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DrugDelivery |Hot Paper|
Human Serum Albumin Conjugated Nanoparticles for pH and
Redox-Responsive Delivery of aProdrug of Cisplatin
Hongdong Shi, Qinqin Cheng,Siming Yuan, Xin Ding, and YangzhongLiu*[a]
Abstract: Platinum anticancer drugs are particularly in need
of controlled drug delivery because of their severe side
effects. Platinum(IV)agentsare designed as prodrugs to
reduce the side effects of platinum(II) drugs;however, pre-
mature reduction could limit the effect as aprodrug. In this
work, ahighly biocompatible, pH and redox dual-responsive
delivery system is prepared by using hybrid nanoparticles of
human serum albumin(HSA) and calcium phosphate (CaP)
for the PtIV prodrug of cisplatin. This conjugate is very stable
under extracellular conditions, so that it protects the
platinum(IV) prodrug in HSA. Upon reaching the acidic and
hypoxicenvironment, the platinum drug is released in its
active form and is able to bind to the target DNA.The Pt–
HSA/CaPhybrid inhibits the proliferation of various cancer
cells more efficiently than cisplatin. Different cell cycle ar-
rests suggest different cellularresponses of the PtIV prodrug
in the CaP nanocarrier.Interestingly,this delivery system
demonstrates enhanced cytotoxicity to tumor cells, but not
to normal cells.
Introduction
Cisplatinisafirst-line chemotherapeutic agent effective for
alarge varietyofsolid cancers. It is well known that cisplatin
targets DNA, and hence,leads to cell apoptosis.[1,2] Due to the
nonspecific reaction of cisplatin, only asmall portion of cellular
cisplatin forms DNA cross-links.[3] Nonspecific reactions reduce
the drugefficacy andare also associated with the side effects
of cisplatin.[1, 4] To solve this problem, many delivery systems
have been designed to improve the drug efficacy of cisplatin,
such as the use of liposomes,[5] polymers,[6] inorganic nano-
materials,[7–9] and metal–organic frameworks.[10, 11]
Human serum albumin(HSA) is the mostabundant plasma
protein (35–50gL
¢1human serum),and is essential for the
transport and metabolism of fatty acids.[12] HSA accumulates
highly in activatedcells, such as those in malignant and in-
flamed tissues,tocover their increased need for amino acids
and energy.[13] Such accumulationresults from the enhanced
retention of macromolecules in tumor tissue, primarily caused
by alack of lymphatic drainage due to an impaired or absent
lymphatic system.[13] In addition, the enhanced permeability of
the vascular system and reduced clearanceinsolid tumors are
also responsible for the accumulationofmacromolecules over
40 kDa.[14] Therefore, HSA is proposed as an effective drug
carriertosites of inflammationormalignancy.[13]
Platinum anticancerdrugs are particularly in need of con-
trolled drug delivery because of their nonspecific reactions and
relatedsevere side effects. Platinum(IV) prodrugs exhibit ad-
vantages in platinum drug delivery because they are kinetically
more inert in coordination substitution than cisplatin. However,
these platinum(IV) compounds could suffer from the
disadvantage of premature reduction prior to cell uptake,[15]
resultinginundesired bindingtoplasma protein molecules,
and thus, limiting their effectiveness as prodrugs. HSA hasalso
showntoprotect platinum(IV) prodrugs in the reducing
environment of the blood, resulting in enhanced circulation
time in the blood.[15] Severalrecent works indicate that HSA
also significantly improves drug targetingtosolid tumors.[16–20]
Cell-responsive delivery is an attractive strategy for en-
hancing drug efficacy. In recent years, various cell-responsive
delivery systems, such as redox active, pH dependent,and en-
zymes, have been intensively investigated. Glutathione(GSH),
which is acellular reductant, is presentmuch more abundantly
in cells (1–10 mm)than in plasma ( 2mm).[21] Some cellular
environments, such as endosomes (pH 5.0) and lysosomes
(pH4.5), are relatively acidic.[22] In addition, solid tumors are
known to have an alteredredox state in comparison with
normaltissues due to tumor hypoxia, lower pH, and elevated
levels of GSH.[23, 24] Therefore, cellular environment responsive
delivery systemscould enhancedrug efficacy and specificity.
Nevertheless, complicated chemical syntheses are often
neededtogenerate responsive delivery systems, andthe
biological functions of thesechemical groups are barely
predictable.
