CLUSTERED VS. UNIFORM DISPLAY OF GALA-PEPTIDES ON CARRIER

NANOPARTICLES: ENHANCING THE PERMEATION OF ENDOSOMAL

MEMBRANES

By

TREVAN LOCKE

A dissertation submitted to the

School of Graduate Studies

Rutgers, The State University of New Jersey

In partial fulfillment of the requirements

For the degree of

Doctor of Philosophy

Graduate Program in Chemical and Biochemical Engineering

Written under the direction of

Stavroula Sofou

And approved by

_______________________

_______________________

_______________________

_______________________

New Brunswick, New Jersey

October 2017

ii

ABSTRACT OF THE DISSERTATION

Clustered Vs. Uniform Display of GALA-peptides on Carrier Nanoparticles: Enhancing

the Permeation of Endosomal Membranes

by TREVAN LOCKE

Dissertation Director:

Stavroula Sofou

GALA-peptide is a random coil in neutral pH; in acidic pH it becomes an

amphipathic alpha helix that aggregates in solution, possibly via its hydrophobic facet,

that runs along the helix's long axis. In the presence of fluid lipid membranes, the GALA-

helix exhibits membrane-active properties that originate from the same hydrophobic

facet; these properties make GALA a candidate for inclusion in drug delivery systems

requiring permeation of the endosomal membrane to enable drug escape into the

cytoplasm. Previous work has shown that uniform functionalization of carrier

nanoparticles with GALA-peptides improved their membrane activity and enhanced the

endosomal escape of delivered therapeutics. The present study aims to evaluate the

potential role of altering membrane activity via cluster-displayed GALA-peptides (for

higher local valency) on the surface of carrier nanoparticles. The presentation of GALA-

peptides on carrier nanoparticles was designed to also be pH-dependent. The peptide

display on the surface of the carrier nanoparticles was uniform in neutral pH; in the acidic

endosomal pH, the surface of nanocarriers formed domains (patches) with high local

densities of GALA-peptides.

iii

The interactions between GALA-functionalized carrier nanoparticles and target

lipid vesicles, utilized as endosome membrane surrogates, were studied as a function of

pH. At endosomal pH values, ranging from 5.5 to 5.0, greatest permeability of target

membranes was induced by nanocarriers with clustered and not with uniformly displayed

GALA. This enhancing effect had an optimum; at even more acidic pH values, too close

approximation of GALA peptides residing within the same patches resulted in

preferential intrapatch peptide interactions rather than interactions with the apposing

target lipid membranes. This behavior could have the same physicochemical origin as the

aforementioned GALA-peptide aggregation, observed in solution with lowering pH at

increasing peptide concentrations.

On the translational front, GALA functionalized liposomes were loaded with

doxorubicin and were functionalized with folate in order to specifically target folate-

receptor expressing cancer cells. The findings suggest advantages in drug delivery in the

presence of GALA that potentially mediates fast transmembrane delivery of therapeutics.

The findings of this study support the potential of utilizing the clustered display of

GALA-peptides on carrier nanoparticles to increase the permeation of endosomal

membranes, and to, therefore, improve the endosomal escape of delivered therapeutics,

enhancing therapeutic efficacy.

iv

ACKNOWLEDGMENTS

First and foremost I want to thank my advisor, Dr. Stavroula Sofou, for allowing

me into her lab and for working with me to get as much as we could out of this incredibly

interesting, but complex project. I am a much better scientist thanks to her guidance and

without her mentorship I would not have been able to complete this work. I also want to

thank my committee members, Dr. Martin Yarmush, Dr. Charles Roth, and Dr. Vikas

Nanda. Over the past several years they have all offered much appreciated help and

guidance.

One of the greatest blessings of this PhD has been my fellow Sofou lab

colleagues. I could not have asked for a better group of smart, funny, hardworking people

to work alongside. I want to thank Dr. Amey Bandekar and Ana Gomez for their help in

training me and getting this project rolling. I also want to thank Dr. Charles Zhu, Dr.

Michelle Sempkowski, Sarah (Sally) Stras, Alaina Howe, Thomas Linz, and Aprameya

Prasad for countless instances of good discussion, experimental help, and general advice.

I also want to thank the various funding sources and other agencies that have

allowed me to complete this work or otherwise supported me. This work was supported

in part by the National Institute of Health Grant T32GM8339 (Rutgers Biotechnology

Training Program), the American Cancer Society Research Scholar Grant RSG-12-044-

01, and the National Science Foundation Grants DMR1207022 and CBET1510015. The

Biotechnology Training Program along with the iJOBS program, both NIH funded

initiatives, have broadened my horizons as a student scientist and an employable person

by exposing me to the wide world of biotech, industry, and how to survive outside of the

lab.

v

The staff and students of Rutgers CBE and BME departments and Johns Hopkins

ChemBE department in addition to Mary Ellen Presa and Mary Createau with the

Biotechnology program have also been instrumental in navigating administrative needs

and/or gaining access to needed equipment. In that vein, I also want to thank Dr. Vikas

Nanda and Dr. Jose James for use of CD facilities, and Dr. Zoltan Szekely and Mr.

Stephen Johnson for their assistance in the lipopeptide conjugation.

Last, but certainly not least, I want to thank my amazing friends and family that

have been a constant support. My parents, Missy and Terry Locke, though occasionally

questioning my choice to spend over 20 years in school, have always been there and have

shaped me into who I am today. I could not have done this without them. Apparently I

made graduate school look good, as my wonderful sister, Sarah Locke, is pursuing her

own PhD and I wish her all the best in that endeavor. I am lucky to have too many friends

to acknowledge specifically, but a heartfelt thanks to them, whether they are near or far,

for making life entertaining and fun.

vi

TABLE OF CONTENTS

ABSTRACT OF THE DISSERTATION ii

ACKNOWLEDGMENTS iv

LIST OF FIGURES vii

LIST OF TABLES viii

CHAPTER 1: INTRODUCTION 1

1.1 Doxorubicin as a chemotherapeutic 1

1.2 Liposome-based delivery systems 1

1.3 Endosomal Escape 3

1.4 GALA as an endosomal escape agent 4

1.5 Dissertation Summary 5

CHAPTER 2: CLUSTERED VS. UNIFORM DISPLAY OF GALA-PEPTIDES ON

CARRIER NANOVESICLES 8

2.1 Introduction 8

2.2 Methods 12

2.3 Results 18

2.4 Discussion 31

CHAPTER 3: EFFICACY OF DOXORUBICIN LOADED GALA LIPOSOMES 36

3.1 Introduction 36

3.2 Methods 36

3.3 Results 39

3.4 Discussion 49

CHAPTER 4: DISSERTATION SUMMARY 51

4.1 Key Findings 51

4.2 Limitations 52

4.2 Future Directions 53

REFERENCES 55

vii

LIST OF FIGURES

2.1 Graphical demonstration of uniform versus clustered presentation

2.2 Tunable surface topography of lipid bilayers and of GALA-display using pH as a

trigger.

2.3 Characteristic HPLC elution profile of GALA lipopeptide

2.4 Representative mass spectrum that confirms expected MW of GALA lipopeptide

2.5 Size distribution of uniform vesicles

2.6 Size distribution of clustered vesicles

2.7 Size distribution of endosome surrogate target vesicles

2.8 Half-lives of release rates from endosome surrogate target vesicles induced by GALA

carrying liposomes

2.9 Half-lives of release rates from endosome surrogate target vesicles induced by free

GALA peptide

2.10 Phase transition thermographs of base membranes versus pH

2.11 Binding extents of GALA-peptide to lipids after incubation of free GALA-peptide

2.12 Wavelengths (max) of maximum emission intensities of tryptophan's fluorescence

spectra

2.13 Expanded wavelengths (max) of maximum emission intensities of tryptophan's

fluorescence spectra

2.14 FRET intensities of clustered and uniform carrier vesicles

2.15 Phase transition thermographs including DPPE-GALA and DSPE-PEG

2.16 Maximum tryptophan fluorescence in various solvents

3.1 Temperature effect on folate binding activity

3.2 Doxorubicin/cell on 22rv1 cells for 3% DSPE-PEG uniform liposomes and free

doxorubicin

3.3 Doxorubicin/cell on LNCaP cells for 3% DSPE-PEG uniform liposomes and free

doxorubicin

3.4 Doxorubicin/cell on 22rv1 cells for 5% DSPE-PEG uniform liposomes and free

doxorubicin

3.5 Doxorubicin/cell on LNCaP cells for 5% DSPE-PEG uniform liposomes and free

doxorubicin

viii

3.6 Doxorubicin/cell on 22rv1 cells for clustered liposomes and free doxorubicin

3.7 Doxorubicin/cell on LNCaP cells for clustered liposomes and free doxorubicin

3.8 Percentage of liposomes bound and internalized to 22rv1 cells after six hours at 37⁰C.

LIST OF TABLES

2.1 Nomenclature and compositions of the carrier lipid vesicles.

2.2 Extents of alpha helicity of GALA peptide

2.3 Appearance of GALA peptide aggregates with decreasing pH

3.1 Loaded concentration of doxorubicin (DXR) by liposome type.

3.2 Apparent binding affinity/dissociation constant, KD, on 22rv1 cells

3.3 Apparent binding affinity/dissociation constant, KD, on LNCaP cells

3.4 Killing efficacy of 3% DSPE-PEG doxorubicin loaded liposomes on 22rv1 reported

as LD50

3.5 Killing efficacy of 3% DSPE-PEG doxorubicin loaded liposomes on LNCaP reported

as LD50

3.6 Killing efficacy of 5% DSPE-PEG containing doxorubicin loaded liposomes on

LNCaP reported as LD50

3.7 Retention of doxorubicin loaded 3% DSPE-PEG liposomes after six hours on 22rv1

cells

3.8 Retention of doxorubicin loaded 5% DSPE-PEG liposomes after six hours on 22rv1

cells

1

1 CHAPTER 1: INTRODUCTION

1.1 DOXORUBICIN AS A CHEMOTHERAPEUTIC

Doxorubicin is an anthracycline, a common class of molecules used in the

treatment of solid tumors1. It has proven to be one of the most effective drugs for the

treatment of human malignancies to date. Several mechanisms contribute to its cytotoxic

effects. Among these are: 1) the intercalation into DNA, both nuclear and mitochondrial,

resulting in inhibition of DNA synthesis, 2) binding to and stabilization of the

topoisomerase II enzyme, resulting in single- and double-strand DNA breaks, due to the

inhibition of the enzyme’s ability to relax DNA structures, 3) the direct release of

cytochrome c from mitochondria, resulting in an increase in apoptotic signaling

pathways, and 4) the formation of free radicals resulting in oxidative stress2. Despite its

strength as a cytotoxic agent, doxorubicin use has been hindered because of significant

cardiotoxicity among other less severe side effects such as vomiting, bone marrow

suppression, alopecia, and mucositis3.

