FIELD OF INVENTION:
This invention relates to a method for preparing a water dispersible
glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) formulation
and use of the same.
BACKGROUND OF THE INVENTION:
Magnetic nanoparticles (MNPs) are emerging as promising candidates for
their applications in biomedical research encompassing of drug delivery,
magnetic resonance imaging, cell mechanics, hyperthermia, in vivo
tracking of stem cells, tumor progression, nucleic acid (DNA and RNA)
separation and cell separations, due to their ultra fine sizes,
biocompatibility and superparamagnetic behaviour. Another important
property which makes magnetic nanoparticles ideal for biomedical
applications is their low toxicity. The MNPs can have high level of
accumulation in the target tissues or organ due to their host cell tropism
and biophysical nature, which helps for the most promising application
of these magnetic nanoparticles in site-specific drug delivery.
For drug delivery, the magnetic nanoparticles are required to have high
magnetization values, size smaller than 100 nm and narrow distributions
of particle size. To these nanoparticles, a pharmaceutical drug can be
loaded on to the surface which could be driven to the target organ and
released there. An external localized magnetic field gradient may be
applied to a chosen site to attract drug-loaded magnetic nanoparticles
from blood circulation, by reducing their systemic distribution and
offering a possibility of administering lower but more accurately targeted
dose. In this process, the magnetic nanoparticles should bear

superparamagnetic property i.e, they do not retain any magnetic property
when the magnetic filed is removed.
Drug targeting to tumors and its other related pathological conditions, is
desirable since anticancer agents demonstrate nonspecific toxicities that
significantly limit their therapeutic potentials. For these applications, the
size, charge and surface chemistry of the magnetic nanoparticles are
particularly important, which strongly affects both the blood circulation
time as well as the bioavailability of the particles within the body. It is
envisioned that nanoparticles can be surface-modified so that it could
simultaneously function as contrast enhancement agent and drug
carrier, allowing real-time monitoring of tumor response to drug
treatment.
Surface coating is an integral part of all MNP formulations meant for
biomedical applications. The colloidal electro stabilization arising from
repulsion of the surface charge are not sufficient enough to prevent
aggregation in the biological solution due to presence of salts and other
electrolytes that may neutralize the charge. Furthermore, on intravenous
injection the MNP is subjected to the adsorption of plasma protein or
opsonization as a first step of clearance by the reticuloendothelial system
(RES). Accordingly evading the uptake by RES and maintaining a long
plasma half life is a major challenge for many MNP applications in drug
delivery. So, a polymeric coating over the MNPs is required for providing
steric barrier and to prevent nanoparticle agglomeration, thereby
avoiding opsonization. Also these coatings provide a way to functionalize
the surface of MNPs such as surface charge and chemical
functionalization. Therefore, to improve their biocompatibility and
injectibility magnetic nanoparticles are generally coated with hydrophilic

polymers such as starch or dextran, polyethyleneglycol (PEG),
streptavidin, poly-L-lysine (PLL), poly ethylene imide (PEI), and the
therapeutic agents of interest which are chemically conjugated or
ionically bound to the outer layer of polymer. This approach is complex,
involves multiple steps with a very little drug loading capacity, and the
bound drug dissociates within hours. Fast release of drug from the
carrier system may be less effective, especially in the tumor therapy,
where drug retention is required for therapeutic efficacy. Entrapping the
magnetic nanoparticles into other sustained release polymeric drug
carrier systems such as nanoparticles formulated from poly-dl-lactide-co-
glycolide (PLGA), polylactides (PLL), polylactic acid (PLA), or in
dendrimers results in significant loss in magnetization of the core
magnetic material. Also in silica coated magnetic nanoparticles there is
decrease in magnetization which has the limitation for the effective
targeting in drug delivery system.
Various monomeric species such as bisphosphonates,
dimercaptosuccinic acid and aloxysilane have been evaluated to facilitate
the anchoring and attachment of polymers on MNP. But coating of the
particle with monomeric species does not allow colloidal stability at
physiological pH. Coating the particles with large molecules, such as
polymers or surfactants containing long-chain hydrocarbons, helps to
prevent aggregation of the particles in biological solution thereby offering
more effective stabilization. Therefore, different research groups mostly
use long chain polymer such as oleic acid and its salt for the stabilization
of iron oxide nanoparticles. Gupta et al have synthesized magnetic
nanoparticles by coprecipitation method using sodium oleate for forming
stable dispersion of magnetic nanoparticles. Jain et al have developed
oleic acid (OA)-pluronic (F-127) stabilized iron oxide magnetic

nanoparticle formulation where they have entrapped some of the
hydrophobic drugs which partitioned into it without any loss of
magnetization. As in their study they found that after a coating of OA,
still these formulations were not well dispersible in. water, so they have
used pluronic types of surfactants to get water based formulation. The
pluronic acid anchors at the interface of the OA shell and give the
aqueous dispersibility and easy load of hydrophobic anticancer agents.
Experimental evidences show that higher doses of pluronic (F-127) have
toxic effects towards human erythrocytes and there is an elevation of
cholesterol and triglycerides in the blood plasma.
Therefore, with an aim of getting colloidal stability of the magnetic
nanoparticle s without use of any surfactant, a different polymeric lipid
molecule was used for coating of the MNPs. Synthetic lipid glyceryl
monooleate (GMO) approved by food and drug administration (FDA), is
an emulisifier, flavouring agent for the food industry and excipient agent
for antibiotics. The ionic polymer GMO also possesses bioadhesive
properties that can be used to enhance the therapeutic efficacy of the
dosage forms by increasing the contact time at the targeted tissues.
Glyceryl monooleate (GMO) is an unsaturated monoglyceride belonging to
the class of water-insoluble amphiphilic lipids. Depending on the water
content and temperature it forms different types of lyotropic liquid
crystals. As water content and temperature increase, it system forms
cubic phase via reversed micellar and lamellar phases. The cubic liquid
crystalline phase is highly viscous, thermodynamically stable, and
insensitive to salts and solvents and coexists in equilibrium with excess
of water and resistant to physical degradation. The high viscosity of GMO
provides sustained release of drugs due to slow drug diffusion or

increased residence time in its solubilized form. The heterogeneous
structure of GMO in water permits incorporation of both hydrophilic and
hydrophobic drugs or a combination and their presence does not induce
a change in lyotropic phase structure. GMO is a metabolite during
lipolysis of triglycerides. Also, GMO itself is an object of lipolysis due to
different kinds of esterase activity. Hence, the cubic phase made of GMO
is biodegradable and, as such a potential candidate for use in drug
delivery systems. GMO has a similar long chain polymer structure as
that of oleic acid, mainly used in the formulation of MNPs. Keeping in
view of these properties of GMO; we have coated the magnetic
nanoparticles with GMO by replacing OA. We have developed a novel
aqueous based ultrafine stable magnetic nanoparticle formulation with a
coating of GMO without the use of any surfactant. The aqueous solubility
of the particle is achieved by the complete removal of the un-adsorbed
GMO during the washing process with the use of different organic
solvents during the synthesis process. We hypothesize that, GMO coated
MNP will be a ideal delivery system for the treatment of cancer as the
hydrophobic drug would partition into the GMO coating and would
provide aqueous dispersibility of the solutions without any loss of
magnetization and at the same time drug loaded MNPs can be used as a
novel drug delivery system with the help of external magnetic field.
Bioseparation
MNPs are beneficial in biomedical research for separating out the specific
biological entities from their native environment in order to concentrate
the samples for further analysis. It is possible due to attraction between
an external magnetic field and the MNPs which enables the separation of
a wide variety of biological entities. Use of biocompatible MNPs is one of

the ways to achieve this. It is a two step process involving i) tagging or
labeling of the desired biological entities with magnetic material and ii)
separating out these tagged entities via fluid based magnetic separation
devices. Labeling is achieved through the surface modification of
magnetic nanoparticles with dextran, phospholipids and Polyvinyl
alcohol (PVA) which provides the link between the particles and the
target site on a cell or molecules. To aim for specific binding on the
surface of the cells, the help of antibody and antigen specificity action
can be taken into account. For active binding the cells are targeted with
biological molecules such as hormones and folic acid. Precision binding
of antibodies specifically to their corresponding antigens provides an
accurate way to label cells e.g MNPs coated with immunospecific agents
have been successfully bound to red blood cells HIV- tat peptides, lung
cancer, bacteria, urological cancer cells and golgi vesicles. The magnetic
separation of target cells from mixtures, such as peripheral blood,
isolation of cancer cells in blood samples or stem cells in bone marrow
has considerable practical potential in improved diagnosis in biomedical
research. When combined with microfluidic technology, low-field
magnetic separations could enable faster and less expensive processing
of tissue samples for biomarker detection. Furthermore, MNPs can be
biologically activated to allow the uptake of cells via endocytic pathways,
thereby allowing certain cellular compartments to be specifically
addressed. Once taken up, the desired cellular compartments can be
magnetically isolated and accurately studied using proteomic analysis.
There are two main challenges to make all the above-discussed
biomedical applications come true: a) a good synthesis route for
manufacturing monodisperse MNPs with diameters <10 nm; and b) a
good method to functionalize the surface of the nanoparticles. The latter
determines the ability of the MNPs to interact in a well-defined and
controllable manner with living cells and to be used

for the cell separation. We have functionalized acid groups on the surface
of the GMO coated magnetic nanoparticles by the use of DMSA (2, 3
meso mercapto succinic acid) which can be further conjugated with the
primary amine groups of any peptide or protein etc.
OBJECTS OF THE INVENTION:
An object of this invention is to propose a method for the preparation of
glyceryl monooleate (GMO) magnetic nanoparticles (MNPs) formulation;
Another object of this invention is to propose a method for the
preparation of glyceryl monooleate (GMO) magnetic nanoparticles (MNPs)
formulation having good aqueous dispersibility;
Still another object of this invention is to propose a method for the
preparation of glyceryl monooleate (GMO) magnetic nanoparticles (MNPs)
formulation which can effectively be used as a carrier for both the
hydrophilic and hydrophobic drugs;
Further, object of this invention is to propose a method for the
preparation of glyceryl monooleate (GMO) magnetic nanoparticles (MNPs)
formulation which has no toxicity;
Yet another object of this invention is to functionalize the formulation
and then to attach any protein or peptide.

