-like nanoparticle (nanogel) with polysaccharide skeleton and hydrophobic multi cores in dilute aqueous solutions.13,14 Self-assembly of the dextran–CCMs conjugates was accomplished by direct dispersion of the polymers in water. Tyndall phenomena were observed for all the aqueous solutions (data not shown), meaning the presence of colloidal particles in these solutions. These results clearly show that amphiphilic dextran–CCMs conjugates self-assembled into nanoparticles in aqueous solutions. So, the colloidal particles were analyzed by DLS measurement. DC16 and DC30 nanoparticles showed a unimodal size distribution peak, but the distribution peak of DC3 was bimodal, as shown in Table 1. The mean diameter of colloidal particles were ranged from approximately 30 nm to 220 nm depending on the number of conjugated CCM, indicating that the number of conjugated CCM is one of the critical factors to determine their size. Typically, loading contents of anticancer drugs per polymeric nanocarrier for cancer theranostics are less than 10 wt %.15 The CCM content (wt %) in per DC3, DC16, and DC30 were 2.7 wt %, 12.8 wt %, and 21.6 wt %, respectively. This result means that DC16 and DC30 nanoparticles load relatively higher amounts of anticancer drugs as well as imaging agents as compared with those of typical nanoparticles reported, because CCM possess activities as both anticancer drugs and imaging agents. It is well known that CCM exhibits anticancer activity and enough strong fluorescence when the concentration of CCM in solutions is reached up to
Figure 1. Schematic illustration of the formation of dextran–CCMs nanoparticles. (a) Self-assembled dextran–CCMs nanoparticles via intermolecular hydrophobic interactions between CCM side-chains. (b) Synthesis of dextran–CCMs conjugates.
Please cite this article in press as: Nagahama, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.062
K. Nagahama et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Figure 2. (a) UV-vis spectra of dextran–CCMs conjugates measured in DMSO (2 mg/mL). (b) Fluorescent spectra of dextran–CCMs nanoparticles measured in PBS (2 mg/mL).
Table 1 Characterization of a series of dextran–CCMs conjugates Sample
No. of CCMa
Mwb
CCM content (%)
Dhc (nm)
DC3 DC16 DC30
3 16 30
41,100 45,870 51,010
2.7 12.8 21.6
32, 226 41 115
a Average number of CCM per a dextran was calculated from the UV–vis spectrum of dextran–CCMs conjugates in DMSO. b Average weight of molecular weight of dextran–CCMs conjugate was calculated using an equation, as follows; Mw of dextran–CCMs conjugate = Mw of dextran (40,000) + Mw of CCM (368) No. of CCM. c Mean diameter of dextran–CCMs nanoparticles in PBS measured by DLS.
micromolar.16 CCM solutions, however, must be prepared by using organic solvent (methanol and DMSO) to exhibit anticancer activity to cultured cancer cells and strong fluorescence in cells, because solubility of CCM in aqueous solution, including several kinds of buffered solutions and cell culture medium, is extremely low (max. 29.9 nM in water).17,18 Therefore, the low solubility of CCM in aqueous media limits its application in human body. It is noteworthy that DC3, DC16, and DC30 easily dissolved in water at 10 mg/mL at room temperature, these polymer concentrations are corresponding to 0.73 mM, 3.49 mM, and 5.88 mM of CCM in dextran–CCMs conjugates, respectively. Namely, dextran–CCMs conjugates show approximately 24,410–196,700 fold higher solubility in water as compared with free CCM. The results indicate that CCMs conjugated to dextran can be easily dispersed in water as interior components of nanoparticles. These CCM concentrations are obviously higher than the concentration at where CCM exhibits anticancer and fluorescence activities. Indeed, dextran–CCMs nanoparticles showed enough strong fluorescence in PBS at 2 mg/mL in concentration (Fig. 2b), while the fluorescence intensity was similar with the fluorescence intensity of free CCM with the corresponding concentrations measured in DMSO (Fig. S3, Supplemental data). The fluorescence intensity of dextran–CCMs nanoparticles increased with the increase in the numbers of CCM side-chains, indicating that the fluorescence properties of dextran–CCMs nanoparticles can be roughly controlled by varying the numbers of CCM side-chains. The potential of dextran–CCMs nanoparticles as materials available for cancer theranostics was investigated in vitro using HeLa cells (human cervical cancer cell line), HUVEC (normal human umbilical vein endothelial cell line), and HDF cells (normal human fibroblast cell line). Fig. 3a shows fluorescent microscopic images of HeLa cells treated with free CCM and dextran–CCMs nanoparticles for 24 h. It is known that hydrophobic CCM molecules are prone to embedded into cell membrane.16 Weak green fluorescence derived from CCM entrapped in cell membrane was