Herein, we designed and synthesized abiocompatible
system,which was sensitivetopHand cellular reductants, for
[a] H. Shi,+Q. Cheng,+S. Yuan, X. Ding,Prof. Y. Liu
CAS Key LaboratoryofSoft Matter Chemistry
CAS High Magnetic Field Laboratory
DepartmentofChemistry
UniversityofScience and Technology of China
Hefei, Anhui,230026(P.R. China)
E-mail:liuyz@ustc.edu.cn
[+
+]These authors contributed equally to this work.
Supportinginformation for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502756.
Chem. Eur.J.2015,21,16547 –16554 Ó2015 Wiley-VCH Ve rlag GmbH &Co. KGaA, Weinheim16547
Full PaperDOI:10.1002/chem.201502756
the deliveryofthe PtIV prodrug of cisplatin. The PtIV prodrug
was linked to HSA to form aPt–HSA complex. Then the Pt–
HSA complex was conjugated to calcium phosphate (CaP)
nanoparticles with sizes of about 100 nm. It is knownthat
nanoparticles of such sizes can be internalized into cells
throughendocytosis.[25] Upon cellular uptake,C
aP is de-
composed with weak acidity (pH <6.0) in the endosome;thus
Pt–HSA is released from the conjugate.The PtIV prodrug is dis-
sociated from HSA in the form of cisplatin upon reduction in
cells (Scheme 1). Thus, this conjugatepossesses both pH and
redox dual-responsive properties in the cellular environment.
Results and Discussion
Preparation of the PtIV–HSA prodrug loaded CaP
nanoparticles
The PtIV prodrug loadedHSA (Pt–HSA) was prepared by the re-
action of cis,cis,trans-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CO2H)] (1)
with HSA (see the Supporting Information for details). MALDI-
TOF mass spectra showedthe mass increaseofHSA after bind-
ing of the platinum complex (Figure S3 in the Supporting In-
formation).The protein-conjugatednanoparticle (Pt–HSA/CaP)
was prepared by the formation of CaP nanoparticles in the
presenceofPt–HSA.
The preparation of protein-loaded CaP nanoparticles was
optimized with the addition of surfactants and changes to the
reactionconditions. The well-dispersed and protein-rich spheri-
cal particleswere obtained in the presence of polyethylene
glycol (PEG). The use of other surfactants, such as cetrimonium
bromide (CTAB), bis(2-ethylhexyl) sulfosuccinate sodium salt
(AOT), or Tween-80, led to alow protein loading capacity and/
or poor particlemorphology (Figure S4 in the Supporting Infor-
mation).The uniform size of Pt–HSA/CaP nanoparticles can be
obtained at arange of low temperatures (25–40 8C, see
Figure S5 in the Supporting Information).
SEM images revealed that the particlesizes were about 90–
110nm(Figure 1A). The high-resolution TEM image indicates
that the particles are nearly spherical;the contrast difference
within the particles is consistentwith the protein loading. The
DLS measurements showedthat the average hydrodynamic
radius in solutionwas 150 nm with anarrow size dispersion
(polydispersion index (PDI)=0.12;Figure 1B). The HSA content
in the hybrid nanoparticles was measured by means of TGA
with heating to 800 8C. Amass loss of 35.7 %was observed on
Pt–HSA/CaP,whereas only 15 %massloss was observed on
CaP withoutHSA (Figure 1C). These data indicatethat about
24%HSA was loaded in the Pt–HSA/CaP hybrid nanoparticles,
which corresponded to 0.27 %Ptinthe particles. The XRD
resultsfor HSA/CaP show abroad band at 2q308,which
indicates theamorphous phase of CaP in the nanoparticles
(Figure S6 in the Supporting In-
formation).
Stability of the nanoparticles
The stability of Pt–HSA/CaP was
analyzed in water and in DMEM
containing 10 %FBS. DLS meas-
urements showedthat the nano-
particles were stable for only 6h
in water and the size increased
considerably over 64 h(Fig-
ure 1D). However,the nanoparti-
cles exhibited high stabilityin
DMEM/FBS medium andthe par-
ticle sizes remained unchanged
for six days. The lack of either
PEG or HSA in the preparation
considerably reduces the stabili-
ty of CaP nanoparticles (Figure 1E). DMEM/FBS medium is
amimic of plasma and widelyused in cell cultures.The high
stabilityofPt–HSA/CaP particles in this mediumsuggests that
this hybrid is suitable for applications in drug delivery.
