Stability of Liposomal 

Nanomedicines

Emily Blick

Elizabeth Kelley

Yun Liu

1
OUTLINE

1. Introduction

2. Materials and methods

3. Results and discussions

4. Conclusions

2
WHAT ARE LIPOSOMES?

• Lipids in solution form liposomes

• Enclose aqueous solutions

• Vesicles can have one or more bilayers

• Unilamellar vesicles simplest model for drug 

delivery

Hydrophilic head group

Hydrophobic tails

Hydrophilic Hydrophobic

Unilamellar Liposome

3
WHY LIPOSOMES?

• Liposomes are promising candidates for future drug delivery applications

• Similar morphology to the cellular membrane

• Biocompatible, biodegradability, and low toxicity

New cancer drug!

Doxil: Liposomal 

Injection for 

Ovarian Cancer

Exparel: Nonopioid pain relief

Liposome Real 

Toning 

Ampoule: Skin 

Care 

4
PHARMACEUTICAL USE: WHAT WE STILL 

DON’T KNOW

• Shelf life and storage conditions for liposomes are unknown details

• Crucial for pharmaceutical use

• My experiments look closely at vesicle stability

• Chemical Instability

• Structural Instability

Formulation Usage 

?

5
HYDROLYSIS • Break down of lipids 

from water moisture

• Oxidative degradation of 

lipids due to the loss of 

electrons

OXIDATION

CHEMICAL INSTABILITY

Free Fatty Acid

What we can measure!

Free Radicals

What we can measure!

6
STRUCTURAL INSTABILITY

Structural Instability:

• Result of chemical degradation

• Unstable liposomes can form many 

different structures

• Multilamellar?

• Aggregation?

• New Structure?

• ?

unilamellar

multilamellar

aggregation

?

structural change

7
OUTLINE

1. Introduction

2. Materials and methods

3. Results and discussions

4. Conclusions

8
MATERIALS

• Using model system 1,2-Dioleoylsn-glycero-3-phosphocholine 

(DOPC)

• Commonly used lipid in research

• Unilamellar vesicles

Lipid Extrusion:

• Lipids in solution forced through a 

filter

• Defined pore size

• Decrease filter size 

throughout extrusion

Unfiltered Porous Filter

Liposomes Filtered 

Liposomes

9
METHODS

Measures the hydrodynamic radius of 

liposomes

2. Dynamic Light Scattering

1. UV Vis Used to measure chemical instability:

hydrolysis: measure free fatty acid

oxidation: measures peroxidized lipid

4. Small-Angle Neutron Scattering Provide information of vesicle structure 

and size under different storage conditions

3. Cryogenic Transmission Electron 

Microscopy (Cryo TEM) Captured high-resolution structures of 

liposomes in solution

10
SMALL ANGLE NEUTRON SCATTERING

• Probes material structures by interacting with nucleus in sample

• Neutron scattering can be measured by a detector

• Scattering vector Q can tell us about structures in our sample 

to nanometer scale

Neutron beam

Sample

Scattered beam

Detector

Q

11
OUTLINE

1. Introduction

2. Materials and methods

3. Results and discussions

4. Conclusions

12
RESULTS

1. Temperature Effect: Increasing temperature storage conditions increases degradation kinetics

A. Hydrolysis and oxidation assays show more chemical degradation with increased storage temperature

B. DLS shows change in vesicle size occurs at shorter time points at higher temperature

C. SANS shows more significant change to vesicle structure at higher temperature 

2. pH Effect: Vesicles in solution with pH 6.5 degrade faster than pH’s of 9 and 2

A. Chemical assays show more hydrolysis and oxidation at 6.5 

B. DLS showed more change at 6.5

C. SANS data shows more structure change at 6.5 than 2 and 9

3. Size Effect: Smaller vesicles will degrade faster than larger vesicles

A. Smaller vesicles show more hydrolysis than larger vesicles 

B. DLS shows greater change in vesicle size for small vesicles

C. SANS shows more change to vesicle structure for smaller vesicles 

4. Structural evolution: Morphology differs at various stages during vesicle degradation

13
THE TEMPERATURE EFFECT:

THE EXPERIMENT

• Put 3 vials of vesicle solutions at 4 temperatures

• Remove vials at varying time points

• Measure vesicle degradation with DLS and chemical assays

• Use SANS to study structure change over time

22°C 55°C 65°C 80°C

18hrs 36hrs 60hrs 18hrs 36hrs 60hrs 18hrs 36hrs 60hrs 18hrs 36hrs 60hrs

14
HYDROLYSIS • More oxidation at higher 

temperatures!