1.2 LIPOSOMAL-BASED DELIVERY SYSTEMS

Liposomes are nano-sized vesicles composed of a lipid bilayer encapsulating an

aqueous space. They have proven to be effective and adaptable drug delivery carriers2.

Liposomes can be modified to tune properties for increased efficacy. Surface

modifications, such as PEGylation or electrostatic charge can alter pharmacokinetics and

biodistributions to meet the needs of a given therapy2. Functionalization of liposomes

with peptides, small molecules, aptamers or antibodies can improve targeting to disease

sites overexpressing a targeted receptor of interest such as prostate specific membrane

antigen (PSMA)4 or human epidermal growth factor 2 (HER2)

5. Researchers can alter the

2

bilayer composition to have highly stable particles that retain for extended periods of

time or make bilayers responsive to stimuli such as light, temperature, or pH to trigger

release at desired sites6, 7, 8

. Clinically, liposomal formulations of traditional

chemotherapeutics such as doxorubicin, paclitaxel, and cisplatin exhibit improved

toxicity profiles, biodistributions, and pharmacokinetics relative to chemotherapeutic

alone, increasing efficacy while limiting dangerous side effects. Some of these

formulations have received FDA market approval2. Notable among these in relation to

this work are Doxil®, Myocet, and Lipo-dox (a generic version of Doxil

®), three

doxorubicin loaded, FDA approved liposome formulations. Additionally, a temperature

responsive formulation, ThermoDox is in clinical trials9. Doxil

® was the first approved

liposomal drug and has been on the market since 1995. It relies on PEGylation of the

nanoparticles for extended circulation times, stable and high loading of doxorubicin and a

“liquid ordered” membrane. Doxil®

uses passive targeting due to the enhanced

permeability and retention (EPR) effect to increase the drug load in the tumor10

. One key

tunable property of liposomes is pH-responsiveness. As reported before by our group,11,

12 lipid membranes can be developed that consist of two primary types of lipids with

different tail lengths: one type that is negatively charged and titratableand a second that is

non-titratable with the pH range of interest (generally a headgroup such as phosphatidyl

choline). The basic idea describing the mechanism of phase-separation and patch

formation is the following: in neutral pH, the electrostatic repulsion among negatively

charged lipids is expected to largely prevail, resulting in well-mixed bilayer membranes.

In acidic pH, the titratable headgroups become protonated, and the intrinsic hydrogen-

3

bonding among the now protonated headgroups13

is expected to act as a major attractive

force resulting in phase-separated lipid domains (patches)12

.

1.3 ENDOSOMAL ESCAPE

A primary impediment to the success of nanoparticle-based targeted delivery

systems is entrapment of vesicle contents within endosomes during their internalization

pathway14

. Therefore, disrupting endosomal membranes and promoting the escape of

endosomal contents is an attractive option for enhancing the efficacy of delivered

payloads. Several strategies for endosomal escape including pore formation, membrane

fusion, the proton sponge effect, and photochemical disruption have been investigated.

These strategies typically take advantage of the inherent acidity of the endosomal

environment14

.

Pore formation is generally based on affecting membrane tension within bilayer

membranes. Agents that cause pore formation tend to be amphipathic peptides. Peptide

binding to a lipid bilayer cause internal membrane stress as the membrane thins due to

increased interaction as the peptides insert into the hydrophilic region of the bilayer. As

the pore forms, the peptides preferentially bind to the pore edges, reducing the membrane

tension. The interplay between these effects can result in stable pores15

.

Fusogenic peptides can promote membrane fusion, providing another mechanism

for the destabilization of endosomal membranes. Membrane fusion naturally plays an

important role in cellular trafficking and endocytosis. Many viruses incorporate peptides

that allow them to coopt or bypass these natural fusion processes16, 17

. These viral

4

peptides are inspiration for synthetic peptides including GALA18

which will be discussed

in more detail below.

The proton sponge effect is typically mediated by agents with a high buffering

capacity and the ability to swell when protonated. This protonation results in an influx of

water and ions into the endosome when may subsequently lead to the rupture of the

endosomal membrane. Similarly, evidence suggests that protonated tertiary amine groups

with an attached hydrocarbon chain can act as detergents, disrupting endosomal

membranes14

. Polyethylenimine (PEI)19

and other imidazole-containing polymers20

are

popular proton sponge agents, though not all high buffering capacity polymers induce

this effect21

.

There is also evidence that endosomal membranes can be disrupted

photochemically. These approaches use photosensitizers such as disulfonated aluminum

phthalocyanine and sulfonated derivatives of meso-tetraphenylporphine that can localize

in the endosome membrane while delivered contents are retained within the endosome.

When light is shone on these sensitizers reactive singlet oxygens are produced that

damage the endosomal membrane and promote release of the endocytosed contents while

leaving the contents largely unaffected14, 22

.

1.4 GALA AS AN ENDOSOMAL ESCAPE AGENT

One popular agent for endosomal membrane destabilization is the peptide GALA.

GALA (WEAALAEALAEALAEHLAEALAEALAEALAA) is a synthetic 30 amino

acid peptide that exhibits a random coil at neutral pH, and forms an amphipathic alpha

helix at acidic conditions23

. Originally inspired by viral fusion proteins, GALA has been

5

shown to have both pore formation24

and fusogenic25

properties. As a free peptide,

GALA forms a pore composed of 8-12 monomers within a bilayer membrane. Leakage

occurs quickly once a pore forms and is dependent on reaching a critical mass of peptide

monomers to form pores24

. Evidence also suggests that GALA induces membrane fusion

of fluid-phase small unilamellar vesicles (~50 nm diameter) but not larger vesicles (>100

nm)25

. Since its development, GALA has been widely studied as an agent to promote

increased transfection efficiency of nucleotide based agents encapsulated within

liposomes.26, 27, 28, 29, 30

It has also been studied as an addition to other systems including

antibodies31

and caged protein nanoparticles32

.

1.5 LIPOSOMAL TARGETING TO PROSTATE SPECIFIC MEMBRANE ANTIGEN

Liposomes targeted toward specific receptors have the potential to enhance the

delivery of chemotherapeutic agents to tumors33

, however they face an issue in being

entrapped within the endocytic pathway, reducing this increased efficacy34

. Typical

targets are either unique to the type of cell being targeted and/or are significantly

overexpressed in those cells relative to healthy cells33

. A popular target of study for these

delivery systems is prostate specific membrane antigen (PSMA). PSMA is expressed in

many prostate cancers35

and, despite its name, is commonly expressed in the tumor

vasculature of breast36

, mouth37

, and many other solid tumors38

. In addition to this,

healthy vasculature does not express PSMA, limiting off target effects38

. Many different

strategies for targeting PSMA have been studied including aptamers39

, antibodies40

, and

small-molecule based ligands41, 42

. Perhaps the simplest of these is folate. Due to PSMA’s

folate hydrolase activity, folate targeting can be used to promote binding and

internalization of liposomes to PSMA expressing cells43

. In this study, this targeting

6

capability is combined with liposome anchored GALA to promote targeting followed by

destabilization of endosomal membranes and enhancement of endosomal escape of the

delivered payload.

The human prostate cancer cell lines 22rv1 (~30,000 PSMA receptors/cell ) and

LNCaP (~180,000 PSMA receptors/cell)40

were chosen as model systems to determine

PSMA targeted GALA liposome efficacy. These cell lines have a strong history of being

representative prostate cancer models and have been used in previous folate based

targeting studies43, 44

.

1.6 DISSERTATION SUMMARY

The aim of this dissertation is to develop and characterize doxorubicin loaded

liposomes capable of specific targeting via folate targeting to PSMA and inducing

endosomal escape via the membrane active peptide GALA. Such a system has the

potential to be an enhancement over current therapies by specifically targeting cancer

cells and promoting release of the delivered contents from the endosome. We hypothesize

that such liposomes can be an effective cancer therapy.

In Chapter 2, we determined that GALA presented in a clustered formation versus

a uniform formation resulted in enhanced induction of release from fluid vesicles acting

as endosomal analogues at endosomal pH’s (ph 5.5). We observed that this effect is lost

at pH’s 5.0 and below and investigated the potential causes for such a phenomenon. Data

suggest that GALA-GALA interactions play the most important role in determining this

behavior.

7

In Chapter 3, we investigated the efficacy of GALA liposomes functionalized

with DSPE-PEG-folate in targeting to and killing cells from two prostate cancer cell

lines. We observed that GALA increases killing efficacy with or without targeting,

suggesting mechanisms beyond just promotion of endosomal escape.