CD spectra were recorded on the protein released from the
nanoparticles to verify structure perturbation of the protein
during the preparation of nanoparticles. The CD spectrum of
native HSA showsanegative band between 208 and 222 nm
and apositive band between195 and 200 nm ;these indicate
well-folded protein with the formation of secondary structures.
The CD spectra werenot changed with the platinum binding.
In addition, the proteins in CaP were obtained by dissolving
the nanoparticles in MES buffer at pH 4.5 (see next section).
The protein releasedfrom CaP had aCDspectrum nearly the
same as that of the native HSA protein (Figure 1F). Thisresult
indicates that the mild preparation process does not perturb
the secondary structure of the protein.
Cellular uptake and distribution of CaP nanoparticles
The cellular uptake of HSA/CaP was analyzed on hepatocellular
carcinoma(HepG2) cells. The HSA was labeled with rhodamine
B(RhB), so that the uptake of nanoparticlescould be
visualized by fluorescencemicroscopy.The time-dependent
measurements showed that relativelyless red fluorescence
was observed in cells at 0.5 hand it increased greatlyduring
4h of incubation (Figure S7 in the Supporting Information).
Scheme1.Schematicillustration of the synthesis Pt–HSA/CaP nanoparticles and the release of cisplatin in
response to low pH and cellular reductants.
Chem. Eur.J.2015,21,16547 –16554 www.chemeurj.org Ó2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim16548
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The results indicate that the hybrid nanoparticles can efficient-
ly enter cells in atime-dependent manner.Confocal laser scan-
ning microscopy (CLSM) clearly shows that the majority of pro-
tein is located in the cytosol(Figure 2A). The cellular uptake of
RhB–HSA/CaP nanoparticleswas also measured by meansof
flow cytometry on HepG2 and HL7702 cells;this showedthat
cellular uptake increased with increasing concentration of
RhB–HSA/CaP (Figure 2B). In addition, the platinum content in
the cells was directly measured by meansofinductively cou-
pled plasma mass spectrometry (ICP-MS). The result shows
that similar amounts of Pt were detected with the treatment
of cisplatin andPt–HSA, whereas
the treatment of Pt–HSA/CaP (in
the same amount as that of Pt)
resultedinabout 70%more
uptake of platinum in cells(Fig-
ure S8 in the Supporting Infor-
mation). Although cytotoxicity
dependsonmany different
properties of drugs, these data
confirm that the Pt–HSA/CaP
particles can be efficiently inter-
nalized into cancer cells.
pH-responsive release of HSA/
CaP
Protein releasefrom HSA/CaP
nanoparticles wasstudied by
measuring the fluorescence of
HSA in solution.After the incu-
bation of HSA/CaP with buffer at
differentpHvalues, the nanopar-
ticles were removed and the
fluorescenceofHSA in solution
was measured. The result
showedthat the hybrid was
stable in neutralsolutions
(pH 7.0 and 7.4), whereas weak
acidity (pH6.0) led to the com-
plete releaseofprotein from the
nanoparticles (Figure 3). The
treatment of 1 mNaCl caused
very little HSA release from the
nanoparticles. This result implies
that the hybrid nanoparticles are
stable in extracellular media
(0.15mNaCl, pH 7.4) ;hence the
protein and PtIV prodrug are ef-
fectively protected in body
fluids. However,upon cellular
uptake by endocytosis, the hy-
brids in endosomes/lysosomes
(pH 4–6) can be disassembled
and protein is thus released.
Redox-responsive release of PtII drug and the bindingof
DNA
The PtIV–HSA complex released from CaP particles is in the
form of aprodrug, which is inert to DNA binding. Cellular re-
ductionisthe crucial step for the activation of PtIV prodrugs
prior to bindingtotarget DNA. Therefore, the reduction and
DNA binding of the Pt–HSA prodrug was investigatedbythe
reactionofPt–HSA with DNA in the presence of acellular re-
ductantascorbic acid (AsA). The platination of DNA was
analyzed by fluorescencemeasurementswith EtBr as aprobe.
