Pea-like nanocabins enable autonomous cruise and step-by-step drug pushing for deep tumor inhibition

Accepted Manuscript Pea-like Nanocabins enable autonomous cruise and step-by-step drug pushing for deep tumor inhibition

Ming Wu, Jiaojiao Chen, Hanitrarimalala Veroniaina, Subhankar Mukhopadhyay, Ziheng Wu, Zhenghong Wu, Xiaole Qi PII: DOI: Reference:

S1549-9634(19)30058-9 https://doi.org/10.1016/j.nano.2019.02.025 NANO 1976

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Revised date:

10 January 2019

Please cite this article as: M. Wu, J. Chen, H. Veroniaina, et al., Pea-like Nanocabins enable autonomous cruise and step-by-step drug pushing for deep tumor inhibition, Nanomedicine: Nanotechnology, Biology, and Medicine, https://doi.org/10.1016/ j.nano.2019.02.025

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ACCEPTED MANUSCRIPT Pea-like Nanocabins Enable Autonomous Cruise and Step-by-Step Drug Pushing for Deep Tumor Inhibition

Ming Wu 1, Jiaojiao Chen 1, Hanitrarimalala Veroniaina 1, Subhankar Mukhopadhyay , Ziheng Wu 2, Zhenghong Wu 1 ,* and Xiaole Qi 1 ,*

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Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University,

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Nanjing 210009, PR China 2

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Jiangning Campus, High School Affiliated to Nanjing Normal University, Nanjing

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211102, PR China.

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Correspondence: Zhenghong Wu; Xiaole Qi

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Email: [email protected] (Z. H. Wu); [email protected] (X. L. Qi) Corresponding author at: Key Laboratory of Modern Chinese Medicines, China

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Pharmaceutical University, Nanjing 210009, PR China Fax:+0086-025-83179703

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Tel: +0086-15062208341

Word count for Abstract: 148. Word count for Manuscript: 5534. Number of Figures: 8. Number of References: 57. Number of tables: 0. 1

ACCEPTED MANUSCRIPT Number of Supplementary online-only files: 1. Conflicts of interest: There are no conflicts to declare.

Acknowledgements: This work was financially supported by the Top-notch

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Academic Programs Project of Jiangsu Higher Education Institutions [grant numbers

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PPZY2015B164], National Natural Science Foundation of China [grant numbers

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51472115], Double first-class innovation team of China Pharmaceutical University [grant numbers CPU2018GY40], Administration of Traditional Chinese Medicine of

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Zhejiang Province [grant numbers 2017ZA075].

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ACCEPTED MANUSCRIPT ABSTRACT: Pea-like nanocabins (HA@APT§DOX) were designed for deeply tumor inhibition. The AS1411 aptamer (APT) constituted “core shelf” which guaranteed DOX “beans” could be embedded, while the outer HA acted as “pea shell” coating. During the

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circulation (primary orbit), HA@APT§DOX could autonomously cruise until leak

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through tumor vasculature. Upon tumor superficial site, the “pea shell” could be

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degraded by high expressed hyaluronic acid enzymes (HAase) and peel-off, resulting in orbit changing of released APT§DOX to reach the deep tumor tissue. Furthermore,

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APT§DOX could be specifically uptaken into A549 tumor cells (secondary orbit).

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Finally, DOX was released under the acidic environment of lysosome, and delivered into nuclear (targeting orbit) to achieve drug pushing for deep tumor inhibition. More

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importantly, the in vivo imaging and anti-tumor effects evaluations showed that these

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nanocabins could effectively enhance drugs accumulation in tumor sites and inhibit

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tumor growth, with reduced systemic toxicity in 4T1 tumor-bearing mice.

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Graphical Abstract: Figure 1.

Abbreviation

aptamers = APT doxorubicin = DOX hyaluronic acid = HA enhanced permeation and retention = EPR 3

ACCEPTED MANUSCRIPT hyaluronic acid enzymes = HAase aptamers§doxorubicin = APT§DOX hyaluronic acid@aptamers§doxorubicin = HA@APT§DOX

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KEYWORDS: nanocabins; aptamers; hyaluronic acid; tumor inhibition

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ACCEPTED MANUSCRIPT Introduction Currently, multiple stimuli-responsive nanoplatforms, such as nanoparticles, nanogels and nanocages, become more and more noticeable on the stage of drug delivery system, due to their indispensably targeting and intracellular uptake features. However,

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clarifying the in vivo cruise and disassemble behaviors of these complexly designed

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nanostructures was considered to be necessary for the investigation of novel

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nanocarriers. Briefly, the aims for diverse tumor related stimuli could be divided into two categories: (1) the specific enzyme responsive degradation and receptors

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mediated intracellular transport to enhance tumor penetration and tumor cell uptake;

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(2) the pulsed drug release in tumor cells responded to the lysosome acidic microenvironment, high glutathione concentration and reactive oxygen species (ROS)

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effect. Most effort was focused on rational utilization of those different stimuli to

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construct multifunctional nanoplatforms for tumor therapy. As well known, aptamers (APT), selected from systematic evolution of ligands by

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exponential enrichment, are a type of single-stranded DNA or RNA with high affinity

