Fabrication of Cellulose-Nanocrystal-Based Folate Targeted


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Fabrication of Cellulose Nanocrystals-Based Folate Targeted Nanomedicine via Layer-by-Layer Assembly with Lysosomal pH-controlled Drug Release into Nucleus Na Li, Han Zhang, Yi Xiao, Yushu Huang, Mengda Xu, Donglei You, Wei Lu, and Jiahui Yu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01556 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Biomacromolecules

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Fabrication of Cellulose Nanocrystal-Based Folate Targeted Nanomedicine via Layer-by-Layer

2

Assembly with Lysosomal pH-controlled Drug Release into Nucleus

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Na Li a, Han Zhang a, Yi Xiao b, Yushu Huang a, Mengda Xu a, Donglei You a, Wei Lu a, Jiahui Yu a *

4

5

a

6

School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062,

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PR China

8

b

9

Shanghai 200003, PR China

Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,

Department of Radiology and Nuclear Medicine, Changzheng Hospital, Naval Medical University,

10

*

Corresponding authors. Tel.: +86 21 6223 8345; fax: +86 21 6223 8345. E-mail address:

[email protected] 1

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Abstract: To increase the cellular uptake and drug loading of cellulose nanocrystals (CNCs)-based

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nanomedicines, folate/[email protected]@CNCs (FA/[email protected]@CNCs)

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nanomedicines were built up by the building blocks of folate (FA), cis-aconityl-doxorubicin (CAD),

14

polyethylenimine (PEI) and CNCs via the robust layer-by-layer (LbL) assembly technique. The drug

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loading content (DLC) of FA/[email protected]@CNCs hybrids was 11.3 wt %, which was almost 20 folds

16

higher than that of CNC-based nano-prodrug we reported previously. FA/[email protected]@CNCs

17

nanomedicines showed lysosomal pH-controlled drug release profiles over 24 h. In detail, the

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cumulative drug release was over 95 % at pH 5.5, while the cumulative drug release was only 17 % at

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pH 7.4. In vitro, FA/[email protected]@CNCs hybrids nanomedicines had a higher (9.7 folds) mean

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fluorescent intensity (MFI) than that of DOX·HCl, with enhanced cytotoxicity and decreased IC50

21

against MCF-7. Thus FA/[email protected]@CNCs hybrids nanomedicines displayed efficient targetability

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and enhanced cellular uptake. In addition, FA/[email protected]@CNCs nanomedicine could deliver more

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DOX to the nucleus than the control group, due to the β-carboxylic acid catalyzed breakage of the pH-

24

labile cis-aconityl amide linkages in CAD. These results indicated that FA/[email protected]@CNCs

25

nanomedicines were the lysosomal pH-controlled drug release into nucleus, and showed great potential

26

to be high-performance nanomedicines to improve the delivery efficiency and therapy efficacy. This

27

study for CNC-based nanomedicines provided important insights into the bio-application of CNCs

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modified by LbL assembly.

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Keywords: CNCs, LbL assembly, FA, Lysosomal pH-controlled drug release, Enhanced cellular

30

uptake, Nanomedicine.

31 2

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1.

Biomacromolecules

Introduction

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Cellulose nanocrystals (CNCs) are natural and sustainable nanostructured biomaterials 1,2, which

34

are excellent nanomaterials to produce new functional nanovehicles in many scientific fields. CNCs

35

have versatile applications in biomedical fields, such as, wound dressings/healing, tissue engineering,

36

biomedical implants and nanomedicines

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potential to address the challenges of tumor therapies, due to their superior physicochemical properties

38

arising from their shape, nanoscale size and surface functional groups

39

physicochemical properties of CNCs, such as large surface areas, numerous surface hydroxyls,

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renewability, biodegradability, and no cytotoxicity, make CNCs ideal candidate carriers for

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nanomedicines. Simultaneously, the CNCs are origin rod-like shape nanoparticles, and it has been

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proved that the rod-like shape nanomedicines can be easily accumulated in the tumor tissues

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penetrate the tumor microenvironments and enhance the cellular uptake 18–20. It has been proved that

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rod-like shape nanoparticles are internalized faster, compared to spherical nanoparticles 21. CNC-based

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rod-like shape nanomedicines have been studied in many groups in the form of nanovectors 22–24. By

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modifying the surface of oxidized CNCs with chitosan oligosaccharide (CSOS), CNC-based drug

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delivery system had been developed in Kam C. Tam’s group. In detail, CSOS were grafted onto the

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oxidized CNCs via the carbodiimide reaction, and the resultant CNC-based drug delivery systems

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improved the solubility of chitosan at physiological pH with the additional advantages of increased

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antibacterial properties 25. In group of Jie Ma etc 14, redox-responsive gene delivery systems had been

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developed based on the polycation-functionalized cotton CNCs. With disulfide bond-linked poly(2-

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(dimethylamino)ethyl methacrylate) (PDMAEMA), in the CNC-based complexes nanomedicines 3

3–11.

Particularly, CNC-based nanomedicines indicate great

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12–16.

The outstanding

17,

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realized effective cancer treatments. Meanwhile, CNC-based nano-prodrugs were prepared by the

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chemical conjugation between CAD and amidated CNCs in our recent report 26. The nano-prodrugs

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showed enhanced cellular uptake and lysosomal pH-triggered drug release, along with fluorescence-

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visible nature. However, the heterogeneous chemical modifications of CNCs involve complex

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procedures and poor reactive efficiency, which resulted in low drug loading, just as the other

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researchers did 27–30. The low drug loading of CNC-based nano-prodrugs limited their further research.

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It is essential to optimize the CNC-based nanoemdicines to get high-performance nanomedicines with

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simplified preparation method, high DLC and enhanced cellular uptake.

