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Apoferritin Nanocage for Brain Targeted Doxorubicin Delivery Zhijiang Chen, Meifang Zhai, Xiang Yang Xie, Yue Zhang, Siyu Ma, Zhiping Li, Fanglin Yu, Baoquan Zhao, Min Zhang, Yang Yang, and Xingguo Mei Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00341 • Publication Date (Web): 20 Jul 2017 Downloaded from http://pubs.acs.org on July 23, 2017

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Molecular Pharmaceutics

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Apoferritin Nanocage for Brain Targeted Doxorubicin Delivery

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Zhijiang Chen a, b, d #, Meifang Zhai a, b, e, #, XiangYang Xie c, #, Yue Zhang a, b, c, Siyu Ma a, b, Zhiping Li a, b,*,

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Fanglin Yu a, b, Baoquan Zhao a, b,*, Min Zhang a, b, Yang Yang a, b,*, Xingguo Mei a, b

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a

State key Laboratory of Toxicology and Medical Countermeasure, Beijing 100850, China

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b

Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China

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c

Wuhan General Hospital of PLA, Wuhan 430070, China

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d

Hubei University of Science and Technology, Xianning 437100, China

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e

Jiamusi University, Jiamusi 154002, China

#

These authors contributed equally to this work.

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*To whom correspondence should be addressed.

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1

ACS Paragon Plus Environment


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ABSTRACT:

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An ideal brain-targeted nanocarrier must be sufficiently potent to penetrate the blood-brain

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barrier (BBB) and sufficiently competent to target the cells of interest with adequate

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optimized physiochemical features and biocompatibility. However, it is an enormous

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challenge to the researchers to organize the abovementioned properties into a single

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nanocarrier particle. New frontiers in nanomedicine are advancing the research of new

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biomaterials. Herein, we demonstrate a straightforward strategy for brain targeting by

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encapsulating doxorubicin (DOX) into a naturally available and unmodified apoferritin

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nanocage (DOX-loaded APO). APO can specifically bind to cells expressed transferrin

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receptor 1 (TfR1). Because of the high expression of TfR1 in both brain endothelial and

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glioma cells, DOX-loaded APO can cross the BBB and deliver drugs to the glioma with TfR1.

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Subsequent research demonstrated that the DOX-loaded APO had good physicochemical

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properties (particle size of 12.03 ± 0.42 nm, drug encapsulation efficiency of 81.8 ± 1.1%),

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significant penetrating and targeting effects in the co-culture model of bEnd.3 and C6 cells in

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vitro. In vivo imaging revealed that DOX-loaded APO accumulated specifically in brain

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tumour tissues. Additionally, in vivo tumour therapy experiments (at a dosage of 1 mg/kg

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DOX) demonstrated that a longer survival period was observed in mice that had been treated

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with DOX-loaded APO (30 days) compared with mice receiving free DOX solution (19

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days).

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Keywords:

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apoferritin nanocage; blood-brain barrier; brain-targeted delivery; glioma

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Abbreviations: Apoferritin nanocage

APO

Blood-brain barrier

BBB

Confocal laser scanning microscopy

CLMS

Doxorubicin

DOX

Flow cytometry

FCM

Magnetic resonance imaging

MRI

Transferrin receptor 1

TfR1

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1. INTRODUCTION

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The efficient penetration of therapeutic agents through the blood-brain barrier (BBB)

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remains a major challenge for drug delivery. It is estimated that approximately 98% of central

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nervous system (CNS) drugs fail to enter clinical trials due to poor BBB penetration.1 The

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appearance of nanocarriers has attracted many researchers' attention recently.2 Except good

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drug loading, releasing and biosafety, an ideal nanocarrier used in CNS drug delivery should

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be able to cross the BBB, target to specific cells and prolong blood circulation time.3,4 Over

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the past few decades, a wide variety of materials have been used for creating nanocarriers,5

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including liposomes, mesoporous silica nanoparticles, carbon nanotubes, polymeric

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nanoparticles,

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which have several disadvantages, such as difficult biodegradation in vivo, low increase in

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the percentage of transport across the BBB, time-consuming transport, and intolerable

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adverse side effects.10,11 To cross the barriers imposed by nanocarriers consist of synthetic

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materials, natural materials which are considered nontoxic may be a good option.12-14

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However, it may be difficult for some natural nanocarriers to assemble in the diseased sites

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simply based on their nanosize. To enhance the targeting ability, the surface of natural

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materials were conjugated with ligands (e.g., peptide and antibodies).15 However, the usage

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of solvent, chemical reagents, and ligands in the modification process may change the

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binding character of natural materials, influence their in vivo performance and

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biocompatibility, even the reproducibility of production.16,17

etc.6-9 However, most nanocarriers are prepared using synthetic materials,

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Ferritin is an iron storage protein that has a cage-like nanostructure 18, 19 and exist in

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many living organisms with a perfect biocompatibility.20 In iron-free conditions, ferritin can