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for specific receptors1, 2; they have many advantages including low molecular weight, non-immunogenicity and easy to synthesize. Besides, APT have many types of specific receptors, such as small molecules, cells3, 4, tissues5 and organisms6, 7. Therefore, they have been widely used in the diagnosis8-10, imaging

11-13

of tumors,

and targeted drug delivery14-16. Although many studies had demonstrated the superiority of APT in the diagnosis and treatment of cancer, most of these studies were focused on in vitro experiments and lack of in vivo data17-19, this was mainly due 5

ACCEPTED MANUSCRIPT to their inherent disadvantages. For example, APT will degrade rapidly by serum nucleases in the blood20, and easy to be removed by the reticuloendothelial system (RES) and the kidney. Therefore, many researchers had been seeking some ways to overcome these problems.

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Among them, chemical modifications were utilized to protect APT, for example,

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adding special nucleotides at the 3' and 5' ends of APT21, which could successfully

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resist the degradation of serum nucleases, or substituting chemical bond H in the 2'-position of ribose or deoxyribose with -F22, 23, -NH224, -OCH325 to increase the

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stability. However, these chemical modifications had always been complicated and

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inefficient. Besides, APT are mainly combined with the receptors by forming a secondary or tertiary structure, but after chemical modification, the structure might

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change, which would affect the combination of APT with receptors, thereby restrained

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in vivo applications of APT26, 27. In addition, polymeric linkers such as polyethylene glycol (PEG)28-30, had also been used to combine APT with nanoparticles for

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increasing the molecular weight and extending their circulating time in vivo31-33.

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Although using linkers was relatively easier, it only addressed the issue of rapid clearance, instead of the problem of degradation by serum nucleases. And it was reported34 that PEG modification induced the production of anti-PEG antibodies, which would affect the stability of complex. Moreover, we noticed that some assembling three-dimensional DNA nanostructures, including DNA tetrahedron35 and three-way junction pocket DNA nanostructure36, were also utilized to construct nanocomplexes of APT and drug for cancer treatment. Although satisfied antitumor 6

ACCEPTED MANUSCRIPT efficiency were demonstrated for these APT integrated DNA nanostructures, the construction of thoroughly DNA composed nanostructure was too costly37-39. Usually, the multistage stimuli-responsive drug delivery system were composed of nanoparticles with different sizes, and each of them is designed to cross one or more

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biological barriers40. Combining these different sizes of nanocarriers can give full

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play to their advantages respectively 41-43. According to Ding44 and Mei45, hyaluronic

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acid (HA) nanoparticles had relatively larger size and internal space. Furthermore, HA nanoparticles did not affect the properties of encapsulated nanocarriers46, and even

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could improve their stability and prolong their circulating time in the body47.

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Typically, AS1411 is a single-stranded DNA aptamer containing 26 bases in the sequence of 5'-GGT GGT GGT GGT TGT GGT GGT GGT GG-3', which can bind

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with nucleolin specifically and effectively in the cell surface. Different from other

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tumor targeting aptamers, such as MUC1 aptamers35, there are many guanines in the sequences of AS1411, which could spontaneously form G-quadruplex structures

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through the annular hoogsteen hydrogen bond between every four guanines48.Then

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these APT molecules are prone to construct a stacked spiral structure through complex topologies between each other. Here, we designed pea-like nanocabins, hybridized from AS1411 aptamers and HA to realize autonomous cruise and step-by-step drug pushing for deep tumor inhibition. As shown in Figure 1, DOX was embedded in the GC rich spiral structure of AS1411 aptamers to form APT§DOX complex, then packaged into HA nanoparticles to form HA@APT§DOX, a pea-like multistage nanocabins. During the systemic circulation 7

ACCEPTED MANUSCRIPT (the primary orbit), these HA@APT§DOX nanocabins with a hydrophilic HA “shell” could achieve autonomous cruise until leak through tumor vasculature walls, via the enhanced permeation and retention (EPR) effect. Upon the tumor superficial site, these HA@APT§DOX nanocabins were intended to execute autonomous hyaluronic

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acid enzymes (HAase) responsive HA “pea shell” degradation to release the

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encapsulated APT§DOX “core shelf”, followed by the AS1411 receptor-mediated

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endocytosis of APT§DOX into tumor cells (the secondary orbit). Finally, DOX was released from APT§DOX under the acidic environment of lysosome, and delivered

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into nuclear (the targeting orbit) to achieve drug pushing for deep tumor inhibition.

Methods

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APT§DOX complex

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The APT§DOX complex was prepared by the method reported49 earlier. Briefly, APT were heated at 95 °C for 5 min, then cooled immediately at 4 °C for 10 min.