SUMMARY OF THE INVENTION:
The resulted magnetic nanoparticles formulation further can be loaded
with different therapeutic drugs and functionalized with different
chemical groups for further conjugate with different peptides, proteins or
targeting moiety.
According to this invention there is provided a method for preparing a
water dispersible glyceryl monooleate (GMO) magnetic nanoparticles
formulation comprising an iron oxide particle core coated with long chain
polymer for producing an aqueous dispersible magnetic nanoparticle
formulation.
A method for preparing glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs) formulation comprising:
heating a mixture of Fe (III) and Fe (II) with constant stirring under N2
atmosphere;
adding ammonium hydroxide to the said mixture;
adding glyceryl monooleate (GMO) to the suspension drop wise;
subjecting the mixture to the step of stirring under N2 atmosphere;
washing the formulation several times with combination of ethylacetate
and acetone (70:30) to wash the excess glyceryl monooleate (GMO);
subjecting the washed formulation to the step of lyophilization to yield
powder form.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING:
Figure 1: Effect of washing in different organic solvents on size of glyceryl
monooleate (GMO) magnetic nanoparticles (MNPs) in water and n-hexane
measured by laser light scattering (data as mean ± SEM, n = 3).
Figure 2: (a) water solubility test. (i) After sonication
(ii) Settling down of particles after
one month
(10 mg of glyceryl monooleate (GMO) magnetic nanoparticles (MNPs)
dispersed in 10 ml of Milli Q water, sonicated in ice bath for 30 seconds,
kept for one month. Even after one month they demonstrated excellent
colloidal stability in an aqueous phase).
(b) Localization of glyceryl monooleate (GMO) magnetic nanoparticles
(MNPs) near magnet (NdFeB).
(i) Without magnet
(ii) With magnet
(c ) Solubility test of magnetic nanoparticles in oil and water phase. (10
mg of glyceryl monooleate (GMO) magnetic nanoparticle (MNP) dispersed
in 10 ml of Milli Q water, sonicated in ice bath for 30 seconds. To this 10
ml of hexane was added. As the hexane has lower density than water it
goes up and shows a phase separation. As the formulated particles are
water dispersible they remain in the aqueous phase).

Figure 3: (a) Effect of GMO conc. On sedimentation of MNPs in water.
(b) Mean particle size of glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs) in water and n-hexane measured by
laser light scattering (data as mean ± SEM, n = 3).
Figure 4: (a) FT-IR spectra of a) pure GMO and b) GMO-MNPs.
(b) FT-IR spectra of GMO coated MNPs: a) Uncoated MNPs, b)
10% GMO coated MNP, c) 15% GMO coated MNP, d) 20%
GMO coated MNP, e) 25% GMO coated MNP and f) 100%
GMO coated MNP.
(c ) Zoom of the FTIR spectra in the range of 3700 cm1 to 3200
cm"1.
(d) Zoom of the FTIR spectra in the range of 2800 cm4.
(e) Zoom of the FTIR spectra in the range of 1200 cm-1 to 1000
cm-1.
(f) Schematic representation of chemisorption of GMO on to the
MNP surface.
Figure 5: (a) Effect of different surfactants on the dispersibility of glyceryl
monooleate (GMO) magnetic nanoparticles (MNPs) in water.
(b) Effect of different surfactants on the size of glyceryl
monooleate (GMO) magnetic nanoparticles (MNPs) in water.

Figure 6: Comparison of size of different percentage (w/w) GMO coated
MNPs with and without surfactant Span 65.
Figure 7: (a) TEM of Iron oxide particles in water.
(b) TEM of Iron oxide particles in n-hexane.
(c) Particle size distribution of glyceryl monooleate (GMO)
magnetic nanoparticles (MNPs) measured by TEM (Average
values of twenty measurements).
Figure 8: (a) XRD powder pattern of MNPs.
(b) XRD powder pattern of glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs)
Figure 9: Selective Area Diffraction (SAD) pattern of native iron oxide
showing different rings.
Figure 10: Magnetization curve of native Iron oxide nanoparticles as a
function of field, measured at 10 K and 300 K
Figure 11: (a) Schematic representation of functionalization of glyceryl
monooleate (GMO) magnetic nanoparticles (MNPs) with
carboxylic groups.
(b) Effect of DMSA concentration on the number of acid
groups present per gram of DMSA coated glyceryl
monooleate (GMO) magnetic nanoparticles (MNPs).

Figure 12: (a) FT-IR spectra of DMSA.
(b) FT-IR spectra of glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs) modified by DMSA:
a) uncoated MNP, b) 0.2 M DMSA, c) 0.4 M DMSA, d) 1.6 M
DMSA coated MNP.
Figure 13: Schematic representation of drug adsorption in the GMO
coating surrounding the iron oxide core.
Figure 14: (a) Release of rapamycin from glyceryl monooleate (GMO)
magnetic nanoparticles (MNPs) under in vitro condition.
The drug loading in glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs) was 7.3% (data as mean ± SEM, n =
3).
(b) Release of paclitaxel from glyceryl monooleate (GMO)
magnetic nanoparticles (MNPs) under in vitro condition.
The drug loading in glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs) was 7.5%. (Data as mean ± SEM, n =
3).
(c) Release of paclitaxel and rapamycin from glyceryl
monooleate (GMO) magnetic nanoparticles (MNPs) in a
combination drug formulation in vitro condition (data as
mean ± SEM, n = 3).

Figure 15: (a) Antiproiferative effect of drugs in solution and loaded in
glyceryl monooleate (GMO) magnetic nanoparticles (MNPs)
with paclitaxel in MCF-7 cells. Cells were treated with drug
either in solution or in glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs), medium was changed on days 2 and
4 and cell viability was measured using MTT assay on day
5 (data as mean ± SEM, n = 6).
(b) Antiproiferative effect of drugs in solution and loaded in
glyceryl monooleate (GMO) magnetic nanoparticles (MNPs)
with rapamycin in MCF-7 cells. Cells were treated with
drug either in solution or in glyceryl monooleate (GMO)
magnetic nanoparticles (MNPs), medium was changed on
days 2 and 4 and cell viability was measured using MTT
assay on day 5 (data as mean ± SEM, n = 6).
(c) Antiproiferative effect of drugs in solution and loaded in
glyceryl monooleate (GMO) magnetic nanoparticles (MNPs)
with (paclitaxel + rapamycin) in MCF-7 cells. Cells were
treated with drug either in solution or in glyceryl
monooleate (GMO) magnetic nanoparticles (MNPs), medium
was changed on days 2 and 4 and cell viability was
measured using MTT assay on day 5 (data as mean ± SEM,
n = 6).
Figure 16: Antiproliferative effect of hydrophobic drugs in MCF-7 cells: IC
Values of paclitaxel, rapamycin and combination of
paclitaxel and rapamycin (1:1 w/w ratio) in solution (grey
bar) and in glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs) (white bar) data as mean ± SEM, n =
6).

DETAILED DESCRIPTION OF THE INVENTION:
Iron (III) chloride hexahydrate (FeCl3.6H2O) pure granulated, 99%, Iron
(II) chloride tetrahydrate (FeCl2.4H2O) 99%, Ammonium hydroxide, 2, 3
meso mercapto Succinic Acid (DMSA), Tween 80, Pluronic F-127, span
series, stannous chloride, mercuric chloride, orthophosphoric acid,
potassium dichromate and potassium bromide were purchased from
Sigma-Aldrich (St. Louis, MO). Glyceryl monooleate was procured from
Eastman (Memphis, TN). FITC-BSA (Albumin from Bovine Serum
Flurescien conjugated) was procured from Invitrogen Corporation,
Carlsbad, CA, USA. N-(3-Dimethylaminopropyl)-N'-ethyl-Carbomdiimide
hydrochloride (EDC) and N-Hydroxy Succinimide (NHS) were procured
from Fluka, Sigma Aldrich, Belgium. Barium diphenylamine sulphonate
(BDAS) was procured from Acros Organics, Belgium. Paclitaxel,
rapamycin were obtained from Shaanxi Schiphar Biotech Pvt Ltd, China.
Magnet NdFeB (12200 G) procured from Edmund Scientific, Tonawada,
NY). All other chemical used were of reagent grade obtained from Sigma.
MilliQ water purged with nitrogen (N2) gas was used in all steps involved
in the synthesis and formulations of magnetic nanoparticles.
Synthesis of Magnetic nanoparticles.
Synthesis of magnetic particles were done according to the protocol of
Jain et al with little modifications. Accordingly, 0.1M Fe (III) (1.35 g FeCb
dissolved in 50 ml N2 purged water) and 0.1 M Fe (II) (0.99 g FeCb
dissolved in 50 ml N2 purged water) were prepared. 15 ml of 0.1 M Fe (III)
and 7.5 ml 0.1 M Fe (II) were mixed and heated at 80°C for 10 minutes
under constant stirring with a magnetic stirrer in N2 atmosphere. 1.5 ml
of ammonium hydroxide (14.5 M) was added to it. Then it was stirred for

20 minutes. Finally the precipitate was washed with N2 purged water
with centrifugation at 20,000 rpm for 20 minutes at 10°C (Sigma
centrifuge, 3-16PK, Germany). The pellets were dispersed in 5 ml of
MilliQ water and frozen at -80°C and were lyophilized using a lyophilizer
(LABCONCO Corporation, USA) for two days at temperature of -48°C and
0.05 mbar. The MNP yield was determined by weighing the lyophilized
powder and was found to be 110 mg.
Formulations of Magnetic nanoparticles
Different formulations of iron oxide nanoparticles were developed by the
following protocol. 15 ml 0.1 M Fe (III) and 7.5 ml 0.1 M Fe (II) was
mixed and heated at 80°C with constant stirring. 1.5 ml of ammonium
hydroxide (14.5 M) was added drop wise to it. Then GMO was added to
the suspension drop wise. To study the amount of concentration of GMO
required to coat the MNPs, we have prepared different formulations
(different weight percentage of GMO to MNP yield were added i.e, 12-560
pi of GMO was added to get 10-504% of GMO coated MNPs). The mixture
was allowed to stir for 20 minutes at 80 °C under a N2 atmosphere to
evaporate the excess amount of ammonia from the formulation. It was
washed with different solvents and centrifugation for 20 minutes at 10 °C
at 20,000 rpm (Sigma centrifuge, 3-16PK, Germany). Washing was
repeated for three times. The washings of the excess GMO from the
magnetic nanoparticles is critical to get a better aqueous dispersibility.
To study the effect of different solvent washings on the GMO coated
magnetic nanoparticles (GMO-MNPs), different solvents like acetone,
ethyl acetate, diethyl ether, chloroform, and mixture of different solvents
in different ratio like ethyl acetate: acetone (50:50 and 70:30) were used
during the washing steps. The pellets were lyophilized for two days at
temperature of -48°C and 0.05 mbar to get the powder form.