4


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form the hollow apoferritin, the inside cavity of which can load molecular cargos and its

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outer surface can be modified.21 However, the surface modifications may bring several

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adverse influences to the apoferritin, such as biocompatibility, stability,22 yield of final

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products.23-24 Fortunately, it was recently reported that apoferritin can bind to cells that

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expressed transferrin receptor 1 (TfR1),25 and it can deliver iron to the brain via this access.26

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Therefore, this provides a natural pathway to target to the brain. The delivery potential of

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apoferritin has been explored in many payloads, such as radioisotopes,27 antibiotics,28

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alkylating agents,29 anticancer drugs,30 and siRNA31. However, the use apoferritin as a carrier

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for brain-targeted delivery on animal model has not previously been tested.

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Glioma is considered one of the most deadly brain diseases.32 Though the

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glioma-associated BBB is structurally impaired and more permeable compared to the healthy

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BBB, it still represents a significant barrier to drug delivery to the brain.33 Because TfR1 is

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both highly expressed in the cells of glioma34 and brain endothelial35, it is an ideal target for

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glioma-targeted drug delivery.36 Doxorubicin (DOX) is a common chemotherapeutics and

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has a good anti proliferation on many cancer cells. It can kill the glioma cells in vitro but has

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minor effects on the in vivo glioma in the injection form.37

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In this paper, a brain DOX delivery strategy via apoferritin nanocage (APO) without

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ligand modification for glioma therapy was tested. In vitro and in vivo experiments were

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performed to explore the brain-targeting delivery efficiency of APO. This study will help

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elucidate the functions of apoferritin nanocage and allow for its application as a powerful

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nanoplatform for brain tumour diagnosis and therapy.

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2. EXPERIMENTAL SECTION 5



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2.1. Materials. DOX was provided by Haizheng Co. Ltd (Taizhou, China). Apoferritin

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from equine spleen (horse spleen apoferritin) 0.2 µm filtered and all chemicals were provided

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by Sigma-Aldrich.

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Mouse glioma C6 cells and mouse brain endothelial bEnd.3 cells were provided by the Cell Resource Centre of IBMS (Beijing, China).

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2.2. APO loading with DOX. The drug loading process was prepared as described by

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Kilic et al.,38 with modifications. Apoferritin from equine spleen (horse spleen apoferritin)

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was obtained from Sigma-Aldrich. Briefly, dissolve APO (10mg) in 100 µL of 0.15M NaCl,

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add in DOX (1 mg/mL), stir for 30 min. Adjust the pH of the mixture to 2.0 with 0.1 M HCl,

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stir for 10 min. In order to reassemble the protein to its native nanosphere form, the pH of the

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solution was then slowly increased to 7.4 with 0.1 M NaOH solution under constant stirring.

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The resulting solution was then transferred to dialysis bags (molecular weight (MW) cut-off

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12,000-14,000) against in total 600 mL of 0.9% NaCl solution for 24 h (replaced with fresh

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0.9% NaCl solution every 8 h ) to remove the free DOX. Finally, the DOX-loaded APO were

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filtration sterilized by 0.2 µm ﬁlter and subpackaged to aseptic pials.

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2.3. Labeling of APO. This process was performed as described by Liang et al.39

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Briefly, dissolve Cy5.5-NHS ester (Lumiprobe) in dimethyl sulphoxide (DMSO) and then

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add this mixture to APO solution (0.1 M NaHCO3, pH 8.5) at a molar ratio of 10:1. Stir the

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solution for 12 h (4 °C) in a dark place and then purify with a polyacrylamide column (MW

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cut-off 6,000; Thermo Scientific) to separate excess dyes.

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2.4. Characterization of DOX-loaded APO. PBS buffer (0.1 M, pH 7.4) and acetate

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buffer (0.1 M, pH 5.0) were used in dialysis to investigate the in vitro drug release profile of

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DOX-loaded APO. In brief, add 0.5 mL of DOX-loaded APO dispersion in a dialysis bag

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(MW cut-off 12,000-14,000), dialyze the bag in 30 mL of release medium at 37 °C for 60 h

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with stirring. At pre-set time points, withdraw 800 µL of release medium from the flask and

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replenish an equal volume of blank medium. The released free DOX at different incubation

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times was assayed by an HPLC, as previously described.40

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The stability of DOX-loaded APO in serum (10% FBS in PBS) was assessed with

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Turbiscan Lab® Expert (Formulaction, L'Union, France) as we previously described.41

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Around 200 µL of tested sample was mixed with 4 mL of PBS with 10% FBS for the

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analysis.