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Next, a fixed concentration of DOX (3 nM) was incubated for 1 h with APT, which

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had various concentrations, and the molar ratios of APT to DOX were 0, 0.01, 0.03, 0.1 0.3, 1, 3, 5, 7 and 9 respectively. The fluorescence spectrum of DOX was tested by fluorescence spectrophotometry (Shimadzu, Japan). The DOX excitation wavelength and emission wavelength are 485 and 556 nm, respectively. Synthesis and characterization of HA packaged APT§DOX (HA@APT§DOX) Briefly, HA@APT§DOX was prepared by the nanoprecipitation method reported50 with some modifications. HA (10 mg) was dissolved in 4 mL of deionized water, then, 8

ACCEPTED MANUSCRIPT 0.5 mL of APT§DOX (10 nmol/mL) prepared in advanced was added, and this solution was stirred for 15 min (800 rpm) to mix well. Next, 8.8 mL of acetone was added, and stirred the solution for 30 min. Afterwards, 4 mg of EDC·HCl and 2 mg of ADH dissolved in 0.1 mL of deionized water, and was added as a crosslinking agent.

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After stirring the solution for 30 min, another 8.48 mL of acetone was added and the

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mixture was stirred for 12 h. Finally, rotary evaporators (RE-85A Rotary Evaporator,

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Yuhua Instrument, China) were used to evaporate acetone, and sephadex gel chromatography was used to separate and remove the unwrapped DOX. The solution

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was freeze-dried for use later. The fluorescence spectrum of HA@APT§DOX and

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APT§DOX were tested by fluorescence spectrophotometry to ensure that HA had packaged APT§DOX.

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The particle size, zeta potential and distribution of these prepared HA@APT§DOX

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nanocabins was investigated by using a Zetasizer 3000HS (Marvin, Worcestershire, UK) in the medium of deionized water. Moreover, the morphology of

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HA@APT§DOX was characterized by transmission electron micrograph (TEM,

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alpha300R, WI Tec, Ulm, Germany), and the Image J (1.8.0-112) software was also utilized to analyze the TEM images for clarifying the possibly irregular shape of these nanostructures. For the in vitro stability evaluation, HA@APT§DOX nanocomplexes and bare APT§DOX (0.12 μM) solutions were added into 100% murine plasma at pre-determined time intervals (4-24 h). These mixtures were incubated at 37 °C and then analyzed with agarose gel (1%) electrophoresis (AGE). In order to verify the integrity of APT after modification, the HA@APT (2 μM) were incubated with HAase 9

ACCEPTED MANUSCRIPT (50 U/mL) at 37 °C for 24 h and then analyzed with AGE. The band was imaged by Chemiluminescence Imaging System (ChemiDoc XRS+, Bio-rad, USA). Afterwards, the indirect method was used to calculate encapsulation efficiency of HA@APT§DOX. Here, the unwrapped DOX was collected through Sephadex® G100

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column based on the indication of free DOX solution control group; then the

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encapsulation efficiency (EE) of HA@APT§DOX was calculated according to the

(total weight of drug − weight of unloaded drug) total weight of drug

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Release of HA@APT§DOX in vitro

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Encapsulation efficiency(%) =

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following formula:

Typically, after being endocytosed into the lysosome of tumor cells, the structure of

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APT§DOX was prone to change under the acidic environment of lysosome, resulted

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in the release of DOX loaded in APT51. Otherwise, the concentration of HAase in the tumor stroma was extremely higher than that in the systemic circulation52, 53. Thus,

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PBS solutions of different pH values (7.4, 6.5 or 5.5), added with different

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concentrations of HAase (0.01, 0.1, 0.2 and 0.4 μM), were chose as the medium for investigating the release behavior of HA@APT§DOX in vitro. In brief, APT§DOX and HA@APT§DOX with known concentrations were dispersed in 2 mL of 0.01 M PBS solutions, then HAase were added when needed. These solutions were sealed into the dialysis bags which were immersed in 50 mL corresponding dissolution medium. The release process lasted for 12 h in a constant temperature shaker (37 °C, 100 rpm). At following different time points (0.5, 1, 2, 3, 4, 6, 8, 10 and 12 h), 1 mL 10

ACCEPTED MANUSCRIPT of release medium was taken out and then 1 mL of fresh medium was added. Finally, fluorescence spectrophotometer was used to calculate the cumulative release of DOX in different conditions.

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Cell culture

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The human non-small lung cancer cell line A549 and the normal human liver cell

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line LO2 were cultured and maintained by RPMI 1640 medium, supplemented with

Tumor cellular uptake in vitro

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and 90% relative humidity at 37 °C.

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10% FBS and 100 U/mL of penicillin/streptomycin, in a 5% CO2-humidified chamber

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The A549 and LO2 cells were seeded in 24-well plates at a density of 5×104 cells

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per well and cultured for 24 h. Then, cells were respectively treated with fresh media containing the blank control (HA@APT, 10 μM), and a series of known concentration

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(20 μM) of free DOX, APT§DOX, HA@APT§DOX and HA@APT§DOX with

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HAase (50 U/mL). Moreover, the fresh media containing 3.68 μM of fluorescein-5-maleimide labeled AS1411 aptamer (FAM-APT) was also added to verify the specific uptake into A549 cells. Then cells were observed under a fluorescence microscope (Olympus IX 51, Osaka, Japan) at the time interval of 1, 2 and 4 h. After been incubated for 4 h, cells were gently washed two times with PBS and collected through a trypsin solution for digestion. Finally, cell suspensions were diluted with 1.5 mL of PBS and subsequently analyzed by a flow cytometer 11

ACCEPTED MANUSCRIPT (MACSQuant Analyzer 10, Miltenyi, Germany) to investigate the fluorescence intensity of DOX and APT-FAM, respectively.

