Extending half-life of H-ferritin nanoparticle by fusing albumin binding

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Extending half-life of H-ferritin nanoparticle by fusing albumin binding domain for doxorubicin encapsulation Chunyue Wang, Chun Zhang, Zenglan Li, Shuang Yin, Qi Wang, Fangxia Guo, Yao Zhang, Rong Yu, Yongdong Liu, and Zhiguo Su Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01545 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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Extending half-life of H-ferritin nanoparticle by fusing albumin binding domain for doxorubicin encapsulation Chunyue Wang1, 2, §, Chun Zhang1, 2, §, Zenglan Li2, Shuang Yin1, 2, 3, Qi Wang2, Fangxia Guo2, Yao Zhang2, Rong Yu1, *, Yongdong Liu2, *, Zhiguo Su2

1

Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West

China School of Pharmacy, Sichuan University, Chengdu, 610041, China 2

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese

Academy of Sciences, Beijing 100190, China 3

School of Chemical Engineering, The University of Adelaide, Adelaide SA, 5005, Australia

* Correspondences: [email protected] (Y. Liu); Tel/Fax: +86-010-82545028. No. 1, Zhongguancun Beiertiao,

Haidian

District,

Beijing,

China;

[email protected]

(R.

+86-028-85503012. Southern Renmin Road, No. 17, Section 3, Chengdu, China. § These authors contributed equally to this work.

Abstract picture


Yu);

Tel/Fax:

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ABSTRACT Nanoparticles based on the heavy chain of the human ferritin (HFn) are arousing growing interest

in the field of drug-delivery due to their exceptional characteristics. However, the unsatisfied

plasma half-life of HFn substantially limits its application as a delivery platform for anti-tumor

agents. Herein, we fused an albumin binding domain (ABD) variant which basically derives from

the streptococcal protein G and possesses a long-acting characteristic in serum albumin to the

N-terminus of the HFn for the aim of half-life extension. This ABD-HFn construct was highly

expressed and fully self-assembled into symmetrical and spherical structure in E. coli bacteria.

The purified ABD-HFn showed a similar particle size with wild-type HFn and also exhibited an

extremely high binding affinity with human serum albumin. To evaluate the therapeutic potential

of this ABD-HFn construct in terms of half-life extension, we encapsulated a model antitumor

agent doxorubicin (DOX) into the ABD-HFn. Significantly outstanding loading efficacy of above

60 molecules doxorubicin for each ABD-HFn cage was achieved. The doxorubicin-loaded

ABD-HFn nanoparticle was characterized and further compared with the recombinant HFn

counterpart. The ABD-HFn/DOX nanoparticle showed dramatically improved stability, and

comparable cell uptake rate when compared with HFn/DOX counterpart. Pharmacokinetics study

in Sprague-Dawley rats showed that ABD-HFn/DOX nanoparticle possessed significantly



prolonged plasma half-life of approximately 17.2 hours, exhibiting nearly 19 times longer than

that of free doxorubicin and 12 times for HFn/DOX. These optimal results indicated that fusion

with albumin binding domain will be a promising strategy to extend half-life for protein-based

nanoparticles.

Key words: albumin binding domain; human ferritin; protein nanoparticle; pharmacokinetics;

doxorubicin encapsulation

Abbreviations: ABD, albumin binding domain; HFn, human ferritin heavy chain; DOX,

doxorubicin; MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass

spectrometry; CD, circular dichroism; FL, fluorescence; SEC, size exchange chromatography;

DLS, dynamic light scattering; TEM, transmission electron microscopy; HSA, human serum

albumin; AUC, area under curve.


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1. INTRODUCTION

Nanoparticles (NPs) have been shown great potential to address important issues related

drug delivery, including reducing drug toxicity, inhibiting drug degradation/sequestration, retarding circulation times and increasing bioavailability.1,

2

Conventional strategies for drug

delivery nanoparticles mainly based on synthetic polymeric and liposomal substances.3 These,

however, may suffer limitations such as wide size distributions, difficulty in site-specific functionalization, low drug loading, particle material toxicity and instability etc.4-7 Caged proteins represent a novel class of nanomaterial that may withstand many of these concerns.8, 9 Among

them, ferritin is a highly symmetrical and multimeric protein cage consisting of 24 subunits that

naturally self-assemble into a hollow structure with an outer diameter of 12-nm and an interior cavity 8-nm in diameter.10 Nanoparticles based on the heavy chain of the human ferritin (HFn) are

arousing growing interest in the field of drug-delivery due to their exceptional characteristics,

such as good biodegradability, high water-solubility, excellent particle uniformity, amenable to versatile functionalization and remarkable capacity for drug loading.11-13 Of particular relevance,

especially in view of potential applications in cancer diagnosis and therapy, is the ability of HFn

to be efficiently and specifically internalized by many types of cancer cells, making it an ideal delivery platform for anti-cancer agents.14-16