Platinum binding prevents intercalation of EtBr into DNA,
Figure 1. Characterization of Pt–HSA/CaP nanoparticles.A)SEM image of Pt–HSA/CaP nanoparticles (scale bar
200 nm). The inset shows aHR-TEMimage (scale bar 20 nm). B) Size distribution of Pt–HSA/CaP nanoparticles
measured by dynamiclight scattering (DLS). C) TGA of HSA, CaP, HSA/CaP,and Pt–HSA/CaP samples underanair
atmosphere. D) The time-dependent size variation of Pt–HSA/CaP nanoparticles measured in water or Dulbecco’s
modified eagle medium (DMEM) containing10% fetal bovine serum (FBS). E) The stability of CaP nanoparticles
prepared with HSA and/orPEG. The particles were dispersed in DMEM/FBS solution and centrifuged at 3000 rpm
for 3min. F) CD spectraofnative HSA, Pt–HSA, and the protein released from nanoparticles.The proteins in CaP
particles were obtained by dissolution in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 4.5).All samples
were measuredin10mmphosphate bufferat258C, pH 7.4.
Chem. Eur.J.2015,21,16547 –16554 www.chemeurj.org Ó2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim16549
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which results in the stoichiometric quenching of fluorescence
from the EtBr–DNA complex.[26] The controlexperiment on cis-
platin confirmed that DNA platination caused aslow decrease
in the fluorescence(Figure 4). Pt–HSA and PtIV complex 1did
not alter the fluorescenceinthe absence of reductants. The
presence of 5mMAsA reduced the fluorescenceofDNA/EtBr
complextoanextent similar to that of treatment with cispla-
tin. This resultconfirmed the reduction of PtIV in the Pt–HSA
conjugate and subsequent DNA binding in the presenceofcel-
lular concentrationsofAsA (Figure 4). This result is consistent
with areport in the literature on the reduction of PtIV prodrugs
in the cellular environment,[27] and suggests that Pt–HSA can
functionasaprodrug of cisplatin and respondtothe reducing
agent AsA.
In vitro cytotoxicity of Pt–HSA/
CaP
The cytotoxicityofPt–HSA/CaP
was evaluated by 3-(4,5-di-
methylthiazol-2-yl)-2,5- diphenyl-
tetrazolium bromide (MTT) assay
on different human cells, includ-
ing five cancercell lines (hepato-
cellularcarcinomaHepG2 cells,
lung carcinoma A549 cells, cervi-
cal cancer HeLa cells, human
breast cancerMDA-MB-231cells,
and breast carcinoma MCF-7
cells) and three normal cell lines
(liver HL7702cells, human breast
MCF-10A cells, and human em-
bryonickidney HEK293cells).
The cytotoxicity of cisplatin, PtIV
compound 1,and Pt–HSA was
also measured for comparison
(Table 1). The IC50 valuesshowed
that the Pt–HSA/CaP conjugate
exhibited generally higher cyto-
toxicitythan that of cisplatin
and 1to cancer cells. The lower
IC50 value of Pt–HSA/CaP,compared with that of Pt–HSA, to
cancer cells suggeststhat the CaP carrierenhances the cyto-
toxicity of Pt–HSA. It is notable that cisplatin showed lower cy-
totoxicity to HepG2 cells than to HL7702 cells, with an IC50
ratio (HepG2/HL7702) of 2.1. Nevertheless, the conjugates of
Pt–HSA/CaP showedareverse effect with aratio of 0.33, which
suggested that the HepG2 cells could be more sensitive to Pt–
HSA/CaP than to cisplatin. The same effect wasobserved by
comparing MCF-7 cells to MCF-10Acells. Cisplatin showed
Figure 2. Intracellular uptake and distribution of RhB-labeled HSA/CaP nanoparticles.A)CLSM analysis of HepG2
cells treatedwith RhB–HSA/CaP nanoparticles for 4h.The RhB-labeled HSAshowsred fluorescence, the blue fluo-
rescence from the Hoechst 33258 stain shows the cell nuclei,and the green fluorescence from the Alexa 488 stain
shows the cytoskeleton. The mergedimaging showsthe locationofRhB–HSAprotein in cells. DAPI =4’,6-diamidi-
no-2-phenylindole. B) Flow cytometric analyses of the fluorescence intensity in HepG2 and HL7702 cells after the
4hof treatment with RhB–HSA/CaP nanoparticles.
Figure 3. The pH-responsive drug release from Pt–HSA/CaPnanoparticles.