Tumor cytotoxicity experiment in vitro

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The A549 and LO2 cells were seeded in 96-well plates at a density of 5 × 103 cells

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per well and incubated for 24 h. Then, fresh media containing APT, HA@APT, free

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DOX, APT§DOX, HA@APT§DOX and HA@APT§DOX with HAase (50 U/mL) were added into the cells and incubated for 48 h. Afterwards, 20 μL MTT (5 mg/mL)

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was added to each well for 4 h. Finally, the cells were dissolved in 150 μL DMSO.

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The absorbance was measured by microplate reader (EL×800, BioTek Instument, Winooski, VT) at 490 nm. The cell viability was calculated using the following

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formula:

absorbance of sample group × 100% absorbance of control group

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Cell viability(%) =

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Tumor spheroid penetration experiment

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We used the liquid overlay method to build a three-dimensional tumor spheroid model54. Briefly, cells were seeded into 2% low-melting-temperature, pre-coated, 96-well agarose plates at a density of 3000 cells/well and cultured for seven days to form the spheroids. Then, the uniform cell spheroids were selected and cultivated with 15 µM of DOX, APT§DOX, HA@APT§DOX and HA@APT§DOX with HAase (50 U/mL) respectively for 6 h. Finally, the fluorescence distribution of DOX in the cell spheroids was observed by using confocal microscopy (LSM700, Carl Zeiss). 12

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Distribution and retention of HA@APT§DOX in vivo All of the animal experiments were conducted in accordance with guidelines that were evaluated and approved by the Ethics Committee of China Pharmaceutical

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University and the humane care of the animals were carried out for these animal

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studies. Female Balb/c mice (20 ± 2 g) were supplied by the Qinglong Mountain

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Animal Center.

The tumor-bearing mice model was built by inoculating mice breast cancer cells

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into mice. Briefly, 0.1 mL of mice breast cancer 4T1 cells (at a density of 1×107/mL)

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was subcutaneously injected into the right flank breast of mice to establish the tumor model. After the tumor grew to 100 mm3, the tumor-bearing mice were divided into

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three groups randomly and injected intravenously with free DOX, APT§DOX and

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HA@APT§DOX solution (equal dose of DOX 5 mg/kg). Afterwards, imaging system (IVIS Spectrum, PerkinElmer, Massachusetts, USA) was used to observe the

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distribution of DOX in vivo at different time points (2, 4 and 8 h). After 8 h, the mice

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were sacrificed to observe the fluorescence intensity of DOX in the tumors and organs (including heart, liver, spleen, lung and kidney) to study biological distribution.

Evaluation of anti-tumor effects in vivo The mice tumor-bearing model was built following the method mentioned before. After the tumor grew up to 100 mm3, the tumor-bearing mice were divided into five groups randomly and injected intravenously with Saline (the control), HA@APT (the 13

ACCEPTED MANUSCRIPT blank carrier), free DOX, APT§DOX and HA@APT§DOX solution (equal dose of DOX 5 mg/kg) every three days. The tumor volume and body weights of mice were measured on a daily basis. The tumor volume was measured by caliper measurements

Tumor volume(𝑚𝑚2 ) =

L × W2 2

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and calculated by the following formula:

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(L and W respectively refer to the longest and shortest diameters of the tumors).

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After 11 days of treatment, all the mice were sacrificed to measure the weight of

calculated by the following formula:

tumor weight of therapy group × 100% tumor weight of control group

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T𝑢𝑚𝑜𝑟 𝑖nhibition rate(%) = 1 −

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the tumors and to observe their intuitive diagrams. The tumor inhibition rate was

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Finally, the tumors and organs were fixed with the formalin solution, and stained

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with hematoxylin and eosin (H&E) for routine histopathological evaluations.

Statistical analysis

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All data were showed as mean ± SD. Statistical difference was evaluated by T test and Analysis of variance (no repeated two-factor analysis), when p < 0.05 and p < 0.01 were thought to be statistical difference and obvious statistical difference respectively.

Results Formation of APT§DOX complex It was reported that fluorescence intensity of DOX would quench after intercalating 14

ACCEPTED MANUSCRIPT into DNA structure55. Here, this feature was utilized to evaluate the formation of APT§DOX complex. As shown in Figure S1, the fluorescence spectrum of DOX kept decreasing with the increasing of APT concentration, till the molar ratio of APT to DOX increased to 1. The results suggested that DOX was loaded in APT by

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intercalating into DNA structure, and the incubation of DOX with APT resulted in

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maximal quenching of the fluorescence of DOX at approximately 1:1 molar

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equivalence of DOX to APT. Therefore, the molar ratio of APT to DOX was fixed at 1:

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1 for subsequent experiments.

