PHA-L lectin and carbohydrate relationship: conjugationwith CdSe/CdS nanoparticles, radiolabeling and in vitro affinitieson MCF-7 cells

Altan Kara • Perihan Unak • Cenk Selcuki •

Ozlet Akca • E. Ilker Medine • Serhan Sakarya

Received: 12 August 2013 / Published online: 18 October 2013

� Akademiai Kiado, Budapest, Hungary 2013

Abstract This research aims to investigate the interaction

between phytohemagglutinin-L (PHA-L) and sialic acid,

which is abundant on the breast cancer cell (MCF-7) sur-

face and displays monosaccharide characteristics, by

experimental and computational methods. Experimentally,

CdSe/CdS nanoparticles (QDs) were synthesized; PHA-L

was conjugated with QDs and labeled with 125I. Radiola-

beling yield was found to be 97 ± 1.2 %. Afterwards,

in vitro bioaffinities of radiolabeled PHA-L conjugated

QDs have been investigated on MCF-7 cells and it has been

observed that the cell incorporation increased with time.

The results indicated that 125I labeled QD-PHA-L conju-

gates represent significant affinity on MCF-7 cells. In the

second step of the study, the crystal structure of carbohy-

drate interaction surface of PHA-L was extracted from the

crystal structure of PHA-L. The interactions between this

surface and sialic acid were calculated by computational

tools. These calculations revealed specific interactions

between PHA-L and sialic acid. Semi-empirical methods,

PM3 and AM1, were used in these calculations. Significant

outcomes have been obtained from the experimental and

computational studies and these results demonstrated that

PHA-L may be an effective agent for imagining MCF-7

cells.

Keywords CdSe/CdS quantum dot nanoparticles

(QDs) � 125I � PHA-L � PM3 � AM1 � MCF-7

Introduction

Lectin modified quantum dot nanoparticles can be useful for

new applications in determination of intercellular receptor

ligand relations, immune recognition reactions because

lectins are glycoprotein molecules which can be used for

specific reversible recognition reactions. Phytohemaggluti-

nin-L (PHA-L) which is obtained from red kidney bean is

one of the major types of lectins due to its cell recognition

property. PHA-L has strong effects on cell agglutination and

mutagenic activities. It can bind to the cell membranes by

interacting with monosaccarides and oligosaccharides.

Because of these characteristics lectins are primarily used

for identification of animal cells [1]. PHA-L has a tetrameric

structure; thus, it has four surfaces which are specific to cell

surface sugars. These surfaces are specific for monosac-

charides and oligosaccharides. Because of these features,

PHA-L is specific for sialic acids which are located on the

MCF-7 cell membranes. This property allows the use of

lectins in the display of breast cancer [2, 3].

Kikkeri reported nonhuman Sia Neu5Gc causes antigen–

antibody mediated chronic inflammation, which can poten-

tially facilitate disease processes such as tumor progression

and vascular inflammation, as well as provide epitopes for

antibodies as novel cancer biomarkers and immunothera-

peutics. CdSe/ZnS Quantum Dots were used to detect and

quantify different compositions of sialoglycans containing

diverse sialic acid forms in biological by this group [4].

A. Kara � P. Unak (&) � O. Akca � E. I. Medine

Department of Nuclear Applications, Institute of Nuclear

Sciences, Ege University, 35100 Bornova, Izmir, Turkey

e-mail: perihan.unak@gmail.com

C. Selcuki

Department of Biochemistry, Faculty of Sciences,

Ege University, 35100 Bornova, Izmir, Turkey

S. Sakarya

Department of Infectious Diseases and Clinical Microbiology,

School of Medicine, Adnan Menderes University, 09100 Aydin,

Turkey

123

J Radioanal Nucl Chem (2014) 299:807–813

DOI 10.1007/s10967-013-2783-5

For that purpose, it was aimed to investigate CdSe/CdS

nanoparticles (QDs) conjugation with PHA-L and radio-

labeled to obtain an effective agent for the diagnosis of

breast cancer in this report.

Materials and methods

Materials

Chemicals were purchased from Sigma, Merck and Bio-

logical Industries. 125I was purchased from Institute of

Isotopes Co. Ltd. Budapest. Culture Media and Supple-

ments were purchased from American Type Culture col-

lection (ATCC, UK). Bradford Protein Analyses kits were

purchased from Fermentes.

