This document describes the synthesis of magnetoliposomes, which are lipid vesicles that encapsulate magnetic iron oxide nanoparticles. The motivation is for applications in drug delivery, MRI, and cancer hyperthermia treatment. The goals are to encapsulate 10nm iron oxide nanoparticles in liposomes at high yield and efficiency. The methods used are preparing liposomes using solvent evaporation, centrifugation to separate encapsulated from unencapsulated nanoparticles, cryo-TEM imaging to view encapsulation, and a Tiron test to measure encapsulation efficiency spectrophotometrically. The results show cryo-TEM images of encapsulated nanoparticles and efficiencies of 42-58% depending on nanoparticle concentration. The conclusions are that a good encaps

1. Synthesis of Fe3O4
Magnetoliposomes
Sarah Keshishian
May 7, 2015
Bothun Laboratory Group

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Introduction
Magnetoliposomes are particles in which a
phospholipid bilayer envelopes a
magnetisable core (Iron)
• Form sphere through its hydrophilic
(polar) head and hydrophobic (fatty acid)
ends, which allows for encapsulation
• Are able to merge with cells in human
body due to physiological similarity

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Motivation
Synthesis of Magnetoliposomes is desired for:
• Drug delivery through targeted or
controlled release
• MRI applications
• Hyperthermia Cancer treatment
Process Motivation comes from simplification of:
Encapsulation Method-Wijaya, Andy. Hamad-Schifferli, Kimberly. “High-Density Encapsulation of
Fe3O4 Nanoparticles in Lipid Vesicles.” 17 Jul. 2007. [1]
Tiron and Efficiency Method-Pradham, Pallab. “Preparation and Characterization of Manganese
Ferrite-based Magnetic Liposomes for Hyperthermia Treatment of Cancer.” 19 December. 2006. [2]

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Goals
1. Encapsulate 10 nm Iron Oxide Nanoparticles in
DOPC/DOPG liposomes at high yield
2. Separate encapsulated Nanoparticles from
Nonencapsulated through Centrifugation
3. Use Cryo-TEM images to view encapsulation
4. Apply Tiron Test and UV-Vis Spectrophotometer to
measure absorbance and determine concentration of
encapsulated Iron
5. Achieve highest Encapsulation Efficiency Possible

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Experimental Methods
Preparing Liposomes
Specifications
• 4 mL sample, 10 mM stock lipid concentration total
• EMG 705 NP, prepared at 3.9% and 1.95% volume
• Use chloroform as solvent and distilled water
• Sonicate sample
• Use Rotary Evaporator to evaporate the chloroform and prepare the
vesicles, decreasing pressure slowly, but keeping temperature
constant at 50 ºC
Sample containing Chloroform,
Water, NPs
Evaporated Sample of Encapsulated NPs,
Nonencapsulated NPs, and water
Rotavap at 50°C from 450
mbar to 150 mbar

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Experimental Methods
Centrifugation
Once samples are made
• Water added to further dilute and prepare for Centrifuge
• Centrifuge once retaining supernatant, and again
discarding it
• The resulting product is the encapsulated NPs
Centrifuge 5 min
At 1000g, retain
supernatant
Centrifuge 5 min at
1000 g, keep NP
Aggregates

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Experimental Methods
Analysis
Cryo-TEM
• Products from centrifuge
prepared in TEM grid
• Cryogenic-TEM fixes the
real structure of a sample in
the liquid phase through use
of liquid Nitrogen
Tiron Test
• Tests efficiency of encapsulation
• Using Iron Chloride dilutions, absorbance is
taken in a spectrophotometer at wavelength
of 670 nm in order to derive a calibration
curve
• By using the equation of the calibration curve,
actual concentration of iron NP is found
• Finally, encapsulation efficiency of Iron is
found

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Results
Cryo-TEM

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Results
Cryo-TEM

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Results
Tiron Efficiency Test
y = 1.6937x
R² = 0.9993
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6
Absorbtion
Iron Concentration (M)
Calibration Curve
Abs
Linear (Abs)
Absorbance
Encapsulated Fe
Concentration (M)
0.03545142 0.060044069
0.024207585 0.041000386
0.034526773 0.058477995
𝐸𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 =
𝑤𝑒
𝑤𝑖
∗ 100
𝑤𝑒= encapsulated Fe concentration
𝑤𝑖= initial Fe concentration

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Results
Efficiency
𝑤𝑖 (mg/mL) 𝑤𝑒 (mg/mL) EE (%)
3.90% 7.86 3.35 42
1.95% 3.93 2.29 58

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Discussion and Conclusions
• Based on final concentrations of Iron, a good
encapsulation efficiency was achieved
• Literature values of EE % range from 22% to 70% [2],
which agrees with our results
• In the future, it would be recommended to vary NP
concentration more as well as vary lipid concentrations,
while keeping NP concentration constant and analyze
encapsulation efficiency based on these changes

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Thank You
• Dr. Bothun
• Matt Preiss
• Iftheker Khan