However, a major problem for recombinant HFn as a delivery platform is the unsatisfied

plasma half-life (approximately 2 h) after intravenous injection, which is generally suffered by most of protein-based therapeutics.12 To fully harness the great potential of HFn nanoparticle for

anti-tumor drug delivery, many strategies have been examined for its half-life extension. 1)

chemical modification; covalent conjugation with PEG chain on the external surface made the

recombinant HFn benefit from increasing hydration radius, resulting in an comparable half-life

with that of endogenous serum ferritin which contains natural carbohydrate polymers on its surface.17 However, the extra chemical modification step and subsequent down-stream process

always result in both lower yields and increased manufacturing costs. 2) genetic fusion with

repetitive Pro-Ala-Ser (PAS) sequences or XTEN peptides; the HFn with natively disordered and

highly soluble polypeptides can be easily co-expressed in E. coli bacteria and the plasma half-life

also can be modulated by the length of the fused sequences. The fully assembled and highly monodisperse PASylated or XTENylated HFn cage compared favorably with PEGylated HFn.13, 18

Moreover, it was reported that improved drug encapsulation efficacy and drug-loaded

nanoparticle stability are observed although the specific mechanism is still not fully understood.13

The simplified downstream process and more favorable therapeutic benefits for HFn construct

indicated more potential of genetic fusion for half-life extension of recombinant HFn.


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Human serum albumin (HSA), as the most abundant plasma protein, is highly soluble, very

stable and exhibits an extraordinarily long circulation time of about 19 days in blood mainly

because of its relatively large molecular size and pH-dependent FcRn-mediated recycling pathway that prevents intracellular degradation.19 A variety of ways based on albumin have been

developed to increase the circulation half-life of therapeutics, such as albumin fusion, fatty acids

(albumin ligand) modification, drug-albumin conjugation and nanoparticle albumin-bound etc, and successfully expedited a series of therapeutic candidates into clinical evaluation.20-23 Recently,

a kind of serum albumin binding peptide which was shared by many kinds of gram-positive

bacteria’s surface protein, especially like the streptococcal protein G, was screened and optimized,

and this albumin binding domain was shown to possess extremely high affinity to HSA in the sub-nanomolar range.25 This albumin-binding domain is composed of three helical structures and

cysteine-free amino acid residues with a relatively low molecular weight of about 5 kDa. The

highly soluble and structurally easy to fold properties allow ABD to be used as a gene fusion

partner in the production of recombinant therapeutic proteins for extending the circulation half-life of the parent entities through affinity capture of serum albumin.26 Therefore, in this study,

to fully harness the long-acting characteristic of serum albumin for half-life extension of HFn, we

fused an albumin binding domain variant which basically derives from the streptococcal protein



G with extremely high affinity with human serum albumin to the N-terminus of the HFn. This

ABD-HFn construct was highly expressed and fully self-assembled into symmetrical and

spherical structure in E. coli bacteria. After purification, the physicochemical properties of

ABD-HFn were characterized and compared with the recombinant HFn. To evaluate the

therapeutic potential of this ABD-HFn construct in terms of half-life extension, we encapsulated a

model antitumor agent doxorubicin (DOX) which is one of the most widely used antitumor drugs

because of its broad spectrum of antitumor activity into the ABD-HFn. The doxorubicin-loaded

ABD-HFn nanoparticle was characterized and further compared with the recombinant HFn

counterpart, and the pharmacokinetics was also investigated in animals.

2. EXPERIMENTAL SECTION

2.1 Materials

Human ferritin heavy chain (HFn) gene based on the amino acids sequence (UniPort NO.

P02794) was synthesized by Sangon Biotech Shanghai Co. Ltd (Shanghai, China) taking into

consideration the codon optimization for high level expression in Escherichia coli and further

ligated into pET-30a(+) plasmid (NdeI/XhoI) to construct expression vector (pET-30a(+)-HFn) by

polymerase chain reaction (PCR) amplification using appropriate primers in our lab. The

pET-30a(+)-ABD-HFn expression vector was similarly constructed by fusing the albumin binding


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domain sequence derived from the streptococcal protein G (LAEAKVLANR ELDKYGVSDF

YKRLINKAKT VEGVEALKLH ILAALP) to the N-terminal of HFn through a (GGGGS)4 flexible linker. The plasmids were finally transfected into BL21 E. coli bacteria for expression of

intended proteins. Doxorubicin was purchased from Meilun (Dalian, China); human serum

albumin was provided by Nanjing Oddfoni Biological Technology (Nanjing, China). Dulbecco’s

Modified Eagle Media (DMEM), Penicillin-Streptomycin solution (100×), fetal bovine serum

(FBS), and 0.25% trypsin-EDTA (1×) solution were purchased from Mediatech (Manassas, VA).