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Synthesis and characterization of HA@APT§DOX In the synthesis of HA@APT§DOX, EDC activated the carboxyl group on HA, and

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provided an O-acylisourea derivative which could react with two primary amines on

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ADH to form a peptide bond, resulting in the adjacent HA chains to be curved and cross-linked in the acetone-water system46. When the acetone was added for the first

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time, the HA polymer chains packaged APT§DOX by cross-linking, as neither HA nor

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APT§DOX was dissolved in the acetone. However, the particle size was large and the structure of nanoparticles was unstable. At the second time, the particle size of nanoparticles kept decreasing and the structure became stable under the effect of acetone, and eventually nanocabins HA@APT§DOX were formed. According to the supporting information of Tables S1-S4, the best synthesis condition was that the molecular weight of HA was 1400 kDa, the ratio of acetone to water in solvent was 300:80, the molar ratio of HA to APT was 1.5:1 and the reaction time was 12 h. As 15

ACCEPTED MANUSCRIPT shown in Figure 2A, the particle size of HA@APT§DOX nanocabins measured by DLS was 107.97 ± 21.55 nm, which hypothesized that they were spherical particles. However, the TEM images (Figure 2C) showed that the particles are not absolute spherical but oblong. In order to illustrate their exact shapes, we utilized the Image J

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(1.8.0-112) software to analyze the TEM images. The results indicated that the

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average length and width of HA@APT§DOX nanocabins were 111.54 ± 6.31 and

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16.26 ± 2.63 nm, respectively. Moreover, the length to width ratio was calculated to be 7:1 approximately.

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In addition, the results of AGE (Figure S2A and 2B) revealed that the APT§DOX

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alone was mostly degraded in plasma within 24 h incubation, while the HA@APT§DOX nanocabins showed good stability in plasma up to 24 h. Besides,

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Figure S2C presented that the band position of HA@APT was different from that of

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bare APT, revealing that the APT was successfully covered by the HA molecules. Meanwhile, the exposed APT (HA@APT with HAase) displayed the same band as

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bare APT. These results suggested that the APT could remain intact after been

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packaged by HA. After encapsulate APT§DOX complex into HA cover, the detected fluorescence intensity of DOX from the integrated HA@ APT§DOX nanocabins was prone to further decline (shown in Figure 2B). The EE of HA@APT§DOX was 61.02 ± 2.27 (%), and the zeta potential was -37.84 ± 2.54 (mV). Release of HA@APT§DOX in vitro The release behavior of APT§DOX was shown in Figure 3A. When the pH was 7.4 or 6.5, the release of APT§DOX was less than 30%, but when the pH was 5.5, the 16

ACCEPTED MANUSCRIPT release rate reached 93%, which indicated that DOX could release effectively and rapidly only when APT were at pH 5.5 (in the lysosome of tumor cells). The release behavior of HA@APT§DOX was shown in Figures 3B and 3C. When the pH was 5.5 and the concentration of HAase was extremely low, the release rate was only 5%.

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When the pH was 5.5 with higher concentration of HAase, the release rate reached

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82%, indicating that HA@APT§DOX could only begin to release in the tumor stroma

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where the concentration of HAase was high, and the release rate and degree of release depended on the concentration of HAase. However, when the concentration of HAase

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was high and the pH was 6.5 or 7.4, HA@APT§DOX released no more than 25%,

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indicating that the release of HA@APT§DOX not only depended on high concentration of HAase, but also on the pH condition. When the concentration of

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HAase was high in the tumor stroma, the outer coverage of HA nanoparticles could be

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degraded by HAase and exposed the APT§DOX, which could remain stable in the tumor stroma (pH 6.5). After APT§DOX entered into the cell through

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nucleolin-mediated active targeting effect, its structure would change in the lysosome

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(pH 5.5), and release DOX finally.

Tumor cellular uptake in vitro As shown in Figure S3, the intracellular uptake efficiency of fluorescently labeled FAM-APT into A549 cells was significantly higher than with LO2 cells (p < 0.01), which demonstrated the specific uptake into A549 cells. For A549 cells expressed much nucleolin56, the fluorescence microscopy results showed in Figure 4A , 17

ACCEPTED MANUSCRIPT different from the free DOX treatment group, the fluorescence intensity of HA@APT§DOX (with HAase) treated group displayed an obvious increasing trend after incubating from 1 to 4 h. The fluorescence intensity of HA@APT§DOX (with HAase) and APT§DOX treated groups were stronger than the free DOX group at 4 h,

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and these two groups showed similar fluorescence intensity. Meanwhile, we got

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corresponding results by flow cytometry (shown in Figure 4C). In addition, the mean

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fluorescence intensity (MFI) of HA@APT§DOX (with HAase) and APT§DOX treated groups shown in Figure 4E were significantly higher than the free DOX group

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(p < 0.01), after incubated 4 h with A549 cells. In contrast, as shown in Figure 4B,

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4D and 4E, with LO2 cells which expressed little nucleolin57, there was significant reduced MFI of APT§DOX, HA@APT§DOX, and HA@APT§DOX (with HAase)

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treated groups compared with free DOX group (p < 0.01).

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Moreover, for A549 cells, we noticed that the HA@APT§DOX group without HAase showed significant lower MFI (p < 0.01), compared to the HA@APT§DOX

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group with HAase. These results indicated that the HAase promoted degradation of

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the HA outer coverage was necessary for the exposure of shielded APT and further APT mediated endocytosis of DOX into tumor cells.