observed in HeLa cells treated with free CCM. In contrast, DC3, DC16, and DC30 nanoparticles were effectively delivered into HeLa cells and exhibited enough strong fluorescence intensity as probes for live cell imaging. However, most of DC3 and DC16 nanoparticles delivered into HeLa cells were localized at near the cell membrane. In comparison with DC3 and DC16 nanoparticles, DC30 nanoparticles were delivered in HeLa cells and well-dispersed in cytoplasm, suggesting that CCM molecules in DC30 nanoparticles are hard to interact with the cell membrane because the dextran outer layer with highly hydrated state would shield the interactions. Fig. 3b and c show fluorescent microscopic images of HUVEC and HDF cells treated with dextran–CCMs nanoparticles for 24 h. In contrast to HeLa cells, significantly low cellular uptake of dextran–CCMs nanoparticles were observed in both cases of HDF and HUVEC cells, suggesting that dextran–CCMs nanoparticles with optimized compositions and structures possess cellular penetrating property in cancer cell-selective manner. In order to deduce the potential transport pathway of dextran– CCMs, we examined cellular uptake of dextran–CCMs nanoparticles in the presence of endocytosis inhibitors. Significant decrease in cellular uptake of all the dextran–CCMs nanoparticles used in this study by HeLa cells were observed in the presence of chlorpromazine, while no significant changes in the cellular uptake were seen in the presence of genistain and cytochalasin D, as shown in Fig. 4. These results indicate that the dextran–CCMs nanoparticles were internalized into HeLa cells mainly via clathrin-mediated endocytosis. The cellular uptake of dextran–CCMs nanoparticles in the absence of endocytosis inhibitors increased with the increase in the numbers of CCM side-chains, suggesting that the CCM side-chains enhance cellular uptake. In constant to HeLa cells, significantly low cell uptake of dextran–CCMs nanoparticles was observed for both normal cell lines. The cell uptake was not changed in the presence of endocytosis inhibitors. Thus, the obtained cancer cell-selective penetrating property is original character of dextran–CCMs nanoparticles and the character is very important for achievement of cancer theranostics. CCMs have been known to bind several kinds of cell membrane proteins, while target proteins and the binding site have not been identified.19 The cancer cell-selective internalization of dextran–CCMs nanoparticles would be resulted from the binding of CCM molecules in the dextran–CCMs nanoparticles with cancer cell specific membrane proteins. In summary, nanoparticles of dextran–CCMs were fabricated through self-assembly approach to create novel nanobiomaterials for cancer theranostics. Significantly high CCM loading contents in the nanoparticles and the high water solubility were achieved. Importantly, the dextran–CCMs nanoparticles were effectively delivered into HeLa cells and exhibited strong fluorescence
Please cite this article in press as: Nagahama, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.062
4
K. Nagahama et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 3. Cellular uptake of CCM and dextran–CCMs nanoparticles. Fluorescent microscopic images of (a) HeLa cell, (b) HUVEC cell, and (c) HDF cell treated with dextran– CCMs nanoparticles and CCM for 24 h. Scale bar 50 lm. Green: dextran–CCMs nanoparticles, Red: Nuclei.
Figure 4. Cellular uptake of dextran–CCMs nanoparticles by HeLa, HUVEC, and HDF cells in the presence of endocytosis inhibitors. (a) Inhibitor ( ), (b) genistein, (c) chlorpromazine, (d) cytochalasin D.
available for live-cell imaging, although the nanoparticles were not delivered into normal cells. Thus, the dextran–CCMs nanoparticles could be a promising nanobiomaterial for creation of novel CCMbased cancer theranostics with high drugs and imaging agents loading. The mechanism of cell penetrating character was not clear in this paper. We will make clear the points in our next paper. Acknowledgements This work was supported by JSPS KAKENHI Grant Number 24700487 and Grant-in-Aid from the Hyogo Science and Technology Association. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.04. 062. References and notes 1. (a) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Deliv. Rev. 2012, 64, 37; (b) Yokoyama, M. Expert Opin. Drug Deliv. 2010, 7, 145. 2. Shi, J.; Votruba, A. R.; Farokhzad, O. C.; Langer, R. Nano Lett. 2010, 10, 3223.
