© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2627 wileyonlinelibrary.com COMMUNICATION Self-Assembled Autophagy-Inducing Polymeric Nanoparticles for Breast Cancer Interference In-Vivo Yi Wang, Yao-Xin Lin, Zeng-Ying Qiao, Hong-Wei An, Sheng-Lin Qiao, Lei Wang, R P Yeshan J Rajapaksha, and Hao Wang* Y. Wang, Y.-X. Lin, Dr. Z.-Y. Qiao, H.-W. An, S.-L. Qiao, Prof. L. Wang, R. P. Y. J. Rajapaksha, Prof. H. Wang CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety National Center for Nanoscience and Technology (NCNST) No. 11 Beiyitiao, Zhongguancun 100190 Beijing, PR China E-mail: wanghao@nanoctr.cn Y. Wang, Y.-X. Lin, H.-W. An, S.-L. Qiao University of Chinese Academy of Science (UCAS) No.19A Yuquan Road, 100049 Beijing, PR China DOI: 10.1002/adma.201405926 one-pot synthesis method and effectively delivered drugs. [19,20] Although numerous nanomaterials influence the autophagy process, [21] few examples, to the best of our knowledge, have been reported to effectively control autophagic flux and ulti- mately achieve therapeutic potential. Herein, we describe the development of autophagy-inducing peptides engineered into polymeric nanoparticles for highly efficient induction of autophagy and interference of breast cancer in vitro and in vivo. Bec1, as an autophagy-inducing pep- tide, covalently grafts onto pH-sensitive polymers (Ps), which self-assemble into micelle-like nanoparticles (P-Bec1) with poly(ethylene glycol) (PEG) as hydrophilic shells ( Scheme 1). The P-Bec1 nanoparticles improved the stability and enhanced the cellular uptake of Bec1 in vitro. P-Bec1 can dissociate in the weakly acidic lysosomal environment (pH 4–5), which resulted in the alkalization and impairment of lysosomes. [22] The disso- ciated P-Bec1 can escape from lysosomes and effectively induce autophagy, leading to autophagic cell death. Moreover, P-Bec1 is also able to effectively inhibit the growth of tumors and slow down tumor development in vivo. The amphiphilic poly(β-amino ester) copolymer composed of the hydrophobic monomer 1,6-hexanediol diacrylate (HDDA, 1.2 mM), the pH-sensitive monomer 3-(dibutylamino)-1-pro- pylamine (DBPA, 0.7 mM), and hydrophilic amino-terminated PEG (PEG-NH 2 , M W = 2 kDa, 0.3 mM) was synthesized by Michael addition. [19,20] The structure of the polymer was charac- terized by 1 H NMR (Figure S4, in the Supporting Information, SI). The degree of polymerization (DP) and molecular weight ( M W ) of copolymers were deduced to be 27 and ca. 16 kDa, respectively. Furthermore, Bec1 was engineered to have a thiol group at the N terminal to conjugate with the poly(β-amino ester) copolymer (Figures S2–S4, SI). The 1 H NMR spectrum of P-Bec1 showed the disappearance of the acrylate double bonds of the polymer P at 5.8–6.3 ppm and the appearance of Bec1 peaks at 6.7–8.5 ppm ( Figure 1a), indicating that the acrylate groups on both ends of the polymer had completely reacted with Bec1. Circular dichroism (CD) spectra of Bec1 and P-Bec1 were analyzed using standard CONTINLL algorithms, [23] which revealed the secondary structure of Bec1 to be 33.8% α-helix, and P-Bec1, 18.8% α-helix, proving that 56% of the secondary structure of Bec1 was not destroyed during the process of copol- ymer synthesis (Figure 1c). The amphiphilic P-Bec1 polymers simultaneously self-assemble into micelle-like nanoparticles during dialysis from dimethyl sulfoxide (DMSO) into water (Scheme 1). The morphologies and hydrodynamic sizes of P and P-Bec1 were observed by transmission electron microscopy Autophagy is a lysosome-based evolutionarily conserved pro- cess that plays an important role in cellular degradation for the clearance of damaged or superfluous proteins and organelles. [1] Abnormalities in autophagy may directly correlate to various pathologic diseases, including neurodegenerative disease, [2] cardiac disease, [3] and cancer. [4] Recently, researchers have attempted to regulate autophagy and prevent further tumor pro- gress. [5,6] Compared to present cancer therapy strategies, such as chemo-, [7] immuno-, [8] gene, [9] radiation, [10] photothermal, [11] and photodynamic [12] therapies, the autophagy-based concep- tual pilot studies for cancer therapy are of particular – and growing – interest in autophagy-deficient cancers, for example, breast, ovarian, and prostate cancers. [6] Beclin-1 (Bec1) is the peptide encoded by the human gene BECN1, a mammalian homolog of the yeast autophagy- related gene (Atg) 6, and participates in the regulation of autophagy through binding to phosphatidylinositol-4,5-bispho- sphate 3-kinases (PI3Ks, signal transducer enzymes), which are required for initiation of autophagosome formation in autophagy. [13] BECN1 has been identified as a haploinsufficient tumor suppressor, and is commonly deleted in human breast, ovarian, and prostate tumors. [14] Furthermore, upregulation of Bec1 enhances Ras-induced cell death in MCF-7 breast cancer cells. [15] Therefore, it has been suggested that overexpression of Bec1 could inhibit tumor development. [6,16] However, the thera- peutic Bec1 peptide has two major drawbacks that hamper their in vivo application, namely poor chemical stability in vivo and non-specific bio-distribution in tissues. Chemical modification and/or self-assembly are two useful approaches for overcoming these limitations. [17] pH-sensitive polymers have been devel- oped widely to realize the controlled disassembly and release of payloads in biological conditions. [18] Our previous studies dem- onstrated that a pH-sensitive poly(β-amino ester) copolymer self-assembled into a micelle-like nanoparticle by means of a Adv. Mater. 2015, 27, 2627–2634 www.advmat.de www.MaterialsViews.com