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The LbL assembly technologies, first introduced by Decher 31, are easy, efficient, reproducible,

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robust, flexible, and extremely versatile ways to fabricate hybrids. The LbL assembly can easily build

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up hybrids nanomedicines just through mixing together diverse building blocks

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hybrids nanomedicines can be built up by the electrostatic interactions based LbL assembly, due to

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that CNCs prepared from the sulfuric acid hydrolysis method possess anionic sulfate half-ester on their

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surface. CNC-based nanomedicines with the ability of pH-controlled intracellular drug release can be

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prepared by imputing pH-responsive linkers to nanomedicines. Because the pH values in diverse

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tissues and cell organelles vary tremendously. For example, the tumor extracellular microenvironment

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is more acidic (pH 6.5) than that of blood and normal tissues (pH 7.4), and the pH values of the

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intracellular organelles (e.g. lysosome) are even lower (4.5-5.5). Based on the original pH gradients,

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pH-responsive nanomedicines can change their conformation or cleaving pH-labile bonds at a specific

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pH value, resulting in payload release at the desired sites. Cis-aconityl amide linkage is an ideal pH-

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responsive linker for CNCs based hybrid nanomedicine, because of its sensibility to a wee difference 4

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32–36.

CNC-based

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of pH values in diverse cell organelles, and the ability to realize subcellular organelles controlled drug

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release 37,38.

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CNC-based nanomedicines can be optimized through gathering folic acid (FA) on its surface to

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gain the active targeting ability to improve drug delivery efficiency and therapy efficacy. It has been

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confirmed that folate receptors (FR) are overexpressed on many tumor cells, some of which are 100

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folders higher than that in normal tissues. Folate displays extremely high affinity (KD = 10-10 M) for

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FR. Simultaneously, FA has numerous advantages to be a target molecule, such as, low molecular

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weight, easy modification, strong penetration and low immune response. Moreover, FA can be loaded

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onto the surface of CNC-based nanomedicines via electrostatic attraction based LbL assembly.

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The purpose of this article is to provide an insight into the strategies to build CNC-based high-

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performance nanomedicines. In fact, CNCs based nanomedicines, namely FA/[email protected]@CNCs

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hybrids were developed via LbL assembly technique. As expected, the robust FA/[email protected]@CNCs

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nanomedicines had comparatively high DLC, which was a prerequisite feature to ensure a pronounced

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cytotoxic and delivery effects. Meanwhile, FA/[email protected]@CNCs nanomedicines had much higher

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cellular uptake than that of DOX·HCl against MCF-7 cells in 40 min, due to the benign

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physicochemical properties of the nanomedicines (i.e. active targeting ability, rod-like shape and

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positive charge). In addition, with pH-labile bonds in CAD compounds, FA/[email protected]@CNCs

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nanomedicines realized lysosomal pH controlled drug release into nucleus. Therefore,

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FA/[email protected]@CNCs hybrids had great potential to be high-performance nanomedicines with

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improved

delivery

efficiency

and

therapy

efficacy.

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The

structure

of

pH-responsive

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FA/[email protected]@CNCs nanomedicines and illustration of FA mediated cellular uptake and efficient intracellular lysosomal pH controlled DOX release into nucleus was shown in Scheme 1.

Scheme 1. Schematic structure of pH-responsive FA/[email protected]@CNCs hybrids nanomedicnines and illustration of FA mediated endocytosis and efficient lysosomal pH controlled DOX release into nucleus.

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2.

Materials and methods

2.1 Materials

CNCs was from Cellulose Lab (Canadian). Doxorubicin hydrochloride (DOX·HCl 98 %) was bought

from

Dalian

Meilun

Biology

Technology

Co.,

Ltd.

(Dalian,

China).

1-(3-

Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl 99 %), N-hydroxysuccinimide (NHS), FA and PEI were bought from Sigma-Aldrich. Chemical agents (e.g. Cis-aconitic anhydride (CAA), dialysis bag and MTT (98 %), Hochest 33342) were got as we previously reported 26. Solvents (e.g. Ultrapure water, triethylamine (TEA) and so on) were prepared as we previously reported

26.

Reagents not mentioned were used directly as received.

2.2 Preparation of FA/[email protected]@CNCs hybrids via LbL assembly

CAD was prepared as we previous report 26. FA/[email protected]@CNCs hybrids were prepared by the in-situ precipitate method via the LbL assembly. In detail, CNCs (10 mg) were dispersed into the PEI deionized water solution (25 mg/mL, 1mL), ultrasonication 15 min, 30 min later, the mix solution was added into the CAD solution (30 mg/mL, DMSO and water, 1mL) drop by drop, then, treated with centrifugation (14000 rpm,10 min) and washing (three times, deionized water). Finally, the deposition was dispersed into the FA solution (10 mg/mL, ethanol). After the treatments of centrifugation and washing, FA/[email protected]@CNCs nanomedicine was collected by lyophilization. 2.3 Characterization of physicochemical properties

2.3.1

-potential measurements

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-potentials of CNCs and FA/[email protected]@CNCs nanomedicine in aqueous phase were measured at 25 oC by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, UK). The concentration was maintained at 0.1 wt%. -potentials measurements were measured in automatic mode for an average of thirteen runs by Smoluchowski approximation in triplicates.

2.3.2

Thermogravimetric analyses (TGA)

TGA was performed using a Mettler Toledo Thermal Analysis Instruments (TGA/DSC 3+ United Kingdom). TGA balance was calibrated and the freeze-dried samples were analyzed in platinum pans under dry nitrogen purge at a flow rate of 50 ml/min from ambient temperature to 800 oC with a heating rate of 10 oC/min. The experimental conditions for data acquisition and analysis were performed on the STAR System.

2.3.3

Differential scanning calorimetry (DSC)

The thermal properties of CNCs and FA/[email protected]@CNCs nanomedicine were characterized by DSC using a differential scanning calorimeter (PerkinElmer, DSC 800, USA). CNCs and FA/[email protected]@CNCs were heated from − 40 to 200 oC under nitrogen atmosphere at a heating rate of 10 oC / min.

2.3.4

Other measurements

Fourier

transform

infrared

spectroscopy

(FT-IR)

absorption

spectra

and

UV-vis

spectrophotometer (UV-vis) absorption spectra and a UV-1800 spectrometer were the same as reported 26.