3. Riehemann, K.; Schneider, S. W.; Luger, T. A.; Godin, B.; Ferrari, M.; Fuchs, H. Angew. Chem., Int. Ed. 2009, 48, 872. 4. Lo, S.-T.; Kumar, A.; Hsieh, J.-T.; Sun, X. Mol. Pharm. 2013, 10, 793. 5. Krasia-Christoforou, T.; Georgiou, T. K. J. Mater. Chem. B 2013, 1, 3002. 6. Chakraborti, S.; Das, L.; Kapoor, N.; Das, A.; Dwivedi, V.; Poddar, A.; Chakraborti, G.; Janik, M.; Basu, G.; Panda, D.; Chakraborti, P.; Surolia, A.; Bhattacharyya, B. J. Med. Chem. 2011, 54, 6183. 7. Banerjee, S.; Prasad, P.; Hussain, A.; Khan, I.; Kondaiah, P.; Chakravarty, A. Chem. Commun. 2012, 7702. 8. Kumar, A.; Li, L.; Chaturvedi, A.; Brzostowski, J.; Chittigori, J.; Pierce, S.; Samuelson, L.; Sandman, A.; Kumar, D. J. Appl. Phys. Lett. 2012, 100, 203701. 9. Naksuriya, O.; Okonogi, S.; Schiffelers, R. M.; Hennink, W. E. Biomaterials 2014, 35, 3365. 10. Barry, J.; Fritz, M.; Brender, J. R.; Smith, P. E. S.; Lee, D.-K.; Ramamoorthy, A. J. Am. Chem. Soc. 2009, 131, 4490. 11. Puvvada, N.; Rajput, S.; Kumar, B. N. P.; Mandal, M.; Pathak, A. RSC Adv. 2013, 3, 2553. 12. Ulbrich, K.; Konak, C.; Tuzar, Z.; Kopecek, J. Makromol. Chem. 1987, 188, 1261. 13. Akiyoshi, K.; Deguchi, S.; Moriguchi, N.; Yamaguchi, S.; Sunamoto, J. Macromolecules 1993, 26, 3062. 14. Morimoto, N.; Hirano, S.; Takahashi, H.; Loethen, S.; Thompson, D. H.; Akiyoshi, K. Biomacromolecules 2013, 14, 56. 15. Duncan, R. Nat. Rev. Cancer 2006, 6, 688. 16. Esatbeyoglu, T.; Huebbe, P.; Ernst, I. M.; Chin, D.; Wagner, A. E.; Rimbach, G. Angew. Chem., Int. Ed. 2012, 51, 5308. 17. Letchford, K.; Liggins, R.; Burt, H. J. Phram. Sci. 2008, 97, 1179. 18. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Mol. Pharmacol. 2007, 4, 807. 19. Bilmen, J. G.; Khan, S. Z.; Javed, M. U. H.; Michelangeli, F. Eur. J. Biochem. 2001, 268, 6318.
Please cite this article in press as: Nagahama, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.062
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Anticancer drug-based multifunctional nanogels through self-assembly of dextran–curcumin conjugates toward cancer theranostics Koji Nagahama ⇑, Yoshinori Sano, Takayuki Kumano Department of Nanobiochemistry, Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-Minamimachi, Kobe 650-0047, Japan
a r t i c l e
i n f o
Article history: Received 13 February 2015 Revised 15 April 2015 Accepted 18 April 2015 Available online xxxx Keywords: Curcumin Self-assembly Amphiphilic polymer Nanogel Theranostics
a b s t r a c t Curcumin (CCM) has been received much attention in cancer theranostics because CCM exhibits both anticancer activity and strong fluorescence available for bio-imaging. However, CCM has never been utilized in clinical mainly due to its extremely low water solubility and its low cellular uptake into cancer cells. We fabricated novel CCM-based biodegradable nanoparticles through self-assembly of amphiphilic dextran–CCM conjugates. Significantly high CCM loading contents in the nanoparticles and the high water solubility were achieved. Importantly, the dextran–CCMs nanoparticles were effectively delivered into HeLa cells and exhibited strong fluorescence available for live-cell imaging, although the nanoparticles were not delivered into normal cells. Thus, the dextran–CCMs nanoparticles could be a promising for creation of novel CCM-based cancer theranostics with high efficacy. Ó 2015 Elsevier Ltd. All rights reserved.