8

2 CHAPTER 2: CLUSTERED VS. UNIFORM DISPLAY OF GALA-PEPTIDES

ON CARRIER NANOVESICLES

Note: This chapter was adapted from the following manuscript:

T. Locke, S. Sofou, Clustered vs. uniform display of GALA-peptides on carrier

nanoparticles: enhancing the permeation of endosomal membranes, under revision,

August 2017

2.1 INTRODUCTION

How ligands are displayed on the surface of functionalized nanoparticles plays a

critical role in defining the geometries of ligand-target interactions. These display

patterns have been extensively studied. For drug delivery applications, usually the aim

involves the increase of affinities of functionalized nanoparticles for their targets.45

Organizing ligands in molecular-level clusters grafted on the surface of nanoparticles has

been a popular approach towards this goal.46, 47

These chemically organized clusters of

ligands or 'patches' can be thought to be of molecular dimensions (~2-10 ligands)

uniformly distributed over the surface of the carrier nanoparticles covering several

thousands of nm2

(Figure 2.1 A) Our lab5 has recently reported a clustered display of

Figure 2.1 Graphical demonstration of the (A) chemically and uniformly organized 'molecular' size

patches on the surface of nanoparticles, and (B) multilavent patches occupying a significant fraction of

the nanoparticles' surface which becomes highly heterogeneous. Black lines indicate ligands.

9

ligands on larger 'patches' with sizes occupying a significant fraction (10 to 30 %) of the

nanoparticle's total surface (Figure 2.1 B). These larger patches, containing high local

densities of ligands, were demonstrated to enhance the affinity of functionalized

nanocarriers to levels not seen by nanocarriers functionalized with the uniformly

distributed molecular clusters of ligands.48

In contrast to the uniform surface distribution

of chemically organized clusters of ligands, the larger patches form highly heterogeneous

nanoparticle surfaces containing a significantly large, densely functionalized patch area

of a few thousands of nm2-in-size

49 on a nanoparticle surface of essentially minimal

functionalization.

Here we aimed to evaluate the role of display (clustered vs. uniform) of

nanoparticle-conjugated GALA-peptides on affecting their membrane activity; i.e. their

property of increasing the permeability of endosome surrogate target membranes. GALA

is a pH-responsive, membrane active, thirty amino acid peptide

(WEAALAEALAEALAEHLAEALAEALAEALAA)50

whose design was inspired by

viral fusion proteins sequences 23, 50

. GALA-peptide is a random coil in neutral pH but as

pH becomes acidic, protonation of the repeated glutamic acid residues, allows for the

formation of an amphipathic alpha helix. Once this alpha helix forms, it has been shown

to associate with and to affect the permeability of fluid-phase lipid bilayers50

. These

properties make GALA-peptide a candidate for inclusion in drug delivery systems that

require endosomal escape31, 51, 52

. Previous work has shown that uniform functionalization

of lipid nanoparticles with GALA-peptides improved the activity against fluid

membanes53

and enhanced the endosomal escape of delivered nucleotide contents26, 27, 28,

29, 30. Here we evaluated the potential role of clustered GALA-peptides on the surface of

10

carrier nanoparticles (incorporated as GALA-functionalized lipids) in improving

permeation of endosome surrogate target membranes. The implications of this design

could potentially improve the endosomal escape of delivered therapeutics enhancing,

therefore, the therapeutic efficacy. This efficacy will be explored in a later chapter of this

work.In this study, GALA-peptide was incorporated into gel-phase lipid vesicles (the

carrier nanoparticles) in the form of GALA-functionalized lipids. To minimize

interactions of the GALA-peptide with the underlying carrier vesicle membranes, the

latter were designed to be in the gel-phase at working temperatures. In addition to the pH-

responsive conformation of GALA-peptide, the underlying lipid membrane patches

within the carrier lipid nanoparticles were also designed to be pH-responsive. The carrier

nanoparticles' lipid membranes consist of two types of lipids with different lengths of

saturated acyl-tails and with the following headgroups (Figure 2.2): titratable

phosphatidic acid with effective pKa close to 5.0,54

and phosphatidyl choline). To tune

the GALA-peptide display on the surface of these nanovesicles, GALA-peptide was

conjugated on the headgroup of lipids with acyl-tail lengths identical to those of the

titratable lipid type. In neutral pH, the distribution of GALA-functionalized lipids within

the vesicles' membrane is relatively uniform. In acidic pH, following formation of lipid

patches, preferential partition of the GALA-functionalized lipids, due to the hydrophobic-

phase acyl-tail matching, results in higher local densities, and, therefore, in the clustered

peptide display5.

11

In this chapter, carrier lipid nanoparticles were prepared presenting GALA-

functionalized lipids (GALA-lipids) in uniform and clustered display. The membrane

activity of nanoparticle-displayed GALA-peptides was evaluated by monitoring the

permeability induced on endosome surrogate target membranes as a function of pH. The

binding and relative orientation of GALA-peptides with all types of lipid membranes, as

well as the interactions between peptides and between lipid vesicles vs. pH were

systematically studied in an effort to characterize the observed membrane activities.

Figure 2.2 (A) Tunable surface topography of lipid bilayers and of GALA-display using pH as a trigger.

The upper (lower) leaflet represents the outer (inner) leaflet of lipid nanoparticles. The co-localization of the

GALA-functionalized lipids (in green/blue) depends on the extent of phase separation of the underlying lipid

membrane. (see main text for description of utilization of molecular lipid-lipid interactions as a function of

pH to form lipid phase-separated domains - shown in blue - and to display GALA-functionalized lipids in

clusters).

12

2.2 METHODS

GALA-peptide, > 95 % pure as reported by Anaspec based on HPLC, was

conjugated to 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (sodium

salt) (16:0-PE-succinyl) via its N-terminus. Resin bound peptide, lipid, HATU, and

DIPEA were mixed in a 1:2:2:4 molar ratio using 5 mL NMP as the solvent. This mixture

was allowed to react for 8 hours at room temperature under agitation. The solvent was

then drained and rinsed with NMP four times and then with DCM once (5 mL washes)

and the resulting conjugate was cleaved from the resin with a cocktail of

TFA/water/TIPS/DODT at a 94:2:2:2 mole ratio. After one hour at room temperature, the

cleavage cocktail was added to a cold mixture of 40 mL of 1:1 (v/v) Ethyl ether:petrol

ether. This mixture was centrifuged at 1,700 RCF for 15 minutes, the supernatant was

removed, and 40 mL fresh ether was added. Following a second centrifugation and

drying under vacuum, the peptide precipitate was dissolved in 40 mL 10% acetic acid.

After 10 minutes, the dissolved lipopeptide was frozen and freeze dried. The resulting

material was purified in a 7.8 mm x 300 mm C4 column using HPLC (Waters, Milford,

MA) with an 85%/15% H2O/ACN to 100% ACN gradient over 1 hour at a flow rate of 5

mL/min equipped with a Waters 2487 Dual λ Absorbance Detector (Waters, Milford,

MA). The molecular weight of GALA-functionalized lipid was confirmed via MALDI-

TOF spectroscopy, using a SCIEX 4800 (Applied Biosystems/MDS SCIEX, Concord,

ON, Canada).

13

2.2.1 Liposome Preparation

Lipid vesicles were formed using the thin film hydration method.54

Lipids used in

liposome preparation were 1,2-distearoyl-sn-glycero-3-phosphocholine (18:0-PC), 1,2-

dipalmitoyl-sn-glycero-3-phosphocholine (16:0-PC), 1,2-dipalmitoyl-sn-glycero-3-

phosphate (sodium salt) (16:0-PA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt)

(18:0-PA), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14:0-PA), 1,2-

distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]

(ammonium salt) (18:-PE-PEG), the conjugated 16:0-PE-GALA lipid, L-α-

phosphatidylcholine (Egg, Chicken) (EggPC), and cholesterol. Briefly, lipids (5 mM total

lipid) were hydrated (with either PBS, calcein 75 mM, or 1 mM MOPS buffered sucrose,

all at pH 7.4 and 300 mOsm), annealed, and extruded 21 times through two stacked 100

nm-diameter polycarbonate membranes at temperatures at least 10oC above the highest

transition temperature of the constituent lipids. Then, non-encapsulated contents were

removed by size exclusion chromatography with Sephadex G50 eluted with PBS at pH

7.4 and 300 mOsm. Vesicle size distribution was measured using the Zetasizer Nano

ZS90 (Malvern Instruments Ltd., Worcestershire, U.K.). Transition temperatures in lipid

membranes were determined using differential scanning calorimetry (DSC) by scanning

samples (2.5 mM total lipid) from 20 to 80 °C at a scan rate of 8 °C/hour in a Microcal

VP-DSC (Malvern Instruments Ltd., Worcestershire, U.K.). Lipid vesicles with uniform

and clustered display of GALA-lipids (carrier vesicles) were composed of 18:0-

PC:chol:18:0-PE-PEG:GALA-16:0-PE-lipid and 18:0-PC:XX-PA:chol:18PE-

PEG:GALA-16PE-lipid at 90:5:3:2 and 63:27:5:3:2 mole ratio, respectively, where XX

was either 14:0, 16:0, or 18:0. The above lipid membranes are primarily in gel-phase at

the working temperatures of this study. Lipid vesicles (target vesicles) used as endosome

14

analogues were composed of EggPC:chol at 7:3 mole ratio. This composition was chosen

to provide a fluid-phase lipid bilayer independent of the working temperature(s) that

would exclude explicit charge effects - since headgroups are zwitterionic - to avoid

related electrostatic interactions with the GALA-peptide displayed by the carrier lipid

vesicles. Target vesicles were not PEGylated to eliminate interference of steric effects on

the above interactions. Variations in the lipid compositions of the carrier vesicles,

without 18:0-PE-PEG lipid and/or with varying amounts of GALA-16:0PE-lipid, were

also used as indicated. A summary of membrane compositions used in this study is

provided in Table 2.1.

Table 2.1 Nomenclature and compositions of the carrier lipid vesicles. Ratios are shown in % mole.