Tumor cytotoxicity experiment in vitro The MTT analysis was a classical method to evaluate the cytotoxicity of different nanocarriers. The inhibition efficiency of APT and unloaded HA@APT nanocabins on A549 and LO2 cells was shown in Figure S4. Under all tested concentrations of APT 18

ACCEPTED MANUSCRIPT and HA@APT ranged from 0.08 to 20 μM, the A549 and LO2 cell viability were higher than 80%, which indicated that the constructed nanocabins themselves had no obvious toxicity or side effects on both tumor and normal cells. As shown in Figure 5A, the A549 cell viability of the APT§DOX and

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HA@APT§DOX (HAase) treated groups were significantly lower than the free DOX

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group (p < 0.05), when the concentration of DOX was higher than 2.5 μM. These

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results were consistent with the tumor cellular uptake experiments in Figure 4A, which displayed stronger fluorescence intensity of APT§DOX and HA@APT§DOX

Notably,

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different

A549

inhibitory

efficiency

between

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incubation.

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(HAase) groups (CDox = 20 μM) versus free DOX group in A549 cells after 4 h

HA@APT§DOX (HAase) and free DOX groups was prone to be more and more

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obvious, as the concentration of DOX increased. Furthermore, the IC50 of DOX in

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A549 cell lines was 17.38 μM, which was reduced to 5.32 μM with the formulation of HA@APT§DOX (HAase), which had better inhibitory effect directly.

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In contrast, the LO2 cell viability of the HA@APT§DOX (HAase) treated group

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was significantly higher than the free DOX group (p < 0.05), when the DOX concentrations were ranged from 5 to 80 μM (Figure 5B). Moreover, the IC50 of DOX in LO2 cell lines was 10.82 μM, which was enhanced to 59.76 μM with the formulations of HA@APT§DOX (HAase). This result suggested that encapsulating DOX into HA@APT§DOX nanocabins would reduce their cell toxicity to normal cells, which could be explained by their corresponding reduced uptake ability shown in Figure 4B. 19

ACCEPTED MANUSCRIPT Obviously, the A549 cell viability of HA@APT§DOX treated group was significantly higher than the HA@APT§DOX (with HAase) treated group (p < 0.01). And the significant different LO2 cell viability between these two treated groups was also verified when the DOX concentrations were higher than 5 μM. These results

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promoted that the intrinsic HAase enriched in tumor superficial site (the first orbit)

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was necessary and charged of the degradation of the HA coverage, resulted in the

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successful autonomous peeling-off and orbit changing of HA@APT§DOX nanocabins. In other words, these results verified the hypothesis that the first-step

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peeling-off of HA@APT§DOX nanocabins on the first obit (tumor superficial site)

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guaranteed their successful access to the secondary obit (tumor cell).

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Tumor spheroid penetration experiment

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Tumor spheroid penetration experiments were conducted to investigate whether the released DOX could deeply penetrate into tumor tissues. The confocal images taken at

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different depth of each spheroid were shown at Figure 6. When the depth was 40 μm

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and 60 μm (spheroid core), the fluorescence intensity of DOX was low, which indicated that DOX was unable to penetrate the spheroids in short time, but fluorescence intensity of APT§DOX and HA@APT§DOX with HAase was very high, indicating APT§DOX and HA@APT§DOX with HAase were able to penetrate into the spheroid, showing bright fluorescence within the spheroid core. Besides, there was almost no fluorescence in HA@APT§DOX group, these results suggested the ability of APT to bring DOX to penetrate deeply into tumor tissues through active targeted 20

ACCEPTED MANUSCRIPT effect. Also, nanocabins HA@APT§DOX could benefit from HAase triggered degradation.

In Vivo Distribution of HA@APT§DOX

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Typically, the in vivo images experiment was conducted to illustrate and compare

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the distribution characteristic of DOX, APT-guided APT§DOX and designed

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HA@APT§DOX nanocabins. As shown in Figure 7A, the tumor-bearing mice model was successfully built by inoculating mice breast cancer 4T1 cells into the right flank

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breast of mice, and the red circle indicated the site of the established subcutaneous

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tumor. After intravenous injection of free DOX, Figure 7A displayed the reduced trend of fluorescence intensity at the tumor site over time. Meanwhile, the contrast

D

which increased the trend of fluorescence intensity at the tumor site could be observed

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in the HA@APT§DOX and APT§DOX treated groups. These results demonstrated that the introduction of APT would endow HA@APT§DOX nanocabins a satisfied

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tumor active targeting properties, as the APT loaded DOX (APT§DOX).

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In addition, Figure 7B displayed the fluorescence images of the major organs (heart, liver, spleen, lung and kidney) and tumors segregated from the tumor-bearing mice, after administrated of DOX, HA@APT§DOX and APT§DOX for 8 h. It could be observed that the HA@APT§DOX treated group showed higher intensity of fluorescence retention in tumor compared to other organs, which verified the superior in vivo targeting ability of HA@APT/DOX nanocabins. Furthermore, as shown in Figure S5, the quantification of DOX signal accumulated in the organs and tumors 21

ACCEPTED MANUSCRIPT also illustrated the different tissue distributions between DOX (control), APT§DOX and HA@APT§DOX.

