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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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enhanced efficiency. Journal of Biotechnology 2017, 254, 34-42. 30. Albanese, A.; Tang, P. S.; Chan, W. C., The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu. Rev. Biomed. Eng. 2012, 14, 1-16. 31. Di Stefano, G.; Kratz, F.; Lanza, M.; Fiume, L., Doxorubicin coupled to lactosaminated human albumin remains confined within mouse liver cells after the intracellular release from the carrier. Digestive & Liver Disease Official Journal of the Italian Society of Gastroenterology & the Italian Association for the Study of the Liver 2003, 35, (6), 428-433. 32. Mross, K.; Maessen, P.; Vandervijgh, W. J. F.; Gall, H.; Boven, E.; Pinedo, H. M., Pharmacokinetics and Metabolism of Epidoxorubicin and Doxorubicin in Humans. J. Clin. Oncol. 1988, 6, (3), 517-526. 33. Sutradhar, K. B.; Khatun, S.; Mamun, A. A.; Begum, M., Distribution and elimination of protein therapeutics: A review. Stamford J. Pharm. Sci. 2011, 4, (2), 1-12. 34. Tencer, J.; Frick, I. M.; Oquist, B. W.; Alm, P.; Rippe, B., Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways. Kidney Int. 1998, 53, (3), 709-15. 35. Holt, L. J.; Basran, A.; Jones, K.; Chorlton, J.; Jespers, L. S.; Brewis, N. D.; Tomlinson, I. M., Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Engineering Design & Selection Peds 2008, 21, (5), 283-8.
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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.
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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).
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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.
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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.
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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).
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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).
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