The fluorescence intensity of DOX·HCl was measured by Fluorescence spectrum (FS). The

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excitation and emission wavelength was 480 nm and 570 nm, respectively. The analysis operation were the same as reported 26 .

Atomic Force Microscopy (AFM) was used to study the morphology of CNCs and FA/[email protected]@CNCs on a Bruker Dimension Icon equipment with an integrated force generated by cantilever/silicon probes. One drop of a 0.01 wt% suspension of CNCs or CNCs based hybrids in water was deposited onto a freshly cleaved mica surface and allowed to dry in air under ambient conditions. Samples were scanned in contact mode in air.

X-ray powder diffractometer (XRD) with Cu Kα (1.5418 Å) radiation was measured on the FACS Calibur Ultima IV (Japan Science Co., Ltd. Japan). The angular of the diffractograms was set from 10º to 80º, and the scan speed was 0.1 s/step with a step size of 0.01º. The anode voltage and current was 50 kV and 40 mA, respectively. The Voigt profile shaped peaks was studied to show the crystal parameters of the CNCs and CNCs based nanomedicines (e.g. crystallinity index, crystallite dimensions and so on). Four of the peaks of (110), (110), (200), and (004) were dealt with Origin Graph software to show the related messages. The diffraction patterns had been adjusted before used. Segal et al had pinpointed the method to calculate the comparative crystallinity of CNCs39. And the equation was as follows:

Where Cr.I. represented the crystallinity index, I200 represented the maximum value of (200) cellulose I reflection, and Iam mean the intensity value at 2θ ≈ 18º. 9

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2.4

DLC and Drug loading efficiency

( DLE

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) of the FA/[email protected]@CNCs hybrids

nanomedicines

The DLC and DLE of FA/[email protected]@CNCs hybrids nanomedicines were confirmed through the uv-vis absorption spectra with a UV-1800 spectrometer. UV-vis spectrometer was tested using dilute suspensions of CNCs and FA/[email protected]@CNCs hybrids, and the content of DOX was calculated according to standard curves as we did in our previous work 26. Dilute solution was scanned at 480 nm (wavelength). Notably, the interference absorption of CNCs at wavelength 480 nm were deducted before the finally calculation of DOX content of each sample.

The DLC and DLE were calculated according to the following formulas:

DLC (wt%) =

DLE (wt%) =

𝑤𝑒𝑖𝑔 ℎ𝑡 𝑜𝑓 𝑙𝑜𝑎𝑑𝑒𝑑 𝑑𝑟𝑢𝑔 𝑤𝑒𝑖𝑔 ℎ𝑡 𝑜𝑓 𝑙𝑜𝑎𝑑𝑒𝑑 𝑑𝑟𝑢𝑔 +𝑤𝑒𝑖𝑔 ℎ𝑡 𝑜𝑓 𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑛𝑎𝑛𝑜𝑐𝑟𝑦𝑠𝑡𝑎𝑙𝑠

𝑤𝑒𝑖𝑔 ℎ𝑡 𝑜𝑓 𝑙𝑜𝑎𝑑𝑒𝑑 𝑑𝑟𝑢𝑔 weight of drug in feed

× 100%

× 100%

(2)

(3)

2.5 Drug release of FA/[email protected]@CNCs hybrids nanomedicines

The DOX release profiles from FA/[email protected]@CNCs hybrids was studied at 37 ºC in three different media, i.e. (a) acetate buffer, pH 5.5; (b) acetate buffer, pH 6.5; and (c) phosphate buffer, pH 7.4. The concentration of the release medium was 10 mM. FA/[email protected]@CNCs hybrids were divided into three groups (each 1 mL), and speedily devolved to dialysis tubes (molecular-weight cutoff = 1000). The dialysis tubes were dipped into 50 mL homologous buffer solution, which were stirred at 37 ºC. At pre-set time point, 200 μL of release media were taken out for FS, then an equal volume of fresh media was added. The fluorescence intensity was measured. 10

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2.6 Cell culture

MCF-7 cells cells were purchased from Chinese Academy of Sciences, which were cultured in minimum essential medium (MEM). All of the media were supplemented with 10 % fetal bovine serum (FBS, HyClone, Logan, UT), streptomycin (100 µg/mL) and penicillin (100 g/mL). All cells were incubated at 37 ºC in a humidified 5 % CO2 atmosphere. The confluent cells were dissociated using a pre-warmed trypsin solution at 37 ºC.

2.7 Cell proliferation inhibition

The cell proliferation inhibition of DOX·HCl and FA/[email protected]@CNCs was estimated by MTT assay against MCF-7 cells that were pre-incubated (8 × 103 cells / well) in 96-well plates. The cells with 180 μL of culture medium per well were laid in constant temperature incubator (Thermo, USA). Overnight, the same amount of fresh medium substituted the old one. Subsequently, 20 μL culture media containing of DOXHCl and FA/[email protected]@CNCs hybrids with different concentrations (final equivalent DOXHCl concentration 0.034, 0.067, 0.135, 0.270, 0.539, 1.079, 2.157, 4.314, 8.628 and 17.256 mg·L-1) were added. Thereafter, cells were incubated with DOX·HCl and FA/[email protected]@CNCs nanomedicines for another 48 h. New culture media containing MTT solution (10 μL/well 5 mg·mL-1) was added. 4 h later, 50 μL/well of pyrolysis solution was added to the 96well plates. An automated BIO–TEK microplate reader (Powerwave XS, USA) was used to test the absorbance (wavelength = 570 nm). Wells treated with 200 μL of PBS were used as a blank (ODblank), and cells only treated with 200 μL of culture medium were used as a control (ODcontrol). The cell viability was calculated as follows: 11

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Cell viability (%) =

ODsample - ODblank ODcontrol - ODblank

Page 12 of 47

×100%

(4)

2.8 Fluorescence Microscope Images (FMI) and Flow cytometry (FCM)

MCF-7 cells were pre-incubated in a 6-well plate (2×105 cells/well) with 2 mL of complete culture medium per well in constant temperature incubator (Thermo, USA), separately. After incubation for 24 h, the culture media were replaced with new culture media containing DOXHCl or FA/[email protected]@CNCs hybrids of the same DOXHCl concentration (5 mg·L-1). Forty minutes later, the culture media were withdrawing, which were used for the FMI. Cells were washed with pH 7.4 PBS and stained with Hoechst 33342 (10 mg L-1). The FMI were captured on inverted fluorescence microscope (Olympus, TH4-200 with Olympus U-HGLGPS). The other half of the 6-well plates were dealt for the FCM, which were dealt with digestion, centrifugation and washing to dump the nanomedicines or DOX. The cells were collected and analyzed by FCM (Guava easy Cyte 6HT2L, USA). During the experiments, baseline was obtained from the blank control group (cells cultured with normal medium). Experiments mentioned above were performed for three times to reduce errors.