Cell Counting Kit (CCK-8) and Trypan blue were from Sigma (USA). All of the other reagents

were of analytical reagent quality. The human lung cancer A549 cell line was kindly provided by

Institute of Microbiology, Chinese Academy of Sciences (Beijing, China). The Sprague-Dawley

(SD) rats (males) were purchased from Vital River Laboratory Animal Technology Co. Ltd.

(Beijing, China). 2.2 Expression and purification of recombinant HFn and ABD-HFn The E. coli BL21 bacteria containing HFn and ABD-HFn plasmid were separately cultured

at 37 °C in 2 x L-B medium supplemented with 0.5% glycerol in a 20-L fermentor (BioFlo 415,

NBS) and further induced with 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) for another 4

hours when OD600nm reached to about 5.0. After fermentation, the bacterial cells were harvested



by centrifugation at 4,000 rpm for 30 min at 4 °C. The pellets were suspended in 20 mM PB (pH

7.0) buffer containing 1.0 mM EDTA and then the cell suspensions were disrupted by

high-pressure homogenizer (APV, Germany) for 3 cycles with a pressure of 800 - 900 bar. After

centrifugation at 10,000 rpm for 30 min at 4 °C, the supernatant was collected and diluted 3 times in 20 mM PB, pH 7.0 to be purified through a previously established method.27 In brief, the

disrupted supernatant was heated at 70 °C for 10 minutes and the precipitate was removed by

centrifugation. The residual supernatant was diluted and firstly purified by anion chromatography

(Q-Sepharose), loaded in 20 mM Tris-HCl, pH 8.5 and eluted by 0.35 M NaCl, 20 mM Tris-HCl,

pH 8.5 , and the eluted protein was further polished by gel filtration (Superdex 200). The purified

proteins (HFn and ABD-HFn) were primarily analyzed by SDS-PAGE and stored at 4 °C for

further use. 2.3 Analysis of protein structure 2.3.1 SDS-PAGE analysis

12% sodium dodecyl sulfate (SDS) polyacrylamide gels were prepared as described by

Laemmli method. The samples were mixed with 5x reducing loading buffer. The mixture was treated at 100 ℃ for 5 minutes. The electrophoresis were initially performed at 90 volts for 30

minutes and further carried out at 150 volts until the bromophenol blue reached the bottom of gels.


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Gels were finally stained with Coomassie brilliant blue R250.

2.3.2 Matrix-assisted laser desorption/ionization time of flight mass spectrometry

(MALDI-TOF) analysis MALDI-TOF MS analysis was performed using the Autoflex Ⅲ (Bruker, USA). ABD-HFn

and HFn samples (0.5 mg/ml) were desalted into distilled water and mixed (v/v 1:1) with

α-cyano-4-hydroxycinnamic acid matrix, and then slowly dispensed onto a MALDI plate. After

dried at room temperature for a while, peptide mass were determined in linear mid-mass positive

mode.

2.3.3 Circular dichroism (CD) analysis

HFn and ABD-HFn proteins were dissolved in phosphate buffer (5 mM) and injected into a

1.0 mm path length quartz cuvette. Far-UV CD analysis was performed on J-810 spectrometer

(Jasco, Japan) at room temperature. The spectrum scope was from 260 nm to 190 nm and the

scanning rate was 1200 nm per minute. Every sample was scanned 3 times and the data was

averaged.

2.3.4 Fluorescence (FL) analysis

The intrinsic fluorescence analysis of the HFn and ABD-HFn was performed on F-4500

fluorescence spectrophotometer (Hitachi, Japan). The excitation wavelength was 280 nm and the



emission wavelength was recorded from 310 nm to 400 nm with a scanning rate of 1200 nm/min.

1.0 cm path length cuvette was utilized. Each sample was also subjected to scan for three times

and the results were averaged for further analysis.

2.3.5 Size exclusion chromatography (SEC) analysis

SEC analysis was performed on ÄKTA pure system (GE healthcare, USA). 500 µl diluted

protein with about 0.5 mg/ml was loaded on the column of Superdex 200 (300 x 10 mm ID, GE

healthcare), which was already equilibrated with 200 mM PB, pH 7.4 and eluted at 0.6 ml/min.

Signals were recorded at 280 nm and 480 nm.