Polymer-based nanoparticles are large and fast growing fields, and such nanoparticles have been applied as delivery carriers for therapeutic agents including anticancer drugs and bio-imaging agents, especially for cancer therapy and diagnostics.1,2 Recently, a new field combining both therapy and diagnostics, called theranostics, has been generated for cancer treatment.3 One of the main therapeutic systems in cancer theranostics is imaging-guided drug delivery. The therapeutic system usually employs polymer nanoparticles for their achievement. Polymer nanoparticles for cancer theranostics must be both nano depot and nano carriers for therapeutic and diagnostic agents.4 Common imaging components utilized for cancer theranostics include photoluminescent mostly fluorescent materials, while common therapeutic components are anticancer drugs.2 Nanoparticles consisting of amphiphilic copolymers, such as micelles, polymersomes, and nanogels with hydrophobic domains, are commonly used in cancer theranostics.5 In these cases, both fluorescent materials and anticancer drugs should be loaded in hydrophobic domains of nanoparticles with enough high amounts. However, there are inherent difficulties in achieving enough high drugs and imaging materials loading per nanoparticle because of the limited capacity of hydrophobic domains in nanoparticles for loading.5
⇑ Corresponding author. Tel.: +81 78 303 1328; fax: +81 78 303 1495. E-mail address: [email protected] (K. Nagahama).
Recently, curcumin (CCM), naturally-occurring hydrophobic molecule derived from turmeric, has been received much attention in cancer theranostics because CCM induces apoptosis to various kinds of cancer cells6,7 with a safe manner to healthy cells and exhibits strong fluorescence as biocompatible probes available for bio-imaging.8,9 Thus, CCM can be applied to our concept described above as anticancer drug, imaging material, as well as building block to create nanoparticle for cancer theranostics. However, CCM has never been utilized in clinical mainly due to two problems. The first problem is poor aqueous solubility. CCM is hydrophobic molecule, and thus the maximum water solubility is about 30 nm, whereas CCM induces apoptosis to cancer cells with an IC50 10–75 lM.10 The second problem is low cellular uptake. CCM deeply inserts into cell membrane, anchored by hydrogen bonding to the phosphate group and hydrophobic interaction with fatty acyl group of lipid, and thus only a few parts of CCM translocate into cytoplasm and nucleus whereat it works.11 Thus, intracellular delivery of intact CCM to cancer cells is big issue to achieve clinical application of CCM in cancer theranostics. Herein, we fabricated novel CCM-based polymer nanoparticles by self-assembly approach. Importantly, the obtained CCM-based nanoparticles exhibited not only cell penetrating property in cancer cell-selective manner, but also strong fluorescence in cancer cells enough for visualizing. Therefore, we describe the synthesis of dextran–CCM conjugates, fabrication of their self-assembled nanogels, its fluorescence properties, and the cell penetrating properties. This study is the first example in theranostics studies
http://dx.doi.org/10.1016/j.bmcl.2015.04.062 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Nagahama, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.062
2
K. Nagahama et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
to overcome water-insoluble problems of useful anticancer and fluorescence agents through utilization of polymeric self-assembly. Ulbrich et al. has reported that hydrophilic polymer-hydrophobic drugs conjugates self-assembled into nanoparticles in aqueous media via hydrophobic interactions among the drug molecules.12 Inspired by this paper, we synthesized amphiphilic polymers composed of hydrophobic CCMs and hydrophilic dextran to fabricate CCM-based nanoparticles. Dextran–CCMs conjugates were synthesized through coupling reaction of activated dextran and CCMs (Fig. 1). Dextran–CCMs conjugates are expected to self-assemble into nanoparticles through hydrophobic interactions between CCM side-chains attached to the dextran in aqueous environment (Fig. 1). In this molecular systems, hydrophobic/hydrophilic balance of amphiphilic dextran–CCMs conjugates would affect