3.

Results and discussion

Nanomedicines present the state-of-the-art laboratory, scientific and clinic aspects of nanotechnologies, nanomaterials, and tools for medical applications. Developing high-performance nanomedicines with optimized physicochemical properties (i.e. active targeting ability and robust preparation method) are critical to find new treatments with various vicious diseases, especially, the

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Biomacromolecules

cancer. A new high-performance hybrids nanomedicines named FA/[email protected]@CNCs had been developed in this article, whose molecular-scale stratified structure was displayed in scheme 2.

Scheme 2: Schematic molecular-scale stratified structures of FA/[email protected]@CNCs hybrids nanomedicines.

3.1 Preparation

of

FA/[email protected]@CNCs

hybrids

nanomedicines

and

-potential

measurements.

The successful reparation of CAD was descripted and shown in supporting information Figure S2. FA/[email protected]@CNCs hybrids had been developed via the LbL assembly, whose molecular-scale stratified structure was showed in Scheme 2 and in supporting information Figure S1. CNCs with negative charge were the anchors of the hybrids, PEI with positive charge was the intermediary layer and negatively charged CAD and FA were absorbed at the outermost layer of FA/[email protected]@CNCs hybrids. Notably, large amount of PEI was absorbed onto the surface of CNCs, resulting in charge

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Page 14 of 47

reversal of the CNCs hybrids. Then, chemotherapeutic DOX was loaded to the pH-responsive hybrids in the form of CAD compound.

Scheme 3: Schematic procedure for FA/[email protected]@CNCs hybrids nanomedicines via LbL assembly: dispersion of the charged anchor in correspondent ionic solution, alternating with centrifugation and washing steps.

The electrostatic interaction based LbL assembly method was employed to construct the FA/[email protected]@CNCs hybrids. The constructional routs were illustrated in details in Scheme 3. As shown in scheme 3, FA/[email protected]@CNCs hybrids were constructed through the most exploited buildup mechanism of LbL adsorption driven by electrostatic interaction of oppositely charged building blocks (i.e. CNCs, PEI, CAD and FA). First, CNCs with negative charge were immerged into polycation PEI solution to form [email protected] hybrids. PEI molecules saturated the negatively charged CNCs resulting in charged-reversal from negative charge to positive charge. There is no aggregate formed after putting PEI and CNCs together. Dynamic light scattering (DLS), atomic force microscopy 14

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(AFM) and optical photos were used to confirm the phenomenon. As shown in Figure S14, both DLS and AFM observations confirmed good dispersing of [email protected] nanoparticles. In Figure S15, both the dispersion systems of CNCs and [email protected] showed off-white color fluidic state without any aggregates. Figure S16 is optical photo for the preparation of FA/[email protected]@CNCs hybrids nanomedicines, which confirmed that there were no any aggregations during the whole procedure again. Second, after several times of centrifuge and washing, the deposition (i.e. [email protected] hybrids) was dispersed into the CAD solution. With negative charge, CAD molecules were absorbed onto the surface of the [email protected] hybrids through electrostatic assembly. Then, FA was absorbed onto the surface of the [email protected]@CNCs hybrids as CAD did. Finally, after several times of centrifuge and washing, the targeting hybrids FA/[email protected] [email protected] were collected from the deposition. In addition, their photos under normal conditions as displayed in Figure S3 and S16 had verified the successful preparation of FA/[email protected]@CNCs. As shown in Figure S16, the orange-red precipitate was just the target hybrids nanomedicines.

Table 1. Average -potential values for CNCs, and FA/[email protected] [email protected] hybrids.

Sample names

pH (6.5)

pH (7.4)

CNCs

- 23.0 ± 3.3

- 22.6 ± 2.0

FA/[email protected]@CNCs

25.2 ± 1.6

28.0 ± 2.5

The -potentials played a crucial role in confirming the successful preparation of the CNCs based nanomedicines via the electrostatic interaction based LbL assembly method. The -potentials of 15

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CNCs, and FA/[email protected]@CNCs were measured and depicted in table 1. According to table 1, under the normal condition (pH 7.4), the surface charge studies indicated that the average -potential of CNCs was negative (i.e. -22.6 ± 2.0 mV). In contrast, the absolute average zeta potential value of FA/[email protected]@CNCs hybrids was positive (i.e. 28.0 ± 2.5 mV). The strongly negative zeta potential of CNCs decreased with the addition of PEI. The strongly positive surface charge of these nanoparticles confirmed that the CNCs were capped by large amount of PEI. These results were consistent with the Scheme 3, and the -potential reversion also confirmed the successful preparation of FA/[email protected]@CNCs hybrids via LbL assembly. According to table 1, comparing to the normal condition (pH 7.4), the average -potentials of CNCs and FA/[email protected]@CNCs hybrids stayed relatively stable at the acid condition (pH 6.5). These results should be caused by the proton receptivity of sulfate groups on the CNCs and PEI, which confirmed the successful preparation of FA/[email protected]@CNCs hybrids again. Moreover, it had been determined that the proton sponge polyplex PEI possessed high density of amino groups in the branched scaffold, which were protonable in lysosome (pH 4.5–5.5). The pH buffering behavior named as ‘‘proton sponge” effects would promote the cellular uptake and endosomal escape of nanomedicines. Their pH buffering behavior had been determined by non-aqueous titration (supplementary Figure S12), and their influence on the cellular uptake had been studied in Figure S13 in the supporting information.