2.3.6 Dynamic light scattering (DLS) analysis

DLS analysis was measured using a Zetasizer Nano ZS90 instrument (Malvern, UK). Before

measurement, the samples were diluted to about 0.5 mg/ml and further centrifuged at 10,000 for

10 min, and equilibrated to 25℃.

2.3.7 Transmission electron microscope (TEM) analysis

TEM analysis was performed using a HT7700 transmission electron microscopy (Hitachi,

Japan). 5 µl HFn/ABD-HFn proteins and HFn/ABD-HFn encapsulating doxorubicin (DOX)

nanoparticles (0.1 mg/mL) were dropped onto glow-discharged and carbon-coated, 230-mesh

copper grid and absorbed for 1 min at room temperature. Then, the excess solution was removed


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and the copper grids were washed with ddH2O. Finally, the samples covered in copper grid were negatively stained with 2% uranyl acetate for 30 s and dried in air after removed the excess dye.

2.4 Albumin binding to ABD-HFn

ABD-HFn solution with constant concentration (1.0 mg/mL) was separately mixed with

human serum albumin (HSA) solution with gradually increased concentration with a molar ratio

(ABD-HFn nanoparticle to HSA) of 1:4, 1:8, 1:12, 1:16 and 1:24. Then, the mixture solution was

loaded on the column of Superose 6 (300 x 10mm ID, GE healthcare) which was already

equilibrated with 200 mM PB, pH7.4, and absorbance signals were recorded at 280 nm. The

corresponding peak area was calculated using the inherent software of ÄKTA pure system.

Besides, the Conc-Area standard curve was obtained by analysis of free HSA varying

concentration to roughly estimate the HSA binding number for each ABD-HFn nanoparticle.

2.5 Doxorubicin encapsulation of HFn and ABD-HFn The disassembly/reassembly method was performed to load drug.12, 28 Firstly, the protein

particles (HFn or ABD-HFn) were dissociated by adjusting to pH2.0 using 0.1 M HCl. Then,

doxorubicin was added into the solution and maintained for another 10 min at room temperature.

The mass ratio of protein particles and doxorubicin was 5:1. Subsequently, the pH value of the

mixture was readjusted up 7.4 by 0.1 M NaOH for reassemble. After stirring at room temperature



for 2 h, the mixture solution was centrifuged at 1,0000 rpm for 10 minutes at 4℃ to remove

aggregated protein and the free DOX was removed through size exclusion chromatography

(Sephadex G25, GE Healthcare, USA). The resulted encapsulated DOX was further verified by

Superdex 200 column. The protein concentration of ABD-HFn/DOX (or HFn/DOX) complex was

determined using Brandford method and the doxorubicin content was measured by the specific

absorbance of doxorubicin at 480 nm. The final HFn/DOX and ABD-HFn/DOX nanoparticles

were further characterized by size exclusion chromatography and TEM analysis which the

methods were described as above.

The loading capacity (N) was calculated as follow:

N=

=

N: loading capacity, C: molarity, MW: molecular weight.

2.6 Leakage of doxorubicin from the HFn/DOX and ABD-HFn/DOX nanoparticles The DOX loaded nanoparticles were dissolved in PBS (pH7.4) at 4 ℃. After 0, 1, 3 and 14

day, samples were taken and centrifuged to remove the aggregated protein. Then, the free DOX

was removed by desalting. Finally, the protein and doxorubicin content of nanoparticles were

measured to calculate the loading capacity as above mentioned.

2.7 Cytotoxicity assay


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The cytotoxicity of HFn/DOX, ABD-HFn/DOX, and free doxorubicin against human lung

tumor cell A549 was evaluated by CCK-8 assay. A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37 ℃ in 5% CO2 atmosphere as before. Exponential growth cells were digested and the final cell density was adjusted to about 1 x 105

cells per milliliter by complete medium. 100 µl cells for each well were seeded in a 96-well plate.

After incubation for 24 hours, mediums were replaced with new complete medium separately

contained free DOX, HFn/DOX, and ABD-HFn/DOX which concentration ranged from 0 µM to

62.5 µM (equivalent DOX). After incubation for another 60 hours, the mediums were removed

and cells were washed once by PBS. Then, 100 µl new complete medium with 10 µl CCK-8

solution was added into each well for another 2 hours. Finally, the absorbance of the solution was

measured at 450 nm (background: 630 nm) by a VersaMax microplate reader (Molecular Devices,

USA).

2.8 Cellular uptake assay

A549 cells in the exponential growth phase were seeded in 96-well plates at a density of 1 × 105 cells per well and cultured for 24 h at 37 ℃ in 5% CO2 atmosphere. Then the culture mediums were replaced by fresh mediums separately containing free DOX, HFn/DOX and

ABD-HFn/DOX (5 µM equivalent DOX). The cells were further incubated at 37 °C for 1 h, 2 h, 4



h and 8 h, respectively. After that, the cells were washed twice with PBS and subsequently

disintegrated by cell lysis buffer. Finally, the cellular fluorescence intensity of the cell lysate was

recorded at λex = 480 nm and λem = 580 nm using a fluorescence microplate reader. The results

were the average of 3 experiments.