To study the effect of different surfactants on aqueous dispersity of
GMO-MNPs, 10 mg of glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs) were taken and dissolved in 10 ml of MilliQ water and was
sonicated for 1 minute at 55 watt (VC505, Sonics Vibracell, Sonics and
Materials Inc., USA). To this different surfactants were added in the ratio
of particle: surfactant (1:1) and was allowed for over night stirring in a
closed container to minimize exposure to atmospheric oxygen to prevent
oxidation of the MNPs. These particles were washed three times with
water to remove the surfactants which were not bounded to the MNPs by
magnetic decantation and lyophilized to get the powder form for further
use.
Characterization of glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs)
Particle size determination by Dynamic Light Scattering and ζ potential
Measurements.
Dynamic light scattering (DLS) was used to measure the hydrodynamic
diameter and Laser Doppler Anemometry (LDA) was used to determine
the zeta potential (mV) of glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs). The DLS and LDA analysis were performed using a
Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The particle size
measurement was done by dispersing MNPs (~1 mg/ml) in MilliQ water
using water bath sonicator for 1 minute and then the suspension was
diluted (100 μl to 1 ml) and the size was measured in polystyerene
cuvette using the Zetasizer Nano ZS. To compare the size of the MNPs in
organic solvent, the measurement of particle size in n-hexane was made
following the same procedure using the quartz cuvette. To further see the

effect of size in respect to the different surfactants added to the glyceryl
monooleate (GMO) Magnetic nanoparticles (MNPs) (~1 mg/ml) surfactant
coated glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) were
suspended in MilliQ water and sonicated using water bath sonicater for 1
minute at 55 watt (VC505, Sonics Vibracell, Sonics and Materials Inc.,
USA) and further diluted (100 μl to 1 ml) for particle size measurement.
The same suspension in MilliQ water was used for measuring the zeta
potential of MNPs.
Transmission Electron Microscopy (TEM).
The internal structure of MNPs were determined by TEM measurements
for which a drop of diluted solution of the glyceryl monooleate (GMO)
Magnetic nanoparticles (MNPs) (either in water or n-Hexane) was placed
in carbon-coated copper TEM grid (150 mesh, Ted Pella Inc, rodding, CA)
and was allowed to air-dry. The samples were imaged using a Philips 201
transmission electron microscope (Philips/FEI Inc, Barcliff, Manor, NY).
The TEM photograph was taken by using the NIH imaged software. To
calculate the mean particle diameter, 50 particles were taken for
measurement.
X-ray Diffraction (XRD)
XRD analysis was carried out to know the crystallinity of the MNPs
formed. The lyophilized samples (~ 500 mg) of native iron oxide particles
and 100% glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs)
were carried out using a Brucker D4 Endeavour, with Bragg-Brentano-
Brentano parafocusing geometry. The analysis was done with copper

target X-ray tube with Cu Ka radiations. The parameters chosen for the
measurement were 2θ steps of 0.08°, 1 second of counting timer per
step, and 2θ range from 10.01° to 69.53°.
Determination of Iron content in the magnetic nanoparticle formulations.
To determine the percentage of iron present in the MNP formulations, the
chemical analysis of the samples was carried out by recommended
analytical procedure. Different glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) formulations (in triplicate) were subjected to di-acid
digestion for wet chemical analysis. The MNP formulations (~ 50 mg)
were first digested by adding 2 ml concentrated HC1 followed by heating
at 60°C for 10 minutes. Then the digested product was diluted to 25 ml
with MilliQ water. To the above diluted sample (5 ml), 2 ml of
concentrated HC1 was added and heated at 60°C for 10 minutes. 4 ml of
0.25 M stannous chloride was added drop wise to the digested product
up to decolouration. Then the sample was cooled to room temperature
and 2 ml of saturated mercuric chloride was added and was mixed well
by shaking. To the mixture, 10 ml of Zimmerman-Reinhard reagent (5 ml
of 5% sulphuric acid and 5 ml of orthophosphoric acid) was added
followed by addition of 10 ml of MilliQ water. Finally, the iron content in
the formulation was analyzed volumetrically by titrating against 0.01 N
potassium dichromate solution using barium diphenylamine sulfonate
(BDAS) indicator.
Fourier Transform infrared spectroscopy (FT-IR).
FT-IR measurement was carried out to know the chemical interactions in
the MNP formulations. FT-IR (Perkin Elmer, FTIR Spectrometer,
SPECTRUM RX 1) was used to characterize the surface composition of

the different formulations of MNPs. Each spectrum was obtained by
averaging 32 interferograms with resolution of 2 cm-1 in the range of 400
to 4000 cm-1. A small amount of MNPs (either native or formulated) were
milled with KBr, and a mixture of them was pressed into a pellet for
analysis with a pressure of 150 kg/cm2.
Magnetization Studies
In order to quantify the amount of magnetism present in the formulated
MNPs magnetization study was carried. The Magnetic properties were
investigated by a Superconducting Quantum Interference Device (SQUID)
magnetometer (MPMS5, Qunatum Design) with fields upto 1.5 T and
temperatures of 10 K and 300 K respectively. Zero-field-cooled (ZFC) and
field-cooled (FC) magnetization measurements were carried out as a
function of temperature. To determine the ZFC measurements the
samples were cooled from 300 K to 10 K in zero fields as a function of
temperature at 100 Oe field strength as gradually warmed. To take the
FC measurement, the sample as cooled in the measuring field. The
magnetization was determined as a function of field M (H) at 10 and 300
K. By putting the magnetization curve in an analytical ferromagnetic
model and by normalizing the diamagnetic contribution (x) due to the
background the saturation magnetization (Ms) and the Coercive field (Hc)
were determined.
Loading of anticancer drugs in Magnetic Nanoparticles.
To exploit the MNP formulations as a drug delivery vehicle, anticancer
drugs were taken into account. For the incorporation of anticancer drugs

in glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs), paclitaxel,
rapamycin and a combination of both (paclitaxel and rapamycin) were
used. We have used 100% GMO coated MNPs for drug loading. 100 mg of
the glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) were
dispersed in 10 ml MilliQ water and was sonicated for 1 minute. The
drugs were dissolved in organic solvent acetonitrile either individually or
in combination (10% w/w to the polymer i.e, 10 mg of either of the drugs
dissolved in 1 ml or 1 ml of combined drugs, 5 mg each). The drug was
added drop wise to the glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) suspension and kept for overnight stirring with a
magnetic stirrer to allow the partitioning of the drug into the GMO shells
surrounding the magnetic nanoparticles. The un-partitioned drugs were
washed with water and were separated by centrifuging the particle
suspension at 13, 800 rpm for 10 minutes at 10°C (Sigma centrifuge, 3-
16PK, Germany). Washing was repeated for three times for the complete
removal of the un-entrapped drug. The pellets were lyophilized for
quantification of entrapment efficiency of different drugs through reverse
phase high performance liquid chromatography (RP-HPLC).
Quantification of drug by RP-HPLC
Quantification of the drug incorporated in the MNPs, was carried out
through RP-HPLC. The estimation of the amount of drug entrapped in
the glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) was done
by direct method. To the lyophilized nanoparticles solvent acetonitrile (1
mg/ml) was added and sonicated in an ice bath for 1 .minute, at 55 watt
and kept in shaker for 24 hours for the drug to come out from the
particles. Then the nanoparticles were centrifuged for 10 minutes at 13,
800 rpm at 10°C (Sigma microcentrifuge, 1-15PK, Germany).

Supernatants were taken out for the estimation of drug entrapped. The
analysis of sample was done by reverse phase isocratic mode of HPLC
with little modification using Agilent 1100 (Agilent technologies,
Waldbronn Analytical Division, Germany) which consists of a column
(Zorbax Eclipse XDB-C18, 150 X 4.6 mm, i.d). 20 μl of different drug
samples were injected manually in the injection port and were analyzed
with the mobile phase of acetonitrile: water (80:20 v/v), which was
delivered at flow rate of 1 ml/min with a quaternary pump (Model No-
G1311A) at 25°C with thermostat (Model No-G1316A). The drug levels
were quantified by UV detection at 228 nm for paclitaxel and 278 nra for
rapamycin with a detector (DAD, Model -G 1315A). The amount of drug
(paclitaxel and rapamycin) in samples was determined from the peak
area correlated with the standard curve. The standard curves of
paclitaxel and of rapamycin were prepared under identical conditions.
The entrapment efficiency was calculated from the following formula
reported earlier.
% of Entrapment Efficiency = (drug loaded in nanoparticles/drug added
in formulation) x 100
Kinetics of Paclitaxel and Rapamycin Release from Magnetic
nanoparticles.
To know the amount of drug released in in vitro condition a kinetics
measurement was done. The release of drugs from glyceryl monooleate
(GMO) Magnetic nanoparticles (MNPs) was carried out by dissolving 10
mg of nanoparticles in 3 ml of PBS (pH = 7.4, 0.01 M, containing 0.1%
w/v of Tween 80). Tween 80 was used in the buffer to maintain the sink
condition during the release study. It was mixed properly by vortexing

and then was divided into three parts, 1 ml each. All the samples were
kept in an orbit shaking incubator (Wadegati Labequip, India) at 37°C,
rotating at 150 rpm. The samples were removed at predetermined time
intervals and centrifuged at 13, 800 rpm for 15 minutes at 10°C (Sigma
microcentrifuge, 1-15 PK, Germany) to get the supernatant. Then the
pellets were dispersed with the same volume of fresh PBS (pH= 7.4, 0.01
M PBS, containing 0.1 % w/v of Tween 80) and vortexed and kept in
shaker. The collected supernatants were lyophilized for 48 hours, and
then were dissolved in acetonitrile and centrifuged at 13, 800 rpm for 10
minutes at 4 °C (Sigma microcentrifuge, 1-15PK, Germany). The obtained
supernatant was taken out and injected in the RP-HPLC to determine the
amount of drug released either paclitaxel, rapamycin or combination of
both with respect to different time intervals.
Cell Culture
The cell culture experiments were carried out in MCF-7 (breast cancer)
cell line purchased from American Type Culture Collection (ATCC,
Manassas, VA) were grown in RPMI 1640 medium (Himedia Laboratories
Pvt. Ltd., Mumbai, India) supplemented with 10% fetal bovine serum
(Himedia Laboratories Pvt. Ltd., Mumbai, India) and 100 ug/ml
penicillin G and 100 μg/ml streptomycin (Gibco BRL, Grand island, NY)
at 37°C in a humidified and 5% CO2 atmosphere (Hera Cell, Thermo
Scientific, Waltham, MA).