Preparation of CdSe/CdS quantum dot nanoparticles

(CdSe/CdS QD)

CdSe/CdS QDs were prepared as previously described [5].

According to this method 3.5 mg of selenium tetra chloride

and 7.5 mg of sodium borono hydrate (NaBH4) in 5 mL

distilled water were put in round bottom flask. Then

1.5 mL of absolute ethanol was added. Reaction has got

begin under nitrogen (N2) on mixer at 45 �C. Colorless

solution of NaHSe in ethanol was formed about 15 min

later. H2Se gas was formed with dilution of NaHSe in

ethanol on mixer under N2 with 50 mM (3–4 mL) H2SO4.

After weighing, 0.0710 g of trisodium citrate dihydrate

was dissolved in 50 mL tris–HCl buffer (pH: 9). 0.266 g of

Cd(CH3COO) 2 2H2O was added. Reaction temperature

was set at 75 �C. H2S was formed after adding sodium

sulfide (Na2S) dilution with sulfuric acid 3–4 mL of H2SO4

at room temperature.

Preparation of QD-PHA-L conjugates

CdSe/CdS quantum-dot nanoparticles (1 mg) were dissolved

in 1 mL de-ionized water. Then, 0.5 mg of l-ethyl-3-(3-

dimetilaminopropil) carbodiimide hydrochloride (EDC) was

added to the QDs solution (pH * 7). The solution was

stirred for 2 hours and then 5 lg PHA-L was added to the

solution. After this step solution was stirred one more hour.

125I labeling QD-PHA-L conjugates

Iodogen was used for labeling of QD-PHA-L conjugates

under the same conditions as earlier described [6–13].

Certain amount of iodogen (250 or 500 lg) was dissolved

in 1 mL dichloromethane (CH2Cl2)and transferred to

closed glass tubes. CH2Cl2 was evaporated by air flow

and iodogen was deposited on the walls of glass tubes as

a thin film then these tubes were stored at -25 �C until

use.125I (1.8 lL) was added to QD-PHA-L conjugate solu-

tion. Incubation period was 20 min. After the labeling

reaction, 125I labeled QD-PHA-L was purified from unre-

acted samples with Sephadex G-50 column.

Chromatographic analyses and quality control

processes

Thin layer radio chromatography (TLRC) method

Labeling yield was controlled by TLRC. TLC silica coated

aluminum sheets with 0.1 mm thickness and 20 9 20 cm

dimension (Merck, 5554) were used. The sheets were cut

as 1.5 9 10 cm sized and labeled products were dropped

0.5 cm above from bottom of the plates using a micropi-

pette. The plates were put in a TLC tank (Sigma) without

application point not touch, after the samples dried on the

plates. ITLRC strips were developed by isopropyl alcohol-

n-butanol-0.2 N NH4OH (4-2-1). After the mobile phase

run to close point from top of the plate, the plate took over

the tank and dried at room temperature. The developed

TLC plates were scanned with using Bioscan 2000. The Rf

values and 125I labeling efficiency of QD-PHA-L conju-

gates were determined.

High performance liquid chromatography (HPLC)

HPLC studies were performed with the Shimadzu low

pressure LC-system equipped with LC-10ATvp quaternary

pump and RF-10AXL fluorescence detector. The column

was Aqua-OH 40-8 (Nucleogel). The samples were eluted

at a flow rate of 1 mL/min. Excitation and emission

wavelengths were achieved at 347 and 474 nm, respec-

tively. Column was eluted with distilled water at 25 �C.

Characterization of nanoparticles

Determination of particle morphology

The morphology of PHA-L and CdSe/CdS quantum dot

nanoparticles were determined by scanning electron

microscope (SEM). The samples were diluted with distilled

water and measured at room temperature. A carbon-coated

200 mesh copper specimen grid was glow-discharged for

1.5 min. One drop of sample suspensions were deposited

on the grid and allowed to stand for 1.5 min. After any

excess fluid was removed with filter paper, the grid was

later stained with one drop of 2 % phosphotungstic acid

and allowed to dry in air for 10 min before examination.

808 J Radioanal Nucl Chem (2014) 299:807–813

123

It vitro bioaffinities

Cell culture studies were performed using MCF-7 cells.

