University of Groningen

DNA nanotechnology as a tool to manipulate lipid bilayer membranesMeng, Zhuojun

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Chapter 1

Functionalization of

Lipid Bilayer Membranes

Chapter 1

10

1. 1 Lipid bilayer membranes

Lipids play an important role in the physiology and pathophysiology of

living systems why they are produced, transported, and recognized by the

concerted actions of numerous enzymes, binding proteins, and receptors.1

Micelles are formed by the aggregation of single-chain lipids in a polar

solvent (such as water) beyond a particular concentration, known as

Critical Micelle Concentration (CMC) (Fig. 1.1A). Therefore, the micelle

formation and stability are highly dependent on the lipid concentration and

solvent composition (Fig. 1.1B).

Two-chain lipids can hardly be packed into micelles due to the bulky

hydrophobic part. They usually form a lipid bilayer membrane, which is a

thin polar sheet made of two layers of lipid molecules and is characterized

by hydrophobic tails facing inwards towards each other and hydrophilic

head groups facing outwards to associate with aqueous solution.2 At this

moment, the hydrophobic parts of the molecules are still in contact with

water, which leads to an energetically unfavorable state of the bilayer. This

is overcome through folding of the bilayer membrane into a liposome with

closed edges (Fig. 1.1C).3,4

Functionalization of Lipid Bilayer Membranes

11

Fig. 1.1 (A) Surface tension as a function of the surfactant concentration. Schematic structure

of a micelle (B) and a liposome (C). (Fig. 1.1 C was adapted from reference 4)

Chapter 1

12

1.2 Classification and Preparation of Liposomes

Depending on the number of bilayers, liposomes can be classified into two

categories: unilamellar vesicles (ULV) and multilamellar vesicles (MLV).

Unilamellar vesicles can also be classified into three categories on the basis

of their sizes, which can vary from nanometer to micrometer range: small

unilamellar vesicles (SUV), large unilamellar vesicles (LUV) and giant

unilamellar vesicles (GUV). GUVs also include other morphologies such as

multilamellar vesicles (MLV), which consist of SUVs or multiple concentric

bilayers (Fig. 1.2).

Fig. 1.2 Schematic structure of unilamellar and multilamellar liposomes.

There are four classical methods to prepare liposomes, differing in the way

how the lipids are dried from organic solvent and then redispersed in

aqueous buffer.5 These steps can be performed individually or jointly.6

These four methods are:

1. Hydration of a Thin Lipid Film.7

2. Reverse-Phase Evaporation Technique. 8

3. Solvent (Ether or Ethanol) Injection Technique.9,10

4. Detergent Dialysis.11

Functionalization of Lipid Bilayer Membranes

13

Since the “Hydration of a Thin Lipid Film” method, is widespread used and

easy to handle, it is explained here in more details. Firstly, the lipids are

dissolved and mixed in an organic solvent to assure a homogeneous

mixture. Once the lipids are thoroughly dispersed in the organic solvent,

the solvent is removed using a dry nitrogen stream in a fume hood to yield

a lipid film. The lipid film is dried to remove residual organic solvent by

using a vacuum desiccator overnight. Afterwards, hydration of the dry lipid

film is accomplished by stirring in an aqueous buffer. The temperature of

the hydrating buffer should be higher than the gel-liquid crystal transition

temperature (Tc) of the lipid. Subsequently, several stirring (above the Tc)

and freeze-thawing cycles of the swelling multilayer sample results in

MLVs. Finally, the sample is extruded multiple times using an extruder and

polycarbonate membranes to obtain unilamellar vesicles (LUVs or SUVs).

Fig. 1.3 shows the classical hydration method of liposome preparation.

Fig. 1.3 Schematic diagram of liposome preparation method. (Schematic obtained from www.

avantilipids.com)

Chapter 1

14

1.3 Modification and Applications of liposomes

The first discovery of liposomes in 1964 by A. D. Bangham12 was the

starting point for these self-assembled containers to become a

multifunctional tool in biology, biochemistry and medicine today. Because

of the structure, charge, chemical composition and colloidal size can be well

controlled by preparation methods, liposomes can be useful in various

applications. Vesicles can also be prepared from natural substances and are

therefore in many cases nontoxic, biodegradable, biocompatible, targetable

and non-immunogenic.13 Due to these properties, liposomes can be used as

drug14-16 protein, plasmid17 and gene18-21 delivery vehicles in medicine and

diagnosis.