Evaluation of Anti-tumor effects in vivo

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The mice 4T1 tumor-bearing model was used to investigate the antitumor activity

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of saline, HA@APT, APT§DOX, DOX, and HA@APT§DOX in vivo. As shown in

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Figure 8A, compared with saline treated group, the free DOX, HA@APT and APT§DOX treated groups displayed a significant reduced trend of relative body

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weights (p < 0.01). However, no significant different relative body weights (%) could

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be observed in HA@APT§DOX treated group versus the saline treated group, which indicated the low systemic toxicity and good biocompatibility of DOX loaded

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nanocabins. Besides, the relative tumor volume (%) curve shown in Figure 8B gave

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that saline and HA@APT treated groups exhibited similar tumor growth trend, indicating that the unloaded nanocomplexes have no therapeutic effect on the

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tumor-bearing mice. In contrast, for those treated with free DOX, APT§DOX and

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HA@APT§DOX groups all exhibited obvious tumor inhibition efficiency compared to saline group. Surprisingly, the HA@APT§DOX group showed significant reduced relative tumor volume and anti-tumor therapeutic efficiency (p < 0.01), compared to the free DOX group. Furthermore, the intuitive diagram of isolated tumors from the tumor-bearing mice was shown in Figure 8C at the end of anti-tumor treatment, which was in accordance with the results of Figure 8B. As shown in Figure 8D, the HA@APT§DOX treated group gave significantly 22

ACCEPTED MANUSCRIPT reduced tumor weight (g), compared to the DOX and APT§DOX treated groups. The calculated tumor inhibition rate of tumor treated with HA@APT§DOX was 41.5%, but for DOX and APT§DOX groups were 29.9% and 17.1%, respectively. Furthermore, we utilized H&E staining images to illustrate the potential pathological

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changes in the subcutaneous tumor and other organs isolated from the tumor-bearing

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mice after anti-tumor therapy. As shown in Figure S6, obvious morphological

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changes could be observed in the hearts of free DOX treated group. However, the HA@APT§DOX and APT§DOX treated groups showed minor changes in the heart

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slice compared to the free DOX, which indicating that encapsulating DOX into these

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nanocomplexes could efficiently alleviate the inherent cardiotoxicity brought by systemic administration of free DOX.

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In addition, these H&E staining images of isolated tumors from tumor-bearing mice

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(Figure 8E) showed that tumor cells were sparsely arranged with large cell gaps, in APT§DOX, DOX, and HA@APT§DOX treated groups. According to all these above

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results, the in vivo anti-tumor effects evaluations had shown that these

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HA@APT§DOX nanocabins could effectively enhance the accumulation of drugs in tumor sites and inhibit tumor growth, with reducing systemic toxicity in 4T1 tumor-bearing mice.

Discussion In this work, we created a pea-like multistage nanocabins (HA@APT§DOX) for deeply tumor therapy. Here, we demonstrated that the DOX “beans” cargos could be 23

ACCEPTED MANUSCRIPT successfully embedded in the APT constituted “core shelf”, and then encapsulated into the outer layer “pea shell” composed of HA. The TEM images illustrated that these HA@APT§DOX nanocabins were not absolute spherical but oblong. The in vitro stability results of HA@APT§DOX demonstrated that the outer layer of HA, as

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the shell of the nanocabins, provided satisfied protection to the inner APT§DOX core,

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which was beneficial for the further in vivo stability during their cruise in the systemic

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circulation. Moreover, the in vitro drug release studies verified that HA@APT§DOX nanocabins could achieve the HAase-responsive release of the cargoes, under the

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tumor microenvironment (pH 5.5).

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The in vitro cell uptake studies demonstrated the specific uptake of APT§DOX released from the integrated HA@APT§DOX into A549 cells. Then, we verified the

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embedding of DOX into APT, which was beneficial for the intracellular uptake of

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DOX, via the specific nucleolin-mediated endocytosis of A549 cells. In contrast, the reduced uptake ability into LO2 cells could be explained by the different

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internalization pathway of DOX, which meant that the possibly increased hydrated

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membranes and particle size generated from HA coverage would be not conducive to the passive diffusion of DOX. Moreover, the in vitro cytotoxicity assays demonstrated that HA@APT§DOX nanocabins would be beneficial for the uptake of the released APT§DOX from the integrated HA@APT§DOX, resulting in satisfied in vitro anti-tumor efficiency. Meanwhile, encapsulating DOX into HA@APT§DOX nanocabins would reduce their cell toxicity to normal cells, which could be explained by their corresponding reduced 24

ACCEPTED MANUSCRIPT uptake ability. All these results promoted that the intrinsic HAase enriched in tumor superficial site (the first orbit) was necessary and charged of the degradation of the HA coverage, to gain the successful autonomous peeling-off and orbit changing of HA@APT§DOX nanocabins.