2.9 Pharmacokinetics study

All animal experiments were performed with the approval of the Institute of Process

Engineering, Chinese Academy of Sciences (IPE, CAS). All animals were raised under Specific

Pathogen Free (SPF) conditions. SD rats (male, 200 g) were randomly assigned to four groups (3

rats in each group) and administrated with PBS, free DOX, ABD-HFn/DOX, HFn/DOX (3.0

mg/kg DOX equivalents) separately via intravenous injection at tail vein. After injection, blood

samples were collected from the retro-orbital sinus at fixed time points, and followed by clotting for at least 0.5 h. Serum was obtained by centrifugation at 6000 rpm for 30 min at 4 ℃. Finally,

100 µl serum of each sample was transferred into a 96-well black plate and the DOX contents

were determined by Varioskan Flash microplate reader (Thermo Fisher Scientific, USA) (λex was

set at 480 nm and λem at 580 nm). Meanwhile, the standard curve of the fluorescence intensity

with varying concentration of doxorubicin in rat serum was also measured for further quantitative

analysis.


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2.10 Statistical analysis

All data were presented as mean ± standard deviation (SD). The statistical significance was

assessed via unpaired T test or one-way ANOVA and was defined as *p > 0.05, **p < 0.05. The

parameters in the pharmacokinetic assay were obtained by fitting the data in a

single-compartment model and calculated using DAS 2.0 software. The IC50 values in the CCK-8

assay were calculated by GraphPad Prism v5.0.

3. RESULTS

3.1 Expression, purification and molecular identification

Both of HFn and ABD-HFn were successfully and highly expressed in E. coli in a soluble

form. After purification by anion exchange chromatography following by gel filtration, most of

impurity proteins were removed and the final ABD-HFn and HFn proteins were obtained with

purity of above 95% in SDS-PAGE analysis (Figure 1A). MALDI-TOF-MS analysis showed an

almost identical molecular weight of 21093.1 Da for HFn (theoretical value: 21094.45 Da) and a

MW of 27489.1 Da for ABD-HFn (theoretical value: 27447.61 Da), about 42 Da larger than the

theoretical value, perhaps an acetylation was happened to ABD-HFn (Figure 1B).

3.2 Structural characterization of HFn-ABD and HFn

Far-UV (260-190 nm) CD analysis was performed

and the CD spectrum profiles of HFn



and ABD-HFn were almost of one, both exhibiting a pronounced double minimum at around 209

and 222 nm which indicated a major second structure of α-helix in both proteins (Figure 2A).

Crystal structures show that both HFn protein and albumin binding domain mainly exist in second structure of α-helix.10, 23 These CD results indicated that the interposition of ABD at the

N-terminal of HFn does not obstruct each other’s structure folding. Fluorescence analysis was

also performed to further investigate the folding of HFn and ABD-HFn (Figure 2B). Compared

with the HFn (λmax = 329 nm), about 5-nm red-shift was occurred to ABD-HFn (λmax= 334 nm), indicating more exposure of aromatic amino acids to solvent which could be ascribed to the fused

albumin binding domain exposing in the particle surface. Size exclusion chromatography was

used to compare the apparent molecular size and the results showed the similar elution profiles

between HFn and ABD-HFn. (Figure 2C).

It was reported that the natural HFn cage possesses a hollow structure with an outer diameter of 12-nm.10 Thus, DLS was performed to determine the molecular size of HFn and ABD-HFn, and the result showed that HFn exhibited a diameter of about 12.9 ± 0.763 nm, similar to the previously reported 12.0 nm. A larger diameter about 14.1 ± 1.432 nm of ABD-HFn was

observed, and this result should be ascribed to the introduced albumin binding domains located in

the particle surface (Figure 2D). To observe the morphology of HFn and ABD-HFn, TEM


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analysis was conducted and the results showed that both HFn and ABD-HFn existed in a

spherical and hollow structure (Figure 3).