OXIDATION

TEMPERATURE EFFECT: 

CHEMICAL ASSAYS

0

1

2

3

4

5

6

7

55C 65C 80c

Absorbance

50nm Vesicles after 60hrs 

?

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

22c 90 days 55c 30 days 65c 2.5 days 80c 2.5 days

Fatty Acid Concentration(mM/ul)

Fatty Acid Concentration(mM/ul) at 

Different Conditions

15
TEMPERATURE EFFECT: 

DLS

• Vesicles at higher temperatures change size 

quicker

• Degradation kinetics increase with 

temperature

• Initially see an increase in vesicle size, followed 

by a decrease and then eventually an increase 

again

• At 22c vesicles will remain unchanged even after 

30 days!

• Note: DLS only tells us about vesicle size not 

structure

40

50

60

70

80

90

100

110

0 0.5 1 1.5 2 2.5 3

Radius(nm)

Time(days)

Short Term Temperature Effects

80

50nm

22c

50nm

65c

50nm

55c

50nm

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25 30

Radius(nm)

Time(days)

Long term Temperature Effects

22c

55c

Time

16
TEMPERATURE EFFECT: 

SANS

0

20

40

60

80

100

120

50 55 60 65 70 75 80 85

Radius(nm)

Temperature(C) 

Radius of Gyration at Different Temperatures

Vesicle size Intermediate Structure Bilayer

• Significant change at low q and Rg showing change 

in vesicle size

• Intermediate structure change 

• At high q samples look similar

• Similar bilayer length scale

• Intermediate structure change 

17

55c

65c

80c
RESULTS

1. Temperature Effect: Increasing temperature storage conditions increases degradation kinetics

A. Hydrolysis and oxidation assays show more chemical degradation with increased storage temperature

B. DLS shows change in vesicle size occurs at shorter time points at higher temperature

C. SANS shows more significant change to vesicle structure at higher temperature 

2. pH Effect: Vesicles in solution with pH 6.5 degrade faster than pH’s of 9 and 2

A. Chemical assays show more hydrolysis and oxidation at 6.5 

B. DLS showed more change at 6.5

C. SANS data shows more structure change at 6.5 than 2 and 9

3. Size Effect: Smaller vesicles will degrade faster than larger vesicles

A. Smaller vesicles show more hydrolysis than larger vesicles 

B. DLS shows greater change in vesicle size for small vesicles

C. SANS shows more change to vesicle structure for smaller vesicles 

4. Structural evolution: Morphology differs at various stages during vesicle degradation 18
THE PH EFFECT: THE EXPERIMENT

• Put 3 vials at different pH values at 22C and 80C for 24 hours

• Measure vesicle degradation with DLS and chemical assays

• Use SANS to study structure change over time

22°C 80°C

pH 2 pH 6.5 pH 9 pH 2 pH 6.5 pH 9

19
HYDROLYSIS

• Slowest hydrolysis 

rates at neutral pH’s

• Faster hydrolysis 

rate at 3 and 10

HYDROLYSIS RATE AND PH IN LITERATURE

PH EFFECT: CHEMICAL ASSAYS

0

0.02

0.04

0.06

0.08

0.1

0.12

22c ph2 22c ph6.5 22c ph9 80c ph2 80c ph6.5 80c ph9

Fatty Acid 

Concentration(mM/ul)

Fatty Acid Concentration (mM/ul) vs pH and 

Temperature Conditions

“Liposome Stability” 

https://encapsula.com/hydrolysis-and-oxidationof-liposomes/

Rate of hydrolysis of HSPC in 0.05 M buffer.