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2.5. In vitro uptake studies. C6 and bEnd.3 cells (1× 105 cells/well) were seeded in

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12-well plates for flow cytometry (FCM) (BD FACSCalibur, USA) and glass-bottomed

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dishes for confocal laser scanning microscopy (CLMS) (UltraVIEW Vox, USA). Cells were

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incubated with Cy5.5-labeled APO at 37 °C for 4 h and stained by Hoechst 33258 for

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imaging or gently suspended in PBS for quantitative analysis by FCM. An antibody blocking

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assay was performed to confirm that TfR1 is the binding receptor of APO to C6 and bEnd.3

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cells. Briefly, 1 µM Cy5.5-labeled APO was incubated with C6 or bEnd.3 cells in the

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presence or absence of a 10-fold molar excess of anti-TfR1 mAbs (clone M-A712; BD

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Bioscience). After incubation for 4 h, the cells were washed by cold PBS and examined by an

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FCM.

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2.6. In vitro cytotoxicity studies. Cytotoxicity of DOX-loaded APO against C6 cells

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was evaluated with an MTT assay as previously described.41 The cell concentration was 4000 7



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cells/well and the samples were determined by a plate reader (Model 680, BIO-RAD, USA).

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2.7. Transport across the BBB and targeting of brain cancer cells. To assess

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potential penetrating and targeting effects, a bEnd.3/C6 co-culture model was established

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according to a previous report.42 The BBB transport assay was performed when the

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transendothelial electrical resistance (TEER) reached 200 Ωcm2.43

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CLSM was applied to investigate the penetration ability of APO. The culture medium in

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the upper chambers was changed by 50 µM Cy5.5-labeled APO or free Cy5.5 in 10% FBS

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containing DMEM. At definite time points, the C6 cells on the bottom of the compartments

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were immediately analyzed by the CLSM. To assess the anti-proliferative effect of the

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DOX-loaded APO against C6 cells after penetrating the BBB model, a sulphorhodamine-B

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staining assay was applied. Free DOX and DOX-loaded APO were added to the apical

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compartments at a final DOX concentration of 1000 ng/mL, respectively. After 48 h, the

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surviving glioma C6 cells in the basolateral compartment were analyzed by the

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sulforhodamine-B staining assay.42

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2.8. Penetration of the BBB/tumor spheroid co-culture model. C6 tumour

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spheroids were built as the following: coat a 48-well plate with 50 µL of 2% low melting

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point agarose, plate 100 µL of culture media (2× 103 cells) onto the well, move the tumour

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spheroids to the lower chamber of the bEnd.3/C6 tumour spheroids co-culture model, add

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Cy5.5-labeled APO or free Cy5.5 (50 µM) to each apical chamber, and incubate for 4 h. Then,

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wash the tumour spheroids with PBS, fixed the samples with 4% paraformaldehyde for 30

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min; observe the tumour spheroids under the CLSM.

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2.9. Penetrating ability in zebrafish. Zebrafish were incubated according to a

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report.44 The fish were divided into groups, and 10 nL of Cy5.5-labeled APO (0.1 mg/mL)

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was injected into the heart of zebrafish by a micro-sprinkler. At definite time points, the brain

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of fish was observed by a CLSM (100× magnification, 673 nm for the excitation

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wavelength).

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2.10. Establishment of intracranial glioma-bearing mice model. Glioma-bearing

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mice model was established as our research team previously reported.45 Around 4×105 C6

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glioma cells (suspended in 4 µL PBS) was injected into the brain.

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2.11. In vivo imaging. The mice in glioma-bearing model were injected with

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Cy5.5-labeled APO (0.5 mg/kg) via tail vein 8 days after the tumour implantation. The

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control mice were injected with physiological saline. Thirty minutes after the injection, the

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IVIS® Lumina II in vivo imaging system (IVIS® Lumina II In Vivo Imaging System, Caliper

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life sciences, USA) was utilized to perform the in vivo imaging at the preset time points.

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After the imaging, the animal were sacrificed, and their organs were removed and observed.

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2.12. Glioma distribution. Eight days after surgery, the Cy5.5-labeled APO (0.5

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mg/kg) was injected to the mice via tail vein. After 30 min, the glioma-bearing mice were

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anesthetized, with their hearts perfused by saline and 4% paraformaldehyde. Then, their

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brains were removed for frozen sections (5-µm-thick), in which the nuclei were stained by

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DAPI. The frozen sections were observed using CLSM.

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2.13. In vivo Antiglioma effect. The glioma model mice were divided into three

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groups (n=10): DOX-loaded APO group, free DOX solution group and physiological saline

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group. Eight days after cell injections, mice in DOX-loaded APO and free DOX solution

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groups were dosed 1 mg/kg DOX related formulations four times every 2 days. At day 16,

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four mice from each group were anesthetized and brain cancer was assessed by magnetic

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resonance imaging (MRI) (Siemens, Munich, Germany) with measurement of the tumour

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diameter. Glioma inhibition was calculated using the formula: Rv=(Vdrug/ Vsaline) × 100%.

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Vdrug is presented the tumor volume after drug treatment, and Vsaline is presented the tumor

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volume after physiological saline treatment. The remaining 6 mice in each group were used

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to performed survival tests. The glioma cell injected day was defined as day 0.

