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End-Sealed High Aspect Ratio Hollow Nanotubes Encapsulating an Anticancer Drug: Torpedo-Shaped Peptidic Nanocapsules ACS Nano Downloaded from pubs.acs.org by UNIV OF BRITISH COLUMBIA on 01/04/19. For personal use only.
Motoki Ueda,*,†,‡ Siyoong Seo,†,‡,§ Baiju G. Nair,‡ Stefan Müller,†,∥ Eiki Takahashi,∥ Takashi Arai,∥ Tomonori Iyoda,⊥ Shin-ichiro Fujii,⊥ Satoshi Tsuneda,§ and Yoshihiro Ito*,†,‡ †
Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science (CEMS), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ‡ Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research (CPR), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan § Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan ∥ Research Resources Division, RIKEN Center for Brain Science (CBS), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ⊥ Laboratory for Immunotherapy, RIKEN Center for Integrative Medical Science (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan S Supporting Information *
ABSTRACT: Nanomaterial morphology is important for the targeted delivery of drugs to tissues as well as subsequent cellular uptake. Hollow nanotubes composed of peptides, with a diameter of 80 nm and various lengths (100, 200, 300, 600 nm), were successfully capped and sealed with a peptide hemisphere to encapsulate the anticancer drug, cisplatin. The torpedo-shaped nanocapsules with an aspect ratio (length/diameter) of 2.4 showed more rapid cellular uptake and accumulation at the tumor site compared with spherical analogues. Successful delivery of cisplatin to tumors was achieved in a mouse model and tumor growth was efficiently suppressed. KEYWORDS: morphology, peptide nanocapsule, aspect ratio, cellular uptake, drug delivery carrier
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a hollow AR space, completely shielding the cargo, encapsulating the material itself, and containing the drug for long enough for it to be released in the cell. The preparation of phase-separated peptide assemblies using two different kinds of amphiphilic polypeptides has been reported, including a round-bottom flask shaped assembly, which was named the “assembly-patchwork system”.15−20 The two kinds of amphiphilic polypeptides formed nanotubes of 200 nm in length and vesicle-forming nanosheets, respectively. Hydrophobic interactions between the edges of the nanosheet and the nanotube enabled the nanosheet to fuse and round up on the open ends of the nanotube, resulting in a round-bottom flask shaped assembly. These amphiphilic peptides and assembly patchwork system may be useful tools for the preparation of a high AR carrier. In this study, we focused on the development of completely sealed nanotubes loaded with an anticancer drug by combining versatile self-assembled peptide nanostructures.15−18 Using these peptide assemblies as
ntravenous injection is a favored mode of administration of cancer chemotherapy drugs because this route enables direct accumulation in the target tissue. Carriers that can entrap drugs and then circulate in the blood to arrive specifically at the target site, thereby reducing side effects, are promising tools for intravenous dosing. For efficient cancer therapy, such drug carriers are required to be stable in the bloodstream, are able to accumulate at the tumor site, and have rapid cellular uptake. During the past decade, it has been reported that the shape of the material can affect cellular uptake,1−3 intracellular transport,4 and biodistribution.5−7 However, there is currently insufficient information about the relationship between the carrier shape and cell functions. It has been reported that gold nanorods,8−10 tobacco mosaic virus,2,11 polystyrene particles,12 and silica particles13,14 with medium and high aspect ratios (length/diameter) (ARs) showed a more rapid uptake of greater particle numbers than spherical particles with an AR of 1. However, it is difficult to completely encapsulate drugs in a hollow nanosized carrier with a high AR, and there are currently no examples of drug delivery systems (DDSs) using high AR carriers. There are many challenges in developing DDS carriers, including creating © XXXX American Chemical Society
Received: August 14, 2018 Accepted: December 21, 2018
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DOI: 10.1021/acsnano.8b06189 ACS Nano XXXX, XXX, XXX−XXX
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Cite This: ACS Nano XXXX, XXX, XXX−XXX
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ACS Nano
Figure 1. Schematic concept of peptide nanocapsules prepared by integrating peptide assemblies. Chemical structures of amphiphilic polypeptides GSL12 and GSL16, which are composed of the same hydrophilic sarcosine chain with a hydrophobic helical block of different lengths. Hollow peptide nanocapsules were prepared by self-sealing of peptide nanotubes with GSL16 molecules.