Lipid

membrane

18:0-

PC

14:0-

PA

16:0-

PA

18:0-

PA

16:0-

GALA-lipid

Chole-

sterol

18:0-

PEG

pH-

responsive

Clustered 1 63 - 27 - 2 5 3

Clustered 2 63 - - 27 2 5 3

Clustered 3 63 27 - - 2 5 3

Uniform 1 90 - - - 2 5 3 X

all one

domain 87 - - - 6 4 3 X

2.2.2 Content Release Studies

To evaluate the ability of free GALA-peptide or GALA-lipid functionalized

carrier vesicles to cause content release from fluid-bilayer target vesicles, the self-

quenching relief of calcein (ex/em 495/515 nm using a Fluorolog-3-22, Horiba Scientific,

Edison, NJ) encapsulated in endosomal-membrane analogue vesicles was monitored.11

Change in self-quenching efficiency, Qt, over time within the pH range from 7.4 to 4.0

was monitored for calcein-containing target vesicles incubated with free GALA-peptide

15

or GALA-functionalized carrier vesicles at 1:25,000 GALA: target lipid mole ratio or

1:100 GALA-lipid: target lipid mole ratio, respectively. Qt was defined as the ratio of the

completely relieved fluorescence intensity divided by self-quenched fluorescence

intensity at each time point. Triton-X 100 (5% w/v) was added to completely relieve

fluorescent quenching. The normalized % change in quenching efficiency was calculated

as (Qt-1)/(Qmax-1)*100, where Qmax was the quenching efficiency at t = 0. The rate of

release was fitted with a single exponential decay in time. To monitor the effects of

GALA-lipid on altering the permeability of the membranes of the supporting carrier

vesicles, similar studies were conducted on relief of self-quenching efficiency of calcein

encapsulated in carrier vesicles functionalized with GALA-lipid in the absence and

presence of target vesicles which did not contain calcein. In all the above studies, pH

was adjusted with HCl.

2.2.3 Free GALA-peptide binding to Lipid Membranes

Lipid vesicles encapsulating 1 mM MOPS and sucrose (300 mOsm) were

incubated with free GALA-peptide at approximately 1:100 free GALA peptide:total lipid

mole ratio in PBS at different pH values for two hours at 37 °C. Unbound free GALA-

peptide was separated by centrifugation (Optima L-90K Ultracentrifuge with an SW-41

rotor (Beckman Coulter, Brea, CA)) at 30,000 rpm for 90 minutes at 22 °C. The

supernatant, ninety percent of the total volume, was carefully removed using a Pasteur

pipette. The remaining volume was removed with the pellet. For quantitation assays, the

pH on both fractions was restored to 7.4.

16

On each fraction, lipid content was determined via Stewart’s Assay55

. Peptide

content was determined by measuring tryptophan's fluorescence (ex/em: 290/356).

Briefly, samples were mixed with ACN to reach a final v/v ratio of 2:1 PBS buffer:ACN,

and samples' tryptophan concentration was quantified using a standard curve. Peptide-

and lipid-content on the supernatant and pellet were corrected for the 10% liquid volume

that was removed with the pellet.

2.2.4 Peptide Characterization

The fluorescence spectra of the tryptophan residue were acquired in order to

investigate tryptophan's local environment upon free GALA-peptide or GALA-

functionalized lipid interaction with lipid membranes (ex 290 nm/ em 310-400 nm).

To evaluate the alpha-helicity of GALA, circular dichroism (CD) spectra of

samples in PBS at variable pH values (in capped quartz optical cells with a 1mm path

length) were obtained on a J-710 Spectropolarimeter (Jasco Analytical Instruments,

Easton, MD) in a sample chamber flushed with nitrogen at 25°C. The θ values, expressed

as degrees centimeter squared per decimole, were recorded, and percent alpha helicity at

222nm was calculated using an empirical relation derived from completely helical poly-L-

lysine56

following spectra correction by background subtraction (of spectra of buffer

alone or of lipid-containing buffer). All samples were prepared to have final peptide

concentration of 0.05 mg/mL and 65 µM total lipid in the case of liposomes containing

GALA-lipid.

17

2.2.5 Fluorescence Resonance Energy Transfer (FRET) measurements

To capture the GALA-lipid surface distribution on uniform and patchy carrier

vesicles, lipid vesicles composed of 16:0-PC:chol:16:0-PE-RhD:16:0-PE-NBD and

18:0-PC:16:0-PA:chol: 16:0-PE-RhD:16:0-PE-NBD at mole ratios of 94.7:4.7:0.3:0.3

and 66.3:28.4:4.7:0.3:0.3, respectively, were used in order to detect the relative distances

between RhD- and NBD-labeled lipids. The fluorescent lipids were chosen to have 16:0-

acyl-tails and were used as surrogates of the GALA-functionalized lipids which were also

conjugated on lipids with 16:0-acyl tails. The preferential partitioning of fluorescent

lipids within phase-separated domains formed primarily by protonated 16:0-PA lipids

with lowering pH was expected to increase the extent of FRET. In vesicle suspensions,

the value of pH was adjusted with 0.2 N HCl, followed by vesicle incubation at 60oC for

two hours. Upon completion of incubation, lipid vesicles were allowed to cool to room

temperature and FRET (ex/em: 460/590 nm) from NBD-labeled lipids (ex/em: 460/530

nm) to Rhd-labeled lipids (ex/em: 550/590 nm) was measured. The measured FRET

intensities were normalized with respect to the corresponding Rhd-lipid fluorescence

intensities for two reasons: first, to correct for potential pipetting errors in sample

preparation, and, second, to correct for the effects on the fluorescence intensities of Rhd-

lipids due to pH change.

2.2.6 Statistical analysis

Results are reported as the arithmetic mean of n independent measurements ± the

standard deviation. Student’s t-test was used to evaluate differences in behavior between

different forms.

18

2.3 RESULTS

2.3.1 Characterization of GALA-functionalized lipid and of carrier- and target-

lipid vesicles

Following purification of GALA-lipid, by HPLC (Figure 2.3), 80 ± 10% yield by

weight was obtained. The molecular weight of the conjugate (MW = 3,827 Da) was

confirmed, within error of measurement, via MALDI-TOF spectrometry (Figure 2.4).

Carrier lipid vesicles, which were designed to be in gel-phase at working temperatures,

exhibited sizes of 90 ± 2 nm (PDI 0.08 ± 0.06, n=12) and 67 ± 6 nm-in-diameter (PDI 0.2

± 0.04, n=12) in the absence and presence of functionalization with GALA-lipid,

respectively. Endosomal-analogue (target) vesicles, which were composed of fluid

membranes, exhibited sizes of 110 ± 14 nm (PDI 0.12 ± 0.05, n = 6). The size of target

and carrier vesicles was stable at physiologic pH for the duration of studies described

herein. For some compositions, acidification of pH resulted in formation of aggregates

(Figure 2.5, 2.6, and 2.7).

19

Figure 2.3 Characteristic HPLC elution profile of the purification of GALA-functionalized lipid

following the conjugation reaction. The boxed fractions indicate the eluents used as the free GALA-

peptide and the GALA-functionalized lipid.

Figure 2.4 MALDI-TOF spectra that confirm the expected MW of GALA-functionalized lipid.

20

Figure 2.5 Size distributions of vesicle suspensions at different pH values. (left) uniform 1 carrier lipid

vesicles with uniform GALA-lipid display; (right) uniform 1 carrier vesicles mixed with endosome

surrogate target vesicles (at lipid ratios identical to studies on content released from the latter shown in

Figure _ of the main text). Different line types correspond to independent measurements of the same

sample.

Figure 2.6 Size distributions of vesicle suspensions at different pH values. (left) clustered 1 carrier lipid

vesicles with pH-triggered clustered display of GALA-lipids; (right) clustered 1 carrier vesicles mixed with

endosome surrogate target vesicles. Different line types correspond to independent measurements of the

same sample.

21

Figure 2.7 Size distributions of vesicle suspensions at different pH values. Endosome surrogate target

vesicles. Different line types correspond to independent measurements of the same sample.

Three compositions of phase-separating carrier vesicles were studied (clustered 1,

2, and 3) which were functionalized with GALA-lipids for pH-dependent clustered

display (Table 2.1). Across all three compositions, the size of the phase-separated domain

was designed to not vary by including the exact same amount of phase-separating lipid

(30 % mole of total phospholipid). The composition clustered 1 was chosen to represent

the case of greatest partition of GALA-lipid within the phase-separated domain due to the

identically chosen lipid acyl-tails between domain-forming lipids (16:0-PA) and GALA-

functionalized lipids (GALA-16:0-PE). For compositions clustered 2 and 3, the acyl-tails

of domain forming lipids (18:0-PA or 14:0-PA) were two carbon atoms shorter or longer

than the GALA-lipid (16:0 carbon atoms), respectively. For the pH-independent, uniform

display of GALA-lipid on carrier vesicles, uniform 1 vesicles were included containing

GALA-lipid at exactly the same overall molar concentration as all clustered compositions

(2 % mole of the total lipid). In an effort to exclude the effect of domain size (patch size),

22

a ‘all one domain’-carrier vesicle composition was also studied. The ‘all one domain’-

carrier vesicles were functionalized throughout the entire vesicle surface at GALA-lipid

density equal to the maximum possible local density of GALA-lipid within the phase-

separated lipid domains when in clustered display in clustered carrier vesicles (i.e. with

all of the 2 % mole functionalized lipid partitioning in the phase-separated domain). The

‘all one domain’-carrier vesicles were not composed of pH-responsive lipid bilayers.

Free GALA-peptide exhibited pH-dependent shift in alpha helicity, showing

maximum helical content at pH 5.5 followed by a slight decrease at pH 4.0 (Table 2.2).

Incorporation of GALA-lipid on carrier vesicles resulted in almost constant levels of

alpha-helicity with weak pH dependence (Table 2.2). These findings were not affected by

the presence or absence of PEGylated lipid.

Table 2.2 Extents of alpha-helicity of GALA-lipids in carrier lipid vesicles and of free GALA-peptide as a

function of pH. Errors correspond to standard deviations of two independent measurements.