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2.14. In vivo safety evaluation. Haemogram analysis was performed to evaluate the

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in vivo safety properties of the nanocarriers. Mice were treated according to the procedure

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described above for the in vivo anti-glioma growth experiment. On day 16, the mice blood

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were collected via the orbit and assayed. Meanwhile, the body weight of each mouse was

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measured daily.

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2.15. Statistical analysis. All data are displayed as the means ± standard deviation

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(SD). The difference between any two groups was processed via ANOVA method. The value

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of P less than 0.05 was considered to be statistically significant.

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3. RESULTS AND DISCUSSION.

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3.1. Characterization of DOX-loaded APO. The subunits of APO can be

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disassembled at strong acidic environments (pH 2.0) and reassembled by returning the pH to

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physiological conditions (pH 7.4) in a shape-memory fashion. Therefore, the payload

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encapsulation/ release in /from APO depends on the pH.38 In this study, we used the pH 10


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method for DOX loading into APO. The DOX encapsulation efficiency of APO was

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approximately 81.8 ± 1.1%. After entering the intracellular region, DOX-loaded APO is

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engulfed by lysosomes. Lysosomal acidification significantly contributes to the disassembly

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of APO and release of DOX. To verify the pH-dependent drug release of DOX-loaded APO,

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the pH 7.4 PBS (0.1 M) and pH 5.0 acetate buffers (0.1 M) were employed at 37 °C to mimic

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the physiological conditions and lysosomal situation, respectively.38 As seen in Fig. 1 A, at

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pH 7.4, a small amount of DOX was released during 60 h of incubation. While, at pH 5.0, the

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release of DOX was relatively fast and reached its maximum release of 89.58 ± 2.71% at 60 h.

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This pH dependent release feature of DOX-loaded APO provides a potential lysosome-based

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drug release mechanism for the nano-vehicle of APO.

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For nanocarriers, the particle size is a key factor that determines their in vivo and in vitro

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fate. After an in vitro releasing study, the particle size of DOX-loaded APO was further

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analysed by a laser particle analyser. As displayed in Fig. 1 B, the mean particle size of the

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DOX-loaded APO was 12.03 ± 0.42 nm (Supporting Information: 4. Size analysis report),

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and it had a narrow size distribution (the polydispersity index was 0.11 ± 0.01). This particle

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size of APO was appropriate for drug targeting as it was sufficiently small to penetrate into

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tissues, reach the receptors on cell surface and assist intracellular transport.46 TEM

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observation verified the formation of DOX-loaded APO (Fig. 1 C). The DOX-loaded APO

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was uniformly dispersed in solution with a round shape. In addition, the TEM image of

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DOX-loaded APO confirmed the particle size values obtained from the laser particle

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analyser.

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The nanocarriers' stability in physiological conditions significantly influences the

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delivery efficient of the carriers, therefore, 10% FBS in PBS that mimicked physiological

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conditions was usually used to predict the nanocarriers' stability in vivo.41 Here, Turbiscan

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Lab® Expert was utilized to assess the in vitro stability of DOX-loaded APO. According to

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this evaluation,47 the transmission or back-scattering profiles (less than 0.5%) obtained (Fig.

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1 D) indicated there was no notable aggregation or sedimentation of DOX-loaded APO in the

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culture medium over 24 h. Similar stability results of DOX-loaded APO were also found in

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full fresh human serum (Fig. S1).

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3.2. In vitro cellular uptake and cytotoxicity assay. bEnd.3 cells are considered as

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the major component of BBB. They can form the tight junctions of BBB that will impede

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most ectogenic molecules cross the BBB into cerebral parenchyma.48 Glioma C6 cells were

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usually selected as the cells of brain tumour model because of its many similarities to human

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multiform glioblastoma. In addition, TfR1 are highly expressed on the surfaces of C6 and

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bEnd.3cells,49, 50 which provides conveniences for further study. Therefore, C6 and bEnd.3

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cells were used in this paper.

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The affinity of APO for C6 and bEnd.3 cells was qualitatively and quantitatively

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analysed by CLSM and FCM, respectively. Hoechst 33258 was used to stain the nucleus

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(blue). As displayed in Fig. 2 A, compared with the control group, remarkable intracellular

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fluorescence (red) were both found in C6 and bEnd.3 cells with APO (represents for

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Cy5.5-labeled APO in this section) treatments. While, the red fluorescence was not found in

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APO and anti-TfR1 mAb treated C6 and bEnd.3 cells, suggested the binding between APO

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and TfR1 on both cells may be specifically inhibited by anti-TfR1 mAb. Furthermore, based

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on the quantized results of FCM, the fluorescence ratio of APO was 529.23 ± 35.75% in C6 12


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cells compared with the control (Fig. 2 B). In bEnd.3 cells, the fluorescence ratio of APO was

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565.14 ± 23.28% (Fig. 2 C). By contrast, the competitive binding of APO to C6 or bEnd.3

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cells was performed by adding anti-TfR1 mAb to the media before the adding of nanocarriers.