Statistical Analysis
Statistical analyses were performed using a Student's t test. The
differences were considered significant for p values of <0.05.
Mitogenic Assay.
To find out the cytotoxicity of the anticancer drugs, mitogenic assay was
carried out. The MCF-7 cells were seeded at 5,000 per well in 96 well
plate (Corning, NY, USA) and kept in the incubator for 24 hours for
better cell attachment. Different concentrations of paclitaxel, rapamycin
or combination of the drug (0.1 uM to 1000 uM), either in solution or
loaded in glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) were
added. Glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs)
without drug and medium were used as respective controls. The medium
was changed on 2nd and 4th days following the drug treatment; no further
dose of drug was added. Viability of the cells was determined at 5th day.
After the specified incubation time, 10 μl MTT (Sigma) was added, and
the plates were incubated for 3 hours at 37 °C in a cell culture incubator
(Hera Cell, Thermo Scientific, Waltham, MA), following which the
intracellular formazan crystals were solubilized in dimethyl sulfoxide and
the color intensity was measured at 540 nm using a microplate reader
(Synergy HT, BioTek Instruments, Inc., Winooski, VT). The
antiproliferative effect of different treatments was calculated as a
percentage of cell growth with respect to respective controls.

Surface Functionalization of Magnetic Nanoparticles
MNPs are difficult to bond with biomoleculs in aqueous solution.
Therefore, to attach any biomolecule on to the surface of the MNPs, the
surface should be functionalized with different functional groups like
carboxylic or amine group. To attach any peptide or protein on to the
surface of the MNPs, the particles should be surface functionalized with
carboxylic groups. Therefore, 2, 3 meso mercapto succinic acid (DMSA)
was used to functionalize the glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) with carboxylic acid groups. 500 mg of glyceryl
monooleate (GMO) Magnetic nanoparticles (MNPs) was added to 5 ml of
0.2 M DMSA dissolved in DMF and kept for 24 hours stirring in a
magnetic stirrer. The sample was washed with ethanol three times by
centrifuging at 13, 800 rpm at 10°C for 20 minutes and the pellets were
lyophilized. To find out the effect of DMSA in the functionalization of
glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs), we have used
different concentrations of DMSA solutions (0.4 - 3.2 M) and followed the
above procedure to get the lyophilized powder.
Acid Number Determination.
For the quantification of free carboxylic acid groups attached on the
surface of MNPs, acid numbers of the glyceryl monooleate (GMO)
Magnetic nanoparticles (MNPs) were determined by the experimental
protocol 20 mg of the different concentration of DMSA coated glyceryl
monooleate (GMO) Magnetic nanoparticles (MNPs) were initially treated
with 5 ml NaOH (1 N) for 30 minutes to cleave some of the surface ester
bonds to generate free carboxylic ends. Then the samples were washed
three times with MilliQ water by centrifuging at 13,800 rpm at 10°C for

20 minutes. Then all the samples were vacuum dried by lyophilizer. Free
acid groups present on the glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) surface were quantified by taking nanoparticle
solution 1 mg/ml and diluting to 50 times. Then the diluted solution was
titrated against NaOH (0.0005 N). NaOH solution is to be standardized
before by titrating against oxalic acid. Acid number was calculated by the
following formula.
Volume required during titration * Normality of NaOH * 40 (Mol. Wt. of NaOH)
A=
Weight of nanoparticles (g)
Conjugation of FITC-BSA
FITC BSA was conjugated to the carboxyl groups, which were
functionalized on the surface of glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs). For conjugation, 10 mg of functionalized glyceryl
monooleate (GMO) Magnetic nanoparticles (MNPs) were added to 5 ml of
PBS (pH = 7.4, 0.02 M). 250 ul of EDC and 250 ul of NHS in PBS (pH =
7.4, 0.02 M, 1 mg/ml) was added to it. The sample was left in room
temperature under magnetic stirring for 4 hours. Then the sample was
magnetically decanted to remove free EDC and NHS. To the pellet 3 ml of
PBS (pH = 7.4, 0.02 M) and 100 ul of FITC-BSA (1 mg/ml) was added.
The solution was left for 2 hours and then incubated at 4 °C overnight.
Next day magnetic decantation was done and the pellets were washed
two times with PBS (pH =7.4, 0.02 M) to remove any unconjugated FITC-
BSA. A standard plot for FITC-BSA was prepared taking concentrations
2.5-20 μg/ml at λex= 488 nm and λem =520 nm using a flouroscence

microplate reader (Synergy HT, BioTek Instruments, Inc., Winooski, VT).
The percentage of conjugation of FITC-BSA to the glyceryl monooleate
(GMO) Magnetic nanoparticles (MNPs) was calculated by indirect method.
First, the amount of un-conjugated FITC-BSA present in the supernatant
was determined by taking the fluorescence measurement and using the
standard plot of FITC-BSA. Then the amount of un-conjugated FITC-BSA
was deducted from the total FITC-BSA amount added to get the amount
of conjugated FITC-BSA.
Physical characterization of Magnetic Nanoparticles (MNPs).
Due to the hydrophilicity nature of the native iron oxide particles, they
preclude dispersibility in organic solvents. During coating of GMO to the
magnetic nanoparticles, GMO gets chemisorbed on the surface of the
iron oxide particles. The hydrophobic nature of the GMO makes the GMO
coated magnetic particles easily dispersible in the organic solvents. The
use of nanoparticles for the drug delivery purpose, it is better to have a
water dispersible formulation. For getting a good water dispersible
formulation, excess amount of GMO has to be washed off from the
surface of the MNPs. Therefore different organic solvents like acetone,
ethyl acetate, diethyl ether, chloroform, and mixture of different solvents
in different ratio like ethyl acetate: acetone (50:50 and 70:30) was tried
as the washing solvent. These solvents were used for washing during the
centrifugation to remove the excess un-adsorbed coating from the
surface of iron oxide particles. It has been found that when acetone,
diethylether, ethylacetate and chloroform were used alone as the washing
solvent during the processing of the MNPs, the resulted nanoparticles
were having a considerable size of around 130 nm in organic solvent but

they posses a higher size range and poor dispersibility in water (Fig. 1).
Therefore a combination of two organic solvents i.e, ethyl acetate and
acetone (having inter miscibility behavior) with varying ratios (50:50 and
70:30 v/v) were employed in the washing steps to remove the excess
amount of GMO. It has been found that with washing in ethyl acetate:
acetone (70:30 v/v) , the resulted MNPs were with good particle diameter
around 144 nm (Fig. 1 and Table 1) and also having better water
dispersibility (Fig. 2c). Therefore further works were carried out with a
mixture of ethyl acetate and acetone in 70: 30 v/v ratios. Both ethyl
acetate and acetone are dipolar aprotic solvents. They help to remove the
excess hydrophobic coating from the magnetic nanoparticle surface. This
in turn results in better aqueous dispersibility.
For the absolute covering of iron oxide nanoparticles with GMO, it is very
much critical to know the optimum percentage of coating for their
eventual dispersion in hexane or water. Particles were prepared with an
increase in GMO concentration (of the total formulation content). With an
increase in GMO concentration there is less particle sedimentation and
good dispersibility in water (Fig 2a) giving the MNPs a better colloidal
stability up to around one month (Fig 2a). With the increase in GMO
concentration, the obtained glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) gives narrow range of particle diameter both in
water and hexane (Fig 3b). Thus our experimental outcomes reveal that
the GMO coating on the surface of the iron oxide particles is required to
give stable liquid crystals.

FTIR
To analyze the surface chemistry of the native MNP and glyceryl
monooleate (GMO) Magnetic nanoparticles (MNPs), the FT-IR
measurements of the nanoparticles were taken and shown in (Fig 4a).
The spectra of pure GMO shows the peaks at 1730 cm"1, 3400 cm1 and
2937 cm-1 which corresponds to the ester bond, O-H stretch dimmer H
bonded and CH2 stretching modes respectively. The spectra of GMO
coated iron oxide nanoparticles shows absence of the ester bond C=O
stretch present at 1730 cm-1. This suggests the adsorption of ester group
of GMO onto the surface of nanoparticles. Further, in the coated
particles, the vibrational stretch of COO- at 1400 cm-1 gradually
increases with increase in percentage of GMO. Also, a band at 1166 cm-1
due to the absorbance of C-0 stretch gradually increases with increase in
concentration of GMO. This finding indicates that the ester group is
chemisorbed onto the surface in carboxylated form with oxygen atom
coordinated to the nanoparticle surface (Fig 4f). These results reveal
significance of surface interaction between GMO and the iron oxide
nanoparticle surface for preparation of magnetic nanoparticles
completely coated with GMO. The strong IR band at 584 era4 is the
characteristic of the Fe-O vibration found in the native MNPs. After
coating of GMO to the MNPs the peak of Fe-O slightly shifts to ~592 cm-1.
Furthermore, the appropriate amount of GMO required for steric
stabilization of the nanoparticles was also investigated. The (Fig 4b)
represents the infrared spectra of iron oxide nanoparticles coated with
different concentrations of GMO. The peak at around 1165 cm4 (Fig 4e)
corresponding to C-O stretch starts to appear for concentrations of GMO
more than 15% used in the formulation. Furthermore, as the GMO
concentration increases, the intensity of asymmetric CH2 stretch at 2922
cm"1 (Fig 4d) and C-H deformation vibration at 1056 cm-1 (Fig 4e)

increases. Also, the intensity of OH vibration band at around 3400 cm1
(Fig 4c) gets broader as the GMO concentration increases.
Further, to know the role of surfactant over the aqueous dispersibility of
the GMO-MNPs, various range of surfactants such as pluronic (F-127)
and Span series (80, 20, 85, 60, 65) were tested (Fig 5a). The effect of
different surfactants on the aqueous dispersibility of the coated magnetic
nanoparticles revealed that Span 65 gives the best water dispersibility
with particle size about 148 nm (Fig 5b). But if we will compare the size
of native glyceryl monooleate (GMO) Magnetic nanoparticle (MNP) and
glyceryl monooleate (GMO) Magnetic nanoparticle (MNP) with span 65
there is no significant change (Fig 6). So the result revealed that the
GMO coated MNPs give a better water dispersibility and better particle
size even in the absence of any surfactant. The shape, size, and
uniformity of the particles were also determined by TEM images. The
picture shown in (Fig 7a) shows that the particles are shaped spherically
and monodispersed with a size of below 10 nm. In hexane also the
glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) show
spherical shape with monodispersibility (Fig 7b). A statistical analysis of
the size of GMO coated MNP population varies from 6 nm to 11 nm with
a mode value of 7 nm (Fig 7c). The particle size measured by TEM is less
than that measured by laser light scattering because, laser light
scattering measures the hydrodynamic diameter where there is some
aggregation of the MNPs.
X-ray diffraction analysis is a technique which reveals information about
the crystallographic structure, chemical composition, and physical
properties of materials. X-ray diffraction patterns of the native iron oxide