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Figure 1. (a) FT-IR spectra of (A) CNCs, (B) FA, (C) DOX, (D) CAD, (E) PEI, and (F) FA/[email protected]@CNCs. (b) UV-vis absorption spectra of (A) CNCs, (B) FA, (C) DOX·HCl, (D) FA/[email protected]@CNCs, and (E) CAD.

In addition, FA/[email protected]@CNCs hybrids nanomedicines were also determined by the FT-IR and UV (Figure 1). As Figure 1a exhibited, (E) PEI produced the peaks at 3362 cm-1, 1565 cm-1, 1482 cm-1, and 815 cm-1. The medium intensity absorption peaks that arose near 3365 and 3200 cm-1 belonged to primary amine N-H of PEI. The FT-IR spectra of FA/[email protected]@CNCs (F) did not differ obviously, due to the absorption bands of the additional -NH2, and -CH2- groups of PEI (E) were covered by the broad -OH and 18

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-CH stretching bands of cellulose at 3200 ~ 3600 and 2900 cm-1, respectively. The presence of signals at 1425, 1160, 1113, and 895 cm-1 indicated that the CNCs (A) kept their pristine nanocrystal form. The characteristic absorptions of amide bands of CAD (D) were amide I band (1635 ~ 1618 cm-1), amide II band (1578 cm-1) and amide III band (1279 cm-1). FA (B) showed the characteristic absorptions peak at 1656 cm−1. Similarly, the intensity of the characteristic adsorption at 1650 cm−1 in FA/[email protected]@CNCs increased, due to the introduction of FA. The presence of those bands in the FA/[email protected]@CNCs spectrum confirmed that the CNCs based high-performance nanomedicines were successfully prepared. These results were also confirmed by UV-vis spectrophotometer. As displayed in Figure 1b, the characteristic absorption peaks of DOX and FA were at 480 nm and 365 nm 40, respectively. The spectrum of FA/[email protected]@CNCs (F) showed a similar characteristic absorption peaks as DOX did. However, CNCs (A) show no distinct absorption peaks among 200 - 600 nm. Altogether, the robust FA/[email protected]@CNCs hybrids were simply constructed by the easy, efficient extremely versatile way of LbL assembly. According to their characteristic UV absorption peaks at 480 nm (DOX) and 365 nm (FA), standard curves had been made and exhibited in supporting information Figure S17 and S18, respectively, and their molar ratio was 2.40. 3.2 Physicochemical properties of the CNCs based hybrids

3.2.1

The DLC and DLE of the CNCs based hybrids

Table 2. Characteristic drug delivery features of CNC-based hybrids

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Name

DLC

DLE

FA/[email protected]@CNCs

11.3

19.4

a: DLC was the abbreviation of drug loading capacity and DLE was the abbreviation of drug loading efficiency.

DLC and DLE of the FA/[email protected]@CNCs hybrids were determined by the FS method according to the formulations as shown in Equation S4 and Figure S5. The DLC was calculated according to a standard curve, and the average amount of DOX absorbed onto FA/[email protected]@CNCs hybrid was 11.3 wt% (DLC) and 19.4 % (DLE). Traditionally, the modification of CNCs utilized chemical conjugation onto their surface through heterogeneous reactions. The external heterogeneous reactions on CNCs surface were not efficient, which directly restricted DLC of the CNC-based nanomedicines. It had been report that the CNCs had high surface area ranging from 50 to 200 m2/g 2 9 41. Taking advantage of their high surface area, the DLC of CNC-based hybrids had been significantly

increased as expected via LbL assembly method.

3.2.2

Morphology of CNCs, FA/[email protected]@CNCs hybrids

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(A)

(B)

(C)

(D)

Figure 2. Typical AFM micrographs of Height Sensor (A, B) and Peak Force (C, D). CNCs (A, C); FA/[email protected]@CNCs hybrids (B, D).

Table 3. The mean diameters and heights for CNCs, and FA/[email protected]@CNCs hybrids.

Sample name

Length (nm)

Width (nm)

CNCs

147.1 ± 2.5

6.1 ± 1.2

FA/[email protected]@CNCs

182.5 ± 6.2

11.1 ± 2.5

a: the diameters and lengths were determined from AFM height images.

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Figure 3. X-ray diffraction grams of CNCs and FA/[email protected]@CNCs hybrids.

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Table 4. Inter planar Spacing's and Crystallite Dimensions Values for CNCs and FA/[email protected]@CNCs hybrids. (Derived from X-ray diffraction

327

data)

328 329

1, Spacings between lattice planes, calculated from the measured 2θ values with the Bragg equation.

330

2, Crystallite dimension in the direction normal to the lattice planes, calculated from the full-width-at-half-maximum of the fitted peak with the

331

Scherrer equation, using 0.94 for the shape factor.

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Table 5. Cr.I. for CNCs and CNC-based hybrids derived from x-ray diffraction data

CrI1(%)

Sample Names CNCs

70.03

FA/[email protected]@CNCs

58.56

333 334

Cr.I. means crystallinity index

335

The geometrical shape and dimensions of nanovators could affect their delivery process and 42,43,

336

the delivery efficacy

337

uptake, trafficking and so on. Meanwhile, suitable size was beneficial to improve their accumulation

338

in the tumor tissues relying on the enhanced permission and retention (EPR) effect. The morphology

339

(shape and dimensions) of CNCs and the CNC-based hybrids was observed via AFM (Figure 2).

340

The geometrical shape of CNCs and FA/[email protected]@CNCs hybrids was exactly rod-like shape as

341

expected (Figure 2). Their mean diameters and heights were listed in table 3, which were

342

determined from AFM height images. The length and diameter of FA/[email protected]@CNCs hybrids

343

nanomedicines (i.e. 182.5 ± 6.2 nm and 11.1 ± 2.5 nm) were bigger than that of CNCs (i.e. 147.1 ±

344

2.5 nm and 6.1 ± 1.2 nm). Electrostatic interaction based LbL assembly was responsible for their

345

tiny difference of the two samples. These results were consistent with the Scheme 3 and Figure 1.