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In addition, the results of in vivo images successfully verified the targeting property

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of the designed HA@APT§DOX nanocabins, which would be contributed to expected

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in vivo anti-tumor efficiency. Surprisingly, the fluorescence intensity of tumor seemed to be strengthened after being further loaded into the HA@APT§DOX, while

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compared to the APT§DOX. It could be explained by the HA constructed outer shell

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of the nanocomplexes, which was prone to improve their stability and prolong their circulating time in the body47. Moreover, the proved in vivo targeting feature of

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HA@APT§DOX in the tumor-bearing mice also verified that, they could achieve the

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expected orbit changing in vivo from the tumor superficial site (the first orbit) to tumor cells (the second orbit), response to the rich HAase exiting in the tumor-bearing

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mice instead of the additional HAase for other in vitro evaluations.

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More importantly, the in vivo anti-tumor therapeutic results demonstrated that the developed HA@APT§DOX nanocabins were endowed an outstanding anti-tumor therapeutically efficiency, which could be considered as a potential nanoplatform for the delivery of chemotherapy drugs. Instead of successful delivery drug loaded nanocomplexes followed with cargoes releasing, our work was directly focused on delivering the cargoes accurately by themselves.

25

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ACCEPTED MANUSCRIPT Figure Legends

Figure 1. (A) Design of multistage nanocabins HA@APT§DOX (B) Schematic illustration of nanocabins HA@APT§DOX delivered to the target orbit accurately. At

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first, HA@APT§DOX gathered at the tumor stroma (primary orbit) via EPR effect.

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Next, nanocabins were degraded by HAase and exposed APT§DOX packaged inside.

SC

Then, ATP§DOX were positioned accurately in the lysosome of cells (secondary orbit) by nucleolin-mediated endocytosis. Afterwards, the acidic environment of lysosome

NU

destroyed APT structure, resulting in the rapid release of DOX. Finally, DOX

MA

spontaneously localized in the nucleus (targeted orbital).

D

Figure 2. (A) The Fluorescence spectrums of DOX, APT§DOX and HA@APT§DOX.

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(B) The appearance and particle size histogram of nanocabins HA@APT§DOX. (C)

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TEM imagines showed the unique stick-like shape of HA@APT§DOX.

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Figure 3. (A) The release behavior of APT§DOX at different pH values. (B) The release behavior of HA@APT§DOX at pH 5.5 and different concentrations of HAase. (C) The release behavior of HA@APT§DOX at 0.2 μM HAase and different pH values. Data are represented as the mean ± SD (n = 3).

Figure 4. Results of tumor cellular uptake in vitro. The fluorescent microscopy images of A549 cells (A) and LO2 cells (B) after incubating with HA@APT (the 35

ACCEPTED MANUSCRIPT control), DOX, APT§DOX, HA@APT§DOX, and HA@APT§DOX (with HAase) for different hours. The analysis of flow cytometry in A549 cells (C) and LO2 cells (D) after incubating with the preparations for 4 h.

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Figure 5. Results of MTT in A549 cells (A) and LO2 cells (B) after being treated with

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DOX, APT§DOX, HA@APT§DOX, and HA@APT§DOX with HAase. Data were

DOX respectively, # and

##

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represented as the mean ± SD (n = 3,* and ** indicate p < 0.05 and p < 0.01 versus indicate p < 0.05 and p < 0.01 versus HA@APT§DOX

MA

NU

with HAase respectively.)

Figure 6. Fluorescent images of DOX in A549 tumor spheroids upon 6 h incubated

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with DOX, APT§DOX, HA@APT§DOX and HA@APT§DOX (HAase).

Figure 7. In vivo imaging results of 4T1 tumor-bearing female mice. (A) In vivo the

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distribution of DOX for different preparations. From left to right are DOX group,

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HA@APT§DOX group and APT§DOX group. The images were taken at 2, 4 and 8 h after intravenous administration. (B) The tumor-bearing mice were sacrificed after 8 h, and fluorescence images of the major organs (heart, liver, spleen, lung and kidney) and tumors were shown.

Figure 8. In vivo anti-tumor efficiency results of 4T1 tumor-bearing female mice after intravenous injection of Saline, HA@APT, DOX, APT§DOX, and HA@APT§DOX. 36

ACCEPTED MANUSCRIPT (A) Relative body weight of mice during 11 days. (B) Relative tumor volume of mice during 11 days. (C) After mice were sacrificed, representative images of excised tumors. (D) Weights of excised tumors from mice. (F) HE staining of the excised tumors. Data are shown as the means ± SD (n = 5). ** Represent p < 0.01 versus ##

represent p < 0.05 and p < 0.01 versus HA@APT§DOX group,

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saline group, # and

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respectively.

37

ACCEPTED MANUSCRIPT Text for the graphical abstract: Pea-like nanocabins (HA@APT§DOX) were designed for deeply tumor inhibition. The APT constituted “core shelf” which guaranteed DOX “beans” could be embedded, while the outer HA acted as “pea shell” coating. During the circulation, HA@APT§DOX could autonomously cruise until leak through tumor vasculature.

PT

Upon tumor superficial site, the “pea shell” could be degraded by high expressed HAase and peel-off, resulting in orbit changing of APT§DOX to reach the deep tumor

RI

tissue. Furthermore, APT§DOX could be specifically indentified and uptaken into

SC

A549 tumor cells, followed by the drug released under the acidic environment of

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CE

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D

MA

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lysosome, and delivered into nuclear for tumor therapy.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8