3.3 Albumin binding to ABD-HFn

To verify the albumin binding ability of ABD-HFn, we mixed ABD-HFn with different

amount of HSA (molar ratio) and then the incubated samples were determined by size exclusion

chromatography (Superose 6 column). As shown in Figure 4A, when the molar ratio was 1:4

(ABD-HFn nanoparticle to HSA), the peak of HSA was almost entirely diminished, indicating

that ABD-HFn was able to bind to HSA and in this ratio all HSA could completely bound to

ABD-HFn. Meanwhile, the particle size of ABD-HFn/HSA (retention volume, around 11.5 ml)

was notably increased compared with that of ABD-HFn (RV, 14.5 ml). The residual HSA (peak

area) significantly increased with the increase of HSA molar ratio. When increased to 1:8, a peak

of residual HSA was detected (RV, 17.8 ml) and the binding of ABD-HFn and HSA tended to be

saturated. According to the binding curve (Figure 5), we calculated the saturable number of

bound HSA was approximate six molecules for each ABD-HFn nanoparticle. In addition, the

saturated ABD-HFn/HSA conjugates were further analyzed by TEM and the image obviously

showed an asymmetric spherical structure with larger particle size of nearly 30 nm in major

diameter than that of ABD-HFn detected by DLS (Figure 4B). What more, the binding between



ABD-HFn and HSA was extremely firm because little released HSA from the HFn-ABD/HSA

was detected after two days incubation.

3.4 Doxorubicin loaded HFn-ABD and HFn nanoparticles

Doxorubicin was loaded into the HFn-ABD and HFn nanoparticles by pH-induced

disassembly/reassembly methods. To characterize the nanoparticles loaded with drug, size

exclusion chromatography (Superdex 200 column) and TEM analysis were conducted. There

were no difference were observed on the retention volume of nanoparticles whether nude or drug

loaded, indicating the high-order structures of HFn and ABD-HFn are totally regained after the

pH-induced disassembly/reassembly process (Figure 6A, C). This was further presented by the

TEM analysis (Figure 6B, D). The accompanied absorbance of the nanoparticles at 480 nm

revealed the success of doxorubicin loading into the HFn and ABD-HFn nanoparticles (Figure

6A, C). The drug loading efficacy was also calculated according to the absorbance of HFn/DOX

and ABD-HFn/DOX conjugates at both 280 nm and 480 nm. About 62 doxorubicin molecules for

each ABD-HFn nanoparticle were successfully loaded, while only about 34 molecules for HFn

nanoparticle. Besides, the stability of DOX loaded nanoparticles was also studied and the results

revealed that ABD-HFn/DOX nanoparticle was quite stable during two weeks storage, while over

20% of doxorubicin was leaked from HFn/DOX nanoparticle (Figure 7).


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3.3 A549 cell cytotoxicity

To evaluate the cytotoxic activities of the HFn/DOX and ABD-HFn/DOX nanoparticles, the

cell ability was monitored against human lung carcinoma cell (A549) after dosing and the result

was show in Figure 7. Both of the HFn/DOX and ABD-HFn/DOX nanoparticles could inhibit the

growth of A549 cells in a dose-dependent manner and showed lower cytotoxicity than that of free

DOX (IC50, 0.88 ± 0.25 µM). Compared with HFn/DOX nanoparticle with an IC50 value of 4.47

± 0.85 µM, ABD-HFn/DOX showed an increased IC50 value of 10.06 ± 1.25 µM, about 2 times

of the former.

3.4 Cellular uptake

To validate whether there is an efficacy of albumin binding domain introduced to HFn

nanoparticle, the cellular uptake of the both conjugates HFn/DOX and ABD-HFn/DOX was

investigated against A549 cells (Figure 9). With incubation time increasing, the increased

intracellular accumulation of doxorubicin was observed in the both conjugates and free

doxorubicin However, compared to the free doxorubicin, the accumulation rate for both

nanoparticles was notably retarded (p < 0.01), and these results were in line with the cytotoxicity

against to A549 cell. Interestingly, different from the cell cytotoxicity results, ABD-HFn/DOX

was demonstrated with a comparable accumulation rate of doxorubicin concentration with that of



HFn/DXO (p > 0.05).

3.5 Pharmacokinetics study

The pharmacokinetic behaviors of the HFn/DOX and ABD-HFn/DOX nanoparticles were

evaluated through monitoring the drug concentration at fixed time points after ABD-HFn/DOX，

HFn/DOX, DOX were intravenous injected at the tail of normal Sprague-Dawley rats. The serum

doxorubicin concentration-time curves and the pharmacokinetic parameters were shown in

Figure 10 and Table 1. The results showed that serum doxorubicin level of free doxorubicin

group was promptly eliminated with an apparent blood retention half-life of 0.91 ± 0.23 hours. In

contrast, the HFn/DOX nanoparticle showeda blood retention half-life of 1.46 ± 0.44 hours,

exhibiting nearly 1.5 times longer than that of free doxorubicin. ABD-HFn/DOX nanoparticle

showed significantly prolonged blood retention half-life of 17.18 ± 4.30 hours, about 19 times

longer than that of free doxorubicin and 12 times than that of HFn/DOX. Correspondingly, area

under the curve (AUC) values of the ABD-HFn/DOX nanoparticle increased to approximately

21.4 and 12.2 folds of free doxorubicin and HFn/DOX nanoparticle, separately.