Lipid membrane pH 7.4 pH 6.0 pH 5.5 pH 4.0

Clustered 1 – No PEG 39.4 ± 3.2% 53.5 ± 3.7% 51.2 ± 13.4% 67.7 ± 23.9%

Clustered 1 48.7 ± 3.1% 50.6 ± 4.5% 45.8 ± 0.1% 42.7 ± 5.0%

Uniform 1– No PEG 40.5 ± 6.9% 45.1 ± 2.3% 43.4 ± 2.4% 48.7 ± 8.7%

Uniform 1 43.6 ± 0.4% 46.8 ± 5.1% 46.3 ± 2.6% 50.1 ± 11.9%

free GALA-peptide 37.8 ± 4.0 % 44.9 ± 3.8 % 48.4 ± 1.0 % 38.0 ± 2.5 %

2.3.2 Studies on Inducing Content Release from Endosome-surrogate Target

Vesicles

Carrier vesicles exhibiting clustered GALA-lipid display with lowering pH

(clustered 1, clustered 2, clustered 3) or with uniformly high density of GALA-

functionalized lipids (‘all one domain’-carrier vesicles) demonstrated non-monotonic

23

activity in inducing content release from fluid-phase endosome-surrogate target vesicles

(Figure 2.8). The maximum release rates were observed at pH 5.5 and 5.0. However,

carrier vesicles uniformly functionalized (uniform 1) with same amounts of GALA-lipids,

as the clustered compositions, exhibited monotonically increasing release rates with

lowering pH (Figure 2.8). At pH values of 5.5 and greater, clustered carrier vesicle

compositions resulted in faster release rates than uniform carrier vesicles. Half lives of

calcein's self-quenching relief (indicators of content release) were 12.2 ± 0.6 min

(clustered 1) vs. 54.3 ± 23.5 min (uniform) at pH 5.5 (p-value < 0.01). However, at pH

4.0, uniform 1 carrier vesicles exhibited faster content release rates compared to all other

carrier vesicle compositions. Free GALA-peptide exhibited monotonic content release

similar to that seen from the uniform carrier vesicles (Figure 2.9). Additionally,

endosome target vesicles in the absence of GALA exhibited minimal leakage across the

pH range studied.

24

Figure 2.8 Half-lives of release rates (t1/2=ln(2)/krelease) from endosome analogue target vesicles

encapsulating self-quenching concentrations of calcein induced by clustered 1 (white circles),

clustered 2 (grey circles), clustered 3 (black circles), uniform 1 (white squares), and ‘all one domain’

(white diamonds) carrier vesicles. The lines are guides to the eyes. Error bars correspond to standard

deviations of three independent measurements. * indicates p-values <0.01.

25

Figure 2.9 Half-lives of release rates (t1/2=ln(2)/krelease) from endosome surrogate target vesicles induced by

free GALA-peptide at 1:25,000 GALA:lipid mole ratio for different pH values. The line is guide to the

eyes. Error bars correspond to standard deviations of three independent vesicle preparations.

2.3.3 Extents of pH-dependent Lipid Phase Separation on Carrier Vesicles

The thermographs of the pH-responsive, phase-separating lipid membranes that

were utilized to form patches and to support the clustered display, demonstrated that the

phase-separated domains persisted at pH values below the critical values of 5.5-5.0

beyond which inversion of the membrane activity of the clustered peptide was observed

(Figure 2.8). In particular, DSC scans of lipid membranes (18:0-PC:XX-PA:chol)

containing domain-forming titratable lipids (clustered 1, clustered 2, clustered 3; with

XX= 16:0, 18:0, 14:0, respectively) suggested formation of more-tightly packed phase-

separated lipid domains with lowering pH as demonstrated by the formation of new

26

transition peaks at increasingly higher temperatures and/or by the relative preferential

growth of existing transition peaks at the higher temperatures (Figures 2.10A, 2.10B and

2.10C)11, 54

. In some cases, the overall transition demonstrated increased widths with

lowering pH suggestive of formation of additional phases. As expected, the lipid

membrane comprising the uniform 1 (18:0-PC:chol) vesicles - which was used for the

uniform display of functionalized lipids - did not exhibit changes in the thermal scans

with lowering pH confirming lack of pH-induced phase-separated domains (Figure

2.10D).

Figure 2.10 Phase transition thermographs for the main lipid components of (A) clustered 1, (B), clustered

2, (C) clustered 3, (D) uniform 1 membranes at different pH values. Y-axis divided into increments of 2

cal/°C.

27

2.3.4 Interactions between GALA-peptides and the Lipid Bilayers

Regardless of the lipid membrane specific compositions of carrier- and target-

vesicles, increased association of the GALA-peptide with the lipid membranes (both

fluid- and gel-phase) was observed with decreasing pH. Figure 2.11 shows that, the fluid-

phase lipid bilayers (used as target vesicles) facilitated this interaction at a significantly

greater extent for 5.0 ≤ pH ≤ 6.0 compared to gel-phase lipid bilayers (used as carrier

vesicles) and also demonstrates indistinguishable extents of GALA-peptide association

with all types of carrier vesicles which were used for the clustered (clustered 1, 2, 3) and

uniform (uniform 1) displays.

Figure 2.11 Binding extents of GALA-peptide to lipids after incubation of free GALA-peptide with

endosome surrogate target vesicles (white triangles; fluid-phase), clustered 1 carrier vesicles (gel-phase;

white circles), clustered 2 carrier vesicles (gel-phase; grey circles), clustered 3 carrier vesicles (gel-phase;

black circles), and uniform 1 carrier vesicles (gel-phase; white squares). The lines are guides to the eyes.

Error bars correspond to standard deviations of three independent vesicle preparations. * indicates p-values

<0.01.

28

In addition, the wavelength (max) of maximum emission of tryptophan's spectra

in GALA-lipid (Figure 2.12) - which is indicative of tryptophan’s enviroment50

- was

similar among the gel-phase lipid membrane compositions of uniform 1, clustered 1,

and/or ‘all one domain’ with varying pH and exhibited different pH-dependence

functionality when GALA-lipid was incorporated in fluid-lipid membranes (Figure

2.12A). In our studies, the spectra of tryptophan in GALA-lipids incorporated in gel-

phase clustered 1, uniform 1, and/or ‘all one-domain' carrier vesicles showed a

significant increase (p-values < 0.05) in max with decreasing pH from 7.4 to 5.5

independent of the presence or absence of PEG-lipid in the same vesicle compositions

(Figure 2.13), suggesting a less hydrophobic environment for tryptophan with lowering

pH (Figure 2.12A). On the other hand, GALA-lipids incorporated in fluid-phase vesicles

exhibited the opposite behavior: decreasing max values with lowering pH, potentially

Figure 2.12 (A) Wavelengths (max) of maximum emission intensities of tryptophan's fluorescence spectra

(ex 290 nm) from GALA-functionalized lipids incorporated in gel-phase lipid membrane vesicles: clustered

1 (white circles), uniform 1 (white squares), and ‘all one domain’ (white diamonds) as a function of pH. The

max values from GALA-functionalized lipids incorporated in fluid-phase lipid membrane vesicles are shown

in black triangles. (B) Wavelengths (max) of maximum emission intensities of tryptophan's fluorescence

spectra (ex 290 nm) from free GALA-peptides in the absence of lipids (black circles), and in the presence of

the main lipid components of clustered 1 (white dotted circles) and uniform 1 (white dotted squares) carrier

lipid vesicles not containing the GALA-functionalized lipids, and in the presence of endosomal surrogate

target vesicles (white dotted hexagons). Free Tryptophan (white dotted triangles). The lines are guides to the

eyes. Error bars correspond to standard deviations of three (with the exception of ‘all one domain’ vesicles

which is two) independent vesicle preparations. * indicates p-values <0.01.

29

suggesting a more hydrophobic environment for tryptophan. Additionally, the GALA-

lipid content in all of the lipid carrier membranes did not have a measurable effect on

max shifts (Figure 2.13).

Figure 2.13 Wavelengths (max) of maximum emission intensities of tryptophan's fluorescence spectra (ex

290 nm) from GALA-functionalized lipids incorporated in lipid membrane vesicles: clustered 1 (white

circles), uniform 1 (white squares), uniform 1- No PEG (black squares), uniform 1-Low GALA (gray

squares) uniform 1-Low GALA, No PEG (gray/black squares), ‘all one domain’ (white diamonds), ‘all one

domain’ (gray diamonds), fluid (black triangles), fluid-Low GALA (white triangles), fluid-‘all one domain’

(gray triangles) as a function of pH. All fluid membranes did not contain PEG. The lines are guides to the

eyes.

For reference, the emission spectra of free tryptophan did not exhibit pH

dependence in solution (Figure 2.12B). Interestingly, solutions of the free GALA-peptide

- in the absence of lipid bilayers - exhibited decreasing max values of tryptophan with

decreasing pH accompanied by formation of aggregates (as monitored by DLS, Table

2.3). In the presence of both gel- and fluid-phase lipid bilayers (Figure 2.12A), the max of

30

tryptophan of free GALA-peptide decreased with lowering pH contrary to max from

lipid-conjugated GALA-peptides incorporated in gel-phase lipid membranes (Figure

2.12B).

Table 2.3 Appearance of GALA Peptide Aggregates with Decreasing pH

pH Particles? Particle Size

7.4 No --

5.5 Yes >10 µm

4.0 Yes ~2 µm

2.3.5 Interactions between GALA-peptides via FRET probe measurements

FRET measurements (Figure 2.14) on pH-responsive clustered 1 lipid vesicles

with probe lipids used as surrogates of the GALA-lipid showed that as pH decreases the

probes/peptides come closer together within phase-separated domains due to their

preferential partition with the domain. Indirect evidence of inter-peptide attraction was

also supported for free GALA-peptides in solution which exhibited aggregation with

lowering pH (Table 2.3). As expected, there was no measurable change in energy transfer

between probe lipids which were displayed uniformly on the corresponding lipid vesicles

at pH values ranging from 7.4 to 4.0.