the water solubility and their self-assembly behavior to form nanoparticles. Therefore, we synthesized a series of dextran–CCMs conjugates with different CCM contents by varying feed molar ratios of CCM to dextran in the synthesis process. Purification of the obtained products were carried out by precipitation technique using excess amount of methanol as poor solvent for the obtained dextran–CCMs conjugates and as good solvent for uncoupled CCM, CDI, and DMAP. Fig. S1 (Supplemental data) shows gel permeation chromatography (GPC) profiles of the dextran–CCMs conjugates. The dextran–CCMs conjugate each shows unimodal peak with a reasonably narrow molecular weight distribution, indicating the complete removal of uncoupled CCM and the reaction byproducts from the obtained dextran–CCMs conjugates. The conjugation of CCMs to dextran was also confirmed by 1H NMR measured in DMSO-d6 as good solvent for dextran–CCMs conjugates (Fig. S2, Supplemental data). UV–vis spectra of the dextran–CCMs conjugates measured in DMSO were shown in Fig. 2a. We used the absorbance at 434 nm to determine the average number of CCM side-chains conjugated to dextran. The characteristics of the
obtained dextran–CCMs conjugates were summarized in Table 1. We used the abbreviations for dextran–CCMs conjugates as DCx (x means numbers of CCM per dextran molecule). DC3, DC16, and DC30 were synthesized and these polymers were used following experiments. It has been reported that amphiphilic polymers composed of polysaccharides main-chain and hydrophobic side-chains, such as cholesterol-conjugated pullulan, form hydrogel-like nanoparticle (nanogel) with polysaccharide skeleton and hydrophobic multi cores in dilute aqueous solutions.13,14 Self-assembly of the dextran–CCMs conjugates was accomplished by direct dispersion of the polymers in water. Tyndall phenomena were observed for all the aqueous solutions (data not shown), meaning the presence of colloidal particles in these solutions. These results clearly show that amphiphilic dextran–CCMs conjugates self-assembled into nanoparticles in aqueous solutions. So, the colloidal particles were analyzed by DLS measurement. DC16 and DC30 nanoparticles showed a unimodal size distribution peak, but the distribution peak of DC3 was bimodal, as shown in Table 1. The mean diameter of colloidal particles were ranged from approximately 30 nm to 220 nm depending on the number of conjugated CCM, indicating that the number of conjugated CCM is one of the critical factors to determine their size. Typically, loading contents of anticancer drugs per polymeric nanocarrier for cancer theranostics are less than 10 wt %.15 The CCM content (wt %) in per DC3, DC16, and DC30 were 2.7 wt %, 12.8 wt %, and 21.6 wt %, respectively. This result means that DC16 and DC30 nanoparticles load relatively higher amounts of anticancer drugs as well as imaging agents as compared with those of typical nanoparticles reported, because CCM possess activities as both anticancer drugs and imaging agents. It is well known that CCM exhibits anticancer activity and enough strong fluorescence when the concentration of CCM in solutions is reached up to
Figure 1. Schematic illustration of the formation of dextran–CCMs nanoparticles. (a) Self-assembled dextran–CCMs nanoparticles via intermolecular hydrophobic interactions between CCM side-chains. (b) Synthesis of dextran–CCMs conjugates.
Please cite this article in press as: Nagahama, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.062
K. Nagahama et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Figure 2. (a) UV-vis spectra of dextran–CCMs conjugates measured in DMSO (2 mg/mL). (b) Fluorescent spectra of dextran–CCMs nanoparticles measured in PBS (2 mg/mL).
Table 1 Characterization of a series of dextran–CCMs conjugates Sample
No. of CCMa
Mwb
CCM content (%)
Dhc (nm)
DC3 DC16 DC30
3 16 30
41,100 45,870 51,010
2.7 12.8 21.6
32, 226 41 115
a Average number of CCM per a dextran was calculated from the UV–vis spectrum of dextran–CCMs conjugates in DMSO. b Average weight of molecular weight of dextran–CCMs conjugate was calculated using an equation, as follows; Mw of dextran–CCMs conjugate = Mw of dextran (40,000) + Mw of CCM (368) No. of CCM. c Mean diameter of dextran–CCMs nanoparticles in PBS measured by DLS.