4. DISCUSSION

Genetic functionalization of albumin binding domain on the external surface of HFn

nanoparticle was achievable only via N-terminal fusion in our work. C-terminal fusion resulted in


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non-structural aggregates (inclusion bodies) which was hard to refold. The ABD-HFn construct

was highly expressed in soluble form and fully self-assembled into a nanocage structure in E. coli

bacteria. The binding affinity of ABD with HSA was preserved and the process could be finished

just in several minutes, indicating an extremely high binding ability. Although there are 24

albumin binding domains on the ABD-HFn nanoparticle surface, the number of binding HSA can

be saturable with an average maximum of about six HSA for each ABD-HFn nanoparticle. This

phenomenon could probably be ascribed to the saturated steric hindrance of bound albumin

wrapping around the ABD-HFn surface.

Encapsulation of doxorubicin through pH-dependent disassembly-reassembly method

resulted in a significantly higher doxorubicin loading efficacy of above 62 molecules for each

ABD-HFn cage when compared with that of HFn which was achieved about 34 molecules of doxorubicin for each cage in our previous work.29 Genetic functionalization of HFn construct

resulting in improved drug loading efficacy was also reported in other group though the specific mechanism was not fully understood.13 A generally proposed principle for this improved loading

efficacy is that the fused domains probably block the HFn surface pores by which the leakage of

drugs maybe happen during drug encapsulation process and storage. Thus it is reasonable that

dramatically improved stability of ABD-HFn/DOX nanoparticle was manifested compared with



the HFn/DOX counterpart in this work.

Both encapsulated doxorubicin nanoparticles showed decreased cell-based cytotoxicity. The

transport of free doxorubicin across the cell membrane is mainly through a fast passive diffusion

mechanism, whereas ABD-HFn/DOX and HFn/DOX nanoparticles enter into cell mainly through

special/non-special endocytosis route, and thus inevitably result in higher IC50 values for the

ABD-HFn/DOX and HFn/DOX nanoparticles when compared with free doxorubicin. This

phenomenon was also happened to some other encapsulated formulations. HFn was reported to be

taken by many kinds of tumor cells through a transferrin receptor 1 (TfR1) mediated endocytosis pathway14. Interestingly, in this work, cell uptake rate of ABD-HFn/DOX nanoparticle showed

comparable with HFn/DOX counterpart, indicating that the binding of albumin on the HFn

surface does not significantly decrease the uptake efficacy of encapsulated doxorubicin. The cell

uptake of protein-based nanoparticles is always depended on its physicochemical properties including receptor binding ability, particle size, surface chargeability and surface functionality.30

Perhaps, the increased particle size and higher doxorubicin loading for a single HFn nanoparticle

might be the major reasons for offsetting the deceased transport efficacy mediated by TfR1

receptor. Besides, the incompletely covered TfR1 receptor binding domain on HFn surface by

ABD and albumin binding probably still play certain roles during this transport process. The


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discrepancy between cell cytotoxicity and cell uptake results probably should be ascribed to the

different DOX release rate from the nanoparticles in cell cytoplasm.

Free doxorubicin was reported to possess a relatively long terminal half-life in human body,

but most of them are rapidly distributed in many organs including bone marrow, heart, kidney,

lung and spleen, instead of storing in circulatory system or binding to serum albumin

(non-covalent) after intravenous injection. The cumulative doxorubicin mainly contributes to

most of the side effects, such as myeloid toxicity, cardiotoxicity, nephrotoxicity and other clinical manifestations.31,

32

In this work, HFn/DOX nanoparticle showed an apparent blood half-life

about 1.46 ± 0.44 hours which is a bit longer than that of free doxorubicin (0.91 ± 0.23 hours). In

contrast, ABD-HFn/DOX nanoparticle demonstrated significantly prolonged apparent blood

half-life of approximately 17.18 ± 4.30 hours, exhibiting nearly 19 times longer than that of free

doxorubicin and 12 times for HFn/DOX nanoparticle. Correspondingly, AUC value of the