1.3.1 Loading and surface modification

Molecular interactions between the cargo and the lipid bilayer membrane

play an important role on liposome formation and cargo encapsulation.22

Liposomes consist of an aqueous core surrounded by a lipid bilayer,

sectioning off two separate inner areas. They can carry hydrophobic

molecules in their hydrocarbon tail region (between the phospholipid

bilayer), or hydrophilic molecules in the core and direct the cargo to the

required diseased site in the body with some targeting moieties on the

surface.23 The thickness of the lipid bilayer is around 4 to 10 nm, which is a

natural barrier for many substances such as sugars and proteins.24 But

small hydrophilic substances such as water, gases, ammonia and glycerol

can penetrate freely through the bilayer.25-27 Some large hydrophilic

substances can be encapsulated in the water core of the liposome during

liposome preparation using the common thin layer hydration method.

Cationic liposomes, which are made of positively charged lipids, appear to

be better suited for DNA delivery due to the natural charge-charge

interaction between the positively charged lipid head groups and the

negatively charged phosphate groups of the DNA-backbone.28,29 Due to

their favorable interactions with negatively charged DNA and cell

membranes,30-33 cationic liposome–DNA complexes are increasingly being

researched for their use in gene therapy and nucleic acid release.34,35 In

order to increase liposomal drug accumulation in the desired cells and

Functionalization of Lipid Bilayer Membranes

15

tissues, the use of targeted liposomes with surface modification has been

suggested.

Surface modification of liposomes with controlled propertied requires the

chemical conjugation of peptides, DNA, antibodies or other targeting

molecules. Moreover, some “smart” vesicle designs allow the release of the

encapsulated cargo by incorporation of transport channels.36-39 Both

chemical attachment and physical interactions can be used to achieve

surface modification (Fig. 1.4A).

Fig. 1.4 (A) Schematic representation of liposomes surface modifications. (B) Interaction of the

particle with cell surface antigens and receptors.40 (C) Scheme of tetrac tagged liposome and

enhanced delivery by the ligand-mediated targeting strategy.41 (Fig. 1.4 B was adapted from

reference 40. Fig. 1.4 C was adapted from reference 41)

To realize active targeting, the liposome surface can be coated with ligands

or antibodies that will confer cell type-specificity to ensure that the

liposomes are internalized and that their content is released, improving the

efficacy and reducing side effects over non-targeted cells (Fig. 1.4B).40 For

instance, tetraiodothyroacetic acid (tetrac), a small molecule which binds

Chapter 1

16

to integrin αvβ3, was used for the surface modification of liposomes and

successfully enhanced the tumor-targeting ability of PEGylated liposome

(Fig. 1.4C).41 Although the physical properties of liposomes were not

significantly changed, tetrac-tagged liposomes showed significantly higher

cancer cell localization than the unmodified PEGylated liposome, and

tumor growth was effectively retarded. The ligand-mediated targeting

strategy could provide better therapeutic effects with more accurate

delivery of nanoparticles.

1.3.1.1 Membrane fusion

Surface modification of lipid bilayers can also be used for membrane fusion

which is an essential process of life resulting in the highly regulated

transport of bio-molecules both between and within cells.42-44 Membrane

fusion is an essential but not a spontaneous process as free energy is

required to overcome the electrostatic and steric repulsions between two

merging membrane surfaces and to break the hydration shell.45,46 A highly

conserved protein machinery, known as SNARE proteins (soluble N-

ethylmaleimide sensitive factor attachment protein receptors), facilitates

the communication within a cell.47-49 The SNAREs from synaptic vesicles

interact with the SNAREs from the target membrane to form a coiled-coil

bundle of four helices, pulling the membranes tightly together and

initiating fusion.

