ORIGINAL PAPER

Anticancer and immunostimulatory role of encapsulated tumorantigen containing cobalt oxide nanoparticles

Sourav Chattopadhyay • Sandeep Kumar Dash • Totan Ghosh •

Sabyasachi Das • Satyajit Tripathy • Debasis Mandal •

Debasis Das • Panchanan Pramanik • Somenath Roy

Received: 27 February 2013 / Accepted: 30 August 2013 / Published online: 17 September 2013

� SBIC 2013

Abstract The purpose of this study is to evaluate the

prospect of using surface modified cobalt oxide(CoO)

nanoparticles as carriers of cancerantigens to human mac-

rophages. N-Phosnomethyliminodiacetic acid (PMIDA)

was used for surface modification to overcome the toxic

effect of CoO nanoparticles. Here, the phosphonate group

of the PMIDA acts as a surface-anchoring agent and the

remaining –COOH groups bind nonspecifically with tumor

associated antigens. This modification allows the conju-

gation of human oral carcinoma (KB) cell lysate (CL) as an

antigen with PMIDA coated CoO nanoparticles (CL–

PMIDA–CoO). Particle characterization was performed by

dynamic light scattering, atomic force microscopy, and

scanning electron microscopy studies. Fourier transform IR

spectroscopy was used to investigate conjugation of the

protein with nanoparticles. Protein encapsulation was

confirmed by protein gel electrophoresis. Active uptake of

antigen-conjugated nanoparticles by macrophages was

confirmed by fluorescence microscopy. The antitumor

activity of the nanocomplex pulsed macrophages was

investigated on a human oral carcinoma cell line (KB)

in vitro. The modified nanocomplexes upregulate IFN-cand TNF-a and induce an anticancer immune response by

activating macrophages. The use of TNF-a inhibitor con-

firmed the ability of the CL–PMIDA–CoO nanocomplex to

stimulate TNF-a mediated immunostimulation. CL–PMI-

DA–CoO nanoparticles efficiently increased the CD4?

population. Thus, our findings provide insight into the use

of PMIDA coated CoO nanoparticles as antigen delivery

vehicles.

Keywords Cobalt oxide nanoparticle � Antigen

delivery � Macrophage � TNF-a � CD4? �Immunotherapy

Abbreviations

AFM Atomic force microscopy

ASA Acetylsalicylic acid

cAMP Cyclic adenosine monophosphate

CL Cell lysate

CL–PMIDA–CoO Cell lysate conjugated

N-phosnomethyliminodiacetic

acid coated cobalt oxide

COX Cyclooxygenase

DC Dendritic cell

DLS Dynamic light scattering

EDC N-Ethyl-N0-(3-dimethylaminopropyl)-

carbodiimide

FCS Fetal calf serum

IFN-c Interferon-c

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-013-1044-y) contains supplementarymaterial, which is available to authorized users.

S. Chattopadhyay � S. K. Dash � S. Das � S. Tripathy �D. Mandal � S. Roy (&)

Immunology and Microbiology Laboratory,

Department of Human Physiology with Community Health,

Vidyasagar University, Midnapore

721102, West Bengal, India

e-mail: roysomenath@hotmail.com

T. Ghosh � D. Das

Department of Chemistry,

University of Calcutta,

92 A.P.C Road, Kolkata

700009, West Bengal, India

P. Pramanik

Nano Materials Laboratory,

Department of Chemistry,

Indian Institute of Technology,

Kharagpur, West Bengal, India

123

J Biol Inorg Chem (2013) 18:957–973

DOI 10.1007/s00775-013-1044-y

iNOS Inducible nitric oxide synthase

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide

NP Nanoparticle

PBS Phosphate-buffered saline

PMIDA N-Phosnomethyliminodiacetic acid

PMIDA–CoO N-Phosnomethyliminodiacetic acid

coated cobalt oxide

POF Pentoxifylline

RBC Red blood cell

SDS-PAGE Sodium dodecyl sulfate

polyacrylamide gel electrophoresis

SEM Scanning electron microscopy

Th1 Type 1 T helper

Th2 Type 2 T helper

Introduction

The use of nanoparticles (NPs) in biomedical applications

is attracting interest owing to their wide range of biotech-

nological applications in drug delivery systems [1], vaccine

administration [2], and cell separation [3]. Biodegradable

and biocompatible NPs are widely established for delivery

of protein antigens to a variety of cell types, including

dendritic cells (DCs), and have induced immunity in ani-

mals [4]. Some scientists have shown the importance of

ligand density in the outcome of an immune response to a

DC targeted NP-based vaccine delivery system [5].

Poly(lactic-co-glycolic acid) mediated antigen delivery is

more efficient than conventional soluble lysate in evoking a

favorable cytolytic CD8? T cell driven antitumor response

in vitro [6].

Metal NPs are also promising tools for delivery of

protein antigens to antigen presenting cells, including DCs,

and have induced an antitumor immune response. Single

walled carbon nanotubes successfully delivered peptide

antigens and generated an immunoresponse [7]. Metal NPs

are attracting interest especially in advanced biomedical

applications [8], including drug and gene delivery [9–11]

and protein antigen delivery [12].

Cobalt is an organometallic compound and also a bio-

polymer [13]. Cobalt has a physiological role as a cofactor

of vitamin B12. Cobalt cannot be regarded only as an

essential element. Among cobalt-based NPs, particularly

cobalt oxide (CoO) NPs are currently attracting enormous

interest owing to their unique size and shape dependent

properties and potential applications, such as pigments,

catalysis, sensors, electrochemistry, magnetism, and energy

storage [14]. Cobalt-based magnetic fluids have been

designed for possible use in medical applications owing to

the better magnetic properties and greater effects on proton

relaxation [15]. Recent developmental work has largely

focused on the use of new metal NPs such as cobalt and

nickel NPs. Cobalt NPs have the ability to enter to the cell

very rapidly [16], which has drawn the interest of

researchers to biomedical application systems based on

cobalt NPs.

Magnetic cobalt NPs are used in drug delivery in eye

surgery to repair detached retinas [17, 18]. However, the

use of cobalt NPs is restricted because of their toxicity.