ABD-HFn/DOX nanoparticle increased by approximately 21.4 and 12.2 folds compared with that

of free doxorubicin and HFn/DOX nanoparticle, separately. Unlike the majority of small

molecule chemical drugs which are deactivated mainly by hepatic metabolism, protein-based

nanoparticles could be eliminated by renal filtration, proteolytic enzymes degradation and receptors-mediated cell uptake and endocytosis.33 Any ways enlarging the particle size and steric



hindrance and the resistance to cell adhesion are expected to ameliorate the in vivo

pharmacokinetic behaviors. HSA binding to ABD-HFn increases HFn particle size from 12 nm to

nearly 30 nm in diameter, and both are far larger than the limited size of glomerular filtration barrier (about 40 kDa of molecular weight or 3.5 nm in diameter).34 Thus, instead of simply

owing to the enlarged particle size, the improved pharmaceutics behaviors of ABD-HFn/DOX

nanoparticle should be mainly attributed to the decreased HFn receptor-mediated cell uptake and

endocytosis which result from the increased steric hindrance of albumin binding on nanoparticle

surface. This masking mechanism for half-life extension of HFn fusion construct was also referred by other group’s work.18 In addition, it also should be worth noting that rat serum

albumin shows much shorter half-life of about 53 h in rat body when compared with that of HSA with nearly 3 weeks in human body.35 Despite potential metabolism differences and the distinct

binding affinity to these albumins from different species, the 19-day half-life for HSA indicates

that large improvements in the half-life of the ABD-HFn/DOX nanoparticle in human body are

reasonably expected.

CONCLUSION

In this study, a novel nanocarrier based on ferritin protein was generated by genetically

fusing with albumin binding domain. The new construct showed larger molecular size, higher


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drug-loading efficacy and post-loading particle stability, and significant half-life extension.

These optimal results indicated that fusion with albumin binding domain could be a promising

strategy to extend half-life for protein-based nanoparticles.

ACKNOWLEDGEMENT

This work was supported by the National Natural Science Foundation of China [Grant No.

21576267 and 81773623], Beijing Natural Science Foundation [Grant No. 2162041], Open

Funding Project of the National Key Laboratory of Biochemical Engineering [Grant No.

2014KF-05], and the Major State Basic Research Development Program of China (No.

2013CB733604).

The authors declare no conflicts of interest.

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Table 1. Pharmacokinetic parameters of doxorubicin and DOX-loading nanoparticles in SD rats (n = 3). Data were fitted in a single-compartment model. T1/2 a

AUC b

CLc

Vd d

MRT e

(h)

(mg/L*h)

(L/h/kg)

(L/kg)

(h)

DOX

0.91 ± 0.23

1.51 ±0.46

12.39 ± 3.54

16.06 ± 1.35

1.36 ± 0.29

HFn/DOX

1.46 ± 0.44

2.65 ± 0.68

6.96 ± 1.78

13.34 ± 0.90

2.99 ± 0.57

ABD-HFn/DOX

17.18 ± 4.30

32.24 ± 6.21

0.026 ± 0.006

0.52 ± 0.11

19.51 ± 5.56

Group

Note: a, half-life; b, area under curve; c, clearance rate; d, apparent distribution volume; e, mean retention time.



Figure 1. (A) 12% SDS-PAGE analysis, lane 1, standard maker; 2, pre-induction bacteria (ABD-HFn); 3, post-induction bacteria (ABD-HFn); 4, purified ABD-HFn; 5, pre-induction bacteria (HFn); 6, post-induction bacteria (HFn); 7, purified HFn. (B) Molecular identification of the purified ABD-HFn and HFn by MAIDI-TOF-MS.


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Figure 2. Structural characterization of the purified ABD-HFn and HFn proteins by circular dichroism. (A) Intrinsic fluorescence. (B) Size exclusion chromatography. (C) Dynamic light scattering (D).



Figure 3. Morphological characterization of the HFn (left) and ABD-HFn (right) proteins by TEM.


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Figure 4. (A) Human serum albumin binding to ABD-HFn nanoparticle. (B) Morphological observation of saturated human serum albumin binding ABD-HFn nanoparticle by TEM.



Figure 5. Determination of the binding molar ratio of HSA on ABD-HFn nanoparticle.


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Figure 6. Characterization of the DOX-loaded HFn/DOX and ABD-HFn/DOX nanoparticles. (A, C) SEC. (B, D) TEM.



Figure 7. Doxorubicin leakage from HFn/DOX and ABD-HFn/DOX nanoparticles.


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Figure 8. Proliferation inhibition effect on A549 tumor cells examined by CCK-8 assay; data were mean ± standard deviation (n=3).



Figure 9. Intracellular doxorubicin concentration in A549 cells after incubating with free doxorubicin and DOX-loaded nanoparticles; data were mean ± standard deviation (n=3), *p > 0.05, **p < 0.05.


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Figure 10. Pharmacokinetics study of free doxorubicin and DOX-loaded nanoparticles in SD rats; data were expressed as mean ± SD (n=3).


Extending half-life of H-ferritin nanoparticle by fusing albumin binding

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