Design and construction of simplified artificial model systems mimicking

natural systems are one of the most promising approaches for studying

complex biological mechanisms.50 Several of these systems have been

reported for realizing membrane fusion, such as DNA51-53, peptides54, 55,

enzymes56 and polymers57. Yang et al. designed an artificial biorthogonal

targeting system that was able to target liposomes and other nanoparticles

efficiently to the tissue of interest by using coiled coil forming peptides,

E4[(EIAALEK)4] (E4) and K4[(KIAALKE)4] (K4) (Fig. 1.5C), which are

known to trigger liposomal membrane fusion when tethered to lipid

vesicles in the form of lipopeptides.58 The same group proved that E4

peptide-modified liposomes could deliver far-red fluorescent dye TOPRO-3

iodide (E4-Lipo-TP3) and doxorubicin (E4-Lipo-DOX) into HeLa cells

Functionalization of Lipid Bilayer Membranes

17

expressing K4 peptide (HeLa-K) on the surface. Then, E4-Lipo-TP3 and E4-

Lipo-DOX were injected into zebrafish xenografts of HeLa-K (Fig. 1.5A, B).

The results showed that E4-liposomes delivered TP3 to the implanted

HeLa-K cells (Fig. 1.5D), and E4-Lipo-DOX could suppress cancer

proliferation in the xenograft when compared to nontargeted conditions.

These data demonstrated that coiled-coil formation enables drug

selectivity and efficacy in vivo.

Fig. 1.5 Drug Delivery by E4/K4 Coiled-Coil Formation in Cells (A) and Zebrafish (B). (C)

Schematic representation of coiled-coil structure between peptides E and K. (D) E4/K4 coiled-

coil formation allows delivering the content in the liposome to cancer cells in the xenograft

zebrafish.58 (This figure was reproduced with permission from reference 58)

1.3.1.2 Controlled release

Conventional liposomes (Fig. 1.6A) are easily recognized by the

mononuclear phagocyte system and are rapidly cleared from the blood

stream.59 Many methods have been suggested to achieve long circulation of

liposomes in vivo by modification of the liposomal surface with hydrophilic

Chapter 1

18

polymers to delay the elimination process, such as coating the surface of

liposomes with biocompatible polymers like poly(ethylene glycol) (PEG)

linked phospholipids. These can be incorporated into the liposomal bilayer

to form a hydrophilic polymer shield over the liposome surface, protecting

the liposome from penetration or disintegration by plasma proteins60-65

(Fig. 1.6B). While many varieties have been synthesized by using

chemically modified forms of PEG, in some cases it’s necessary to make the

liposomes shed their cloak of modified PEG molecules when they reach

their target (Fig. 1.6C). In this way they can interact with the target and

release their payload. Using imaging technologies, visual evidence of the

effect of PEGylation on the circulation kinetics of the liposomes was

provided (Fig. 1.6D).66 The images clearly demonstrate that PEGylation

significantly enhances the persistence of liposomes in the blood stream. At

the same time, the uptake of PEGylated liposomes in organs (liver and

spleen) responsible for particle clearance decreased.

Fig. 1.6 Schematic representation of (A) conventional liposome, (B) PEG-liposome and (C)

chemically modified PEG-liposome. (D) The effect of PEGylation on the circulation persistence

of liposomes. The liposomes were labeled with Tc-99m, administered in rats, and the rats were

imaged with a gamma camera over 24 h. As is evident from the heart (H) image signal, the

PEG-liposomes remained in circulation even 24 h post-injection. The accumulation in the liver

(L) and the spleen (S) was also lower in the case of PEG-liposomes, as compared to the plain

liposomes.66 (Fig. 1.6 D was adapted from reference 66)

Functionalization of Lipid Bilayer Membranes

19

Ligands conjugated with hydrophobic molecules form amphiphiles. The

hydrophobic part can insert into the liposome bilayer, exposing the ligand

outside of the liposomes for being recognized or for other interactions. For

instance, DNA-b-polypropyleneoxide (DNA-ppo) has proven to be stably

anchored into the lipid membrane for over at least 24 h. In this way, the

containers are encoded with sequence information. The DNA-ppo present

on the surface was used for anchoring a photosensitizer by hybridization.

Upon light irradiation the PPO was oxidized leading to cargo release (Fig.