HSA release from HSA/CaPnanoparticles was measured by fluorescence. The
particles were incubated in 100 mmsolution of MES buffer at different pH
values,inwater or in a1msolution of NaCl for 1, 4, and 8h.
Figure 4. Redox-responsive release of PtII drug from Pt–HSA and DNA plati-
nation.The reactionswere conductedonplatinumcomplexes in the ab-
sence or presence of reductant AsA.The platinationofDNA was measured
by fluorescencedecline.All reactionswere conducted with herring sperm
DNA (0.01 mg) at aPt/nucleotide ratio of 1in10mmNaClO4and 10 mm
phosphate buffer (pH 7.4) at 37 8C. EtBr (0.04mg) and 0.4 mNaCl was added
before fluorescence measurements. The excitation wavelengthwas
l=530 nm and the emission was observedatl=592 nm.
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high cytotoxicity to MCF-10Acells, whereas Pt–HSA/CaP dem-
onstrated high cytotoxicity to MCF-7 cells. Although various ef-
fects can cause different responses of cells to platinum agents
(such as cellular uptake or redox environmentfor PtIV reduc-
tion), the higher cytotoxicity of HSAconjugates is consistent
with areport that HSA can enhance the efficacy of anticancer
drugs more efficiently to cancer cells than to normalhealthy
cells.[16]
Cell cycle and apoptosis analysis
Cell cycle analysis was conducted on A549 cellswith the treat-
ment of Pt–HSA/CaP for 48 or 72 h. Apoptotic cells with
degraded DNA fragments were accumulated in the sub-G1
region.[28] Cisplatin and Pt–HSA displayed time-dependent S
and G2arrest with very little increase in the sub-G1fraction
(Figure 5). Interestingly,the Pt–HSA/CaP conjugate induced cell
cycle arrest different from that of cisplatin or Pt–HSA. Pt–HSA/
CaP did not cause much G2arrest;however,asignificantly
higher sub-G1fraction was observed for Pt–HSA/CaP (14.08 %
at 48 hand 21.05 %at72h)compared with cisplatin (2.66 %at
48 hand 5.65%at72h)and Pt–HSA (1.11% at 48 hand 1.50 %
at 72 h). This observation indicates differentcell cycle perturba-
tions of Pt–HSA/CaP,which suggests that the presence of CaP
nanoparticles alters the cellular response of platinum drugs.
This alteration could be attributed to different cell uptake and
metabolism of Pt–HSA/CaP conjugates. The CaP control in-
duced aslight G2phase arrest, probably due to the nonsponta-
neous influx of calcium by the uptake of CaP nanoparticles
throughendocytosis.[29, 30]
The tumormicroenvironmental response is an attractive
strategy for controlled drug delivery and release. These drug-
delivery systems are more stable in blood and normaltissues;
however,the specific microenvironment triggers drug release
in tumors,which results in enhanced drug efficacy andre-
duced side effects.[31] In recent years, varioustypes of pH- and
redox-responsive chemical groups have been introduced into
drug-delivery systems, such as hydrazones, esters, orthoesters,
acetals,imines, and cis-aconityls for pH responsiveness and di-
sulfide bonds for redox responsiveness.[32, 33] Complicated
chemicalsyntheses are typically required to introduce these re-
sponsivegroups into drug-delivery system. The harsh reaction
conditions might not be applicable for protein-associated sys-
tems and chemical modificationscould limit the clinic applica-
tion of these materials. Herein, we prepared apHand redox
dual-responsive delivery system by using highly biocompatible
materials with afacile and mild preparation procedure. CaP is
essential for making teeth and bones,[34] and it forms pH-
tunable particlesfor the intracellular release of drugs.[35] The
biomineralization of cisplatin, which formed with CaP and
carbonate, was found to overcome drug resistance.[36] CaP is
known to have ahigh affinity to protein ;[37] thus, we can take
advantage of this to preparePt–HSA/CaP conjugates for the
delivery of PtIV prodrugs.