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The result showed that the cellular binding of APO in the presence of excess anti-TfR1 mAb

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in C6 or bEnd.3 cells was significantly suppressed and became almost equivalent to that of

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control. This result implied that APO could not effectively discern and bond with the target

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cells with the low expression level of TfR1. Therefore, because of the high expression of

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TfR1 in both C6 and bEnd.3 cells, a higher fluorescence intensity was found in both C6 or

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bEnd.3 cells treated with APO compared with control, which was consistent with the results

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found in Fig. 2 A.

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The cytotoxicities of free DOX and DOX-loaded APO were assayed by the MTT after

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incubation with C6 cells for 72 h. As seen in Fig. 2 D, the anti-proliferative effects of

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DOX-loaded APO was concentration depended. In addition, the results indicated that the

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difference in cytotoxicities between free DOX and DOX-loaded APO was not as obvious at

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low concentrations (0.1, 1 and 10 ng/mL). Free DOX demonstrated a stronger inhibition

244

effect than DOX-loaded APO at high concentrations (100 ng/mL and 1000 ng/mL) with the

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strongest inhibitory effects on C6 cells at 1000 ng/mL. This results suggested that small

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molecular DOX (free DOX) may quickly penetrate into cells via passive diffusion under a

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high concentration gradient in vitro. While the DOX-loaded APO may underwent a

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lysosome-based drug release process after entering the intracellular region. Therefore, free

249

DOX exhibited a stronger inhibitory effect on C6 cells than DOX-loaded APO under the

250

same dosage.

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3.3. Penetration studies in the BBB cell model. The BBB is a major physiological

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barrier that prevents drugs or drug delivery systems from entering the brain targeted region.

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Therefore, the in vitro BBB co-culture model consisted of bEnd.3/C6 cells was constructed to

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estimate the penetration efficiency of APO in mimicking conditions in vivo. Cellular uptake

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of Cy5.5-labed APO or free Cy5.5 in C6 cells was analyzed by CLSM. As shown in Fig. 3A,

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the red fluorescence (Cy5.5) in APO group was observed at each time point, indicating that

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APO could cross the BBB and promote uptake in C6 cells. In contrast, no red fluorescence

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were found in the co-culture model with free Cy5.5 treatment during the experimental period.

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Anti-proliferative results were also consistent with the above findings in the bEnd.3/C6

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cell co-culture model. As a result, after the addition of free DOX and DOX-loaded APO at

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the final DOX concentration of 1000 ng/mL, the survival rates (%) of C6 cells after crossing

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bEnd.3 cells was almost 100% (97.46 ± 2.53%) and 40.25 ± 1.79%, respectively. The

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DOX-loaded APO exhibited a significant inhibitory effect by aiding the DOX cross the BBB

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and finally targeted into the glioma cells. Although the free DOX had a stronger inhibition

265

effects on C6 cells than DOX-loaded APO in the in vitro cytotoxicity assay at high

266

concentrations (100 ng/mL and 1000 ng/mL, respectively) (Fig. 2 D), DOX-loaded APO

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could more strongly inhibit the growth of C6 cells after crossing bEnd.3 cells than free DOX

268

in the bEnd.3/C6 cell co-culture model, which could be attributed to APO delivery.

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Three-dimensional tumour spheroids, considered as a perfect in vitro tool mimicking

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solid tumours, have been frequently utilized to predict the anti-cancer effects of tested drug.

271

To simulate the in vivo environment of glioma more effectively, a bEnd.3/C6 tumour

272

spheroid co-culture model was constructed. The co-culture model was incubated with free

14


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273

Cy5.5 or Cy5.5-labeled APO in the upper chamber, and the C6 tumour spheroids in the basal

274

chamber were imaged by CLSM. APO was observed to be efficiently internalized in a

275

bEnd.3/C6 cell co-culture model, which might also influence delivery in the bEnd.3/C6

276

tumour spheroid co-culture model. As displayed in Fig. 3 B, fluorescence was only found in

277

APO treated group, suggesting that APO could cross the tight BBB monolayer and penetrate

278

the cores of the C6 tumour spheroid. Of note, the free Cy5.5 group did not cross the BBB

279

model and penetrate the tumour spheroids. This result further supported the data of cellular

280

uptake analysis in a bEnd.3/C6 cell co-culture model (Fig. 3 A).

281 282

Overall, these results demonstrated that the APO has the possible abilities to across the BBB and approach glioma cells in vitro.