Recently, it has been understood that polymeric or inor-

ganic coatings on magnetite/maghemite NPs can overcome

the toxicity issue [19]. This modification involves the

chemical modification of the surface of the magnetic NPs

with organophosphorus compounds, which offer a prom-

ising alternative in the coupling of organic components to

metal oxides [20]. The bonding of organophosphorus

molecules to the inorganic phase results from the formation

of strong M–O–P bonds through heterocondensation and

coordination. Homocondensation with the formation of P–

O–P bridges is unlikely, and such bridges are not stable in

the presence of water. Organophosphorus coupling agents

react specifically with metal oxide surfaces and assist only

monolayer formation. The resulting monolayers are highly

stable under physiological conditions [21, 22].

This study aimed to develop a potent antigen delivery

vehicle using N-phosnomethyliminodiacetic acid (PMIDA)

coated CoO (PMIDA–CoO) NPs. Our previous report

showed that PMIDA–CoO NPs have anticancer activity

without affecting normal cells [23]. In this article, we

intend to establish that PMIDA–CoO NPs have the ability

to bind with tumor-associated antigens and act as an

effective antigen delivery carrier to stimulate macrophages

and to evaluate the macrophage-mediated killing of oral

cancer cells in vitro.

Materials and methods

Chemicals and reagents

PMIDA, crystal violet, 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT), histopaque 1077,

acetylsalicylic acid (ASA), indomethacin, pentoxifylline

(POF), N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide

(EDC), and rhodamine 123 were procured from Sigma (St.

Louis, MO, USA). Anti-CD4? antibody was purchased

from Partec (Germany). Minimum essential medium,

RPMI 1640, fetal calf serum (FCS), penicillin, streptomy-

cin, sodium chloride, sodium carbonate, sucrose, Hanks

balanced salt solution, ethylenediaminetetraacetate, and

dimethyl sulfoxide were purchased from Himedia (India).

Tris(hydroxymethyl)aminomethane–HCl,

958 J Biol Inorg Chem (2013) 18:957–973

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tris(hydroxymethyl)aminomethane buffer, KH2PO4, K2HPO4,

HCl, formaldehyde, alcohol, and all other chemicals of the

highest purity grade were procured from Merck (Mumbai,

India).

Culture of the cancer cell line

The oral carcinoma cell line (KB) was obtained from the

National Centre for Cell Sciences (Pune, India) and was

cultured in MEM (Minimum essential medium) medium

supplemented with 10 % FCS, 100 U/ml penicillin,

100 lg/ml streptomycin, and 4 mM L-glutamine under a

5 % CO2 and 95 % humidity atmosphere at 37 �C for

in vitro experiments.

Cell lysate preparation of the KB cell line

The cell suspension was collected in a centrifuge tube and

centrifuged at 1,500 rpm for 5 min. The supernatant was

decanted, and the cell pellets were resuspended in ice cold

phosphate-buffered saline (PBS) at concentrations from

2 9 105 cells per milliliter and were subjected to four

freeze thaw cycles (alternating liquid nitrogen and 37 �C

water bath treatment) followed by sonication (ultrasonic

processor, Tekmar, Cincinnati, OH, USA) for 20 s on ice.

Lysates were centrifuged at 12,000 rpm for 20 min at 4 �C

to remove cellular debris. Supernatants were collected and

stored at -20 �C [6]. The protein content of the lysate

preparations was measured according to the method

described by Lowry et al. [24] using bovine serum albumin

as a standard.

Synthesis of cell lysate conjugated PMIDA–CoO NPs

PMIDA–CoO NPs were prepared by a thermal decompo-

sition technique, and were characterized by X-ray diffrac-

tion, dynamic light scattering (DLS), and transmission

electron microscopy studies as described in our previous

report [23]. PMIDA–CoO NPs (10 mg/ml) were suspended

into 5 ml PBS (pH 7.4). Six hundred microliters of lysed

cell suspension containing a protein concentration of 5 mg/

ml and 5 mg/ml EDC was added to the solution. The entire

solution was stirred in the dark for 12 h, and the particles

were separated by centrifugation at 10,000 rpm for 12 min.

Assessment of protein encapsulation and protein release

rate

The rate of release of proteins from NPs was measured

during incubation under controlled conditions [25]. A

sticky mass of 5 mg of NPs was suspended in 1 ml of PBS

and was incubated at 37 �C for different time intervals with

continuous agitation in an orbital shaker incubator. After

the incubation, the suspension was centrifuged at

10,000 rpm for 5 min at 37 �C. The supernatant was ana-

lyzed for the total protein content according to the method

described by Lowry et al. [24]. The release efficiency was

calculated using the following formula:

Cumulative protein released=total protein content of NPsð Þ� 100 %:

Sodium dodecyl sulfate polyacrylamide gel

electrophoresis analysis

To assess whether a representative selection of tumor-

associated proteins was encapsulated and released, the

protein compositions of preencapsulation and postencap-

sulation samples were compared on sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) gels by

using Coomassie brilliant blue staining. A portion of the

tumor lysate suspension served as the preencapsulation

sample. Protein samples were then boiled with 69 sample

buffer run on 12 % SDS-PAGE gels and stained with

Coomassie brilliant blue to visualize protein bands [6].

Characterization of cell lysate conjugated

PMIDA–CoO NPs

Dynamic light scattering

DLS analysis was done with a Zetasizer Nano ZS (Malvern

Instruments) according to the method of Chakraborty et al.

with some modifications. The concentration of the CoO

NPs was 100 lg/ml, the CoO NPs were sonicated for

2 min, and dynamic particle sizes were measured by sus-

pending two drops of an aqueous suspension of NPs in

10 ml of Millipore water. When the NPs had completely

dispersed in water, they were analyzed with a DLS ana-

lyzer. The experiments were repeated several times to

obtain the average size of the NPs [23].

Atomic force microscopy

The size and surface topography of a drop-coated film of

cell lysate (CL) conjugated PMIDA–CoO (CL–PMIDA–

CoO) NPs were investigated by atomic force microscopy

(AFM; NanoScope, 111a MultiMode, Veeco Instruments,

USA), and high-resolution surface images were produced.

In AFM characterization, the contact mode (NP10) with a

silicon probe over scan sizes of 10 lm was used [26].