31

Figure 2.14 Normalized FRET intensities (with respect to Rhodamine's fluorescence at each pH value)

between NBD-labeled 16:0-PE lipids and RhD-labeled 16:0-PE lipids, used as GALA-16:0-lipid

surrogates, as a function of pH for clustered 1 carrier lipid vesicles (composed of 18:0-PC / 16:0-PA, white

circles) and uniform 1 carrier lipid vesicles (composed of 18:0-PC, white squares). The lines are guides to

the eyes. Error bars correspond to standard deviations of three independent vesicle preparations. * indicates

p-values <0.01.

2.4 DISCUSSION

The finding of reduced membrane activity of carrier nanoparticles with GALA-

lipids displayed in clusters relative to those with uniform GALA display at pH lower than

5.0 was unexpected (Figure 2.8). This finding could be potentially attributed to the

following factors. (1) decrease in the extents of lipid-phase separation - supporting the

patch formation and peptide clustering - of the underlying lipid membrane of the

clustered carrier vesicles, and/or (2) preferential interaction of GALA-peptides with the

underlying clustered lipid membrane of the carrier vesicle, and/or (3) preferential

32

interactions - due to their close proximity - between GALA-peptides conjugated on lipids

residing within the same patch.

The first hypothesis above is excluded based on the thermograph data in Figure

2.9 which shows no loss of membrane phase separation with respect to pH. This was

further shown by thermographs of vesicles containing PEG and/or GALA also not losing

phase separation with respect to pH as shown in Figure 2.15.

As shown in Figure 2.11, for all membranes considered, increased association of

GALA peptide with the lipid membranes was observed with decreasing pH. This was in

agreement with previous studies.18, 57, 58

Previous reports, which were mostly conducted

using fluid-phase lipid vesicles, attributed the increased peptide-lipid interactions with

lowering pH to the reduced electrostatic repulsion between the peptide and the lipid

headgroups combined with the preferential orientation/association of the hydrophobic

facet of the alpha-helical GALA-peptide with the membrane's hydrophobic region.57

Most importantly, the finding that there was no distinguishable difference between the

carrier vesicle membranes provides evidence do exclude the second hypothesis above.

Furthermore, when looking at the maximum emission of tryptophan’s spectra in GALA-

lipid, no differences in max values were observed across the different carrier vesicle

compositions for pH values between 5.5 and 4.0 Figure 2.12A. Tryptophan is the 1st

amino acid from the GALA peptide-lipid junction point (Figure 2.2),18

and, in general,

lower max values were found to correlate with increasing hydrophobic environments as

shown by measuring tryptophan's spectra in solvents with varying polarity (Figure 2.16).

33

Figure 2.15 Phase transition thermographs for lipid membranes containing DPPE-GALA, DSPE-PEG,

or both at pH 7.4 left and pH 5.5 right. A) clustered 1, B) clustered 2, C) clustered 3, D) uniform 1

34

Potentially different peptide orientations with respect to the supporting lipid

membrane would be expected to also affect the max values. Since there was no

difference, this finding suggests that the observed different membrane activity between

clustered (clustered 1) and uniform (uniform 1) display of GALA lipids on nanocarriers

at pH 4.0 (Figure 2.8) was probably not due to different orientations of GALA relative to

the supporting lipid bilayer of the carrier vesicles at pH 4.0. The above findings,

therefore, suggest that the reduced membrane activity observed at pH values lower than

5.0 by carrier nanoparticles with GALA-lipids displayed in clusters relative to those with

uniform GALA display could not be attributed to different orientations or extents of

association of the peptide with the different supporting lipid membranes.

Ruling out both of the above hypotheses leaves the preferential interaction

between adjacent lipid-conjugated GALA-peptides clustered in patches a plausible

explanation of the observed inversion of membrane activity by carrier vesicles with

Figure 2.16 Wavelength (λmax) corresponding to

maximum of fluorescence emission spectra (ex 289

nm) of Tryptophan in solvents with variable polarity

index (PI). Errors correspond to standard deviations

of three independent measurements. * indicates p-

values < 0.01.

35

clustered GALA display at pH values lower than 5.0. FRET measurements shown in

Figure 2.13 confirm the portioning of GALA peptide within phase-separated domains

with decreasing pH. Furthermore, the release induced by the ‘all one domain’-carrier

vesicles followed a trend similar to the clustered membranes. The interactions between

lipid-conjugated GALA-peptides could potentially be driven by intermolecular histidine-

histidine attractive forces as has been previously reported on different helical

sequences59

. Alternatively, alignment of the hydrophobic facets of adjacent GALA

helices may have also contributed to these molecular complexes60

. Indirect evidence of

inter-peptide attraction was also supported, as reported before,58

for free GALA-peptides

in solution which exhibited aggregation with lowering pH (Table 2.3) or with increasing

GALA concentration57

.

In summary, this study demonstrated that the type of display (clustered vs.

uniform) of membrane-active peptides on the surface of nanoparticles may strongly

influence their effectiveness against apposing target membranes. Nanoparticles

functionalized with membrane-active ligands (such as the peptide GALA) have a role to

play in drug delivery18, 26, 27

. This is because of a key challenge related to the escape of

therapeutic agents from the endosomes into the cytoplasm usually observed after ligand-

receptor mediated endosomal internalization of drug delivery carriers. Clustering on the

surface of carrier nanoparticles of membrane-active peptides may have the potential to

become an alternative to established approaches for endosomal escape such as the

inclusion on nanocarriers of polyethylenimine which acts via the proton sponge effect61

.

Additionally, clustering could be beneficial for other reported fusion inducing or pore

forming peptides14

which are regularly included on the surface of nanocarriers.

36

3 CHAPTER 3: EFFICACY OF DOXORUBICIN LOADED GALA

LIPOSOMES

3.1 INTRODUCTION

In this study, the targeting capability (via measurements of the equilibrium

dissociation constant, KD) and killing efficacy of clustered 1 and uniform constructs

functionalized with GALA, folate conjugated to PEG-lipid, both, or neither is examined

on the human prostate cancer cell lines 22rv1 (~30,000 PSMA receptors/cell ) and

LNCaP (~180,000 PSMA receptors/cell)40

. The causes for efficacy or in some cases the

lack thereof will be discussed along with future directions to be explored based on these

findings.

3.2 METHODS

3.2.1 Liposome Preparation

Clustered and uniform lipid vesicles were formed using the thin film hydration

method, as described above.54

Lipids used in liposome preparation were 1,2-distearoyl-

sn-glycero-3-phosphocholine (18:0-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

(16:0-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (16:0-PA), 1,2-

distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]

(ammonium salt) (18:-PE-PEG), the conjugated 16:0-PE-GALA lipid, and cholesterol to

make the clustered 1 and uniform membranes described in Chapter 2. To add targeting

functionality, 1% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[folate(polyethylene glycol)-2000] (ammonium salt) (18:0-PE-PEG-folate) was

incorporated into liposomes. Liposomes were hydrated with PBS at pH 8.5 or 250 mM

ammonium sulfate at pH 8.5, were annealed at 60 °C for 2 hours, and were extruded 21

37

times through two stacked 100 nm-diameter polycarbonate membranes at 70oC. Then,

non-encapsulated contents were removed by size exclusion chromatography with

Sepharose 4B eluted with PBS at pH 8.5 and 300 mOsm. Liposomes encapsulating

ammonium sulfate were then loaded with doxorubicin, following an adaptation of

published protocol2. Briefly, liposomes were mixed with either 0.25 or 0.5 mM

doxorubicin in 150 mM NaCl. This mixture was incubated at 60 oC for 90 minutes under

agitation. After completion of incubation, unencapsulated doxorubicin was removed by

size exclusion chromatography with Sephadex G50 eluted with PBS at pH 8.5 and 300

mOsm. Before addition to cells, loaded liposomes were passed through a 0.22 µm syringe

filter to sterilize the liposome solution. At each stage of the loading process, small

aliquots were taken to track loading efficiency.

3.2.2 Cell Lines

22rv1 and LNCaP human prostate cancer cell lines were obtained from the

American Type Culture Collection (ATCC, Rockville, MC, USA). Both cell lines were

cultured in RPMI 1640 Media supplemented with 10% fetal bovine serum and 100

units/mL penicillin and 100 μg/mL streptomycin solution at 37°C with 5% CO2. Media

was purchased from ATCC, fetal bovine serum was purchased from Omega Scientific

(Tarzana, CA), and penicillin streptomycin was from Corning Life Sciences (Corning,

NY).

3.2.3 Characterization of Targeted Constructs

The apparent equilibrium dissociation constant KD was evaluated on 22rv1 and

LNCaP cell lines by measuring cell-associated liposomal fluorescence using serial

dilutions of targeted liposomes incubated with 500,000 cells per liposomal concentration

38

incubated for 6 hours at 4 oC to minimize bound liposome internalization. The extent of

non-specific binding was measured by blocking cells with a one hour pre-incubation of

500x free folate at 4 o

C, followed by the primary 6 hour incubation. At the end of the

incubation, cells were washed 3x with ice cold PBS to remove unbound liposomes prior

to measurement and aliquots were removed for cell counting.

3.2.4 Determining Efficacy of Loaded Constructs

22rv1 and LNCaP cells were plated in 96-well plates at 20,000 cells/well and

allowed to incubate overnight at 37 oC. Following this incubation, doxorubicin loaded

liposomes were diluted in media to achieve nine different concentrations of delivered

doxorubicin (including zero/ no treatment). To each well in the 96 well plates, 150 μL of

mixed media and liposomes were added, such that there were three wells for each

construct at each concentration on each of the two cells lines. Following addition of all

doxorubicin constructs, plates were incubated for six hours at 37 oC. At the end of this

incubation, cells were washed with sterile PBS and fresh media was added. Cells were

allowed to incubate in these conditions for two doubling times (68 and 80 hours for 22rv1

and LNCaP cells, respectively). Following the two doubling time incubation, cells were

washed with sterile PBS and MTT dye was added to determine cell viability. After three

hours, detergent solution was added and plates were allowed to sit overnight at 37 oC.