To achieve redox-responsivedrug release, we take the
advantage of the PtIV prodrug strategy.Itisknown that PtIV
complexes are reduced in cells, which generates the active
form of the PtII drug for binding to target DNA. However,the
presence of extracellular reductants leads to the premature re-
ductionofPt
IV agents and limits their function as prodrugs.[38]
HSA aloneisknown to protect the PtIV agentsinthe reducing
environment of the blood.[15, 39] The formation of HSA/CaP par-
ticles furtherprotects the platinum compound from the
mediumand enhances the stabilityofplatinum agents. The
conjugation of HSA could also improve drug efficacy against
solid tumors.[40]
Bovine serum albumin (BSA) has also been used as amodel
protein for adelivery system. Thereversibleformation of BSA
particles is applicable for the delivery of therapeutic agents.[41]
The loading of the hydrophobic drug ibuprofen on BSA/CaP
particles in organic solvent re-
sults in the sustained release of
the drug.[42,43]
Herein, the PtIV prodrug conju-
gated to HSA/CaP nanoparticles
allows pH and redox dual-re-
sponsivereleaseofthe drug.
The stabilityofCaP nanoparti-
cles is important for biological
applications. We found that the
addition of PEG significantly im-
provedthe stability of HSA/CaP
nanoparticles. It is worth noting
that all materials used in the
preparation of the delivery
Table 1. Inhibitoryeffect (IC50 in Pt [mm])ofcisplatin, 1,Pt–HSA, and
Pt–HSA/CaPondifferentcells.
Cisplatin 1Pt–HSAPt–HSA/CaP
HepG2 5.340.79 13.452.87 8.29 1.34 2.91 0.56
A549 6.711.06 30.67 5.87 13.742.612.35 0.31
HeLa 5.081.64 8.76 2.477.503 0.851.66 0.12
MCF-79.840.95 72.10 8.31 50.49 6.2113.71 3.14
MDA-MB-231 2.660.54 8.27 1.373.09 0.63 1.360.15
HL7702 2.530.23 8.76 1.785.22 1.04 8.87 1.85
MCF-10A 6.771.04 55.93 3.57 46.53 5.2117.17 0.92
HEK293 6.531.12 18.95 3.87 15.14 2.016.33 1.04
Figure 5. Apoptosis of HepG2 cells by using an annexinV/PIassay treatedwith CaP, cisplatin, 1,Pt–HSA, and
Pt–HSA/CaP.
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system are highly biocompatible, including PEG. The mild con-
ditions used for the preparation of hybrid nanoparticles, such
as low temperature, short preparation time, and absence of or-
ganic solvents, are highly desirable for protecting the native
protein from denaturation.Thus,the hybrid CaP system could
also be applicable for delivering other native medicinal
proteins.
Conclusion
We designed abiocompatible, dual-responsive, drug-delivery
system by using HSA protein and CaP hybrid nanoparticles to
deliver aprodrug of cisplatin. The hybrid system was stable in
the extracellularenvironment;however,upon cellular uptake
by endocytosis, the CaP nanoparticles decomposed in the
weakly acidic environment of endosomes/lysosomes and the
HSA protein was then released.The prodrug loaded on HSA
could be reduced by cellular reductants, such as AsA, and the
active form of cisplatin be generated. The PtIV prodrug loaded
on HSA reacted with the target DNA only in the presence of
reductant.The formation of HSA/CaP nanoparticle protected
the PtIV prodrug from premature reduction in the extracellular
environment;hence, it was an ideal platform for controlled
drug delivery.Moreover,the Pt–HSA/CaP hybrid inhibited the
proliferation of various cancer cells more efficiently than cispla-
tin. Different cell cycle arrests of Pt–HSA/CaP,relative to Pt–
HSA and cisplatin, suggested adifferent cellular response of
the PtIV prodrug in the CaP nanocarrier.All materials used in
the preparation were highly biocompatible and the mild prep-
aration process led to unaffected protein folding. Therefore,
this methodcould have potential utility for the preparationof
delivery systems for proteins and drugs.
Experimental Section
Materials
MTT,AsA, and herring sperm DNA were purchased from Sangon
Biotech (Shanghai)Co.,Ltd. 1-Ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide
(NHS) were obtained from Aldrich. Ultrapure water (18.2 MW)was
obtained from aMillipore Milli-Q Biocel purification system with
a0.22 mmfilter.All other reagents and solvents were purchased
commercially and used without further purification.