MCF-7 is an established cell line derived from human

breast (American Tissue Type Cu1ture Collection) and

cultured in RPMI- 640 incubated in 5 % CO2 humidified

atmosphere at 37 �C. MCF-7 cells were grown in minimum

essential medium (Eagle) 2 mM glutamine, 1.5 g/L sodium

bicarbonate, 0.1 mM non-essential amino acid, 1 mM

sodium pyruvate and 10 % fetal bovine serum (FBS).The

cells were maintained in exponential growth by subcul-

turing the cells using trypsin–EDTA (0.25 % by w/v in

Hanks’ balanced salt solution) after breeding up to covered

80 % of the plate surface and 1 9 105 cells were planted

each of the 24 well plate.

To see optimum incorporation time of 125I labeled

ligands to MCF-7 cells, time parameters were defined as

30, 60 and 120 min. Specific activity of labeled ligands for

the cells was adjusted as 3 lCi/mL.125I labeled QD-PHA-L conjugates were added to wells

of MCF-7 cells (0.75 9 105). Incubation periods were

applied as 30, 60 and 120 min as previously set. Before

labeled medium was put into flasks, the medium was dis-

carded by vacuum pump and the cells were washed three

times with PBS at the end these periods. 200 lL of RIPA

lyses buffer was put each well of the plates in order to lysed

cells. The cells were excavated using pipette tip while

solution a few times repippetting thus cells were taken to

the Ripa buffer. 100 lL of suspended solution which

contain the cells were taken to the eppendorf vial for

radioactivity analyses and 900 lL scintillator was added

(1, 2, 4-trimethylbenzene) (LSC-cocktail) to each vial.

Eppendorf vials were taken specials vials and then they

were counted in Packard Tri-carb-1200 liquid scintillation

counter at Ege University Faculty of Medicine Physiology

Department.

25 lL of remaining solution were taken for protein

analyses. Protein analyses were done using with Bicinch-

oninic acid method at 560 nm using Thermo Scientific

Multiscan Spectrum spectrometer in the Ege University

Biochemistry Department. Radioactivities per unit amount

of cells were calculated using protein analyses results. By

dividing control to these results incorporation ratios were

calculated.125I labeled QD-PHA-L incorporation to MCF-7 cells

was calculated and the results were expressed as the per-

centage of the administrated dose for per gram of pro-

tein(AD %/g protein).

Statistical analyses

One way variance analyses were applied to determine

incorporations for each parameter to understand the

statistically significant differences. P values were checked

to see 0.95 reliability between the incorporation values.

Computational methods

Semi-empirical methods

Semi-empirical methods are used in quantum mechanics

and they solve electronic Schrodinger equations [14].

These methods give results, especially for medium-sized

and large systems which are close to experimental values

because some of the parameters and assumptions were

obtained from very high quality experimental and compu-

tational data [15]. In this work, semi-empirical methods

AM1 [16] and PM3 [17, 18] as implemented in Spartan 08

software [19] are used to optimize the studied systems.

In order to calculate interactions between reactants,

quantum mechanical methods were used. To form smaller

models for lectin-carbohydrate systems, crystal structures

of studied lectins were taken from Research Collaborator

for Structural Bioinformatics Protein Data Bank (RCSB

PDB). For reliability, the most abundant form of the sialic

acid structure was taken from the same source. Carbohy-

drate specific sites on the PHA-L lectin is extracted from

the crystal structure and used in the investigation of the

interactions with the sialic acid. These structures were

optimized to obtain the most stable form of the compounds

separately. The most stable structures have been used to

analyze the interactions.

Results and discussion

In this study, we evaluated the potential of using 125I

labeled QD-PHA-L conjugates for imaging of MCF -7

breast cancer cells by using computational and experi-

mental methods.

Thin layer radio chromatography analyses of 125I

labeled conjugates

The radiochemica1 yield of 125I1abeled QD-PHA-L was

found to be 96 ± 0.8 while stayed at the origin

(Rf = 0.082). The iodination mechanism is commonly

based on the oxidation of iodide (I-). Iodogen was used for

oxidizing 125I because it is one of the most commonly used

oxidizing agent (1, 3, 4, 6-tetrachloro-3a, 6a-diphenyl-

glycouril). Nevertheless, the solid-state surface reactions

probably have very important roles on the oxidation of I-

to I? that reacts electrophilically with the substituent.