Scanning electron microscopy

The particle size and microstructure were studied by high-

resolution scanning electron microscopy (SEM; instrument

from Nikon, Japan) according to the method of Ghosh et al.

J Biol Inorg Chem (2013) 18:957–973 959

123

with some modifications. In brief, CL–PMIDA–CoO NPs

were suspended in deionized water at a concentration of

1 mg/ml and then the sample was sonicated using a soni-

cator bath until the sample formed a homogenous suspen-

sion. For size measurement, the sonicated stock solution of

CL–PMIDA–CoO NPs (0.5 mg/ml) was diluted 20 times.

SEM was used to characterize the size and shape of the

PMIDA–CoO NPs [26].

Surface morphology analysis by Fourier transform IR

spectroscopy

The conjugation of CL with PMIDA–CoO NPs was

investigated by Fourier transform IR spectroscopy with a

PerkinElmer Spectrum RX I Fourier transform IR system

with a frequency ranging from 500 to 4,000 cm-1 and a

resolution of 4 cm-1. The KBr pellet method was used to

prepare the samples [26].

Isolation of macrophages and lymphocytes from peripheral

blood mononuclear cells

Fasting blood samples were collected from all groups of

individuals satisfying the Helsinki protocol. The peripheral

blood mononuclear cells were isolated from heparinized

blood samples according to the method of Hudson and Hay

[27]. Blood was diluted with PBS (pH 7.0) in an equal ratio

and then layered very carefully on the density gradient

(histopaque 1077) in a 1:2 ratio and centrifuged at

1,400 rpm for 20 min. The white milky layer of mononu-

clear cells was carefully removed and cultured in RPMI

1640 medium supplemented with 10 % FCS, 100 U/ml

penicillin, 100 lg/ml streptomycin, and 4 mM L-glutamine

under a 5 % CO2 and 95 % humidity atmosphere at 37 �C

for 2 h. After 2 h, the nonadherent layer of the cultured

cells was washed twice with PBS and centrifuged at

2,000 rpm for 10 min to obtain the required pellet of

lymphocytes. The adherent portion was cultured for 8 days

to obtain monocyte-derived macrophages.

Macrophage pulsing

Macrophages (104 cells per milliliter) were pulsed with

25 lg/ml CL–PMIDA–CoO NPs and 25 lg/ml ovalbumin

antigen in complete RPMI 1640 medium for 24 h at 37 �C

[8]. The dose of NPs was selected on the basis of our

previous report [23].

The cytotoxicity of protein–CoO NPs toward macrophages

and lymphocytes

Normal human lymphocytes and macrophages were seeded

into 96 wells of tissue culture plates containing 180 ll of

complete medium and were incubated for 48 h. Ovalbumin

antigen and CL–PMIDA–CoO NPs were added to the cells

at different concentrations (1, 5, 10, and 25 lg/ml), and the

mixtures were incubated for 48 h at 37 �C in a humidified

incubator (NBS) maintained at 5 % CO2. The cell viability

was estimated by MTT assay according to our previous

report [23]. The plates were read on a microplate reader

(model 550, Bio-Rad, Tokyo, Japan) at a wavelength of

570 nm. Cytolysis was calculated as a percentage.

Hemolysis assay

Ethylenediaminetetraacetic acid stabilized human blood

samples were freshly obtained from healthy subjects

according to the Hay protocol. First, 5 ml of blood sample

was added to 10 ml of PBS, and then red blood cells

(RBCs) were isolated from serum by centrifugation at

10,016g for 10 min. The RBCs were further washed five

times with 10 ml of PBS. The purified RBCs were diluted

to 50 ml with PBS. RBCs were incubated with deionized

water and with PBS and were used as the positive and

negative controls, respectively. Then, 0.2 ml of diluted

RBC suspension was added to 0.8 ml of CL–PMIDA–CoO

NP solutions at systematically varied concentrations and

mixed by gentle vortexing. The CL–PMIDA–CoO NPs

suspended in PBS solutions with different concentrations

were prepared immediately before RBC incubation by

serial dilution. All the sample tubes were kept in a static

condition at room temperature for 12 h. Finally, the

mixtures were centrifuged at 10,016g for 3 min, and

100 ll of the supernatant of all samples was transferred to

a 96-well plate for measurement of the hemoglobin con-

centration. The absorbances of the supernatants at 570 nm

were determined using a microplate reader with the

absorbance at 655 nm as the reference [28]. The con-

centration of hemoglobin was calculated using the fol-

lowing formula:

Hemoglobin gm=dlð Þ ¼ ðsample absorbancesÞ=ðHb standard absorbanceÞ � concentration of Hb standard:

ð1Þ

Cancer cell co-culture with pulsed macrophages

The pulsed macrophages were collected by trypsinization

and were centrifuged at 1,500 rpm for 5 min. The pellet

cells were washed with PBS at room temperature. After

they had been washed three times, the cells were resus-

pended in RPMI 1640 medium with 5 % FCS. KB cells

were plated in culture plates at a density of 4 9 104 cells

per well and were incubated in minimum essential medium

with 5 % FCS for 24 h at 37 �C. After the adherence of KB

cells to the plate, pulsed macrophages were added to each

960 J Biol Inorg Chem (2013) 18:957–973

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well in a ratio of 5:1 or 10:1 (macrophages to cancer cells)

and were then incubated for 1, 3, and 5 days at 37 �C [29].

Cancer cell viability assay

After co-culture, each well was washed with sterile saline

to remove the macrophages and dead KB cells. The sur-

viving KB cells were stained with 0.1 % crystal violet/

methanol at room temperature for 10 min. The plates were

read on a microplate reader (model 550, Bio-Rad, Tokyo,

Japan) at an wavelength of 570 nm. Cytostasis was cal-

culated as a percentage. The absorbance of surviving KB

cells in the absence of macrophages (control absorbance)

was set at 100 %, and the experimental absorbance was

divided by the control absorbance [29].

Uptake of protein-CoO NPs by macrophages

Macrophages (104 cells per milliliter) were pulsed with

25 lg/ml rhodamine 123 tagged CL–PMIDA–CoO NPs in

complete RPMI 1640 medium for 3 h at 37 �C. After they

had been washed twice with ice cold PBS, the cells were

incubated with 0.25 % trypsin in PBS for 3 min at 37 �C.