Cell culture
The human cancer cells, including cervical HeLa, breast carcinoma
MDA-MB-231, hepatocellular carcinoma HepG2, breast carcinoma
MCF-7, and lung carcinoma A549 cells, were obtained from the
American Type Culture Collection (ATCC). The normal liver cells
HL7702, normal human breast cells (MCF-10A), and human embry-
onic kidney cells (HEK293) were obtained from the cell bank of the
Chinese Academy of Sciences. The cells were maintained in DMEM
containing 10%FBS (for HeLa, MDA-MB-231, HepG2 cells, MCF-7,
HL7702);RPMI1640 (for A549);DMEM-based medium containing
sodium pyruvate, 2mml-glutamine, and 10%FBS (for HEK293) ;or
DMEM-based medium containing 2.5 mml-glutamine, 20 ng mL¢1
epidermal growth factor,0.1 mgmL¢1cholera toxin, 10 mgmL¢1in-
sulin, 500 ng mL¢1hydrocortisone, and 5% FBS (for MCF-10A) in
ahumidified atmosphere containing 5% CO2at 37 8C.
Synthesis of Pt–HSA and RhB–HSA
The PtIV prodrug of cisplatin, 1,was synthesized according to apro-
cedure reported in the literature.[44] 10 mmPtIV prodrug 1was pre-
pared in HEPES buffer (50 mm)before EDC (50 mm,5equiv) and
NHS (20 mm,2equiv) were added. The mixture was incubated at
room temperature for 30 min before adding HSA (1 mm). After stir-
ring at room temperature for 24 h, the solution was dialyzed
against distilled water before the solution of Pt–HSA was
lyophilized. The product was characterized by MALDI-TOF mass
and ICP-MS.
RhB–HSA was prepared by using the same method as that for
Pt–HSA with RhB instead of 1.
Synthesis of protein-loaded CaP nanoparticles
The HSA-containing CaP nanoparticles were synthesized by the re-
action of Ca(NO3)2with (NH4)2HPO4in the presence of HSA. HSA
(20 mg) and PEG (4 g; MW =4000) were added to asolution of
Ca(NO3)2(100 mL, 2.5 mm). The pH of this solution was adjusted to
10.0 with asolution of ammonia (1 m). The mixture was stirred at
room temperature for 10 min.
A2.5 mmsolution of (NH4)2HPO4(100 mL) was prepared and the
pH was adjusted to 10.0 with asolution of ammonia, which was
added dropwise to asolution of HSA/Ca(NO3)2at arate of
10 mLmin¢1with stirring. The precipitate was collected by centrifu-
gation at 10 000 rpm for 10 min and washed twice with ultrapure
water.The precipitate of HSA/CaP nanoparticles was dispersed in
2mLofultrapure water or DMEM/FBS medium.
The Pt–HSA/CaP and RhB–HSA/CaP nanoparticles were prepared
by means of the same procedure by replacing HSA with Pt–HSA or
RhB–HSA.
To test the effect of surfactant on the formation of HSA/CaP,the
nanoparticles were prepared with different surfactants, including
CTAB, AOT,ortween-80, instead of PEG.
pH-response study
The pH-response study was conducted by measuring HSA released
from the hybrid nanoparticles in MES buffer at pH 4.5, 5.0 6.0, 6.5,
7.0, or 7.4. Buffer (200 mL) was added to asolution of HSA/CaP
nanoparticles (50 mL), then the mixture was incubated at 37 8C. Su-
pernatants were collected at 1, 4, or 8h by centrifugation at
12000 rpm for 10 min. HSA in the supernatant was quantified by
measuring the fluorescence on aHitachi F-4600 spectrophoto-
meter.
Reaction of Pt–HSAwith DNA
Herring sperm DNA was dissolved in 10 mmphosphate-buffered
saline (PBS) and the DNA concentration was measured by deter-
mining the UV/Vis absorption at l=260 nm with an extinction co-
efficient of 6600m¢1cm¢1.The platinum concentration was mea-
sured by ICP-MS. The reaction was carried out on 0.04 mg mL¢1
DNA and 0.1 mmplatinum complex in 10 mmPBS (pH 7.4) contain-
ing 10 mmAsA and 10 mmNaClO4at 37 8Cinthe dark. Samples at
different reaction times (0, 2, 4, 8, 12, 24, 36, 48 h) were taken for
DNA platination measurements by adding 0.025 mg mL¢1EtBr and
0.4mNaCl. Then the fluorescence spectra were recorded on aHita-
chi F-4600 fluorescence spectrometer by using a1cm path length
cuvette under the following conditions:scan speed 1200 nmmin¢1,
Chem. Eur.J.2015,21,16547 –16554 www.chemeurj.org Ó2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim16552
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excitation slit width 5nm, and emission slit width 10 nm. The
excitation and emission wavelengths were l=530 and 592 nm,
respectively.