Iodogen has been used for radioiodination of some kind

molecules [5–13]. Regarding with lectins Barrientos et al.

[3] has used iodojen for radioiodination of V.album

J Radioanal Nucl Chem (2014) 299:807–813 809

123

agglutinin and Akca et al. [5], prepared 125I labeled Sam-

bucusnigra agglutinin (SNA) lectin conjugated CdSe/CdS

using iodogen method.

Determination of conjugate and PHA-L morphology

by scanning electron

Microscopy (SEM)

Figure 1 shows the SEM images of CdSe/CdS quantum

dots and QD-PHA-L conjugates. According to figures

CdSe/CdS quantum dots morphological images appear in

the form of cauliflower morphology small crystal clusters.

Mean sizes of the quantum dots were the range of

7–12 nm. However, morphology of QD-PHA-L conjugates

clearly seems to be different from PHA-L and CdSe/CdS

quantum dots. This morphological different represents the

affinity between PHA-L and CdSe/CdS quantum dots.

Time dependent study of 125I labeled QD-PHA-L

conjugates affinity to MCF-7 cells

In order to study time dependent incorporation of 125I

labeled QD-PHA-L conjugates to MCF-7 breast cancer

cells, MCF-7 cells were incubated with 125I labeled sam-

ples for various lengths of time (30, 60 and 120 min.). At

the end of the incubation periods, wells contain cells were

washed extensively and results were evaluated to observe125I labeled QD-PHA-L conjugates affinity to MCF-7

breast cancer cells. In this study, four different radiolabeled

samples (125I PHA-L, 125I CdSe/CdS quantum dots, 125I

QD-PHA-L conjugate and 125I) used to observe specificity

of 125I labeled QD-PHA-L conjugates to MCF-7 cells.

Figure 2 shows time dependent incorporations of 125I, 125I

labeled PHA-L, 125I labeled CdSe/CdS quantum dots, 125I

labeled QD-PHA-L conjugated MCF-7 cell. According to

the results, samples represented different incorporation

profiles. 125I labeled PHA-L’s represented the highest

incorporation for 30 min (12.3 ± 1.2, 12.4 ± 1.6 and

8.94 ± 0.7 for 30, 60 and 120 min) and incorporation did

not changed significantly by time. We also examined 125I

labeled CdSe/CdS incorporation and they changed about

7.2 ± 0.4, 5.0 ± 0.6 and 6.2 ± 1.0, respectively depend

on incubation periods. Lectin conjugation increased

incorporation ratios besides higher incorporations were

observed at the end of 2 h. According to these results,125I

labeled PHA-L QDs have highly elevated affinities to

MCF-7 breast cancer cells while nonlabeled 125I showed

insignificant (around 0.4 ± 0.2) affinity to MCF-7 cells.

Calculation of PHA-L affinity to MCF-7 cell

In this part of the study, we tried to calculate the interac-

tions between PHA-L and sialic acid. MCF-7 cell surface

contains excess sialic acid which is a monosaccharide [20].

PHA-L is a macromolecular protein which is specific for

monosaccharide. Because of these characteristics, it is

predicted that PHA-L can be used for MCF-7 cell recog-

nition and then calculations have been carried out to

investigate the interactions between PHA-L and sialic acid.

In these calculations we used carbohydrate specific

sequence on the surface of PHA-L. This sequence consists

of twenty one amino acids: GFSATTGINKGN-

VETNDWLSW. Some parts of this carbohydrate specific

surface correspond to the cavities in the main structure

which are more open for interactions. These parts with

sequences GINKGNV (shortened as GIN for simplicity),

GFS and LSW have been investigated separately for lectin-

Fig. 1 a Morphological image of CdSe/CdS quantum dots b QDs-PHA-L conjugates at 50,000 magnification

810 J Radioanal Nucl Chem (2014) 299:807–813

123

carbohydrate interactions. In this way, we calculated spe-

cific interactions and kept the system smaller than the main

structure in order to get more detailed results in a shorter

time. As a result of calculations we observed energy,

geometry changes and molecular interactions (i.e. hydro-

gen bond formation) in the lectin-sialic acid system. All the

calculations have been carried out with PM3 and AM1

semiempirical methods as implemented in Spartan08

software.

Table 1 displays some of the calculated properties with

AM1 and PM3 for lectin-carbohydrate systems. It is

observed that PM3 calculations overestimate the hydrogen

bonding in studied systems; therefore, the following dis-

cussion will mainly depend on the AM1 results unless

otherwise stated.