283

3.4. APO mediated delivery across the BBB in zebrafish. Several anticancer

284

agents can inhibit glioma cells in vitro and later fail in clinical therapy. The major obstacle is

285

the BBB, which is a selective hindrance. After in vitro evaluation of penetrating ability, we

286

examined the ability of APO to cross the in vivo BBB by employing zebrafish which has a

287

similar BBB structure as mammals. This provides a quick, effective, and cost-efficient model

288

for screening the brain-targeted efficacy of nanocarriers in vivo. In this study, the transport in

289

the zebrafish of Cy5.5-labed APO or free Cy5.5 was studied from 0 min to 15 min. The

290

distribution of Cy5.5 fluorescence signals in the brain area and outside of the vessels

291

represents the ability of samples to cross the BBB into brain. As shown in Fig. 4, free Cy5.5

292

(red) was not found in the brain area, but it only found in the vessels of zebrafish (green) after

293

the injection during the experimental period. The results indicated that free Cy5.5 was

294

confined to the vascular system and did not across the BBB into the brain area in the 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

295

zebrafish. As expected, the fluorescence signals (red) of Cy5.5 in APO were observed in the

296

brain region but without the brain vessels (green). As shown in confocal images (Fig. 4), even

297

at 10 min, the fluorescence signals could be observed in the brain area. This implied that

298

APO could rapidly cross the BBB. We conclude that the APO could significantly increase the

299

delivery efficiency of brain targeting in vivo. These results from zebrafish were consistent

300

with the in vitro results shown in Fig. 3. The APO could be used for further study in the

301

treatment of brain cancer in vivo.

302

3.5. Distribution of APO in mice with intracranial glioma. The chemotherapies

303

for glioma in clinic is dissatisfactory largely due to the existence of BBB and non-targeted

304

nature of drugs. The selective distribution of drug loaded nanocarrier in tumour sites would

305

enhance the anticancer efficiency of chemotherapy in vivo. To estimate the in vivo targeting

306

ability of APO, in the present experiment, the in vivo biodistribution of Cy5.5-labeled APO

307

was imaged by collecting fluorescence signals of the whole body in mice with intracranial C6

308

glioma. As shown in Fig. 5 A, high accumulation of Cy5.5-labeled APO was detected in the

309

brain area even at 30 min after injection, and this was subsequently verified by the strong

310

fluorescence found in the isolated brain (Fig. 5 B). The trend observed for in vivo distribution

311

analysis in mice was consistent with the results of the zebrafish (Fig. 4). This phenomenon

312

indicated that APO can cross the BBB into the brain in vivo. Furthermore, 4 h after the

313

injection, the major organs of the mice were isolated and observed (Fig. 5 B). Obviously, the

314

brain accumulation was much higher for the APO group. The results implied that the APO

315

could across into the brain and reduce the non-target distribution in other organs as heart,

316

lung and spleen. In addition, mild fluorescence was found in the liver and kidney of APO, 16


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317

this phenomenon was consistent with a previous report.39 This may because the liver and

318

kidney are the major filtering organs in body, drugs are easily trapped in these filters. This is

319

also a shortcoming of APO carrier to be overcome in the future.

320

To evaluate its in vivo glioma-targeting capability, an immunofluorescence assay was

321

conducted after treatment with Cy5.5-labeled APO in mice bearing intracranial glioma. As

322

shown in Fig. 5 C, no obvious fluorescence was observed in the glioma of the control group.

323

A significant high distribution of Cy5.5-labeled APO was observed in the glioma region,

324

indicating the precise glioma targeting property of APO because of the high expression level

325

of TfR1 on both the BBB and glioma cells. The results of immunofluorescence assay are

326

consistent with the in vivo imaging findings (Fig. 5 A and B) and in line with our

327

expectations that the APO brain-targeting delivery system could not only penetrate the BBB,

328

but it could also target to the glioma area and accumulate in the tumour cells.

329 330

These preliminary data of the in vivo distribution studies strongly demonstrated that the APO carriers could efficiently across the BBB into the target cells.

331

3.6. In vivo anti-tumor efficacy. To investigate whether DOX-loaded APO displays

332

anti-glioma activity in vivo, the inhibition effects of the drug loaded nanocarriers on tumour

333

growth in mice bearing intracranial C6 glioma were studied. After treatment with the control

334

formulations (physiological saline or free DOX) or DOX-loaded APO, the overall

335

anti-glioma efficacy was observed by magnetic resonance imaging (MRI) to monitor the

336

cancer volume and was confirmed using survival curves.

337

Consistent with the results found in the in vivo distribution, tumour inhibition analysis

338

confirmed the significant brain glioma-targeting effect of DOX-loaded APO in mice with 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

339

intracranial C6 glioma. As shown in Fig. 6 A, the glioma diameter in the brain at day 16 was

340

clearly reduced according to MRI after treatment with the DOX-loaded APO compared with

341

those after treatment with control formulations. This indicates DOX-loaded APO crossed the

342

BBB and targeted glioma cells. Tumour inhibition at day 16 (Fig. 6 B) was 100.00 ± 15.28%

343

for physiological saline, 88.34 ± 17.65% for free DOX, and 35.31 ± 8.52% for DOX-loaded

344

APO. These results suggest that the anti-tumor potency of DOX-loaded APO is remarkably

345

better than that of free DOX in intracranial C6 glioma-bearing mice. Similar results were also

346

found in intracranial U87 (human glioblastoma cells) xenograft glioma-bearing nude mice

347

(Fig. S2).