346

Recent investigations showed that rod-like nanovectors tended to follow along the vascular wall and

347

have an easier margination in the tumor tissues than the spheres ones. Meanwhile, with enough

348

margination, the internalization of rod-like nanomedicines was better than the sphere ones

349

addition, it had been reported that rod-shaped nanomedicines exhibited better pharmacokinetics and

24

for example, rod-like nanomedicines were beneficial to the cellular

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In

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350

efficiency than spherical ones 44. Thus, the FA/[email protected]@CNCs rod-like hybrids with targeting

351

group of FA had great potential to act as high-performance nanomedicines.

352

Besides the observation of their morphology, their crystalline feature (i.e. inter planar spacing's

353

and crystallite dimension values) and Cr.I. of CNCs and FA/[email protected]@CNCs hybrids were

354

studied via XRD. As shown in Figure 3, XRD analysis of the CNCs and FA/[email protected]@CNCs

355

indicated that the rod-like FA/[email protected]@CNCs hybrids kept their primary cellulose nanocrystal

356

form. These results were consistent with morphologic observation on AFM in Figure 2. The

357

diffraction pattern of FA/[email protected]@CNCs (Figure 2. B and D) displayed the primary elements

358

of cellulose nanocrystal form, which was labeled in the diffraction pattern of CNCs (Figure 2. A

359

and C). A tabulation of interlayer free spacings, crystallite size and Cr.I. values for CNCs and

360

FA/[email protected]@CNCs were made from the X-ray diffraction data, which were displayed in Table

361

4 and 5, respectively.

362

3.2.3

25

Thermal stability of the CNCs and FA/[email protected]@CNCs.

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364 365

Figure 4. (A) DSC thermograms of CNCs (a) and FA/[email protected]@CNCs (b). (B) Thermal

366

gravimetric analysis (TGA) of CNCs (a) and FA/[email protected]@CNCs (b).

367

TGA and DSC were carried out to further confirm and elucidate the physicochemical

368

characteristics of CNCs and the FA/[email protected]@CNCs hybrids. The thermal stability of CNCs and

369

FA/[email protected]@CNCs were investigated and the analysis was shown in Figure 4. According to

370

Figure 4 (A) DSC thermograms of CNCs and the FA/[email protected]@CNCs hybrids, the Tg does not

371

deeply vary among the two samples, in detail, the glass transition temperature (Tg) of CNCs (a) and

372

FA/[email protected]@CNCs (b) were 47.2 oC and 45.3 oC, the FA/[email protected]@CNCs hybrid

373

nanomedicine was original the mixture of FA,CAD,PEI and CNCs. So its Tg was less than CNCs

374

(a). The difference of Tg also reflected some changes during the crystalline regions and amorphous

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375

region of the CNCs. The results were consistent with the Cr.I. data in Table 4. According to the

376

TGA curves of CNCs (a) and FA/[email protected]@CNCs (b) hybrids in Figure 4 (B), the decomposition

377

temperatures (Td) of CNCs and FA/[email protected]@CNCs were 295.8 oC and 310.0 oC, whose first-

378

order derivative form DTG was shown in Figure S7 in the supporting information. These results

379

indicated that FA, CAD, and PEI were absorbed tightly outside the CNCs nanoparticles, resulting

380

in the changing of the Td values. This difference in thermal behaviors of CNCs and

381

FA/[email protected]@CNCs further supported the conclusions stretched from their -potential values

382

and the average diameters and heights in Table 1 and Table 3.

383

3.2.4

In vitro drug release

384 385

Figure 5. In vitro behavior of DOX release from FA/[email protected]@CNCs hybrid in PBS with

386

different pHs at 37 ºC. Data represent the mean ± SD (n = 3).

387

FS method was used to minor the drug release of the CNCs based hybrids, which was explained

388

in Equation S4 and Figure S5 in the supporting information. The release deed of DOX from the

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389

pH-responsive FA/[email protected]@CNCs nanomedicines was carried out at different pHs. As

390

anticipated, the release deed was pH-dependent, and the accumulated DOX increased accompanying

391

the decrease of pH values. FA/[email protected]@CNCs nanomedicines were rather stable at pH 7.4 (i.e.

392

normal physiological conditions) but tended to rid DOX at pH 5.5 (i.e. lysosomal conditions). As

393

shown in Figure 5, no obvious explosive DOX fleeing from FA/[email protected]@CNCs hybrids was

394

observed in pH 7.4 PBS. However, significantly DOX fled FA/[email protected]@CNCs hybrids in pH

395

5.5 PBS, and almost 95 % DOX fled at pH 5.5 in 24 h. The degradation half-life periods (T1/2) of

396

FA/[email protected]@CNCs hybrids at different pHs were as follows: T1/2(pH=7.4) = 321.3 h, T1/2(pH=6.5) =

397

15.5 h, T1/2(pH=5.5) = 1.3 h. These results revealed faster release rate of DOX from

398

FA/[email protected]@CNCs hybrids at lysosomal pH (Figure 5), in agreement with the hydrolysis

399

results. It had been reported that under low pH β-carboxylic acid could catalyze the hydrolysis of

400

the cis-aconityl amide

401

nanomedicines in lysosomes, due to the catalyzed breakdown of pH labile cis-aconityl amide

402

linkages at lysosomal pH. Under acid conditions, DOX could be protonated easily resulting in

403

positively charged DOX. Because of the repulsive interaction with positively charged PEI, DOX

404

was abandoned from FA/[email protected]@CNCs hybrids in acidic conditions

405

confirmed that DOX would expeditiously flee FA/[email protected]@CNCs hybrids at lysosomal pH (pH

406

5.5), while hardly flee at normal physiological condition (pH 7.4) which impede the discharge of

407

DOX. Therefore, FA/[email protected]@CNCs hybrids nanomedicines were lysosomal pH-triggered drug

408

release, which could be confirmed by the co-location of drug-related red DOX fluorescence and the

409

lysosome-related green fluorescence in section 3.5 and Figure 11.