micromolar.16 CCM solutions, however, must be prepared by using organic solvent (methanol and DMSO) to exhibit anticancer activity to cultured cancer cells and strong fluorescence in cells, because solubility of CCM in aqueous solution, including several kinds of buffered solutions and cell culture medium, is extremely low (max. 29.9 nM in water).17,18 Therefore, the low solubility of CCM in aqueous media limits its application in human body. It is noteworthy that DC3, DC16, and DC30 easily dissolved in water at 10 mg/mL at room temperature, these polymer concentrations are corresponding to 0.73 mM, 3.49 mM, and 5.88 mM of CCM in dextran–CCMs conjugates, respectively. Namely, dextran–CCMs conjugates show approximately 24,410–196,700 fold higher solubility in water as compared with free CCM. The results indicate that CCMs conjugated to dextran can be easily dispersed in water as interior components of nanoparticles. These CCM concentrations are obviously higher than the concentration at where CCM exhibits anticancer and fluorescence activities. Indeed, dextran–CCMs nanoparticles showed enough strong fluorescence in PBS at 2 mg/mL in concentration (Fig. 2b), while the fluorescence intensity was similar with the fluorescence intensity of free CCM with the corresponding concentrations measured in DMSO (Fig. S3, Supplemental data). The fluorescence intensity of dextran–CCMs nanoparticles increased with the increase in the numbers of CCM side-chains, indicating that the fluorescence properties of dextran–CCMs nanoparticles can be roughly controlled by varying the numbers of CCM side-chains. The potential of dextran–CCMs nanoparticles as materials available for cancer theranostics was investigated in vitro using HeLa cells (human cervical cancer cell line), HUVEC (normal human umbilical vein endothelial cell line), and HDF cells (normal human fibroblast cell line). Fig. 3a shows fluorescent microscopic images of HeLa cells treated with free CCM and dextran–CCMs nanoparticles for 24 h. It is known that hydrophobic CCM molecules are prone to embedded into cell membrane.16 Weak green fluorescence derived from CCM entrapped in cell membrane was
observed in HeLa cells treated with free CCM. In contrast, DC3, DC16, and DC30 nanoparticles were effectively delivered into HeLa cells and exhibited enough strong fluorescence intensity as probes for live cell imaging. However, most of DC3 and DC16 nanoparticles delivered into HeLa cells were localized at near the cell membrane. In comparison with DC3 and DC16 nanoparticles, DC30 nanoparticles were delivered in HeLa cells and well-dispersed in cytoplasm, suggesting that CCM molecules in DC30 nanoparticles are hard to interact with the cell membrane because the dextran outer layer with highly hydrated state would shield the interactions. Fig. 3b and c show fluorescent microscopic images of HUVEC and HDF cells treated with dextran–CCMs nanoparticles for 24 h. In contrast to HeLa cells, significantly low cellular uptake of dextran–CCMs nanoparticles were observed in both cases of HDF and HUVEC cells, suggesting that dextran–CCMs nanoparticles with optimized compositions and structures possess cellular penetrating property in cancer cell-selective manner. In order to deduce the potential transport pathway of dextran– CCMs, we examined cellular uptake of dextran–CCMs nanoparticles in the presence of endocytosis inhibitors. Significant decrease in cellular uptake of all the dextran–CCMs nanoparticles used in this study by HeLa cells were observed in the presence of chlorpromazine, while no significant changes in the cellular uptake were seen in the presence of genistain and cytochalasin D, as shown in Fig. 4. These results indicate that the dextran–CCMs nanoparticles were internalized into HeLa cells mainly via clathrin-mediated endocytosis. The cellular uptake of dextran–CCMs nanoparticles in the absence of endocytosis inhibitors increased with the increase in the numbers of CCM side-chains, suggesting that the CCM side-chains enhance cellular uptake. In constant to HeLa cells, significantly low cell uptake of dextran–CCMs nanoparticles was observed for both normal cell lines. The cell uptake was not changed in the presence of endocytosis inhibitors. Thus, the obtained cancer cell-selective penetrating property is original character of dextran–CCMs nanoparticles and the character is very important for achievement of cancer theranostics. CCMs have been known to bind several kinds of cell membrane proteins, while target proteins and the binding site have not been identified.19 The cancer cell-selective internalization of dextran–CCMs nanoparticles would be resulted from the binding of CCM molecules in the dextran–CCMs nanoparticles with cancer cell specific membrane proteins. In summary, nanoparticles of dextran–CCMs were fabricated through self-assembly approach to create novel nanobiomaterials for cancer theranostics. Significantly high CCM loading contents in the nanoparticles and the high water solubility were achieved. Importantly, the dextran–CCMs nanoparticles were effectively delivered into HeLa cells and exhibited strong fluorescence
Please cite this article in press as: Nagahama, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.062
4
K. Nagahama et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 3. Cellular uptake of CCM and dextran–CCMs nanoparticles. Fluorescent microscopic images of (a) HeLa cell, (b) HUVEC cell, and (c) HDF cell treated with dextran– CCMs nanoparticles and CCM for 24 h. Scale bar 50 lm. Green: dextran–CCMs nanoparticles, Red: Nuclei.