20
PH EFFECT: 

DLS

• Vesicles at neutral pH grew larger 

than vesicles at pH of 2 and 9

• Opposing what is suggested in 

literature

• Different membranes may have 

80 changing stability properties

82

84

86

88

90

92

94

ph 2 80c ph 6.5 80c ph 9 80c

Radius after 24 hours at 80C

Original 

Vesicle 

Size

21
PH: SANS

• Temperature has a much larger 

impact on vesicle structure than 

pH 

• See slight change in structure at 

different pH values at 80C Varying pH values at 80C

Vesicles kept at 22c

22
RESULTS

1. Temperature Effect: Increasing temperature storage conditions increases degradation kinetics

A. Hydrolysis and oxidation assays show more chemical degradation with increased storage temperature

B. DLS shows change in vesicle size occurs at shorter time points at higher temperature

C. SANS shows more significant change to vesicle structure at higher temperature 

2. pH Effect: Vesicles in solution with pH 6.5 degrade faster than pH’s of 9 and 2

A. Chemical assays show more hydrolysis and oxidation at 6.5 

B. DLS showed more change at 6.5

C. SANS data shows more structure change at 6.5 than 2 and 9

3. Size Effect: Smaller vesicles will degrade faster than larger vesicles

A. Smaller vesicles show more hydrolysis than larger vesicles 

B. DLS shows greater change in vesicle size for small vesicles

C. SANS shows more change to vesicle structure for smaller vesicles 

4. Structural evolution: Morphology differs at various stages during vesicle degradation 23
THE SIZE EFFECT: THE EXPERIMENT

• Repeated temperature effect experiments with duplicates at 50nm and 100nm

22°C 55°C 65°C 80°C

18hrs 36hrs 60hrs 18hrs 36hrs 60hrs 18hrs 36hrs 60hrs 18hrs 36hrs 60hrs

18hrs 36hrs 60hrs 18hrs 36hrs 60hrs 18hrs 36hrs 60hrs 18hrs 36hrs 60hrs

50 nm

100 nm

24
HYDROLYSIS OXIDATION

SIZE EFFECT: CHEMICAL ASSAYS

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

50 65 2 day 100 65 2 day

Fatty Acid Concentration(mM/ul)

Fatty Acid Concentration(mM/ul) at 

Different Conditions

0

0.5

1

1.5

2

2.5

3

3.5

100nm 65 C 50nm 65 C

Absorbance

Oxidation of Liposomes at 65 C

25
SIZE EFFECT: 

DLS

0

20

40

60

80

100

120

0 5 10 15 20

avg radius (nm)

days

Radius over Time at 55c with Varying Vesicle Sizes

• Smaller vesicles change size more 

quickly at the same temperature!

• Data shown here is 55c but 

reproducible at other 

temperatures

26
100NM VESICLES AT 65C

• Minimal change in vesicle scattering 

between 18 hours and 60 hours

• Greater change in vesicle size and 

structure between 18 hours and 60 hours

50NM VESICLES AT 65 C

SIZE EFFECT: SANS

Low Q giving 

us size 

information

27
RESULTS

1. Temperature Effect: Increasing temperature storage conditions increases degradation kinetics

A. Hydrolysis and oxidation assays show more chemical degradation with increased storage temperature

B. DLS shows change in vesicle size occurs at shorter time points at higher temperature

C. SANS shows more significant change to vesicle structure at higher temperature 

2. pH Effect: Vesicles in solution with pH 6.5 degrade faster than pH’s of 9 and 2

A. Chemical assays show more hydrolysis and oxidation at 6.5 

B. DLS showed more change at 6.5

C. SANS data shows more structure change at 6.5 than 2 and 9

3. Size Effect: Smaller vesicles will degrade faster than larger vesicles

A. Smaller vesicles show more hydrolysis than larger vesicles 

B. DLS shows greater change in vesicle size for small vesicles

C. SANS shows more change to vesicle structure for smaller vesicles 

4. Structural evolution: Morphology differs at various stages during vesicle degradation 28
MORPHOLOGY:

LITERATURE RESULTS

Why is this similar to my work?