RESULTS/DISCUSSION The synthesized amphiphilic peptides, GSL12 and GSL16, which have 12 and 16 hydrophobic residues forming an αhelical block, respectively (Figure S10), spontaneously selfassembled into nanotubes (peptide amphiphile nanotube, PANT) having an 80 nm diameter, and vesicles (peptide amphiphile nanosphere, PANS) having an 80 nm diameter in saline, respectively (Figure 2A,B and Figure S11). The different morphologies of the GSL12 and GSL16 assemblies were most likely because of differences in the packing between neighboring helices in the hydrophobic layer. The alignment of leucine residues, which have large side chains, was considered a key factor in this difference (Figure S12). The leucine (Leu) and aminoisobutyric acid (Aib) alignment produces a tilted ridge and groove of side chains along the helix axis. The sidechain ridges of Leu are found on the back and front faces of the helix, and the grooves of Aib alignment exist on the other right and left faces. Thus, an anisotropy in the GSL12 membrane is induced and the curvature is determined by the tilted packing of GSL12 with a ridge-groove interaction. As a result, the ridge-groove packing facilitates tubular formation. However, GSL16 is not packed with a tilt angle because the long ridges and grooves of the side chains cover across the two
a base material has the advantages of biocompatibility and structural stability because of the nature and secondary structure of peptides. In addition, the peptides are easily modified using a combination of different amino acids21−23 or bioactive molecules.24−26 Furthermore, the rapid biodegradation of peptide assemblies enabling drug release is expected because of the abundance of proteases in cancer cells. Here, we found that hollow nanotubes composed of peptides, with a diameter of 80 nm and various lengths (100, 200, 300, 600 nm), could be successfully capped and sealed with a peptide hemisphere to encapsulate the anticancer drug cisplatin. Furthermore, a high capping yield was achieved by a new preparation method wherein vesicle-forming peptides selfassembled on the edge of nanotubes. Using these torpedoshaped peptide amphiphile nanocapsules (PANCs) (Figure 1) with various ARs, the relationship between cellular uptake and the AR of the material was determined and compared with the spherical analogue. Finally, in vivo experiments using nearinfrared fluorescent (NIRF) cancer imaging showed successful anticancer drug delivery to the tumor site and efficient suppression of tumor growth by accumulation of the torpedoshaped PANCs at the tumor site. B
DOI: 10.1021/acsnano.8b06189 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 2. Peptide assemblies and nanocapsule formation by sealing the open mouths of nanotubes. Negative stained TEM images of peptide nanotubes from GSL12 (A), peptide nanosphere from GSL16 (B), and peptide nanocapsule formed by capping the open mouth of PANT with GSL16 (C). Cryo-TEM images of a PANC showing complete fusion at the sealing site (D, red arrow). TEM images of peptide assemblies prepared by adding GSL16 nanosheet dispersion (E) or GSL16 nanosphere dispersion (F) to GSL12 PANT dispersion and heating the mixture. Schematic illustration of nanocapsule formation mechanism from PANT and GSL16 solution, GSL16 nanosheet, and GSL16 PANS dispersion (G). Scale bars are 200 nm (A−C, E, and F) and 100 nm (D).
neighboring faces. Thus, GSL16 is packed in a parallel manner to form an isotropic sphere. In contrast to the nanotubes, further self-assembly and shape change did not occur after vesicle formation because of the lack of a hydrophobic edge. The peptide amphiphile nanocapsules (PANCs) were prepared by adding GSL16 solution to a PANT dispersion and heating the mixture at 90 °C for 1 h (Figure 2C, D and Figure S16) to give a high yield of 76% (capped nanotubes/ total assemblies) (n = 186). The PANT has a hydrophobic open mouth, which is imperfectly covered by the hydrophilic polysarcosine chain of the amphiphilic polypeptide. Thus, when the vesicle-forming amphiphile GSL16 was added to a PANT dispersion and heated, the hydrophobic block clusters, self-assembles, and then forms a spherical assembly on the open mouth of the PANT (Figure 2G). This is the mechanism of PANC formation. In a previous report showing the success of one-end-capping of nanotubes, nanotubes with both mouths capped were observed as a minor fraction.18 By changing the preparation method from a combination of a PANT and GSL16 nanosheet to a PANT and GSL16 molecules, we successfully improved the yield of PANCs. Here, this PANC, a phase-separated assembly of GSL12 and GSL16, had stable morphology and was not an intermediate structure because it was stable enough to maintain the morphology for at least 6 months (Figure S25). This structural stability is a very important property for industrial and clinical use. GSL16 also spontaneously formed a nanosheet assembly after injection of GSL16 into saline without heat treatment (Figure S11D). When this GSL16 nanosheet dispersion was mixed with a PANT dispersion and heated at 90 °C for 1 h, PANCs were also observed in the transmission electron microscope (TEM) images (Figure 2E) because the nanosheets also have a hydrophobic edge. The GSL16 nanosheet fused to a PANT between the hydrophobic edges and rolled up, with the result that a PANC was formed by sealing with a
GSL16 nanosheet to decrease the hydrophobic area (Figure 2G). In contrast, combining PANS and PANT dispersions did not result in PANC formation under the same conditions because the PANSs do not have hydrophobic edges (Figure 2F). The sealing force is the hydrophobic interaction between the hydrophobic edges of the assemblies. Thus, both PANTs and GSL16 nanosheets can be regarded as amphiphilic structures. Through the hydrophobic interactions between the assemblies (or amphiphiles), nanostructures were integrated to build a higher-order structure. As amphiphilic structures, PANTs can fuse to each other. In fact, PANTs elongated upon heat treatment of the dispersion (Figure 3A). As a result, PANTs of various lengths were obtained by adjusting the heating conditions: 115 ± 12 nm (80 °C, 1 h, n = 212); 198 ± 6 nm (80 °C, 3 h, n = 184); 377 ± 43 nm (80 °C, 5 h, n = 249); 306 ± 28 nm (90 °C, 1 h, n = 200); 561 ± 80 nm (90 °C, 3 h, n = 103); and 869 ± 124 nm (90 °C, 5 h, n = 51) (Figure 3A, Figure S13, and Table 1). The PANTs having lengths of 115, 198, 306, and 561 nm were named PANT1.4, PANT2.4, PANT3.8, and PANT7.0, which had mean aspect ratios (ARs) (length/diameter) of 1.4, 2.4, 3.8, and 7.0, respectively, estimated by dividing the length by the diameter (80 nm) of the PANT. These PANTs had a small polydispersity index (PDI) of