410

45–47.

Therefore, DOX could be release from CAD, and flee the

3.3 Cell cytotoxicity

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48.

Those results

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411

The biological activity of pH-responsive FA/[email protected]@CNCs hybrids nanomedicine was

412

depicted in Scheme 1. As shown in Scheme 1, the schematic structure of FA/[email protected]@CNCs

413

hybrids and illustration of FA mediated cellular uptake and efficient intracellular release of DOX

414

triggered by lysosomal pH, which were the prerequisite properties for its biological activities.

415

Benefiting from their rod-like shape and FA targeting, ligand-bearing FA/[email protected]@CNCs

416

nanomedicines had high affinity to tumor cell-surface receptors, which resulted in high-affinity

417

uptake of FA/[email protected]@CNCs nanomedicines

418

promoted the release of FA (or the FA/[email protected]@CNCs hybrids) from its receptor FR. FR would

419

be translate back to the surface of the tumor cells to mediate another uptake of

420

FA/[email protected]@CNCs nanomedicines 49,50.

42.

Meanwhile, the lysosomal acid conditions

421 422

Figure 6. Cell viability of MCF-7 cells after incubation with FA/[email protected]@CNCs hybrids and

423

DOX·HCl for 48 h. Data were presented as mean ± SD.

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424

Table 6. IC50 of MCF-7 cells incubated with DOX·HCl and FA/[email protected]@CNCs hybrids for

425

48h.

IC50 (mol·L-1)

Groups DOX·HCl

0.86 ± 0.21

FA/[email protected]@CNCs

0.12 ± 0.10

426 427

IC50 is the half-maximal inhibitory concentration.

428

The cell viability was determined by MTT assay against MCF-7 cells (Figure 6). As shown in

429

Figure 6, FA/[email protected]@CNCs hybrids showed a greater cytotoxicity than the control group at

430

relative low concentration, which may be caused by the combination of FA targeted and rod-like

431

shape effected enhancement of cellular uptake. Indeed, FA displayed extremely high affinity (KD =

432

10

433

FA/[email protected]@CNCs displayed high affinity to the FR positive MCF-7 cells. According to the

434

FA targeting, FA/[email protected]@CNCs nanomedicines should be rapidly bind to FR and become

435

internalized via an endocytic process, leading to enhanced cellular uptake. In addition, it had been

436

reported that rod-like nanomedicines with well-defined dimensions and known initial surfaces

437

chemistries could promote cellular uptake

438

targeting FA, rod-like shape of the nanomedicines and the pH triggered DOX release together were

439

responsible for the high cytotoxicity of FA/[email protected]@CNCs hybrids against MCF-7 cells. The

440

cytotoxicity of two sample drugs was consistent with their IC50 values in table 6. These cell

441

cytotoxicity conclusions were also supported by the other two cellular lines whose results were

442

displayed in Figure S8 and S9.

31

-10

M) for its cell surface-oriented receptor FR

51–53,

50.

That should be the reason why

resulting in great cytotoxicity

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19 54.

Actively

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443

3.4 Cellular uptake

444 445

Figure 7. FMI of MCF-7 cells incubated with FA/[email protected]@CNCs hybrids (a) and DOXHCl

446

(b) hybrids at 40 min were captured on an IFM. The scale bars corresponded to 20 m. The images

447

showed bright light, DOX fluorescence in cells (red), cell nucleus stained by Hoechst 33342 (blue),

448

and mergers of three images (from left to right).

449 450

Figure 8. MFI of MCF-7 cells incubated with FA/[email protected]@CNCs hybrids and DOXHCl at 40

451

min measured by FCM.

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452

Fluorescence microscope images was used to investigate the cellular uptake of

453

FA/[email protected]@CNCs and DOX·HCl against MCF-7 (Figure 7). DOX was a spontaneous

454

fluorescent substance whose emission wavelength was about 575 nm. Therefore, DOX was used as

455

a fluorescent probe to monitor cellular uptake quantitatively and qualitatively. As shown in Figure

456

7, the blue regions were nucleus, which was stained by Hoechst 33342. The red regions were caused

457

by the DOX fluorescent intensity, which reflected the location and amount of DOX dispersed in the

458

cellular. In FA/[email protected]@CNCs group, the obviously red regions in the cells confirmed that large

459

amount of FA/[email protected]@CNCs hybrids were endocytosed into the cells. These results should be

460

benefit from the FR receptor of the MCF-7 cells, as while as the rod-like shape and surface charge

461

of FA/[email protected]@CNCs hybrids 55,56. In had been reported that each FR-positive tumor cell could

462

mediated(1~2) × 105 FA molecules or FA loaded nanomedicines endocytosis every hour, which

463

ensure the strong MFI of FA/[email protected]@CNCs nanomedicines. The obviously overlapping of the

464

red and blue regions (as shown in the yellow circles in Figure 7) confirmed that DOX released from

465

the hybrids and move to the nucleus, due to the breakdown of pH labile chemical bind in CAD,

466

which caused the disruption of layer interactions in the FA/[email protected]@CNCs hybrids. These

467

results had also been confirmed on the other cellular lines whose results were showed and explained

468

in supporting information Figure S10 and S11. All of the observations were consistent with

469

Scheme 1 and Figure 5. These FMI of FA/[email protected] @CNCs hybrids were also consistent with

470

the high cytotoxicity and relatively low IC50 in Table. 6. As shown in Figure 7, palpable DOX

471

fluorescent intensity showed relatively less cellular uptake of DOXHCl than the

472

FA/[email protected]@CNCs hybrids, which should be caused by short co-incubation time with the cells.

473

The investigations of the time effects on DOXHCl showed that the highest cellular uptake happened

474

after 4 h co-incubation (supplementary Figure S6).