348

The cancer patients' quality of life and prolonged survival time are the major clinical

349

evaluating indicators to estimate the anti-tumor therapy. In further investigation of the

350

potential of DOX-loaded APO in anti-glioma therapy in vivo, the Kaplan-Meier survival

351

curve was utilized in the intracranial C6 glioma-bearing mice (Fig. 6 C). Although free DOX

352

expanded the median survival time from 17 to 19 days, no significant difference was

353

observed between the free DOX and physiological saline groups, which may be explained by

354

the poor glioma-targeting efficiency of free DOX. As expected, treatment with DOX-loaded

355

APO significantly prolonged the median survival time (30 days), which was 1.76- and

356

1.58-fold higher than that of physiological saline and free DOX, respectively. The longer

357

survial time of APO group could be chiefly attributed to the fine targeting delivery ability of

358

APO, which was demonstrated by in vivo imaging (Fig. 5 A, B and C).

359

3.7. Toxicity studies. The goal for the designing of a targeting nanocarrier is to

360

achieve an optimal therapeutic efficacy with desired safety profiles during the in vivo 18


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361

applications. For the preliminary safety assessment, the body weight changes of tested mice

362

was recorded during the drug treated period (Fig. 7 A). For the free DOX group, more than

363

15% of body weight loss was found at the end of the experiment, which may be due to the

364

non-targeted characteristics of free DOX and tumour cachexia of the mice. In contrast, the

365

weight loss of DOX-loaded APO group was smaller of than that of the free DOX group,

366

which indicated APO reduced nonspecific cellular uptake through brain-targeted delivery.

367

Moreover, haemogram analysis was performed to further evaluate the in vivo safety

368

properties of the nanocarriers. As shown in Fig. 7 B and C, there was no obvious decrease in

369

the red blood cell (RBC) and white blood cell (WBS) levels in free DOX and DOX-loaded

370

APO, respectively. In Fig. 7 D, the mean corpuscular volume (MCV) of the free DOX group

371

was significantly lower than the other two groups. As showed in Fig. S3, free DOX displayed

372

histological damages in the organs of heart, liver and kidney, while the DOX-loaded APO

373

only displayed mild liver toxicity. These results showed that APO could significantly

374

decrease the toxicity of DOX from negligible weight loss and haematological indicators, and

375

it was relatively safe at the present test dose.

376

Overall, the naturally existed APO without any ligand functionalization, and does not

377

contain any potential hazardous material, which would not activate immunological or

378

inflammatory responses. Therefore, APO demonstrates perfect biocompatibility when used in

379

vivo. This study displays that APO is small (Fig. 1) and can bind to cells via interacting with

380

TfR1 (Fig. 2), which can greatly break through the brain physiological barriers (Fig. 4) and

381

actively, deeply penetrate into the glioma tissues (Fig. 3). Combined with their intrinsic

382

glioma-targeting properties, APO exhibited a notably longer median survival time (Fig. 6) 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

383

and lower toxicity (Fig. 7) when compared with the free DOX, which is more likely due to

384

the changes in the bio-distribution of DOX given by APO (Fig. 5). Therefore, APO is a good

385

drug delivery system for transporting drugs into the brain.

386

The brain drug targeting is a very complex problem. We should admit that the tumor

387

selecting ability of the apoferritin nanocage here was very limited. This is because the

388

nanocarrier prepared in this paper will delivery drugs to any cells that over-expressed TfR1s,

389

it cannot distinguish the kinds of tumor cells. In other words, the apoferritin nanocages are

390

partly glioma targeted. This research was only a small step forward in the drug targeting of

391

glioma. The future improvement of this nanocarrier may depend on the modifications of its

392

surface. Further researches are urgently in need.

393

4. CONCLUSIONS

394

These research data clearly demonstrate that apoferritin nanocages serve as an excellent

395

platform for delivering drugs into the brain in a targeted fashion, and they have a substantial

396

anti-glioma effect. The improved anti-glioma effects of DOX-loaded APO than free DOX

397

may be owing to the combined contribution of excellent physicochemical features, efficient

398

BBB penetration, active tumour targeting, effective endocytosis and good biosafety of APO.

399

This study was the first to evaluate apoferritin nanocage loaded with drug targeted to the

400

brain tumour in vivio. Although the DOX-loaded APO can effectively inhibit the glioma in

401

mice model, there remain some challenges for the APO carriers to overcome. For example,

402

the distribution of APO in the liver and kidney is very high, which needs further improvement.