The cells were then fixed with 1 % paraformaldehyde in

ice-cold PBS. A fluorescence image of the cells was taken

with a phase-contrast fluorescence microscope at 9400

magnification [25].

NO release assay

The NO concentration was measured by a microplate assay

method with Griess reagent (1 % sulfanilamide, 0.3 %

naphthylethylenediaminedihydrochloride, 7.5 % H3PO4).

Briefly, culture supernatants (100 ll) were mixed with

100 ll of Griess reagent. The nitrite concentration in the

culture supernatant was measured at a wavelength of

550 nm after 10 min of mixing [30].

Co-culture of pulsed macrophages with lymphocytes

The pulsed macrophages were plated at 2 9 103 cells per

well. Lymphocytes were then added to the culture plate in a

ratio of 5:1 (macrophages to lymphocytes) and the mixture

was then incubated for 24 h at 37 �C [31]. After co-culture,

each well was washed with sterile saline. The cells were

stained with 0.1 % crystal violet/methanol at room tem-

perature for 10 min. The plates were read on a microplate

reader (model 550, Bio-Rad, Tokyo, Japan) at a wave-

length of 570 nm. Cytostasis was calculated as a percent-

age. The absorbance of surviving lymphocytes in the

absence of macrophages (control absorbance) was set at

100 %, and the experimental absorbance was divided by

the control absorbance [30].

Flow-cytometric analysis of CD4? cells

Lymphocyte surface markers were detected using mono-

clonal antibodies against CD4? conjugated with different

fluorochromes (Partec, Germany). After treatment, lym-

phocytes were incubated for 30 min at room temperature

with fluorescein isothiocyanate conjugated anti-human

CD4? monoclonal antibody. After three washings, samples

were resuspended in PBS and analyzed with a BD FAC-

SCalibur flow cytometer. The results are given as the

percentage of positively stained cells. The percentage of

CD4? T cells from different groups are shown as repre-

sentative histograms [6].

Cytokine analysis

To investigate the effect of CL–PMIDA–CoO NPs on

cytokine production, an ELISA technique was used for the

determination of interferon-c (IFN-c), TNF-a, and IL-12

production. Freshly prepared human-monocyte-derived

macrophages were cultured at 1 9 105 cells per milliliter

and were treated with various concentrations of CL–PMI-

DA–CoO (1–25 lg/ml) for 24 h. After treatment of the

NPs, cell-free supernatants were harvested via successive

10-min centrifugations (2,000, 7,000, and 13,000 rpm) and

were stored at -80 �C until analysis. ELISA was per-

formed by the protocol of eBiosciences kits, with all

samples analyzed in triplicate.

Incubation with POF

Human blood macrophages were isolated from heparinized

blood by histopaque density centrifugation as previously

described. Macrophages were co-cultured with cancer cells

in a ratio of 1:10 for 1, 3,and 5 days in RPMI medium

alone, and with 1 mM POF [32]. After incubation, cell

viability was measured by crystal violet cell proliferation

assay [30].

Incubation with ASA and indomethacin

In another experiment, ASA and indomethacin at final

concentrations of 1 mM and 50 lM [33], respectively,

were included in the reaction mixture of pulsed macro-

phages and target cells, and the protocol described in the

previous section was followed [30].

Protein estimation

Protein content was determined using bovine serum albu-

min as a standard according to the method of Lowry et al.

[24].

J Biol Inorg Chem (2013) 18:957–973 961

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Statistical analysis

The data were expressed as the mean ± the standard error

of the mean (n = 6). Comparisons between the means of

control and treated groups were made by one-way analysis

of variance (using the statistical package Origin 6.1;

OriginLab, Northampton, MA, USA) with multiple com-

parison t tests, and p \ 0.05 as the limit of significance.

Results and discussion

NPs have been used successfully to deliver macromole-

cules such as drugs, protein, genes, and human growth

factors [6]. For these purposes, different NPs have been

used, such as poly(lactic-co-glycolic acid)-coated NPs,

poly(ethylene glycol)-coated NPs, and metal NPs. Metal

NPs have been used successfully for separation of proteins,

drug delivery [34], and vaccination [7]. Recently, Cho et al.

[35] proved that CoO NPs could be a very useful adjuvant

where both type 1 T helper (Th1) and type 2 T helper

(Th2) responses are needed to clear pathogens. In our study,

we have tried to deliver antigen (CL) against oral cancer

cells (KB) to macrophages by PMIDA–CoO NPs, to

establish a potent anticancer immunotherapy.

Synthesis of PMIDA–CoO and CL–PMIDA–CoO NPs

We synthesized CoO NPs by a calcination method using

COCl2 as raw material. It was modified by conjugating

0 5 10 15 20 25

0.0

0.5

1.0

1.5

2.0

2.5

*

*

*

Pro

tein

con

cent

ratio

n (m

g/m

l)

Time point in hour

% of Protein release

Protein release assay

0 5 10 15 20 25

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

*

*

*

*

% of Protein concentration in sup

Con

cent

ratio

n of

pro

tein

(m

g/m

l)

Time point in hourProtein concentration in sup

0 5 10 15 20 250

10

20

30

40

50

60

70

*

*

*

Pro

tein

con

cent

ratio

n (m

g/m

l)

Time point in hour

% of Protein encapsulation with NP

Protein encapsulation with NP

a b

c

Fig. 1 a The percentage of protein encapsulated by nanoparticles

(NPs), b the percentage of protein in the supernatant (sup), and c the

percentage of protein released from the NPs. n = 6; values are

expressed as the mean ± the standard error of the mean. Asterisks

indicate a significant difference as compared with the control group

962 J Biol Inorg Chem (2013) 18:957–973

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PMIDA, which led to the formation of a stable aqueous

dispersion nanocluster. The formation of NPs was con-

firmed by X-ray diffraction, DLS, and transmission elec-

tron microscopy [23]. Conjugation of PMIDA with CoO

NPs was determined by Fourier transform IR spectroscopy

[23]. Then, the PMIDA–CoO NPs were stirred with cancer

cell lysate following incubation for 12 h. The pellet was

collected by centrifugation at 14,000 rpm for 20 min. It

was washed with deionized water three times and dried for

further experiments.