particles revealed diffraction peaks at 110, 220, 311, 400, 422 and 511
which are the characteristic peaks of the Fe3O4 crystal with a cubic
spinel structure. The position and relative intensity of all diffraction
peaks were identical with standard spectra of magnetite. Here, no peaks
corresponding to γ- Fe2O3 and a Fe2O3 like 210, 213 etc are observed.
This suggests that there are no impurities like a ferric oxide in the
glyceryl monooleate (GMO) Magnetic nanoparticle (MNP) formulations.
The XRD pattern of GMO coated MNP showed the same peaks at the
same position, but, the intensity of the magnetite peak at 311 is less
than that of native iron oxide particle (Fig. 8a and 8b). This lowering of
intensity of the magnetite peak is due to the GMO coat over the MNP
surface. The rings in the selected Area Diffraction (SAD) image shown in
(Fig 9) were consistent with a cubic inverse spinal structure of magnetite
and it indicates the good crystallinity of the nanoparticles. The
characteristic d spacing corresponds to the hkl values, {111}, {220}, {311},
{400}, {422}, {511}. These results showed a good coincidence with the
XRD data. The SAD values also correspond to the standard atomic
spacing for Fe3O4 along with respective hkl indexes from the Joint
Committee on Powder Diffraction Standards (JCPDS) card number (19-
0629) (Table 2).
Iron content analysis
The iron content in the formulated glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) must be sufficient to respond to an external
magnetic field. Therefore the amount of iron content in the nanoparticles
was determined. The determination of iron content in the GMO coated
magnetic nanoparticles revealed that the Iron content in native iron oxide
particles was found to be 70.37%. Iron content in 100% w/w glyceryl

monooleate (GMO) Magnetic nanoparticles (MNPs) was found to be
67.57%. When the percentage of GMO was increased to 504% w/w the
iron content in the nanoparticles was decreased to 63.11%.
SQUID
The saturation magnetization Ms at 10 K and 300 K and the coercivity
He of native MNPS and glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) are shown in (Table 3). The Ms values were
normalized assuming 100% magnetite for the simplicity using iron mass.
The SQUID analysis shows typical hysteresis curves at 10 and 300 K for
the optimized nanoparticles formulation as depicted in (Fig 10). The
hysteresis loop have negligible coercivity at room temperature, and the
magnnetisation at 1.5 T (after subtracting the diamagnetic background)
were 50.4 ± 0.3 emu/g magnetite for 504% glyceryl monooleate (GMO)
Magnetic nanoparticles (MNPs), 52.2 ± 0.7 emu/g magnetite for 100%
glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) and 63.73 ±
0.7 emu/g magnetite for uncoated MNPs at 300 K. The nanoparticles were
not superparamagnetic at 10 K. The saturation magnetizationat 10 K for
glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) were higher
than that of uncoated MNPs and hysteresis developed. From the
magnetization values it can be measured that 100% w/w glyceryl
monooleate (GMO) Magnetic nanoparticle (MNP) has actually only 18.0%
w/w GMO coating where as 504% w/w glyceryl monooleate (GMO)
Magnetic nanoparticle (MNP) has only 20.4 % w/w GMO on the surface.

Functionalization and characterization of MNPs.
The most unique feature of magnetic nanoparticles is their response to a
magnetic force (Fig 2b), and this feature has been utilized in applications
such as drug targeting and bioseparation including cell sorting. Since
magnetic nanoparticles are attracted to a high magnetic flux density, it is
possible to manipulate cells labeled with magnetic nanoparticles using
external magnets. To make the magnetic nanoparticles usable for cell
sorting or bioseparation purpose first the particle should be
functionalized through a coating or encapsulation of specific chemical
group or charge because the MNPs are difficult to bond directly with
biomolecules in an aqueous solution. To attach any biomolecule like
peptide or protein or any primary amine on to the surface of the
magnetic nanoparticles, the particles should be surface functionalized
with carboxylic group or amine groups. In this study, we have
functionalized -COOH groups on to the surface of the glyceryl
monooleate (GMO) Magnetic nanoparticles (MNPs) (Fig lla). The
functionalization of magnetic nanoparticles with carboxylic group was
achieved by coating with DMSA which was confirmed by FTIR and acid
number determination from the nanoparticles.
In the FTIR spectra of pure DMSA (Fig 12a), the peak at 1701 cm-1
corresponds to C=O stretch. The peaks around 2550 cm-1 and 3850 cm"1
corresponds to the S-H stretch and OH stretch respectively. The (Fig 12b)
depicts that the uncoated glyceryl monooleate (GMO) Magnetic
nanoparticle (MNP) and the DMSA coated glyceryl monooleate (GMO)
Magnetic nanoparticles (MNPs) have a strong absorbance of Fe-O bond at
around 580 cm1 and absorbance of O-H stretch at around 3400 cm"1.
After coating of DMSA to the glyceryl monooleate (GMO) Magnetic

nanoparticles (MNPs), the peak at 1701 cm-1 corresponding to C=O
stretch in pure DMSA can be located in DMSA coated glyceryl
monooleate (GMO) Magnetic nanoparticles (MNPs) with a shift to around
1650 cm1 and the intensity increases as we go on increasing the
concentration of DMSA from 0.2 to 1.6 M. Another vibrational mode at
around 1376 cm"1 is assigned to C-O stretch which also increases with
increase in concentration of DMSA. So the FTIR data suggests the
attachment of carboxylic group to the surface of the glyceryl monooleate
(GMO) Magnetic nanoparticles (MNPs). The attachment of carboxylic
groups on to the surface of the glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) can also be confirmed by chemical analysis
through acid number determination by acid base titration method. Acid
number is an important parameter to quantify the free carboxylic acid
groups present on the surface of the MNPs. The (Fig lib) shows the
effect of concentration of DMSA on the acid number of the MNPs. There
is an increase of acid number from 8 to 130/gm of MNPs with an
increase of concentration of DMSA from 0.2 M to 1.6 M. But further
increase in concentration of DMSA does not significantly change the acid
number, which shows a saturation binding of DMSA on the MNPs
surface. Therefore, glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs) with 1.6 M DMSA coating were selected for further experiments.
FITC-BSA was taken as a model protein to determine the efficacy of the
carboxylic group functionalized glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs). The conjugation of FITC-BSA was done to the
glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) coated with
1.6 M DMSA. The conjugation efficiency was calculated by taking the
absorbance at λex= 488 nm and λem =520 nm using a flouroscence
microplate reader. It was found that with addition of 100 ug of FITC BSA

to 10 mg of magnetic nanoparticles; about 91% of FITC-BSA was
conjugated.
Characterization of drug loaded MNPs
The mean hydrodynamic diameter of the formulated glyceryl monooleate
(GMO) Magnetic nanoparticles (MNPs) with and without drugs was found
to be in the range of 150-200 nra with a polydispersity index (PI) of ~ 0.2
(Table 4). The zeta potential is another important parameter to know the
stability of the MNP formulations. It measures the magnitude of the
repulsion and attraction between the particles. The zeta potential of
glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) was found to
be ~ 36 mV. Incorporation of drugs showed a decrease in zeta potential of
the glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) to 22-26
mV as depicted in (Table 4). But the overall high positive zeta potential of
~ 30 mV shows the stability of the different glyceryl monooleate (GMO)
Magnetic nanoparticle (MNP) formulations. The amount of drugs
incorporated in the glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs) was quantified by measuring the encapsulation efficiency.
Paclitaxel loading in glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs) showed an encapsulation efficiency of 75% (i.e, 75% of the added
drug was entrapped in the formulation). Similarly, rapamycin loaded
glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) showed an
encapsulation efficiency of 73% (i.e, 73% of the added drug was
entrapped in the formulation). When both the drugs were used in the
glyceryl monooleate (GMO) Magnetic nanoparticle (MNP) formulation,
encapsulation efficiency of paclitaxel was 98% and rapamycin was 99%.
A sustained release of the drugs was observed from the in vitro release
profiles (Fig 14). The release of paclitaxel and rapamycin from the

glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) was 73%, 80%
respectively in two weeks showing a sustained release profile. In the
combined drug formulation (paclitaxel + rapamycin), the release of
rapamycin drug was around 91% in two weeks whereas the release of
paclitaxel was around 81%.
Antiproliferative activity of drug loaded glyceryl monooleate (GMO)
Magnetic nanoparticles (MNPs)
The cell viability percentage due to the different drug loaded glyceryl
monooleate (GMO) Magnetic nanoparticle (MNP) formulations were
determined by MTT assay. The result showed the typical dose dependent
sigmoidal antiproliferative effect on the MCF-7 cells. As the concentration
of paclitaxel was increased from 1 ng to 1000 ng/ml the cell viability
percentage decreased from 95% to about 30% in case of drug in solution.
But, in case of drug in nanoparticles as the concentration of paclitaxel
was increased from 1 ng to 1000 ng/ml the cell viability percentage
decreased from 85% to about 35 % (Fig 15a). Similarly, in case of
rapamycin in drug solution the percentage of cell viability was decreased
from 65% to 37% and the drug in nanoparticles showed a decrease from
63% to 40% (Fig 15b). In case of combinational drug formulation, the free
drug showed in decrease from 75% to 37% whereas the drug in
nanoparticles showed a decrease in cell viability percentage from 70% to
27% (Fig 15c). The paclitaxel loaded glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) and the paclitaxel in solution showed similar IC50
values. But the rapamycin loaded glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) showed a significantly higher IC50 value than that
of the drug in solution (58.8 ng/ml vs. 84.2 ng/ml). But the combination

drug formulation showed similar IC50 values as that of the combination
drugs in solution (Solution- 21.99 ng/ml vs. MNP- 16.6 ng/ml) (Fig 16).
The MNPs have a proven candidacy for its biocompatibility and its wide
application in the field of medical sciences. Therefore, a novel
monodispersed water soluble MNP formulation was developed in which
hydrophobic anticancer drugs can be loaded efficiently. To prevent MNPs
from aggregation and opsonization in a biological solution, it must have a
polymeric coating on its surface. Before loading of drugs the magnetic
nanoparticles are generally surface modified with hydrophilic polymers
such as starch or dextran, PEG, PLL, PEI, and the therapeutic agents of
interest is either chemically conjugated or ionically bound to the outer
layer of polymer so as to improve their biocompatibility and stability.
To obtain better aqueous dispersibilty, several groups have used
surfactants like pluronic, as the amphiphilic molecule which forms a
coating on the surface of the iron oxide nanoparticles. Using the
surfactant pluorinc F-127, Jain et al have successfully developed an
aqueous based formulation of iron oxide with hydrophobic drug loading.
The aqueous dispersibility of the MNPs is achieved by the anchorage of
the pluronic F-127 at the interface of OA shell surrounding the iron oxide
particles. Experimental studies show that higher doses of pluronic F-127
has the toxic effect to human erythrocytes. When the pluronic was used
as emulsifying agent for the drug amphotericin-B, there is detectable cell
lysis of human erythrocytes at the concentration of 16 ug/ml. It has also
been experimented that use of Pluronic F-127 at higher doses shows an
elevation of cholesterol and triglycerides in the blood plasma.