Figure 3 displays the most stable complex optimized for

sialic acid-GIN system (Sialic A-GIN-2). As seen from the

figure, the complex formed mainly by the help of a

hydrogen bond network which include both intra- and

intermolecular hydrogen bonds. The complex is more sta-

ble compared to the reactants by 48.7 kJ/mol. This exo-

thermic energy differences explained strong interactions

and new bonds formed between two molecules.

In Fig. 4 optimized complex for the sialic acid-GFS

system (Sialic A-GFS) is shown. Although new hydrogen

bonds form between the two molecules [21] similar to the

sialic acid-GIN system, AM1 results indicate that this

system does not form a stable complex. On the other hand,

Fig. 2 Time dependent

incorporations of 125I, 125I PHA-

L, 125I CdSe/CdS QDs, 125I QD-

PHA-L conjugated MCF-7 cells

Table 1 Calculated heat of formation (DH) values and complexation

energies (DECOMP) of lectin-carbohydrate systems with PM3 and

AM1 methods (Bold values indicate the values obtained by summing

the values for separated lectin and sialic acid, all other values refer to

optimized complexes)

PM3 AM1

DH

(kJ/mol)

DECOMPa

(kJ/mol)

DH

(kJ/mol)

DECOMPa

(kJ/mol)

Sialic A. 1 GIN -3664.6 -3906.7

Sialic A-GIN-1 -3682.7 -18.1 -3932.0 -25.3

Sialic A-GIN-2 -3710.0 -45.4 -3955.4 -48.7

Sialic A-GIN-3 -3685.4 -20.8 -3944.4 -37.7

Sialic A. 1 GFS -2086.1 -2304.6

Sialic A-GFS -2121.6 -35.5 -2298.8 5.8

Sialic A. 1 LSW -2097.7 -2234.0

Sialic A-LSW-1 -2116.2 -18.5 -2292.2 -58.2

Sialic A-LSW-2 -2119.3 -21.6 -2283.7 -49.7

a DECOMP = DH(complex) - (DH(lectin) ? DH(sialic acid))

Fig. 3 AM1 optimized structure of GINKGNV-sialic acid complex

Fig. 4 AM1 optimized structure of GFS-sialic acid complex

J Radioanal Nucl Chem (2014) 299:807–813 811

123

PM3 results show a very stable complex formation. This

discrepancy requires higher level calculations but the

results do not affect the general outcomes of the

calculations.

In Fig. 5, we observed new hydrogen bonds which are

shown by green dashed lines in the sialic acid-LSW system

(Sialic A-LSW-1). Thus, this part of PHA-L also showed

affinity to sialic acid which is the highest among all studied

systems. According to the calculated results, exothermic

energy difference represents strong interactions and new

bond formation between LSW and sialic acid molecules for

both of the optimized complexes. AM1 results indicate that

this tripeptidic fragment is the most probable sialic acid

binding site.

Conclusions

The size of the particles have the potential to reach many of

the body instead of quantum dot nanoparticles, they

thought that they are suitable for use in applications such as

imaging and radiotherapy.125I labeled PHA-L conjugated QDs presented elevated

in vitro bioaffinities to MCF-7 cells. It is known that MCF-7

which specific to breast cancer cells uses cell surface sialic

acid molecules for recognition according to experimental

and computational works. According to this information

after the optimization of PHA-L, sialic acid on the surface

of MCF-7 breast cancer cells, was extracted and the surface

of the crystal structure of carbohydrate in different posi-

tions were interacted with molecular modeling.

Computational methods have also been used to support

experimental results. Crystal structure of PHA-L which has

been taken from protein data bank has been used in mod-

eling to understand specific interactions between lectin

sites and sialic acid. Calculations have revealed that intra-

and intermolecular hydrogen bonds are mainly responsible

for the interactions and sialic acid can bind to lectins

effectively at more than one site.

As consequences computational results have promising

outcomes as it may be possible to design and synthesize

specific peptide sequences selectively binding to target

molecules replacing the very large lectin molecules.

It is concluded that this computational and experimental

(in vitro) study indicated the potential use of 125I labeled

QD-PHA-L conjugates as a useful tool for imaging and

therapy for MCF -7 breast cancer cells.

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