Surfactant

Literature has studied the effects of adding 

surfactants to liposomal solutions

Increasing Surfactant Concentration

Break down of lipids from water 

moisture

Free Fatty Acid

• Early hydrolysis 

product similar 

to surfactant 

and may have 

similar effects 

on liposomes!

No known studies showing this pattern without 

causing liposomal structure change with addition of 

surfactant

Almgren, Mats, “Mixed micelles and other structures in the 

solubilization of the bilayer lipid membranes by surfactants”

29
SMALL ANGLE NEUTRON SCATTERING

• Able to generate a 2D scattering pattern on detector

• Generate 1D plots that contain structural information

Neutron beam

Sample

Scattered beam

Detector

Q

30
MORPHOLOGY:

SANS DATA

22C Sample at 30 days

-2 slope

80C Sample at 2.5 days

-1 slope

55C Sample at 90 days

-4 slope

Scattering curve can tell us 

about vesicle structure!

22c 30 Days 80c 2.5 Days 55c 90 Days

Increasing degradation

31
MORPHOLOGY:

CRYO TEM

Multilamellar Unilamellar

• Overall 22C samples remained unchanged 

and unilamellar after 30 days!

32

22C SAMPLE 30 DAYS
CONCLUSIONS

• Temperature Effect: Increasing temperature storage conditions increases 

degradation kinetics

• Temperature increases the hydrolysis and oxidation rate

• pH Effect: Vesicles in solution with pH 6.5 degrade faster than pH’s of 9 and 2

• Opposing what is reported in literature

• Showing various properties will differ between membranes

• Size Effect: Smaller vesicles will degrade faster than larger vesicles

• More hydrolysis and oxidation products present in smaller liposome 

solutions

• Structural evolution: Morphology differs at various stages during vesicle 

degradation

• Vesicles undergo transitions into 1D structure and eventually large 3D 

structure

33
ACKNOWLEDGMENTS

• Center for High Resolution Neutron Scattering

• NIST Center for Neutron Research

• Yun Liu

• Elizabeth Kelley

• Joe Dura

• Julie Borchers

34
DYNAMTIC LIGHT 

SCATERING • Popular light scattering technique 

• Laser beam shot at sample and detector 

senses changes in scattering light

• Particles scattered at known angle

• Can provide diffusion coefficient 

• Can be related to particle size

• Only valid at low concentrations

• All samples at 1mg/ml

Radius!

35
MY PREVIOUS CONCLUSIONS

• Last summer used DLS to study vesicle size 

change at different temperatures over time

• Saw significant change in vesicle size at 55c

• Initial expansion and then following contraction of 

vesicle

• Surprising results not well understood

• Saw dependency on vesicle size with the rate of 

degradation

Future Work

• Run longer-term stability experiments

• Study the pathways of chemical decomposition 

• Use small angle neutron scattering to 

understand liposome structure at different 

degradation time points at different 

temperatures

40

60

80

100

120

0 5 10 15 20

avg radius(nm)

days

Radius over Time at varying Temperatures

0

20

40

60

80

100

120

0 5 10 15 20

avg radius (nm)

days

Radius over Time at 55c with Varying Vesicle Sizes

36
LONG-TERM STABILITY EXPERIMENTS

• Ran the same experiments as last summer twice 

over longer time periods

• Changes:

• Controlled for light by completely wrapping everything 

in foil

• Ran chemical assays to check for degradation

• Checked pH measurements

• Decided to not continue with 4c because so similar to 

22c

• Again saw significant change in vesicle size at 55c

• Initial expansion and then following contraction of 

vesicle

• Slower than last summer

• Conclusions:

• Vesicles are degrading at 55c and forming another 

structure

• Exposure to light accelerates this process

• Vesicle size contributes to degradation kinetics

0

20

40

60

80

100

120

0 20 40 60 80 100

Radius(nm)

Time(days)

Batch One

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25 30

Radius(nm)

Time(days)

Batch Two

22c

55c

37