Hierarchical Nanoassemblies-Assisted Combinational Delivery of

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Hierarchical Nanoassemblies-Assisted Combinational Delivery of Cytotoxic Protein and Antibiotic for Effective Cancer Treatment Meng Liu, Shiyang Shen, Di Wen, Mengru Li, Teng Li, Xiaojie Chen, Zhen Gu, and Ran Mo Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04976 • Publication Date (Web): 16 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018

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Nano Letters

Hierarchical Nanoassemblies-Assisted Combinational Delivery of Cytotoxic Protein and Antibiotic for Effective Cancer Treatment

Meng Liu1,a, Shiyang Shen1,a, Di Wen2, Mengru Li1, Teng Li1, Xiaojie Chen1, Zhen Gu2,*, Ran Mo1,*

1

State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for

Metabolic Diseases, Center of Advanced Pharmaceuticals and Biomaterials, China Pharmaceutical University, Nanjing 210009, China 2

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill

and North Carolina State University, Raleigh, NC 27695, USA.

a

These authors contributed equally to this work.

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Abstract

Protein therapeutics hold increasing interest with promise of revolutionizing the cancer treatment by virtue of potent specific activity and reduced adverse effect. Nonetheless, the therapeutic efficacy of anticancer proteins is highly compromised by multiple successive physiological barriers to protein delivery. In addition, concurrent elimination of bulk tumor cells and highly-tumorigenic cancer stem-like cells (CSCs) as a promising strategy has been evidenced to significantly improve cancer therapy. Here we show that a hierarchicallyassembled nanocomposite can self-adaptively transform its particulate property in response to endogenous tumor-associated signals to overcome the sequential barriers and achieve enhanced antitumor efficacy by killing CSCs and bulk tumor cells synchronously. The nanoassemblies

preferentially

accumulate

in

tumor,

and

dissociate

under

tumor

microenvironmental acidity accompanied by the extracellular release of small-sized ribonuclease A (RNase A)-encapsulated nanocapsule (R-rNC) and small-molecule anti-CSC doxycycline (Doc), which exhibit increased tumor penetration and intracellular accumulation. The endocytosed R-rNC rapidly release RNase A within both CSCs and tumor cells at intracellular reductive condition, causing cell death by catalyzing RNA degradation, while Doc eradicates CSCs by inhibiting mitochondrial biogenesis. The hierarchical assemblies show superior cytotoxicity on the CSC-enriched MDA-MB-231 mammospheres and enhanced antitumor efficacy on the xenograft tumor mouse model.

Keywords: drug delivery; protein therapy; radical polymerization; cancer stem-like cells; combination cancer therapy

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ToC figure

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Protein-based therapeutics, such as enzymes, antibodies, cytokines, and transcription factors, play an increasingly essential role in biomedical and pharmaceutical applications,1, 2 which show considerable advantages of highly specific bioactivity and minimized side effects. A number of protein therapeutics have been used for cancer treatment in preclinical and clinical trials3-5 due to their potent anticancer activities by cell proliferation blockage, apoptosis activation, and anti-angiogenesis. However, the majority of protein drugs and drug candidates suffer from low stability, plasma proteolysis, and poor membrane permeability. Spurred by the fast development of nanotechnology, many nanosystems, such as liposomes, polymeric nanoparticles, nanogels and inorganic nanocarriers, have been employed to improve the robustness and effectiveness of proteins.6-9

For in vivo therapeutic applications, the nanocarrier-mediated delivery of proteins is required to overcome successive physiological barriers, including blood circulation, tumor microenvironment, cell membrane and endo-lysosomal degradation, to ultimately strike their targets inside the cells.10-12 Each transport step affects the final therapeutic efficacy of anticancer proteins of note.12 The property of large particle size (~100 nm) and negatively charged surface has been proved to be appropriate for nanoparticles to preferentially extravasate outside the blood vessels and accumulate at the tumor site, but unfortunately turns into a major limitation to subsequent tumor penetration and cellular internalization.10, 13, 14 Conversely, small-sized nanoparticles ( 50 µm) treated with the released 28 ACS Paragon Plus Environment

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R-rNC, Doc and their mixture for 10 days (n = 6). *P < 0.05,

***

P < 0.001. f) Representative

images of the mammospheres treated with the released R-rNC (0.16 µM), Doc (12.5 µM) and their mixture for 10 days. Scale bar represents 50 µm. g) Flow cytometric analysis of the CD133+ cell population in the mammospheres treated with the released R-rNC (0.64 µM), Doc (50 µM) and their mixture for 48 h. h) Flow cytometric analysis of the side population in the mammospheres treated with the released R-rNC (0.64 µM), Doc (50 µM) and their mixture for 48 h.

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Figure 5. a) Fluorescent images of the tumor-bearing mice at 2, 6, and 24 h post administration of different Cy5.5-labeled formulations. Red arrows indicate the tumor sites. b) Fluorescent images of the harvested tissues at 24 h post administration. The numbers from (1) to (6) represent heart, liver, spleen, lung, kidney, and tumor in order. c) Fluorescent images of the tumor sections at 24 h post administration of Rho-R-rNC/aNG and Rho-R-rNC/nNG. Scale bar represents 100 µm. d) Tumor growth curves of the tumor-bearing mice treated with Doc/R-rNC/nNG, R-rNC/aNG, and Doc/R-rNC/aNG (n = 6 for the saline group or n = 7 for the drug-containing formulations). *P < 0.05,

**

P < 0.01. e) Inhibitory ratio of the tumor

growth 22 days after onset of treatment (n = 7). *P < 0.05, **P < 0.01. f) Percentage of the CD133+ cell population in the tumor 22 days after onset of treatment (n = 3). *P < 0.05, ***P < 0.001.

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67x51mm (300 x 300 DPI)

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