Polyplex micelle installing intracellular self-processing functionalities without free catiomers for safe and efficient systemic gene therapy through tumor vasculature targeting.
Both efficiency and safety profiles are crucial for promotion of gene delivery systems towards practical applications. A promising template system was previously developed based on block catiomer of poly(ethylene glycol) (PEG)-b-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide}-cholesteryl [PEG-PAsp(DET)-cholesteryl] with strategies of ligand conjugation at the α-terminus for specific affinity to the targeted cells and cholesteryl conjugation at the ω-terminus for structural stabilization to obtain systemic retention. Aiming for advocating this formulation towards practical applications, in the current study, the binding profile of this polymer to plasmid DNA (pDNA) was carefully studied to address an issue of toxicity origin. Quantification of free polymer composition confirmed that the toxicity mainly results from unbound polymer and polyplex micelle itself has negligible toxicity. This evaluation allowed for identifying an optimal condition to prepare safe polyplex micelles for systemic application that possess maximal polymer-binding but exclude free polymers. The identified polyplex micelles then faced a drawback of limited transfection efficiency due to the absence of free polymer, which is an acknowledged tendency found in various synthetic gene carriers. Thus, series of functional components was strategically compiled to improve the transfection efficiency such as attachment of cyclic (Arg-Gly-Asp) (cRGD) peptide as a ligand onto the polyplex micelles to facilitate cellular uptake, use of endosome membrane disruptive catiomer of PAsp(DET) for facilitating endosome escape along with use of the conjugated cholesteryl group to amplify the effect of PAsp(DET) on membrane disruption, so as to obtain efficient transfection. The mechanistic investigation respecting the appreciated pH dependent protonation behavior of PAsp(DET) permitted to depict an intriguing scenario how the block catiomers manage to escape from the endosome entrapment in response to the pH gradient. Subsequent systemic application to the pancreatic tumor demonstrated a capability of vascular targeting mediated by the cRGD ligand, which was directly confirmed based on in situ confocal laser scanning microscopy observation. Encouraging this result, the vascular targeting to transfect a secretable anti-angiogenic gene was attempted to treat the intractable pancreatic tumor with anticipation that the strategy could circumvent the intrinsic physiological barriers derived from hypovascular and fibrotic characters. The obtained therapeutic efficiency demonstrates promising utilities of the proposed formulation as a safe systemic gene delivery carrier in practical use.
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Qixian
Q
Chen
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Japan Science and Technology Agency, PRESTO, Japan. Electronic address: osada@bmw.t.u-tokyo.ac.jp.
Kensuke
K
Osada
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, China.
Zhishen
Z
Ge
Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Satoshi
S
Uchida
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Innovation Center of NanoMedicine (iCONM), Institute of Industrial Promotion - KAWASAKI, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan.
Theofilus A
TA
Tockary
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Innovation Center of NanoMedicine (iCONM), Institute of Industrial Promotion - KAWASAKI, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan.
Anjaneyulu
A
Dirisala
Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Akitsugu
A
Matsui
Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Innovation Center of NanoMedicine (iCONM), Institute of Industrial Promotion - KAWASAKI, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan.
Kazuko
K
Toh
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Kaori M
KM
Takeda
Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Innovation Center of NanoMedicine (iCONM), Institute of Industrial Promotion - KAWASAKI, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan.
Xueying
X
Liu
Polymer Chemistry Division, Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan.
Takahiro
T
Nomoto
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Tekihiko
T
Ishii
Department of Molecular Medicinal Sciences, Division of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Makoto
M
Oba
Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Yu
Y
Matsumoto
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Innovation Center of NanoMedicine (iCONM), Institute of Industrial Promotion - KAWASAKI, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan. Electronic address: kataoka@bmw.t.u-tokyo.ac.jp.
Kazunori
K
Kataoka
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics / Q000627:therapeutic use; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008823:Micelles; D009369:Neoplasms / Q000098:blood supply* / Q000235:genetics / Q000628:therapy*; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000628:therapy; D010190:Pancreatic Neoplasms / Q000098:blood supply / Q000235:genetics / Q000628:therapy; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics / Q000627:therapeutic use; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D014162:Transfection / Q000379:methods
D008823:Micelles; D011506:Proteins; C571441:poly(ethylene glycol)-poly(N'-(N-(2-aminoethyl)-2-aminoethyl)aspartamide) block copolymer; D011092:Polyethylene Glycols; D004247:DNA
Endosome escape; Polyplex micelle; Systemic administration; Toxicity; Tumor gene therapy; Tumor vasculature targeting