Characterization of CL–PMIDA–CoO NPs

Encapsulation of tumor-associated antigens in polymeric

NPs is a promising approach to enhance the efficiency of

antigen delivery for antitumor vaccines [6]. Here, we report

that PMIDA–CoO NPs were successfully bound with

cancer cell lysate antigens. Encapsulation of cancer cell

lysate antigens by NPs depends on the exposure time,

because increased protein encapsulation was observed with

increasing exposure time (Fig. 1). The concentration of

cancer cell lysate bound NPs was 63.23 % after 24 h

(Fig. 1a).

Protein (CL antigens) release was found to be time-

dependent. This finding was due to the large concentration

gradient between the CL–PMIDA–CoO complex and the

outer water phases [36, 37]. The biphasic release pattern is

potentially useful for delivery of antigens to DCs, as it

provides a continuous supply of antigens to the DCs [38].

CL–PMIDA–CoO NPs contain tumor lysate antigens of

different molecular weights. We observed the protein

release rate increased with the incubation time (Fig. 1c).

The size distribution of CL–PMIDA–CoO NPs in

aqueous medium was characterized by DLS. The mean size

of the NPs in aqueous solution was 120 ± 20 nm, as

shown in Fig 2a. This study of the NPs further confirmed

the presence of stable protein-tagged CoO NPs.

The morphology of the CL–PMIDA–CoO NPs was

studied by AFM and SEM. The AFM image and SEM

morphology of CL–PMIDA–CoO NPs showed they have

nearly spherical geometry with a mean size of

90 ± 15 nm. This finding is represented in Fig. 2b and c.

The presence of some bigger particles is attributed to

aggregation or overlapping of some small particles. The

observed NP size was approximately larger than the

hydrodynamic diameter obtained from the DLS experi-

ment. SEM describes the size in the dried state of the

sample, whereas DLS measures the size in the hydrated

state of the sample, so the size measured by DLS was a

hydrodynamic diameter and was larger. However, one has

to bear in mind that by SEM analysis we measured the

image of dried particles, whereas DLS gives an average

size estimation, which is biased toward the larger-size end

of the population distribution.

The conjugation of cancer cell lysate with PMIDA–CoO

NPs was investigated by Fourier transform IR spectros-

copy, which showed a sharp peak at 648 cm-1, indicating

the presence of M-O-M vibration, and it was shifted from

the characteristic IR band of CoO NPs at 566 cm-1 [23].

The bands in the regions of 1,078 and 1,238 cm-1 were

60 80 100 120 140 1600

20

40

60

80

Num

ber

(%)

Meandiameter (nm)

a

b

c

Fig. 2 a Dynamic light scattering histogram of the cell lysate (CL)-

conjugated N-phosnomethyliminodiacetic acid (PMIDA)-coated

cobalt oxide (CL–PMIDA–CoO) NPs, b Atomic force microscopy

image of CL–PMIDA–CoO NPs, and c scanning electron microscopy

picture of CL–PMIDA–CoO NPs

J Biol Inorg Chem (2013) 18:957–973 963

123

mainly due to the symmetric and asymmetric stretching

modes of phosphodiester groups, respectively [39, 40]. The

peaks at 1,216, 1,440, 1,666, and 3,464 cm-1 were clearly

separated and were assigned to the stretching vibrations of

the amide group and the hydroxyl group, respectively,

which were the typical protein absorption peaks. The peaks

observed at 1,072 and 2,922 cm-1 correspond to C–H

bonding due to the formation of a coordination bond

between CL and CoO (Fig. 3a, b) [41].

To confirm the protein encapsulation and release of

tumor lysate tagged CoO NPs, whole cell lysate was sub-

jected to electrophoretic separation on SDS-PAGE gels

followed by staining. Prominent bands were observed

which indicate tumor protein encapsulation (Fig. 3c). The

conjugations of protein with NPs were facilitated by EDC

and N-hydroxysuccinimide. EDC and N-hydroxy-

succinimide reacted with the surface carboxylate (–COOH)

group on the NP to yield an O-acylisourea active

intermediate. This intermediate was then attacked by a

primary amine (–NH2) group of the protein’s lysine side

chain, forming a stable covalent bond between the protein

and the NP [42].

Effect of CL–PMIDA–CoO NPs on macrophages

and lymphocytes

The toxicity of the CL–PMIDA–CoO NPs toward normal

human macrophages and lymphocytes in vitro was

checked. CL–PMIDA–CoO NP mediated cytotoxicity

toward these normal cells was measured by MTT assay

[34]. It was found that there was no significant difference in

cell viability between the cells treated with CL–PMIDA–

CoO NPs and the cells treated with ovalbumin antigen. The

cell proliferation assay showed that ovalbumin antigen

(25 lg/ml) increased the macrophage population up to

1.02-fold and the lymphocyte population up to 1.14-fold

Fig. 3 Fourier transform IR spectra of CL (a) and CL–PMIDA–CoO (b). c Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis

of the marker (lane 1), cancer cell lysate (lane 2), and CL–PMIDA–CoO complex (lane 3)

964 J Biol Inorg Chem (2013) 18:957–973

123

compared with the negative control. Hence, these NPs are

safe for biomedical applications (Fig. 4a).

Hemolytic activity

The hemolytic potentials of CL–PMIDA–CoO NPs were

assessed by incubating NPs with human RBCs for 12 h,

followed by colorimetric analysis of the hemoglobin

released. The CL–PMIDA–CoO NPs displayed no hemo-

lytic activity. The surface functionalization with cellular

protein made these metal NPs biocompatible nanocom-

plexes (Fig. 4b, c).

Effect of CL–PMIDA–CoO NP pulsed macrophages

on KB cells

The cytotoxicity of CL–PMIDA–CoO pulsed macrophages

toward KB cells was considerable (Fig. 5). We compared

all the results by using ovalbumin antigen as a positive

control. The result showed a high level of cytotoxicity

when KB cells were co-cultured with CL–PMIDA–CoO

pulsed macrophages and ovalbumin pulsed macrophages.