Figure 4. Cellular uptake of dextran–CCMs nanoparticles by HeLa, HUVEC, and HDF cells in the presence of endocytosis inhibitors. (a) Inhibitor ( ), (b) genistein, (c) chlorpromazine, (d) cytochalasin D.
available for live-cell imaging, although the nanoparticles were not delivered into normal cells. Thus, the dextran–CCMs nanoparticles could be a promising nanobiomaterial for creation of novel CCMbased cancer theranostics with high drugs and imaging agents loading. The mechanism of cell penetrating character was not clear in this paper. We will make clear the points in our next paper. Acknowledgements This work was supported by JSPS KAKENHI Grant Number 24700487 and Grant-in-Aid from the Hyogo Science and Technology Association. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.04. 062. References and notes 1. (a) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Deliv. Rev. 2012, 64, 37; (b) Yokoyama, M. Expert Opin. Drug Deliv. 2010, 7, 145. 2. Shi, J.; Votruba, A. R.; Farokhzad, O. C.; Langer, R. Nano Lett. 2010, 10, 3223.
3. Riehemann, K.; Schneider, S. W.; Luger, T. A.; Godin, B.; Ferrari, M.; Fuchs, H. Angew. Chem., Int. Ed. 2009, 48, 872. 4. Lo, S.-T.; Kumar, A.; Hsieh, J.-T.; Sun, X. Mol. Pharm. 2013, 10, 793. 5. Krasia-Christoforou, T.; Georgiou, T. K. J. Mater. Chem. B 2013, 1, 3002. 6. Chakraborti, S.; Das, L.; Kapoor, N.; Das, A.; Dwivedi, V.; Poddar, A.; Chakraborti, G.; Janik, M.; Basu, G.; Panda, D.; Chakraborti, P.; Surolia, A.; Bhattacharyya, B. J. Med. Chem. 2011, 54, 6183. 7. Banerjee, S.; Prasad, P.; Hussain, A.; Khan, I.; Kondaiah, P.; Chakravarty, A. Chem. Commun. 2012, 7702. 8. Kumar, A.; Li, L.; Chaturvedi, A.; Brzostowski, J.; Chittigori, J.; Pierce, S.; Samuelson, L.; Sandman, A.; Kumar, D. J. Appl. Phys. Lett. 2012, 100, 203701. 9. Naksuriya, O.; Okonogi, S.; Schiffelers, R. M.; Hennink, W. E. Biomaterials 2014, 35, 3365. 10. Barry, J.; Fritz, M.; Brender, J. R.; Smith, P. E. S.; Lee, D.-K.; Ramamoorthy, A. J. Am. Chem. Soc. 2009, 131, 4490. 11. Puvvada, N.; Rajput, S.; Kumar, B. N. P.; Mandal, M.; Pathak, A. RSC Adv. 2013, 3, 2553. 12. Ulbrich, K.; Konak, C.; Tuzar, Z.; Kopecek, J. Makromol. Chem. 1987, 188, 1261. 13. Akiyoshi, K.; Deguchi, S.; Moriguchi, N.; Yamaguchi, S.; Sunamoto, J. Macromolecules 1993, 26, 3062. 14. Morimoto, N.; Hirano, S.; Takahashi, H.; Loethen, S.; Thompson, D. H.; Akiyoshi, K. Biomacromolecules 2013, 14, 56. 15. Duncan, R. Nat. Rev. Cancer 2006, 6, 688. 16. Esatbeyoglu, T.; Huebbe, P.; Ernst, I. M.; Chin, D.; Wagner, A. E.; Rimbach, G. Angew. Chem., Int. Ed. 2012, 51, 5308. 17. Letchford, K.; Liggins, R.; Burt, H. J. Phram. Sci. 2008, 97, 1179. 18. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Mol. Pharmacol. 2007, 4, 807. 19. Bilmen, J. G.; Khan, S. Z.; Javed, M. U. H.; Michelangeli, F. Eur. J. Biochem. 2001, 268, 6318.
Please cite this article in press as: Nagahama, K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.062