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475

As shown in Figure 8, the quantitative cellular uptake of FA/[email protected]@CBCs and

476

DOXHCl against MCF-7 cells were analyzed by the FCM. Cells incubated in the culture medium

477

with no drugs were control group, whose MFI was adjusted to less than 10. The MFI of

478

FA/[email protected]@CNCs hybrids and DOXHCl were 552.1 ± 44.2 and 56.8 ± 21.5, respectively.

479

MFI of FA/[email protected]@CBCs hybrids was much higher than that of DOXHCl against MCF-7

480

cells, which was consist with the fluorescence images in Figure 7. It had been reported that MCF 7

481

cells were FR-positive cells, so, specific binding of FA to FR could assist the entrapping of

482

FA/[email protected]@CNCs. The high affinity of FA for FR leads to the specific binding of the

483

nanomedicines to the FR-positive cells, resulting in enhanced cellular uptake. That also explained

484

the high cytotoxicity of the FA/[email protected]@CNCs nanomdicines in Figure 6. Simultaneously, it

485

had been confirmed that the CNC-based nanoparticles had good membrane permeability 41, which

486

was benefit to the cellular uptake. In addition, the positively charged CNCs based nanoparticles can

487

be take-up by cells without affecting the cell membrane integrity

488

could directly illustrate the high MFI results of FA/[email protected]@CNCs hybrids which had great

489

potential to act as high-performance targeted nanomedicines.

490

57.

Those research conclusions

3.5 FA targeted ability and lysosomal pH-triggered drug release

491

It had been proved that FR was frequently overexpressed on cancer cells. FR could be suppressed

492

while co-incubated with FA and made the failure of FA target. To precisely prove the FA targeted, the

493

FR positive tumor cells was incubated with 300 ug/mL folic acid in their media for two weeks to get the

494

FR negative tumor cells. The FR negative cells treated with FA/[email protected]@CNCs nanomedicines were

495

set as one of control groups. The other control group was FR positive tumor cells treated with DOX·HCl.

496

In detail, MCF-7 cells ware selected to verify the targeting ability of FA/[email protected]@CNCs

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497

nanomedicines. After 40 min co-incubation, the cellular uptake of different DOX formulations was

498

qualitatively and quantitatively analyzed through the fluorescence microscopy and automated BIO-TEK

499

microplate reader, respectively. As shown in Figure 9, the fluorescence intensity of cells in

500

FA/[email protected]@CNCs hybrids group was the strongest compared to the other two control groups, which

501

qualitatively confirmed the FA targeting ability of FA/[email protected]@CNCs hybrids. As shown in Figure

502

10, the relative fluorescence unit of cells in FA/[email protected]@CNCs hybrids group was much stronger

503

than that of the other two control groups, which quantitatively confirmed the FA targeted ability of

504

FA/[email protected]@CNCs hybrids nanomedicines.

505 506

Figure 9. Fluorescence microscope images of the three groups: (a) FR positive MCF-7 cells treated with

507

FA/[email protected]@CNCs hybrids, (b) FR negative MCF-7 cells treated with FA/[email protected]@CNCs

508

hybrids and (c) FR positive MCF-7 cells treated with DOXHCl. The scale bars corresponded to 20 m.

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509

The images showed bright light, DOX fluorescence in cells (red), and mergers of two images (from left

510

to right).

511 512

Figure 10. The relative fluorescence unit (RFU) of the three groups: (a) FR positive MCF-7 cells treated

513

with FA/[email protected]@CNCs hybrids, (b) FR negative MCF-7 cells treated with FA/[email protected]@CNCs

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hybrids and (c) FR positive MCF-7 cells treated with DOXHCl.

515

Taking advantage of DOX fluorescence and lysosomal tracker, MCF-7 cells were used to precisely

516

prove the lysosomal pH controlled drug release. As shown in Figure 11, the drug-related red DOX

517

fluorescence and the lysosome related green fluorescence overlapped, which confirmed the co-location

518

of lysosomes and FA/[email protected]@CNCs hybrids in the tumor cells. Those results precisely indicated

519

the lysosomal pH controlled drug release.

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Figure 11. Fluorescence microscope images of MCF-7 cells incubated with FA/[email protected]@CNCs

522

hybrids or DOXHCl. The scale bars corresponded to 20 m. The images showed bright light, drug-

523

related DOX fluorescence in cells (red), cell lysosome stained by LysoTracker Green (green), and

524

mergers of two images (from left to right).

525

4.

Conclusions

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In this study, the robust pH-responsive FA/[email protected]@CNCs hybrids nanomedicines were

527

developed via facile LbL assembly. Compared to the CNC-based prodrug we previously reported,

528

FA/[email protected]@CNCs hybrids exhibited high DLC (i.e. 20 folds higher), which indicated the high

529

surface area of the CNCs and the stable electrostatic interaction between the molecules and

530

adsorbent surfaces. Benefiting from the uniform rod-like shape and folate targeting,

531

FA/[email protected]@CNCs hybrids nanomedicines gained enhanced cellular uptake and increased

532

cytotoxicity. Thus, the CNC-based nanomedicines with well-defined dimensions and known initial

533

surfaces chemistries and folate targeting exhibited great potential as high-performance

534

nanomedicines, which can significantly improve the pharmacokinetics and therapeutic efficiency.

535

The future of CNCs based high-performance nanomedicines, especially the hybrids built up by LbL

536

assembly, is bright and iridescent.

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5.

ASSOCIATED CONTENT

538

Supporting Information

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HPLC, ESI-MS, 1H-NMR, Illustration, Optical pictures, Fluorescence spectrum equations,

540

Standard curves, DTA, MTT, Non-aqueous titration, Fluorescence microscope images and Flow

541

cytometry were enclosed in the supporting information (PDF).

542

AUTHOR INFORMATION

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Corresponding Author

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*E-mail: [email protected]

545

ORCID

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Jiahui Yu: 0000-0002-1215-3851

547

Notes

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The authors declare no competing financial interest.

549

Acknowledgements

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The work was supported by the National Natural Science Foundation of China (No.

551

51573050 and 81871405).

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