Our result suggested that CL–PMIDA–CoO pulsed mac-

rophages have greater cytotoxicity toward KB cells in

comparison with ovalbumin pulsed macrophages. As

shown in Fig. 5a, during the co-culture of pulsed macro-

phages with KB cells in a ratio of 5:1, there was a low

amount of cell killing on day 3 (30.36 %), but on day 5 the

killing was higher (46.22 %). At a ratio of 10:1, a signifi-

cant amount of KB cell killing induced by pulsed macro-

phages was observed on day 3 (35.62 %) and on day 5

(54.82 %) (Fig. 5b). All the results were significant at the

p \ 0.05 level.

Uptake of CL–PMIDA–CoO NPs by macrophages

The cellular internalization of NPs is an endocytic process

[43]. Cargo binds to the plasma membrane and is inter-

nalized into membrane-bound endocytic vesicles that are

transported through the cell by motor proteins moving

along the cytoskeleton [44]. From the fluorescence images

(Fig. 6), the CL–PMIDA–CoO nanocomplex was found to

be distributed in the cytoplasm, leaving a clear zone for the

nucleus, indicating cellular uptake instead of adhesion to

the surface and the NPs preferentially targeted the cancer

cells and were internalized. This internalization might be

due to the receptor-mediated endocytosis [34, 45, 46].

NO release

Stimulated macrophages secreted several cytotoxic factors

such as TNF-a and NO [47–49]. This clearly demonstrated

that the cytotoxic factors were secreted by the macrophages

when stimulated with the CL–PMIDA–CoO complex and

ovalbumin antigen. In the presence of the CL–PMIDA–

CoO complex and ovalbumin antigen, single culture of

macrophages generated a significant amount of NO

(p \ 0.05) in the medium after 1, 3, and 5 days of incu-

bation (Fig. 7a). Our result showed the presence of a high

concentration of NO in the co-culture medium of pulsed

macrophages and KB cells on day 3 (27.65 %, 59.65 %)

and day 5 (45.85 %, 62.85 %) for ratios of 5:1 and 10:1,

respectively (Fig. 7b, c), since the amount of this NO was

(+)v

e Con

trol

(-)ve

Con

trol

1 µg

/ml

5 µg

/ml

10 µ

g/m

l

25 µ

g/m

l

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

* ****

Con

cent

ratio

n of

Hb

(gm

/dl)

CL-PMIDA-CoO NP

0 5 10 15 20 25

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

***

***

* **

**

Cel

l Via

bili

ty

Nanoparticles (µg/ml)

Macrophage + TAA-PMIDA-CoO NP Lymphocytes + TAA-PMIDA-CoO NP Macrophage + OVA Lymphocytes + OVA (-)Ve Control

a

b

Fig. 4 a Cytotoxicity of ovalbumin (OVA) antigen and CL–PMIDA–

CoO NPs toward normal human lymphocytes and macrophages.

b Estimation of hemolytic activity of CL–PMIDA–CoO NPs against

normal human red blood cells. n = 6; values are expressed as the

mean ± the standard error of the mean. Asterisks indicate a

significant difference as compared with the control group. Hb

hemoglobin, TAA tumor-associated antigen

J Biol Inorg Chem (2013) 18:957–973 965

123

correlated with the cytotoxicity toward KB cells. In the

presence of ASA and indomethacin, the NO release was

54.65 % on day 3 and 69.85 % on day 5 for the highest

ratio (Fig. 7d). Several studies have evaluated the rela-

tionship between inducible nitric oxide synthase

(iNOS) and cyclooxygenase (COX)-2. However, most of

the previous works did not evaluate how COX-2 influ-

ences iNOS expression. For instance, NO donor and

NO derivatives such as peroxynitrite (ONOO2) have

been reported to induce COX-2 expression [50, 51]

Peroxynitrite, the coupling product of NO and superoxide,

activates both COX-1 and COX-2 [52]. Another report

[53] demonstrated that low-dose NO induces COX-2

expression in macrophages in which NO was found to

inhibit apoptosis. Moreover, there have been several

observations that suggest an interaction between COX-2

and iNOS. Both enzymes are expressed in the same

population of colonic epithelial cells in ulcerative colitis

patients, as well as in Barrett’s esophagus and esophageal

adenocarcinomas [45, 54]. In vitro, peroxynitrate, which

E: T- 5:1

a

Day 1 Day 3 Day 5

0

20

40

60

80

100

**

*

**

**

*

% o

f cel

l via

blity

Macrophage / KB cell

KB Cells Normal Macrophages+KB Cells

OVA pulsed Macrophages+KB Cells

CL-PMIDA-CoO pulsed Macrophages+KB Cells

E: T- 10:1

Day 1 Day 3 Day 5

0

20

40

60

80

100

**

*

**

**

*

% o

f cel

l via

blity

Macrophage / KB cell

KB Cells Normal Macrophages+KB Cells

OVA pulsed Macrophages+KB Cells

CL-PMIDA-CoO pulsed Macrophages+KB Cells

E: T- 10:1

Day 1 Day 3 Day 5

0

20

40

60

80

100*

*

*

*

*

*

*

% o

f cel

l via

blity

Macrophage / KB cell

KB Cells Normal Macrophages+KB Cells

OVA pulsed Macrophages+KB Cells

CL-PMIDA-CoO pulsed Macrophages+KB Cells

b

c

Fig. 5 Cytotoxicity of CL–PMIDA–CoO NP pulsed macrophages

co-cultured with KB cells at ratios of macrophages to KB cells of 5:1

(a) and 10:1 (b). n = 6; values are expressed as the mean ± the

standard error of the mean. Asterisks indicate a significant difference

as compared with the control group

966 J Biol Inorg Chem (2013) 18:957–973

123

is derived from NO and O2, has been shown to activate

COX [50–52].