Therefore we have developed a novel formulation using long chain
polymer having a little affinity towards the aqueous base, so that the
toxic effect of the surfactant can be avoided. Our formulation gives a well
aqueous dispersibilty without the use of any surfactant. During the
synthesis process of the iron oxide nanoparticles the organic solvents
play a vital role in removing the excess amount of GMO coating and
keeping a balance between the hydrophobicity required for drug
attachment and hydrophilicity required for making a water dispersible
formulation. In our formulation, a mixture of ethyl acetate and acetone in
70:30 (v/v) ratio is helpful to maintain this balance. As both the solvents
are aprotic they help to remove the excess hydrophobic coating from the
magnetic nanoparticle surface.
In our MNP formulation the hydrophobic drug is partitioned in the form
of distribution in the GMO crust surrounding the iron oxide
nanoparticles (Fig 13). This method has the advantage of offering greater
flexibility of loading hydrophobic drug either alone or in combination.
GMO, on the other hand, forms liquid crystal in the presence of water. In
our formulation of glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs), after washing by organic solvent mixture the free GMO gets
removed by leaving behind layers of GMO which are adsorbed onto the
surface of MNPs. Our result demonstrated that the GMO is well
partitioned and chemisorbed as an attachment of carboxylate head
groups on the surface of iron oxide nanoparticles. It is also reported that
the magnetic nanoparticles prepared by co-precipitation method have
enormous hydroxyl groups on the surface. Since the nanoparticles
possess a high surface to volume ratio, therefore the surface hydroxyl
groups readily reacts with the carboxylic groups of the GMO molecules at

higher temperature. During the formulation process the GMO gets
chemisorbed on the surface of the MNPs to make the first layer through
electrostatic interaction between the carboxyl head groups of GMO and
hydroxyl groups of MNPs. Further layers of GMO on the surface of MNPs
are very weak which are only due to adsorption and not by any
electrostatic interaction. Similar chemisorption of drug was achieved with
the cobalt nanoparticles in the presence of fatty acids.
The adsorption of the GMO has been confirmed by FT-IR analysis. In the
pure GMO there is presence of C=O stretch at 1730 cm-1. In the GMO
coated MNPs the vibrational stretch of COO- was observed at 1400 cm1
instead of C=O stretch. This shows the chemisorption of GMO on the
surface of MNPs. Similar results were also observed by other groups
working on OA coated MNPs. In their study, after adsorption of OA on
the MNP surface no peaks relating to C=O was found. Instead, peaks for
asymmetric and symmetric stretches of COO were found which are due
to the chemisorption of the carboxylic group in carboxylate form. In our
formulation, the OH groups of the GMO may have contributed for the
better water dispersibility as the FTIR results show that the vibrational
stretch for OH group at 3400 cm-1 gradually increases with increase in
percentage of GMO coating. This is attributed to the adsorption of GMO
on the surface of the MNPs as explained in (Fig 4, f).
Zeta potential is a bulk property that is not sensitive to the changes in
the surface chemistry. The magnitude of the zeta potential gives an
indication of the potential stability of the colloidal system. If all the
particles in suspension have a large negative or positive zeta potential
then they will tend to repel each other and there will be no tendency for

the particles to aggregate. The zeta potential of glyceryl monooleate
(GMO) Magnetic nanoparticles (MNPs) in our formulation shows a high
positive value ~30 mV. The zeta potential of the MNPs did not change
significantly with the increase of the GMO coating and also after loading
of the drug to the glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs).
The use of polymer coated nanoparticles have limited applications
because of less drug loading capacity particularly with hydrophobic anti
cancer drugs. A group has shown that the mitoxantrone drug was
ionically attached to the starch coated magnetic nanoparticles modified
with phosphate groups. But the dissociation of drug from the particles
comes after -60 minutes under in vitro condition. Also the amount of
drug associated with the formulation is very low (0.8 wt%). In our
formulation, the drug load is quite high around (7.5 wt%). Ideally for the
effective treatment, the drug delivery vehicle should carry the heavy
payload so that it can systemically and effectively dissociate the drugs to
the affected tissues. We have formulated paclitaxel, rapamycin and
combination of drugs in glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) formulations and achieved higher entrapment
efficiency (more than 75%). The drug loaded glyceryl monooleate (GMO)
Magnetic nanoparticles (MNPs) exhibit a sustained release and dose
dependent cytotoxicity activity in the cancer cells. Earlier Rudge et al
have also observed the dose dependant antiproliferative effects of the
magnetically targeted carriers loaded with doxorubicin on SK-Br3 cell.
The in vitro release study was carried out to estimate the amount of drug
releasing from the glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs). The anticancer drug loaded to the MNPs probably diffuses out
from the polymeric shell under the influence of the concentration

gradient, similar observation was observed in OA coated iron oxide
nanoparticles.
Our formulation offers the aqueous dispersibility and the flexibility of
most effective partitioning of hydrophobic drug either alone or in
combination to the glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs) with exclusion of the surfactants. We have prepared paclitaxel
and rapamycin loaded glyceryl monooleate (GMO) Magnetic nanoparticles
(MNPs), and a combination of both. The encapsulation efficiency was
around 75%. From this it seems possible that large amount of drugs is
feasible to be partitioned into the glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs). Thus our formulation can also be used for
combination drug therapy which shows synergistic effect of different
drugs so that low dose drug can be used. A combination of drugs is
significantly more effective than either drug alone having a dramatically
longer effect on cancer. Our result demonstrated that the IC 50 value of
rapamycin was quite higher in the nanoparticles formulation (84.2
ng/ml). In the combined drug formulation the IC50 value has significantly
decreased compared to individual drug (16.6 ng/ml). This decrease in
IC50 value in combined drug formulation gives an opportunity for using
lower doses of drugs, which will minimize the toxicity towards the
healthy cells. The combined drugs in solution also showed a lower IC50
value (21.99 ng/ml). However the glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) formulation allocates delivering same ratios of both
the drugs at the target site.
in our glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs)
formulations it can be observed that the IC50 value of drugs in
nanoparticles are more or less same as that of the drugs in solution. This

probably suggests the sustained release of the drugs from the particulate
system as only a small fraction of the adsorbed drug gets released during
the experimental period of 5 days. This may help in preventing cancer
from relapsing and drug resistance. Also this will prevent degradation of
the drugs before accumulation of the particles at the target site.
Magnetic nanoparticles due to their magnetic property are very useful in
different biomedical applications like cell separation, drug targeting or
targeting of specific biomolecules. For such processes, the surfaces of the
MNPs have to be altered with specific targeting moieties. The targeting
moieties or the biomolecules require specific functional groups like
carboxylic group or amine groups to get attached on the MNP surfaces.
In our formulation, we have functionalized the glyceryl monooleate
(GMO) Magnetic nanoparticles (MNPs) with carboxylic groups by coating
the glyceryl monooleate (GMO) Magnetic nanoparticles (MNPs) with
DMSA. The functionalization of MNPs was confirmed by FTIR analysis.
After coating to the MNPs, the carbonyl stretch at 1700 cm1 of DMSA
gives a COOH stretch at around 1650 cm1. As the concentration of
DMSA increases, vibrational modes at around 1376 cm1 and 1650 cnr-1
assigned to C-O stretch and COOH stretch respectively gets increased.
Also another peak at around 1715 cm-1 can be observed at higher
concentration of DMSA. This refers to the C=O stretch which reveals the
existence of DMSA. These results also coincide with the work of other
groups working on DMSA to get carboxyl group functionalized
nanoparticles. These functionalized glyceryl monooleate (GMO) Magnetic
nanoparticles (MNPs) can be used for different applications like lysozyme
immobilization, uptake by smooth muscle cells.

Therefore, the colloidal carrier system is one of the preference mode of
targeting the tumors by anticancer drugs through enhanced permeation
retention (EPR) effect. As the tumors have leaky vasculature and
impaired lymphatic drainage, the intravenously injected colloidal carrier
extravasate and accumulate in the tumor tissues. For successful
targeting to the tumors the magnetic nanoparticles must escape the
uptake by the RES and circulate in the blood for prolong period of time.
Hydrophilic polymers like Pluronic, polyethylene glycol are used to
change the surface property of the colloidal nanoparticles so that they
can evade the uptake by the RES by making them "stealth". The delivery
of hydrophobic anticancer drugs through the systemic circulation is a
challenge. Various drug delivery vehicles such as micelles, emulsions
and nanoparticle formulations have been investigated to address these
problems. However the iron oxide nanoparticles are well tolerated by
body and degrade with time.
Table 1: Effect of different solvents on synthesis of Iron oxide particles


Table 2. Effect of GMO on Magnetic properties of Iron oxide
nanoparticles.

Table 3: The d-spacing values (nm) calculated from the electron
diffraction pattern in Fig 9 and the standard atomic spacing for Fe2O3
along with respective hkl indexes from the JCPDS card (19-0629).


Table 4: Physical characterization of different drug formulations in water

EXAMPLES:
Example 1: Materials
Iron (III) chloride hexahydrate (FeCl3.6H2O) pure granulated, 99%, Iron
(II) chloride tetrahydrate (FeCl2.4H2O) 99%, Ammonium hydroxide, 2,3
meso mercapto Succinic Acid (DMSA), Tween 80, Pluronic F-127, span
series, stannus chloride, mercuric chloride, orthophosphoric acid,
potassium dichromate and potassium bromide were purchased from
Sigma-Aldrich (St. Louis, MO). Glyceryl monooleate was procured from
Eastman (Memphis, TN). FITC-BSA (Albumin from Bovine Serum
Flurescien conjugated) was procured from Invitrogen Corporation,
Carlsbad, CA, USA. N-(3-Dimethylaminopropyl)-N'-ethyl-Carbomdiimide
hydrochloride (EDC) and N-HydroxySuccinimide (NHS) were procured
from Fluka, Sigma Aldrich, Belgium. Barium diphenylamine sulphonate
(BDAS) was procured from Acros Organics, Belgium. Paclitaxel,
rapamycin were obtained from Shaanxi Schiphar Biotech Pvt Ltd, China.
Magnet NdFeB (12200 G) procured from Edmund Scientific, Tonawada,
NY). All other chemical used were of reagent grade obtained from Sigma.