Well absorbance values were measured in a Beckman Coulter (Brea, CA) DTX 880 plate

reader using a filter centered at 590 nm.

In parallel to the above, 300,000 cells/well were plated in six-well plates and also

allowed to incubate overnight at 37 oC. Following this incubation, solutions of 10 mM

doxorubicin were prepared for each construct and 1 mL of each was added to one of the

39

wells in the six-well plate. Plates were incubated for six hours at 37 oC. Following this

incubation, the media was removed for determining doxorubicin retention. Cells were

washed with sterile PBS and aliquots were removed for cell counting. Cells were then

lysed to detect doxorubicin uptake fluorescently (ex/em: 470 nm/592 nm).

To determine retention the saved doxorubicin loaded liposome containing

media from above was split into two fractions. One fraction was passed through a

Sephadex G50 column eluted with PBS at pH 7.4 to separate leaked doxorubicin. The

second fraction was diluted to achieve an equivalent volume with the first. Trition X-100

(5% w/v) was added to each fraction before incubating them at 80 oC for ten minutes.

After allowing fractions to cool, doxorubicin content was measured via fluorescence

(ex/em: 470nm/592 nm). Dividing the first fraction by the second provided a

measurement of liposomal retention.

3.3 RESULTS

3.3.1 Doxorubicin Loading Optimization

Developing targeted, GALA functionalized liposomes revealed limitations in

loading doxorubicin. Chief among these was the deactivation of folate after prolonged

exposure to 70 °C or higher (Figure 3.1), and aggregation of folate liposomes above 80

°C. Furthermore, the presence of GALA negatively impacted the loading efficiency of

doxorubicin, resulting in aggregation of liposomes. This was partially remedied by

loading at pH 8.5 instead of 7.4 to reduce the amount of protonated histidine residues. A

summary of loaded doxorubicin under various conditions can be seen in Table 3.1.

40

Figure 3.1 Binding of targeted liposomes to 22rv1 cells. Circles indicate liposomes prepared at 60 oC and

squares at 70 oC. Black symbols correspond to unblocked binding of liposomes, gray symbols correspond

to bound liposomes incubated with cells whose folate receptors have been blocked by exposure to excess

free folate.

41

Table 3.1 Loaded Concentration of Doxorubicin (DXR) by liposome type. Errors correspond to standard

deviations of two independent measurements.

Liposome Membrane Loaded DXR

Concentration

(µM) 60 °C 70 °C

Uniform 72 ± 7 72 ± 4

Uniform Targeted 83 ± 10 77 ± 0

Uniform GALA 28 ± 7 48 ± 22

Uniform GALA Targeted 40 ± 10 79 ± 1

Uniform Low GALA 6 ± 1 not

measured

Uniform Low GALA Targeted 9 ± 9 not

measured

Uniform 5% PEG 90 ± 13 not

measured

Uniform Targeted 5% PEG 95 ± 19 not

measured

Uniform GALA 5% PEG 22 ± 6 not

measured

Uniform GALA Targeted 5%

PEG

58 ± 1 not

measured

Clustered 20 48 ± 8

Clustered Targeted 29 65 ± 12

Clustered GALA not

measured

47 ± 20

Clustered GALA Targeted not

measured

49 ± 26

Clustered Low GALA 15 ± 4 not

measured

Clustered Low GALA Targeted 8 ± 7 not

measured

3.3.2 Evaluation of Targeting Efficacy

Folate targeted liposomes with and without GALA exhibited specific binding on

both LNCaP and 22rv1 cell lines, though varying degrees of binding were observed.

Tables 3.2 and 3.3 show the KD values for each construct investigated. As previously

mentioned exposing folate to temperatures greater than 70 °C reduced targeting efficacy

(Figure 3.1). Raising folate-functionalized lipid above 1% mole of the total lipid

membrane content resulted in aggregation of liposomes regardless of total PEG-lipid

42

content. Furthermore, clustered liposomes as described in previous chapters (Table 2.1)

without GALA showed very little specific binding to cells. Lowering the fraction of

titratable lipid by a factor of three resulted in improved binding. This improvement in

binding only translated to GALA bearing liposomes when GALA content was also

reduced by a factor of three.

Table 3.2 Apparent equilibrium dissociation constant, KD, on 22rv1 cells. Errors correspond to errors in the

fit of the data.

22rv1 Uniform

(μM)

Clustered

(30% of area in

patch) (μM)

Clustered

(10% of area in

patch) (μM)

Targeted 31 ± 5.1 60 ± 16 13.9 ± 4.0

Targeted + GALA 9.0 ± 1.0 Undetectable Undetectable

Targeted + Low

GALA

not

measured

Not measured 11 ± 1.2

Table 3.3 Apparent equilibrium dissociation constant, KD, on LNCaP cells. Errors correspond to errors in

the fit of the data.

LNCaP Uniform

(μM)

Clustered

(30% of area in

patch) (μM)

Clustered

(10% of area in

patch) (μM)

Targeted 9 ± 1.5 130 ± 62 24 ± 6.4

Targeted + GALA 13 ± 1.1 Undetectable Undetectable

Targeted + Low GALA not

measured

not measured 14.4 ± 3.1

3.3.3 Evaluation of Killing Efficacy

Loaded liposomes were evaluated by their efficacy in killing 22rv1 and LNCaP

cells. Given the improved binding ability of the smaller patch size, only those liposomes

43

were tested for killing efficacy. Tables 3.4 and 3.5 list the LD50 values for constructs

tested on these cells.

For the uniform presentation, targeted liposomes exhibited more effective killing

than nontargeted liposomes without GALA in both cell lines, as expected. Interestingly,

the addition of GALA to uniform liposomes enhanced both targeted and nontargeted

killing efficacy at two different levels of liposomal GALA. Additional DSPE-PEG was

added to uniform liposomes, to block GALA’s efficacy when not targeted to cells. This

data is shown in Table 3.6. With the exception of targeted liposomes on LNCaP cells,

increasing DSPE-PEG levels resulted in higher LD50's or lower efficacy.

The clustered liposomes exhibited a consistent level of killing on 22rv1 cells

regardless of liposome functionalization. Clustered liposome data for LNCaP cells was

not available due to experimental failures.

Table 3.4 Killing efficacy of 3% DSPE-PEG Doxorubicin loaded liposomes on 22rv1 reported as LD50.

Errors correspond to standard deviations of two (or three for Free DXR) independent measurements.

*Errors correspond to propagations of errors in the fit of the data ap<0.01

22rv1 Uniform

(μM)

Clustered

(10% of area in

patch) (μM)

Nontargeted 70.0 ± 59.0 1.5 ± 88*

Targeted 15.0 ± 23 1.5 ± 85*

Nontargeted GALA 8.3 ± 0.14a not measured

Targeted GALA 3.1 ± 0.14a not measured

Nontargeted Low GALA 2.9 ± 0.6 1.4 ± 0.2

Targeted Low GALA 0.4 ± 65* 1.0 ± 37*

Free DXR 0.61 ± 0.08

44

Table 3.5 Killing efficacy of 3% DSPE-PEG Doxorubicin loaded liposomes on LNCaP reported as LD50.

Errors correspond to standard deviations of two (or three for Free DXR) independent measurements. *Error

corresponds to propagations of errors in the fit of the data

LNCaP Uniform (μM)

Nontargeted 77.2 ± 29

Targeted 4.4 ± 3.4

Nontargeted GALA 10.6 ± 3.4

Targeted GALA 4.5 ± 1.6

Nontargeted Low GALA 1.7 ± 0.8

Targeted Low GALA 0.3 ± 95*

Free DXR 0.28 ± 0.18

Table 3.6 Killing efficacy of 5% DSPE-PEG containing Doxorubicin loaded liposomes on LNCaP reported

as LD50. Errors correspond to standard deviations of two independent measurements. **One of two

liposome preparations; error corresponds to propagations of errors in the fit of the data

5% PEG Liposomes 22rv1 (μM) LNCaP (μM)

Nontargeted Undetectable Undetectable

Targeted 158.9 ± 41.2 1.7 ± 1.0

Nontargeted GALA 12.5 ± 0.6 23.4 ± 1084**

Targeted GALA 6.4 ± 2.0 12.4 ± 11.9

In addition to measuring liposome efficacy, retention of doxorubicin by liposomes

and the amount of doxorubicin per cell were also measured in parallel experiments as

exactly identical incubation conditions (37 °C, six hour incubation). Retention of

liposomes is shown in Table 3.7 (3% DSPE-PEG) and Table 3.8 (5% DSPE-PEG).

45

Liposomes retained well over six hours in the presence of cells. Targeted liposomes

delivered more doxorubicin to 22rv1 cells than nontargeted liposomes, while liposomes

with GALA delivered more than those without (Figure 3.2). The same liposomes on

LNCaP cells showed higher DXR uptake for GALA liposomes than for liposomes with

targeting alone (Figure 3.3). These trends were consistent at both levels of DSPE-PEG-

lipid tested (Figures 3.4 and 3.5). Clustered data, while incomplete (due to low

doxorubicin loading), suggests fewer and smaller differences between the different

liposome compositions in terms of doxorubicin uptake per cell (Figures 3.6 and 3.67).

When observing uptake of free doxorubicin, 22rv1 cells had over twice the uptake of

doxorubicin per cell.

Table 3.7 Retention of Doxorubicin loaded in 3% DSPE-PEG-lipid containing liposomes after six hours of

incubation with 22rv1 cells. Errors correspond to standard deviations of two independent measurements.