Effect of CL–PMIDA–CoO NP pulsed macrophages

on lymphocytes

Macrophages have several important physiological

applications, including initiation of acquired immunity

such as antigen processing and presentation, production of

cytotoxic factors such as oxygen species or NO, activation

of T cells, and secretion of the cytokines that regulate

acquired immunity. The proliferation of lymphocytes with

the co-culture of pulsed macrophages clearly established

that the amount of proliferation of lymphocytes was

comparable to that with CL–PMIDA–CoO pulsed mac-

rophages (Fig. 8a). In this experiment we showed the

viability of lymphocytes but not CD4? or CD8? cells

specifically though as they are the specific marker. From

this study we tried to show the viability profile by co-

culturing freshly isolated lymphocytes with pulsed mac-

rophages, and we reveled that lymphocyte proliferation

was increased (nonsignificant at p \ 0.05) in the presence

of pulsed macrophages in culture medium. The potential

activation of primary immune cells by PMIDA–CoO NPs

is the key parameter that helps in the generation of an

anticancer immune response. After incubation with CL–

PMIDA–CoO NPs at a concentration of 25 lg/ml for 24

h, the percentage of CD4? cells was significantly

increased (Fig. 8b). PMIDA–CoO NPs promoted differ-

entiation of T cells (1.61 % in ovalbumin and 1.56 % in

CL–PMIDA–CoO NPs) and made the Th1/Th2 balance

move to the Th1 type, by significantly increasing the

CD4? cell count.

Cytokine estimation from CL–PMIDA–CoO NP pulsed

macrophages

After treatment, cell-free supernatants were used to quan-

tify cytokine levels using an ELISA. The results demon-

strated significant dose-dependent increases in the levels of

IFN-c and TNF-a at all NP concentrations tested. The

results showed that macrophages increased the production

of IFN-c by 1.44-, 1.69-, 1.75-, and 2.39-fold, TNF-a by

1.91-, 2.18-, 2.5-, and 2.76-fold, and IL-12 by 1.1-, 1.29-,

1.87-, and 2.96-fold compared with negative controls at NP

doses of 1, 5, 10, and 25 lg/ml, respectively (Fig. 9).

Nanomaterials have been shown to modulate expression of

cytokines, which are soluble biological protein messengers

that regulate the immune system. Published studies have

demonstrated the ability of certain nanomaterials to induce

cytokine production, although this appears heavily depen-

dent on a variety of factors, including material composi-

tion, size, and method of delivery [55]. Much remains to be

learned, however, regarding the proinflammatory potential

of CL–PMIDA–CoO NPs. Studies were performed to

evaluate the ability of NPs to modulate IFN-c, TNF-a, and

IL-12 cytokine production in primary human immune cells

(Fig. 9). These particular cytokines were chosen because

they represent critical pathways that are involved in the

inflammatory response and differentiation processes. The

results demonstrated significant dose-dependent increases

in the levels of IFN-c, IL-12, and TNF-a at all NP con-

centrations tested. This results confirmed the activation of

macrophages by pulsing with CL–PMIDA–CoO NPs. CL–

PMIDA–CoO NPs were capable of inducing some key

components of inflammation such as IFN-c, TNF-a, and

IL-12 in vitro. CL–PMIDA–CoO NPs induce high levels of

TNF-a, help to promote Th1 differentiation [56, 57], and

function as a regulator of acute inflammation [58]. The

ability of CL–PMIDA–CoO NPs to induce IL-12, IFN-c,

and TNF-a at NP concentrations below those causing

appreciable cytotoxicity indicates immunomodulatory

effects that may function to bias the immune response

toward Th1-mediated immunity. It is the cytokine profile

that directs the development and differentiation of T helper

cells into the two different subsets, Th1 and Th2 [56].

Relevant to our findings, IL-12 and IFN-c play a critical role in

Th1 development, and help set up a perpetuating loop whereby

more Th1 development is favored. Our findings indicate that

careful titration of CL–PMIDA–CoO NP based therapeutic

interventions may be successful in elevating the levels of a

group of cytokines important for eliciting a Th1-mediated

immune response with effective anticancer actions.

Fig. 6 Internalization of rhodamine 123 tagged CL–PMIDA–CoO

NPs into macrophages by fluorescence imaging: a gray scale and

b fluorescence images

J Biol Inorg Chem (2013) 18:957–973 967

123

Incubation with POF

The production of TNF-a by mononuclear phagocytes is

regulated by the intracellular levels of cyclic adenosine

monophosphate (cAMP) [59]. Exogenous cAMP analogues

and substances such as prostaglandin E, which are capable of

increasing the intracellular level of cAMP, reduce the release

of bioactive TNF-a by downregulating the expression of the

TNF-a gene [59, 60]. Recent studies reported the ability of

theophylline and POF, both phosphodiesterase inhibitors, to

suppress monocyte/macrophage TNF-a production by

increasing the intracellular accumulation of cAMP [61, 62].

POF is a xanthine which has been used clinically for the

treatment of vascular diseases since 1984 [63–66]. There is

increasing evidence that POF may also play a therapeutic role

in the inhibition of inflammatory processes. POF has been

shown to improve resistance against sepsis or endotoxin

challenge in mice, rats, and humans [65, 67], most likely by

decreasing circulating TNF-a levels [29]. POF is able to

inhibit the synthesis of messenger RNA for TNF-a in mouse

Day 1 Day 3 Day 50

5

10

15

20

25

30

35

40

45

50

*

*

*

*

**

µM

/mg

prot

ein

NO release by macrophages / KB cell Co-culture

Control OVA treated CL-PMIDA-CoO treated

E: T- 5:1

Day 1 Day 3 Day 50

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

* *

*

*

*

**

*

*

**

*µM

/ m

g pr

otei

n

NO release by Macrophages

Control Macrophage 1µg protein-PMIDA-CoO 5µg protein-PMIDA-CoO 10µg protein-PMIDA-CoO 25µg protein-PMIDA-CoO

a

Day 1 Day 3 Day 50

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

*

*

**

*

µM

/mg

prot

ein

NO release by Macrophages / KB cell Co-culture

Control OVA Antigens CL-PMIDA-CoO NPs

E: T- 10:1

Day 1 Day 3 Day 50

5

10

15

20

25

30

35

40

45

50

55

60

65

70

*

*

*

**

µM

/mg

prot

ein

NO release by Macrophages / KB cell Co-culture

Control OVA Antigen CL-PMIDA-CoO treated

b

c d

Fig. 7 a Release of NO after co-culture of CL–PMIDA–CoO NP

exposed macrophages. NO release after co-culture of CL–PMIDA–

CoO NP pulsed macrophages with KB cells at ratios of macrophages

to KB cells of 5:1 (b) and 10:1 (c), and in the presence of

acetylsalicylic acid (ASA) and indomethacin at a ratio of 10:1 (d).

n = 6; values are expressed as the mean ± the standard error of the

mean. Asterisks indicate a significant difference as compared with the

control group

968 J Biol Inorg Chem (2013) 18:957–973

123

peritoneal macrophages at the transcriptional level [32]. Also

in humans, POF is able to reduce the release of TNF-a by

peripheral blood macrophages [30]. The present study showed

that use of POF in culture medium increased the cancer cell

viability by 98.15 % and 95.15 % on day 1, by 94.43 % and

82.61 % on day 3, and by 88.52 % and 81.03 % on day 5 after

co-culture with CL–PMIDA–CoO NP pulsed macrophages

(Fig. 10a). The viability was increased more in the presence of

POF than in the presence of ASA. Our findings for human

peripheral blood macrophages show that TNF-a is responsible

for CL–PMIDA–CoO NP mediated anticancer therapy.