MilliQ water purged with nitrogen (N2) gas was used in all steps involved
in the synthesis and formulations of magnetic nanoparticles.
Example 2: Synthesis of Magnetic nanoparticles.
Synthesis of magnetic particles were done according to the protocol of
Jain et al with little modifications. Accordingly, 0.1M Fe (III) (1.35 g FeCb
dissolved in 50 ml N2 purged water) and 0.1 M Fe (II) (0.99 g FeCb
dissolved in 50 ml N2 purged water) were prepared. 15 ml of 0.1M Fe (III)
and 7.5 ml 0.1M Fe (II) were mixed and heated at 80°C for 10 minutes
under constant stirring with a magnetic stirrer in N2 atmosphere. 1.5 ml
of ammonium hydroxide (14.5 M) was added to it. Then it was stirred for
20 minutes. Finally the precipitate was washed with N2 purged water
with centrifugation at 20,000 rpm for 20 minutes at 10°C (Sigma
centrifuge, 3-16PK, Germany). The pellets were dispersed in 5 ml of
MilliQ water and frozen at -80°C and were lyophilized using a lyophilizer
(LABCONCO Corporation, USA) for two days at temperature of -48°C and
0.05 mbar. The MNP yield was determined by weighing the lyophilized
powder and was found to be 110 mg.
Example 3: Formulations of Magnetic nanoparticles
Different formulations of iron oxide nanoparticles were developed by the
following protocol. 15 ml 0.1 M Fe (III) and 7.5 ml 0.1 M Fe (II) was mixed
and heated at 80°C with constant stirring. 1.5 ml of ammonium
hydroxide (14.5 M) was added drop wise to it. Then GMO was added to
the suspension drop wise. To study the amount of concentration of GMO
required to coat the MNPs, we have prepared different formulations
(different weight percentage of GMO to MNP yield were added i.e, 12-560

μl of GMO was added to get 10-504% of GMO coated MNPs). The mixture
was allowed to stir for 20 minutes at 80°C under a N2 atmosphere to
evaporate the excess amount of ammonia from the formulation. It was
washed with different solvents and centrifugation for 20 minutes at 10°C
at 20,000 rpm (Sigma centrifuge, 3-16PK, Germany). Washing was
repeated for 3 times. The washings of the excess GMO from the magnetic
nanoparticles is critical to get a better aqueous dispersibility. To study
the effect of different solvent washings on the GMO coated magnetic
nanoparticles (GMO-MNPs), different solvents like acetone, ethyl acetate,
diethyl ether, chloroform, and mixture of different solvents in different
ratio like ethyl acetate: acetone (50:50 and 70:30) were used during the
washing steps. The pellets were lyophilized for two days at temperature
of -48°C and 0.05 mbar to get the powder form.
The study the effect of different surfactants on aqueous dispersity of
GMO-MNPs, 10 mg of GMO-MNPs were taken and dissolved in 10 ml of
MilliQ water and was sonicated for 1 minutes at 55 watt (VC505, Sonics
Vibracell, Sonics and Materials Inc., USA). To this different surfactants
were added in the ratio of particle: surfactant (1:1) and was allowed for
over night stirring in a closed container to minimize exposure to
atmospheric oxygen to prevent oxidation of the MNPs. These particles
were washed 3 times with water to remove the surfactants which were
not bounded to the MNPs by magnetic decantation and lyophilized to get
the powder form for further use.
Example 4: Characterization of GMO-MNPs
Particle size determination by Dynamic Light Scattering and £ potential
Measurements.

Dynamic light scattering (DLS) was used to measure the hydrodynamic
diameter and Laser Doppler Anemometry (LDA) was used to determine
the zeta potential (mV) of GMO-MNPs. The DLS and LDA analysis were
performed using a Zetasizer Nano ZS (Malvern Instruments, Malvern,
UK). The particle size measurement was done by dispersing MNPs (~1
mg/ml) in MilliQ water using water bath sonicator for 1 minute and then
the suspension was diluted (100 μl to 1 ml) and the size was measured
in polystyerene cuvette using the Zetasizer Nano ZS. To compare the size
of the MNPs in organic solvent, the measurement of particle size in n-
hexane was made following the same procedure using the quartz cuvette.
To further see the effect of size in respect to the different surfactants
added to the GMO-MNPs (~1 mg/ml) surfactant coated GMO-MNPs were
suspended in MilliQ water and sonicated using water bath sonicater for 1
minute at 55 watt (VC505, Sonics Vibracell, Sonics and Materials Inc.,
USA) and further diluted (100 μl to 1 ml) for particle size measurement.
The same suspension in MilliQ water was used for measuring the zeta
potential of MNPs.
Transmission Electron Microscopy (TEM).
The internal structure of MNPs were determined by TEM measurements
for which a drop of diluted solution of the GMO-MNPs (either in water or
n-Hexane) was placed in carbon-coated copper TEM grid (150 mesh, Ted
Pella Inc, rodding, CA) and was allowed to air-dry. The samples were
imaged used a Philips 201 transmission electron microscope (Philips/FEI
Inc, Barcliff, Manor, NY). The TEM photograph was taken by using the
NIH imaged software. To calculate the mean particle diameter, 50
particles were taken for measurement.

X-ray Diffraction (XRD)
XRD analysis was carried out to know the crystallinity of the MNPs
formed. The lyophilized samples (~500 mg) of native iron oxide particles
and 100% GMO-MNPs were carried out using a Brucker D4 Endeavour,
with Bragg-Brentano-Brentano parafocusing geometry. The analysis was
done with copper target X-ray tube with Cu Ka radiations. The
parameters chosen for the measurement were 20 steps of 0.08°, 1 second
of counting timer per step, and 20 range from 10.01° to 69.53°.
Determination of Iron content in the magnetic nanoparticle formulations.
To determine the percentage of iron present in the MNP formulations, the
chemical analysis of the samples was carried out by recommended
analytical procedure. Different GMO-MNP formulations (in triplicate)
were subjected to di-acid digestion for wet chemical analysis. The MNP
formulations (~50 mg) were first digested by adding 2 ml concentrated
HCI followed by heating at 60°C for 10 minutes. Then the digested
product was diluted to 25 ml with MilliQ water. To the above diluted
sample (5 ml), 2 ml of concentrated HCI was added and heated at 60°C
for 10 minutes. 4 ml of 0.25 M stannus chloride was added drop wise to
the digested product up to decolouration. Then the sample was cooled to
room temperature and 2 ml of saturated mercuric chloride was added
and was mixed well by shaking. To the mixture, 10 ml of Zimmerman-
Reinhard reagent (5 ml of 5% sulphuric acid and 5 ml of orthophosphoric
acid) was added followed by addition of 10 ml of MilliQ water. Finally, the
iron content in the formulation was analyzed volumetrically by titrating
against 0.01 N potassium dichromate solution using barium
diphenylamine sulfonate (BDAS) indicator.

Fourier Transform infrared spectroscopy (FT-IR).
FT-IR measurement was carried out to know the chemical interactions in
the MNP formulations. FT-IR (Perkin Elmer, FTIR Spectrometer,
SPECTRUM RX I) was used to characterize the surface composition of the
different formulations of MNPs. Each spectrum was obtained by
averaging 32 interferograms with resolution of 2 cm-1 in the range of 400
to 4000 cm-1. A small amount of MNPs (either native or formulated) were
milled with KBr, and a mixture of them was pressed into a pellet for
analysis with a pressure of 150 kg/cm2.
Magnetization Studies
In order to quantify the amount of magnetism present in the formulated
MNPs magnetization study was carried. The Magnetic properties were
investigated by a Superconducting Quantum Interference Device
(SQUID) magnetometer (MPMS5, Quantum Design) with fields upto 1.5 T
and temperatures of 10 K and 300 K respectively. Zero-field-cooled (ZFC)
and field -cooled (FC) magnetization measurements were carried out as a
function of temperature. To determine the ZFC measurements the
samples were cooled from 300 K to 10 K in zero field as a function of
temperature at 100 Oe field strength as gradually warmed. To take the
FC measurement, the sample as cooled in the measuring field. The
magnetization was determined as a function of field M (H) at 10 and 300
K. By putting the magnetization curve in an analytical ferromagnetic
model and by normalizing the diamagnetic contribution (x) due to the
background the saturation magnetization (Ms) and the Coercive field (He)
were determined.

Example 5: Drug loading in the formulation
Loading of anticancer drugs in Magnetic Nanoparticles.
To exploit the MNP formulations as a drug delivery vehicle, anticancer
drugs were taken into account. For the incorporation of anticancer drugs
in GMO-MNPs, paclitaxel, rapamycin and a combination of both
(paclitaxel and rapamycin) were used. We have used 100% GMO coated
MNPs for drug loading. 100 mg of the GMO-MNPs were dispersed in 10
ml MilliQ water and was sonicated for 1 minute. The drugs were
dissolved in organic solvent acetonitrile either individually or in
combination (10% w/w to the polymer i.e, 10 mg of either of the drugs
dissolved in 1 ml or 1 ml of combined drugs, 5 mg each). The drug was
added drop wise to the GMO-MNPs suspension and kept for overnight
stirring with a magnetic stirrer to allow the partitioning of the drug into
the GMO shells surrounding the magnetic nanoparticles. The
unpartitioned drugs were washed with water and were separated by
centrifuging the particle suspension at 13,800 rpm for 10 minutes at
10°C (Sigma centrifuge, 3-16PK, Germany). Washing was repeated for 3
times for the complete removal of the unentrapped drug. The pellets were
lyophilized for quantification of entrapment efficiency of different drugs
through reverse phase high performance liquid chromatography (RP-
HPLC).
Quantification of drug by RP-HPLC.
Quantification of the drug incorporated in the MNPs, was carried out
through RP-HPLC. The estimation of the amount of drug entrapped in
the GMO-MNPs was done by direct method. To the lyophilized
nanoparticles solvent acetonitrile (1 mg/ml) was added and sonicated in