403

In the subsequent researches, we will perform the in vitro and in vivo evaluations, including

20


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404

the mechanism of brain-targeted delivery, and further explore the application of APO in

405

brain-targeted delivery.

406

407

We are grateful for the financial support from the Beijing Science and Technology New Star

408

(Grant No. Z161100004916162), Beijing NSF(Grant No. 7172162) and Hubei NSF (Grant

409

No. 2016CF198) .

410

411

(1) Karamanos, Y.; Pottiez, G. Proteomics and the blood-brain barrier: how recent findings

412 413 414

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For Table of Contents Use Only

Apoferritin Nanocage for Brain Targeted Doxorubicin Delivery Zhijiang Chen a, b, d #, Meifang Zhai a, b, e, #, XiangYang Xie

c, #

, Yue Zhang a,b,c, Siyu Ma a,b, Zhiping

Li a, b,*, Fanglin Yu a, b, Baoquan Zhao a, b,*, Min Zhang a,b, Yang Yang a,b,*, Xingguo Mei a,b a

State key Laboratory of Toxicology and Medical Countermeasure, Beijing 100850, China Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China c Wuhan General Hospital of PLA, Wuhan 430070, China d Hubei University of Science and Technology, Xianning 437100, China e Jiamusi University, Jiamusi 154002, China b

#

These authors contributed equally to this work. *To whom correspondence should be addressed.

Apoferritin nanocage (APO) can specifically bind to cells expressed transferrin receptor 1 (TfR1). Because of the high expression of TfR1 in both brain endothelial and glioma cells, DOX-loaded APO can cross the blood-brain barrier (BBB) and deliver drugs to the glioma with TfR1.


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Figure 1. Physicochemical characterization of DOX-loaded APO. (A) In vitro release of DOX from APO at pH 5.0 and pH 7.0 at 37 °C. (B) Particle size distribution of DOX-loaded APO. (C) Morphological appearance of DOX-loaded APO based on TEM. (D) Stability of DOX-loaded APO in the presence of 10% FBS. The transmission and backscattering profiles were measured at each time point using a Turbiscan Lab® Expert analyser. The data are presented as the means ± SD (n = 3). 92x70mm (300 x 300 DPI)



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Figure 2. In vitro cellular uptake and cytotoxicity. (A) Confocal laser scanning microscopy (CLSM) analysis of the uptake of various samples by C6 and bEnd.3 cells. (B) Flow cytometry (FCM) measurement of various samples uptake by C6 cells. (C) FCM measurement of various sample uptake by bEnd.3 cells. (D) The cytotoxicity of free DOX and DOX-loaded APO. The data are presented as the means ± SD (n = 3). * indicates P< 0.05. 194x315mm (300 x 300 DPI)


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Figure 3. Uptake of APO on the in vitro co-culture model. (A) CLSM images of C6 cells treated with APO or free Cy5.5 after crossing the co-culture model of bEnd.3/C6 cells, which contain Cy5.5 (red) and Hoechst 33258 (blue, nuclear imaging). (B) The penetration of C6 tumour spheroids was assessed by a CLSM with different depths after crossing the co-culture model of bEnd.3/C6 tumour spheroids. 299x231mm (300 x 300 DPI)



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Figure 4. In vivo brain imaging in zebrafish. Cy5.5 (red) that travelled from vessels (green) after the injection the Cy5.5-labed APO. Free Cy5.5 (red) retained within vessels (green). 230x92mm (300 x 300 DPI)


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Figure 5. Distribution of APO in mice with intracranial glioma. (A) Whole body imaging at different time points after systemic administration. (B) Fluorescence detection of isolated main tissues and organs from mice at the end of observation. (C) Distribution of Cy5.5 in the brain of mice bearing intracranial C6 glioma determined by a CLSM. The yellow line shows the margin of intracranial glioma and arrow indicates the glioma cells. The red represents Cy5.5 and the nuclei were stained by DAPI (blue). 130x171mm (300 x 300 DPI)



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Figure 6. Anticancer efficacy in intracranial C6 glioma-bearing mice. (A) MRI of normal and pathological brains at 16 day after inoculation. (B) Inhibition of the brain glioma volume at 16 day after inoculation. (C) Kaplan-Meier survival curves. The data are presented as the means ± SD (n = 3). * indicates P< 0.05. Notes: Efficacy after treatment with various formulations with a dose of 1 mg/kg DOX at days 8, 10, 12, and 14 from inoculation. 146x97mm (300 x 300 DPI)


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Figure 7. In vivo safety evaluation. (A) Body weight changes in intracranial C6 glioma-bearing mice after treatments with various samples. Haematological indicators of (B) RBC, (C) WBC and (D) MCV on day 16 after inoculation. The data are presented as the means ± SD (n = 3). * indicates P< 0.05. 161x118mm (300 x 300 DPI)


Apoferritin Nanocage for Brain Targeted ... - ACS Publications

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