Uniform Clustered

Nontargeted 87.0 ± 7.1 % 91.0%

Targeted 85.0 ± 7.8 % 91.0%

Nontargeted GALA 75.0 ± 5.7 % not measured

Targeted GALA 75.0 ± 14 % not measured

Nontargeted Low GALA 99.0% 89.0 ± 13 %

Targeted Low GALA 86.0% 80.0%

Table 3.8 Retention of Doxorubicin loaded in 5% DSPE-PEG-lipid containing liposomes after six hours of

incubation with 22rv1 cells. Errors correspond to standard deviations of two independent measurements.

Uniform

Nontargeted 96.0 ± 0.7 %

Targeted 98.0 ± 1.4 %

Nontargeted GALA 75.0 ± 1.4 %

Targeted GALA 72.0 ± 4.2 %

46

Figure 3.2 Doxorubicin uptake per cell for 22rv1 cells incubated with uniform nontargeted (cyan), targeted

(blue), nontargeted with GALA (red) and targeted with GALA (dark red) 3% DSPE-PEG-lipid containing

liposomes and free doxorubicin (black) after six hours. Error bars correspond to standard deviations of two

independent measurements. *:p<0.05

Figure 3.3 Doxorubicin uptake percell for LNCaP cells for uniform nontargeted (cyan), targeted (blue),

nontargeted with GALA (red) and targeted with GALA (dark red) 3% DSPE-PEG-lipid containing

liposomes and free doxorubicin (black) after six hours. Error bars correspond to standard deviations of two

independent measurements. *:p<0.05

47

Figure 3.4 Doxorubicin uptake per cell for 22rv1 cells incubated with uniform nontargeted (cyan), targeted

(blue), nontargeted with GALA (red) and targeted with GALA (dark red) 5% DSPE-PEG-lipid containing

liposomes and free doxorubicin (black) after six hours. Error bars correspond to standard deviations of two

independent measurements. *:p<0.05

Figure 3.5 Doxorubicin uptake per cell for LNCaP cells incubated with uniform nontargeted (cyan),

targeted (blue), nontargeted with GALA (red) and targeted with GALA (dark red) 5% DSPE-PEG-lipid

containing liposomes and free doxorubicin (black) after six hours. Error bars correspond to standard

deviations of two independent measurements. *:p<0.05

48

Figure 3.6 Doxorubicin uptake percell for 22rv1 cells incubated with clustered nontargeted (cyan), targeted

(blue), and nontargeted with GALA (red) 3% DSPE-PEG-lipid containing liposomes and free doxorubicin

(black) after six hours. Due to low drug loading doxorubicin per cell for targeted liposomes with GALA

could not be compared to the other conditions. Error bars correspond to standard deviations of two

independent measurements.

Figure 3.7 Doxorubicin uptake per cell for LNCaP cells incubated with clustered nontargeted (cyan),

targeted (blue), and nontargeted with GALA (red) 3% DSPE-PEG-lipid containing liposomes and free

doxorubicin (black) after six hours. Due to low drug loading doxorubicin per cell for targeted liposomes

with GALA could not be compared to the other conditions. Error bars correspond to standard deviations of

two independent measurements.

49

3.4 DISCUSSION

The above data show that for uniform liposomes adding either targeting

functionality via folate or endosomal escape functionality improve killing of prostate

cancer cells in vitro, while the combination of the two lead to some increase in

effectiveness. While the increase in effectiveness from targeting was expected, the

increase from adding GALA alone was not predicted. It is possible that in addition to

endosomal escape activity at acidic pH that GALA also has some cell membrane

penetrating properties even in a neutral pH environment. This hypothesis is supported by

our CD data in chapter 2, Table 2.2 showing that upon conjugation of GALA peptide on

liposomes (independent of the underlying lipid membrane composition) the extent of

helicity - independent of pH - is greater than the helicity of the free peptide at pH 7.4.

This observation is supported, at least in 22rv1 cells, by the apparent KD’s (Table 3.2)

showing a stronger binding interaction (lower KD) with GALA than without. This

potential membrane penetration is further evidenced by binding experiments at 37 ⁰C

showing increased levels of liposome association with 22rv1 cells by GALA liposomes

as compared to liposomes with GALA (Figure 3.8). This effect is potentially harmful as it

could contribute to off target toxicities. To address this potential challenge, GALA

concentration was lowered in an attempt to minimize GALA’s effect without targeting,

but was not successful. To a degree, this behavior is also observed in the measurements

of delivered doxorubicin per cell, where GALA liposomes deliver more doxorubicin than

their non-GALA counterparts (Figures 3.2 and 3.3). It was originally hypothesized that

GALA-liposomes would deliver similar amounts of drug per cell, but would be more

effective due to enhanced endosomal escape. It appears, that at least in part, GALA

50

increases efficacy by increasing the amount of delivered drug possibly acting not only the

endosomal membrane but also on the plasma membrane of cells. Interestingly, GALA

liposomes deliver more doxorubicin to LNCaP cells than targeted liposomes.

Figure 3.8 Percentage of liposomes bound and internalized to 22rv1 cells after six hours at oC.

For the clustered liposomes, a smaller patch was used to improve targeting

efficacy. As Tables 3.2 and 3.3 show, the original clustered membrane adopted from

Chapter 2 exhibited poor targeting efficacy. It is possible that the properties of this highly

negatively charged membrane resulted in poor partitioning of the folate-PEG lipid into

the liposome membrane resulting in a lower effective amount of targeting ligands.

Despite being well retaining membranes, the new clustered membrane exhibited

similar killing behavior for all constructs. The similarity in this effect is partially

supported by the similar levels of detected doxorubicin per cell delivered by clustered

liposomes. Further investigation will be required to deduce the causes behind this

behavior.

51

Regardless of the challenges in loading these liposomes, even the least efficient

loadings of GALA-functionalized liposomes seem to be significant enough to enable cell

killing within an order of magnitude of free doxorubicin levels within six hours. This is a

promising result that suggests this system may have potential uses a therapeutic delivery

vehicle despite the difficulties in its preparation.

Taken together, this data suggests the liposomes described here have the potential

to be an effective therapy. The addition of GALA enhances delivered doxorubin per cell,

and, therefore, killing. While this result is promising, for this to be a truly attractive

therapeutic option, adjustments will need to be made to limit the effect of untargeted

GALA liposomes. In this work, raising the amount of DSPE-PEG-lipid showed more

potential for limiting this effect than lowering the amount of GALA-lipid per liposome.

Further studies adjusting the levels of these two functionalized lipid types may deliver

even stronger results

52

4 DISSERTATION SUMMARY

4.1 KEY FINDINGS

The key finding of this dissertation is that carrier liposomes presenting the

membrane destabilizing peptide GALA in a clustered presentation induce enhanced

membrane permeability of endosome analogue target vesicles at endosomal pH than

liposomes presenting GALA in a uniform presentation when the amount GALA peptide

is equivalent. This effect is lost at pH less than 5.0. Increasing GALA-lipid concentration

in a uniform membrane results in similar non-monotonic behavior in induced release,

suggesting that GALA-GALA interactions determine the effectiveness of release. This

finding may prove beneficial for developing endosomal escape directed particles or other

particles relying on peptide interactions on the surface of a nanoparticle.

In our studies, this enhanced behavior from clustered GALA did not conclusively

result in enhanced killing in vitro when compared to uniformly presented GALA.

However, GALA-functionalized liposomes did result in more effective targeting, drug

delivery, and killing compared to liposomes without GALA. This could indicate that in

addition to endosomal escape functionality, GALA has a strong potential in disrupting

plasma cell membrane bilayers when anchored to liposomes.

4.2 LIMITATIONS

The inability to more directly measure the behavior of GALA anchored to lipid

membranes limited our ability to study why a clustered presentation of GALA-lipid

behaves differently. In addition to the presented data, proving the nature of GALA-

GALA interactions on lipid membranes conclusively could strengthen efforts to take

53

advantage of this phenomenon. This could potentially be accomplished by preparing

liposomes with two different fluorescently tagged GALA-lipids such that either

quenching or enhancement of signal can be detected based on proximity of one GALA-

lipid to another similar to the FRET studies described in Chapter 2.

The complex nature of the liposomes described in this text present several

challenges as the individual components have differing optimal ranges. There is an upper

limit on the amount of folate-PEG that can be incorporated before aggregation begins to

occur. The activity of folate-PEG is also sensitive to temperature lowering the upper limit

of temperature liposomes can be exposed to weakening the loading ability of

doxorubicin. Under certain conditions significant aggregation also occurs when loading

GALA-functionalized liposomes with doxorubicin. This can be partially controlled by

adjusting the pH and loading concentration of doxorubicin but this adds additional

limitations on the optimal window for loading.

4.3 FUTURE STUDIES

Additional studies on clustered presentation of functional ligands on liposomes

could shine light on why certain levels of titratable PA lipid seem to impair targeting

capability. Also more studies are needed to adequately describe why and how clustered

liposomes behave as seen in the killing efficacy assays shown. Futhermore, repeating

many of the experiments in this text with a version of the GALA peptide with its histidine

residue removed or replaced could reveal interesting facets of not only clustered GALA-

lipid behavior, but also variables affecting the doxorubicin loading of GALA-

functionalized liposomes.

54

Modeling the activity of these liposomes based off the presented data could help

illuminate ideal compositions for the best outcome. Combining constants related to the

targeting of PSMA with release rates from the liposome and rates of endosomal escape

derived from intracellular imaging data of GALA-induced endosomal escape could

present a strong model of the system for determining bottlenecks to effectiveness.

The system described in this text may have some utility as a delivery platform.

Within the realm of PSMA targeting, developing these liposomes as an antivascular

could be an option4. Folate is also overexpressed in inflamed tissue and may be another

avenue where targeted nanoparticles that can induce endosomal escape could be useful62

.

Additionally, folate could be exchanged with other targeting moieties to target other

receptors overexpressed in diseases, like HER2 for breast cancer therapy, extending the

added functionality of endosomal escape and enhanced delivery.

55

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