an ice bath for 1 minute, at 55 watt and kept in shaker for 24 hours for
the drug to come out from the particles. Then the nanoparticles were
centrifuged for 10 minutes at 13, 800 rpm at 10°C (Sigma
microcentrifuge, 1-15PK, Germany). Supernatants were taken out for the
estimation of drug entrapped. The analysis of sample was done by
reverse phase isocratic mode of HPLC with little modification using
Agilent 1100 (Agilent technologies, Waldbronn Analytical Division,
Germany) which consists of a column (Zorbax Eclipse XDB-C18, 150 X
4.6 mm, i.d). 20 μl of different drug samples were injected manually in
the injection port and were analyzed with the mobile phase of
acetonitrile: water (80:20 v/v), which was delivered at flow rate of 1
ml/min with a quaternary pump (Model No-G1311A) at 25°C with
thermostart (Model No-G1316A). The drug levels were quantified by UV
detection at 228 nm for paclitaxel and 278 nm for rapamycin with a
detector (DAD, Model -G 1315A). The amount of drug (paclitaxel and
rapamycin) in samples was determined from the peak area correlated
with the standard curve. The standard curves of paclitaxel and of
rapamycin were prepared under identical conditions. The entrapment
efficiency was calculated from the following formula reported earlier % of
Entrapment Efficiency = (drug loaded in nanoparticles / drug added in
formulation) x 100
Example 6:
Kinetics of Paclitaxel and Rapamycin Release from Magnetic
nanoparticles.
To know the amount of drug released in in vitro condition a kinetics
measurement was done. The release of drugs from GMO-MNPs was

carried out by dissolving 10 mg of nanoparticles in 3 ml of PBS (ph = 7.4,
0.01 M, containing 0.1 % w/v of Tween 80). Tween 80 was used in the
buffer to maintain the sink condition during the release study. It was
mixed properly by vortexing and then was divided into 3 parts, 1 ml
each. All the samples were kept in an orbit shaking incubator (Wadegati
Labequip, India) at 37°C, rotating at 150 rpm. The samples were removed
at predetermined time intervals and centrifuged at 13,800 rpm for 15
minutes at 10°C (Sigma microcentrifuge, 1-15PK, Germany) to get the
supernatant. Then the pellets were dispersed with the same volume of
fresh PBS (pH = 7.4, 0.01 M PBS, containing 0.1 % w/v of Tween 80) and
vortexed and kept in shaker. The collected supernatants were lyophilized
for 48 hours, and then were dissolved in acetonitrile and centrifuged at
13, 800 rpm for 10 minutes at 4 °C (Sigma microcentrifuge, 1-15PK,
Germany). The obtained supernatant was taken out and injected in the
RP-HPLC to determine the amount of drug released either paclitaxel,
rapamycin or combination of both with respect to different time intervals.
Example 7:
Cell culture
The cell culture experiments were carried out in MCF-7 (breast cancer)
cell line purchased from American Type Culture Collection (ATCC,
Manassas, VA) were grown in RPMI 1640 medium (Himedia Laboratories
PVT. LTD., Mumbai, India) supplemented with 10% fetal bovine serum
(Himedia Laboratories Pvt. Ltd., Mumbai, India) and 100 ug/ml penicillin
G and 100 ug/ml streptomycin (Gibco BRL, Grand island, NY) at 37°C in
a humidified and 5% CO2 atmosphere (Hera Cell, Thermo Scientific,
Waltham, MA).

Example 8:
Statistical analyses were performed using a Student's t test. The
differences were considered significant for p values of <0.05.
Example 9:
Mitogenic Assay.
To find out the cytotoxicity of the anticancer drugs, mitogenic assay was
carried out. The MCF-7 cells were seeded at 5, 000 per well in 96 well
plate (Corning, NY, USA) and kept in the incubator for 24 hours for
better cell attachment. Different concentrations of paclitaxel, rapamycin
or combination of the drug (0.1 uM to 1000 uM), either in solution or
loaded in GMO-MNPs were added. GMO-MNPs without drug and medium
were used as respective controls. The medium was changed on 2nd and
4th days following the drug treatment; no further dose of drug was added.
Viability of the cells was determined at 5th day. After the specified
incubation time, lOul MTT (Sigma) was added, and the plates were
incubated for 3 hours at 37°C in a cell culture incubator (Hera Cell,
Thermo Scientific, Waltham, MA), following which the intracellular
formazan crystals were solubilized in dimethyl sulfoxide and the color
intensity was measured at 540 nm using a microplate reader (Synergy
HT, BioTek Instruments, Inc., Winooski, VT). The antiproliferative effect
of different treatments was calculated as a percentage of cell growth with
respect to respective controls.

Example 10:
Surface Functionalization of Magnetic Nanoparticles
MNPs are difficult to bond with biomolecules in aqueous solution.
Therefore, to attach any biomolecule on to the surface of the MNPs, the
surface should be functionalized with different functional groups like
carboxylic or amine group. To attach any peptide or protein on to the
surface of the MNPs, the particles should be surface functionalized with
carboxylic groups. Therefore, 2, 3 meso mercapto succinic acid (DMSA)
was used to functionalize the GMO-MNPs with carboxylic acid group. 500
mg of MNP-GMO was added to 5 ml of 0.2 M DMSA dissolved in DMF
and kept for 24 hours stirring in a magnetic stirrer. The sample was
washed with ethanol 3 times by centrifuging at 13,800 rpm at 10°C for
20 minutes and the pellets were lyophilized. To find out the effect of
DMSA in the functionalization of GMO-MNPs, we have used different
concentrations of DMSA solutions (0.4 - 3.2 M) and followed the above
procedure to get the lyophilized powder.
Acid Number Determination.
For the quantification of free carboxylic acid groups attached on the
surface of MNPs, acid numbers of the GMO-MNPs were determined
following the experimental protocol by Garkhal et al. 20 mg of the
different concentration of DMSA coated GMO-MNPs were initially treated
with 5 ml NaOH (1 N) for 30 minutes to cleave some of the surface ester
bonds to generate free carboxylic ends. Then the samples were washed 3
times with MilliQ water by centrifuging at 13,800 rpm at 10°C for 20
minutes. Then all the samples were vacuum dried by lyophilizer. Free
acid groups present on the GMO-MNPs surface were quantified by taking
nanoparticle solution 1 mg/ml and diluting to 50 times. Then the diluted

solution was titrated against NaOH (0.0005 N). NaOH solution is to be
standardized before by titrating against oxalic acid. Acid number was
calculated by the following formula.
Volume required during titration *Normality of NaOH * 40 (Mol. Wt. Of NaOH)
A=
Weight of nanoparticles (g)
Example 11 :
Conjugation of FITC-BSA
FITC BSA was conjugated to the carboxyl groups, which were
functionalized on the surface of GMO-MNPs. For conjugation, 10 mg of
functionalized GMO-MNPs were added to 5 ml of PBS (pH =7.4, 0.02 M).
250 ul of EDC and 250 ul of NHS in PBS (pH=7.4, 0.02 M, 1 mg/ml) was
added to it. The sample was left in room temperature under magnetic
stirring for 4 hours. Then the sample was magnetically decanted to
remove free EDC and NHS. To the pellet 3 ml of PBS (pH=7.4, 0.02 M)
and 100 ul of FITC-BSA (1 mg/ml) was added. The solution was left for 2
hours and then incubated at 4°C overnight. Next day magnetic
decantation was done and the pellets were washed 2 times with PBS
(pH=7.4, 0.02 M) to remove any unconjugated FITC-BSA. A standard plot
for FITC-BSA was prepared taking concentrations 2.5 - 20 μg/ml at λex
=488 nm and λem=520 nm using a flouroscence microplate reader
(Synergy HT, BioTek Instruments, Inc., Winooski, VT). The percentage of
conjugation of FITC-BSA to the GMO-MNPs was calculated by indirect
method. First, the amount of un-conjugated FITC-BSA present in the

supernatant was determined by taking the fluorescence measurement
and using the standard plot of FITC-BSA. Then the amount of un-
conjugated FITC-BSA was deducted from the total FITC-BSA amount
added to get the amount of conjugated FITC-BSA.

WE CLAIM:
1. A method for preparing a water dispersible glyceryl monooleate
(GMO) magnetic nanoparticle formulation comprising an iron oxide
particle core coated with long chain polymer for producing an
aqueous dispersible magnetic nanoparticle formulation.
2. The glyceryl monooleate magnetic nanoparticle formulation as
claimed in claim 1, further comprises of one or more therapeutic
agent or a functional group.
3. The glyceryl monooleate magnetic nanoparticle formulation as
claimed in claim 1, wherein said therapeutic agent is selected from
hydrophobic and hydrophilic agent.
4. A method for preparing glyceryl monooleate (GMO) magnetic
nanoparticles (MNPs) formulation comprising:
heating a mixture of Fe (III) and Fe (II) with constant stirring under
N2 atmosphere;
adding ammonium hydroxide to the said mixture;
adding glyceryl monooleate (GMO) to the suspension drop wise;
subjecting the mixture to the step of stirring under N2 atmosphere;
washing the formulation several times with different solvents to
wash the excess glyceryl monooleate (GMO);
subjecting the washed formulation to the step of lyophilization to
yield powder form.

5. The method as claimed in claim 1, wherein the mixture was heated
at 80°C.
6. The method as claimed in claim 1, wherein 12-560 μl of GMO was
added to get 10-504% of GMO coated MNPs.
7. The method as claimed in claim 1, wherein the mixture was stirred
for 20 minutes at 80°C under N2 atmosphere to evaporate the excess
ammonia from the formulation.
8. The method as claimed in claim 1, wherein the organic solvent is
ethyl acetate: acetone (70:30).
9. The method as claimed in claim 1, wherein the said formulation was
sonicated for 2 minutes at 55 watt.
10. The method as claimed in claim 1, wherein the step of washing with
different solvents and centrifugation was done for 20 minutes at
10°C at 20,000 rpm.
11. The method as claimed in claim 7, wherein said solvents were
selected from acetone, ethyl acetate, diethyl ether, chloroform, and
mixture of different solvents in different ratio like ethyl acetate:
acetone (50:50 and 70:30).
12.The method as claimed in claim 8, wherein the said step of washing
was repeated for 3 times.

13.The method as claimed in claimed 1, wherein the said glyceryl
monooleate (GMO) magnetic nanoparticles (MNPs) formulation can
effectively be used as a carrier for both the hydrophilic and
hydrophobic drugs.
14. A glyceryl monooleate (GMO) magnetic nanoparticle formulation can
be used in biomedical field such as cell separation.
15. The GMO-MNP formulation as claimed in claim 1, wherein the
functional group can be used by attaching with any protein or any
targeting moiety.

The present invention is an aqueous dispersible magnetic nanoparticle formulation with a high drug loading capacity used for sustained drug
delivery. The formulated magnetic nanoparticles are composed of an iron oxide core coated with a long chain polymer, which provides aqueous
dispersibility without the use of surfactant. A method is developed for the functionalization of magnetic nanoparticles for use in biomedical field.