Incubation with ASA

Cell viability significantly decreased in the presence of

ASA and indomethacin in medium (63.43 % on day 3 and

Day 1 Day 3 Day 50

20

40

60

80

100

120

% o

f cel

l via

blity

Macrophage / lymphocytes

Control Lympo. OVA pulsed Macrophages+Lympo.

TAA-NP pulsed Macrophages+Lympo. a

b

Fig. 8 a Viability of

lymphocytes after co-culture

with CL–PMIDA–CoO pulsed

macrophages in a 1:10 ratio.

b CD4? cell count was

measured by fluorescence-

activated cell sorting; negative

control (I), positive control (II),

CL–PMIDA–CoO treatment

(III)

J Biol Inorg Chem (2013) 18:957–973 969

123

46.03 % on day 5) in a ratio of 10:1 (Fig. 10b). By using

inhibitors of the COX pathway [33], we have been able to

show that NP pulsed macrophages exhibit lower cytotoxic

activity against cancer cells in vitro. We found that the

cytotoxicity was inhibited in the presence of ASA and

indomethacin.

We believe that we have successfully established a

model in which cytotoxicity toward oral cancer is

exhibited by activation of normal human macrophages

with the help of the CL–PMIDA–CoO complex. This

model allows basic studies of delivery of tumor lysate to

macrophages and activated-macrophage-mediated

immunostimulation against oral cancer cells. It has

already been reported that human macrophages lack the

cofactor tetrahydrobiopterin, which is necessary to pro-

duce NO [68]. It is seen that the presence of ASA in

medium increased NO production, which is responsible

for killing cells. Besides, there are other cytotoxic factors

which are responsible for macrophage-mediated killing of

oral cancer cells [69, 70]. To find the factors involved in

anticancer activity in our experiment, we used POF, a

potent TNF-a blocker. It was found that TNF-a was

responsible for killing KB cells as evident from the

increase in KB cell viability in the presence of POF in

a

IFN-gamma

0123456789

1011121314151617 *

* *

*

*

Pg

/ ml

Control OVA Control 1µg/ml NP 5µg/ml 10µg/ml 25µg/ml

TNF-alpha0123456789

10111213141516171819

*

*

*

*

*

Pg

/ ml

Control OVA Control 1µg/ml NP 5µg/ml 10µg/ml 25µg/ml

IL-120

5

10

15

20

25

30

35

40 *

*

*

*

Pg

/ ml

Control OVA Control 1µg/ml NP 5µg/ml 10µg/ml 25µg/ml

b

c

Fig. 9 Estimation of cytokine release from CL–PMIDA–CoO NP

pulsed macrophages at different doses. Release of interferon (IFN)-c(a), TNF-a (b), and IL-12 (c) was estimated by ELISA. n = 6; values

are expressed as the mean ± the standard error of the mean. Asterisks

indicate a significant difference as compared with the control group

970 J Biol Inorg Chem (2013) 18:957–973

123

medium (Fig. 9). Inhibition of COX-2 also increases KB

cell viability, but the viability is lower than that resulting

from blocking TNF-a. From this point of view, we say

that CL–PMIDA–CoO NPs stimulate TNF-a-mediated

anticancer immunotherapy, and a probable pathway is

presented in Scheme 1.

Conclusions

The results show that PMIDA–CoO NPs efficiently bind

with tumor cell lysate antigens and deliver antigens to

macrophages. The pulsed macrophages show good anti-

cancer activity. Such a metal nanocarrier system offers

E: T- 10:1

Day 1 Day 3 Day 50

20

40

60

80

100*

*

*

*

*

*

*

% o

f cel

l via

blity

Macrophage / KB cell

KB Cells Normal Macrophages+KB Cells

OVA pulsed Macrophages+KB Cells

CL-PMIDA-CoO pulsed Macrophages+KB Cells

Day 1 Day 3 Day 50

20

40

60

80

100

**

*

% o

f cel

l via

blity

Macrophage / KB cell (10:1)

KB Cells Normal Macrophages+KB Cells

OVA pulsed Macrophages+KB Cells

CL-PMIDA-CoO pulsed Macrophages+KB Cells a

E: T- 10:1

b

Fig. 10 Viability of cancer cells after co-culture with CL–PMIDA–

CoO NP pulsed macrophage in a 1:10 ratio in the presence of

a pentoxifylline (POF) and b ASA. n = 6; values are expressed as the

mean ± the standard error of the mean. Asterisks indicate a

significant difference as compared with the control group

Scheme 1 Proposed pathway.

COX-2 cyclooxygenase 2, iNOS

inducible nitric oxide synthase,

MØ macrophage, NFjB nuclear

factor jB

J Biol Inorg Chem (2013) 18:957–973 971

123

versatility in that it can simultaneously deliver an adjuvant

with the antigen to trigger macrophages. As the study was

an in vitro study, only the direct effects of cytotoxicity

toward cancer cells were observed. Therefore, in vivo

experiments are needed to observe the influences of other

coexisting factors.

Acknowledgments The authors express their gratefulness to the

Department of Biotechnology, Government of India, for funding. The

authors also express their gratefulness to the Indian Institute of

Technology, Kharagpur and Vidyasagar University, Midnapore, for

providing the facilities to execute these studies.

Conflict of interest The authors declare that there are no conflicts

of interest.

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