Cell uptake imaging
HeLa cells were seeded in 24-well plates at adensity of 5Õ104cells
per well. After 24 hofincubation at 378Cin5%CO
2,the cells
were treated with RhB–HSA/CaP nanoparticles (in 2.6 mgmL¢1RhB).
After further incubation for different times (0, 0.5, 1, 2, 3, or 4h),
the medium was removed and cells were washed three times with
PBS. The fluorescence imaging of cells was analyzed by
fluorescence microscopy.
CLSM imaging was recorded on HepG2 cells treated with RhB–
HSA/CaP nanoparticles for 4h.Then the cells were washed there
times with PBS, and fixed in 4% paraformaldehyde. The nuclei
were stained with DAPI and the cytoskeletons were stained with
Alexa Fluor 488. After staining, the cells were washed three times
with PBS and mounted on microscope slides for imaging by using
aLeica TCS-SP5 spectral confocal laser scanning microscope (Leica
Microsystems, Germany,with two-photon system). Excitation wave-
lengths were l=364, 488, and 560 nm for DAPI, Alexa Fluor 488,
and RhB, respectively.
Cellular uptake measured by flow cytometry
HepG2 cells were seeded in 24-well plates at adensity of 15Õ
104cells per well in DMEM medium (0.5 mL) and incubated in ahu-
midified 5% CO2atmosphere for 24 h. Then the cells were treated
with RhB–HSA/CaP nanoparticles at aseries of concentrations.
After removal of the medium, cells were washed with cold PBS
three times. Then the cells were harvested and the fluorescence of
RhB in the cells was analyzed by means of aFACS Calibur flow
cytometer (BD Biosciences, USA). The results were analyzed with
Flowjo 7.6 software.
Platinum content in cells
HepG2 cells were seeded in 6-well plates and incubated overnight
in ahumidified atmosphere containing 5% CO2at 37 8C. Cells were
treated with drugs (cisplatin, Pt–HSA, or Pt–HSA/CaP at aconcen-
tration of 25 mmPt). After 2h of incubation, cells were washed
three times with PBS buffer and digested with trypsinization. The
harvested cells were digested by nitric acid before ICP-MS
measurements.
Cytotoxicity assay
The cancer cells were seeded at adensity of 1Õ103cells per well in
a96-well plate in medium (100 mL) and incubated for 24 hbefore
the addition of drug. Then the cells were incubated with different
concentrations of drugs for 72 h. Subsequently,the medium was
changed and asolution of MTT (25 mL; 5mgmL¢1in PBS) was
added to each well. After incubation for 4hto allow viable cells to
reduce the yellow tetrazolium salt (MTT) into dark blue formazan
crystals, the culture medium was removed. DMSO (150 mL) was
added to each well and the cells were shaken for 20 min. The ab-
sorbance was measured at l=570 nm by using aBio-Rad 680 mi-
croplate reader.The IC50 values were calculated by using GraphPad
Prism software (version 5.01) based on data from three parallel
experiments.
Cell cycle analyses
HepG2 cells cultured in 12-well plates were treated with cisplatin,
1,Pt–HSA, of Pt–HSA/CaP at aPtconcentration of 10 mmfor 48 or
72 h. Cells were then harvested and the DNA content was analyzed
by using aCycleTEST PLUS DNA Reagent Kit (BD Biosciences, San
Jose, USA) according to the manufacturer’s instructions. Cell cycle
distributions and DNA contents were determined by using aBD
FACS Calibur flow cytometer.The results were analyzed by using
FlowJo 7.6 software. Percentages of cells in G1(resting), S(DNA
synthesis), and G2/M (mitotic cells) phases, as well as of those
undergoing apoptosis (sub-G1), were recorded.
Acknowledgements
This work was supported by the National Basic Research
ProgramofChina (973 Program, 2012CB932502), the National
ScienceFoundationofChina (U1332210, 21171156,21573213),
and the Collaborative Innovation Center of Suzhou Nano
Scienceand Technology.
Keywords: cancer ·drug delivery ·nanoparticles ·platinum ·
proteins
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Received: July 14, 2015
Publishedonline on September 25, 2015
Chem. Eur.J.2015,21,16547 –16554 www.chemeurj.org Ó2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim16554
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