1.7).67

Fig. 1.7 Illustration of selective cargo release from DNA block copolymer (DBC) -decorated

phospholipid vesicles. (1) DNA-ppo is stably anchored in unilamellar lipid vesicles; (2) DBC-

decorated vesicles are functionalized with conjugated DNA-photosensitizers by hybridization;

(3) singlet oxygen is generated by light irradiation; and (4) selective cargo release is induced

by the oxidative effect of singlet oxygen.67 (This figure was reproduced with permission from

reference 67)

1.3.2 Stimuli-responsive liposomes

Liposomes can suspend cargos with their peculiar solubility properties and

act as a sustained-release system for microencapsulated molecules. After

modification, liposomes can be used as stimuli-responsive nanoparticles,

which are visionary concepts to deliver and release a drug exactly where it

is needed.68,69 There are several ways to trigger cargo release, such as

light,70 temperature71,72 and magnetism.73 Often two or more triggers need

Chapter 1

20

to be combined to appropriately improve the cargo release kinetics and

distribution to reduce side effects.

1.3.2.1 Light responsive vesicle systems

Methods of sensitizing liposomes to light have progressed from the use of

organic molecule moieties to the use of metallic plasmon resonant

structures which can be broadly categorized as photochemical or

photophysical release. Photochemical release can be achieved via

photoisomerization, photocleavage and photopolymerization, which all

lead to destabilization of the liposome bilayer and release of encapsulated

contents (Fig. 1.8A-C).

Fig. 1.8 Release from liposomes mediated by photochemical responses: photoisomerization (A),

photocleavage (B), or photopolymerization (C); and photophysical responses: molecular

absorbers (D) and gold nanoparticles (E).

Functionalization of Lipid Bilayer Membranes

21

On the other hand, photophysical release from liposomes does not rely on

any chemical changes of structures within or associated with the bilayer

membrane. Examples of photophysical release discussed here take

advantage of photothermal conversion of absorbed light with ensuing

thermal and/or mechanical processes in the lipid membrane and the

surrounding medium. The methods for achieving photophysical release are

developed around molecular absorbers (Fig. 1.8D) or gold nanoparticles

(Fig. 1.8E).70

1.3.2.2 Temperature responsive vesicle systems

Temperature-responsive liposomes are classified into two types:

traditional temperature-responsive liposomes and liposomes modified

with temperature-responsive polymers. Traditional temperature-

responsive liposomes which are composed of temperature-responsive

lipids show the greatest permeation of the lipid membrane at its gel-to-

liquid crystalline phase transition temperature.

Moreover, liposomes modified with temperature-responsive polymers

exhibit a lower critical solution temperature (LCST) behavior. These

polymers are soluble in an aqueous solution below this temperature but

dehydrate and aggregate if heated above the LCST. This behavior induces

the release of a drug within a polymer-modified liposome. For instance, a

temperature-responsive polymer, poly (N-isopropylacrylamide)-co-N,N'-

dimethylaminopropylacrylamide (P(NIPAAm-co-DMAPAAm)) was

synthesized and used for liposome modification. This research showed that

the polymer underwent dehydration and aggregation above 40 °C and that

temperature-responsive polymer-modified liposomes had faster cellular

uptake and release compared to non-modified liposomes (Fig. 1.9).74

Chapter 1

22

Fig. 1.9 Liposomes modified with temperature-responsive polymers are used for cellular

uptake. The copolymer displayed a thermosensitive transition at a lower critical solution

temperature (LCST) that is higher than body temperature. Above the LCST, the temperature-

responsive liposomes started to aggregate and release their content. The liposomes showed a

fixed aqueous layer thickness (FALT) at the surface below the LCST, and the FALT decreased

with increasing temperature. Above 37°C, cytosolic release from the temperature-responsive

liposomes was higher than that from the PEGylated liposomes, indicating intracellular

uptake.74 (This figure was adapted from reference 74)

1.3.2.3 Magnetic responsive vesicle systems

Magnetoliposomes are composed of a lipid bilayer surrounding

superparamagnetic iron oxide nanoparticles. Due to the biocompatibility,

size, material-dependent physicochemical properties and potential

applications as alternative contrast enhancing agents for magnetic

resonance imaging, magnetoliposomes are ideal candidates to achieve a

spatial and temporal control over drug release.75,76 Superparamagnetic iron

oxide nanoparticles (SPION) can be guided to their site of action using an

externally applied magnetic field. The subsequent accumulation of SPION

in the target site can be exploited for simultaneous drug delivery, MR

imaging or hyperthermia therapy of cancer (Fig. 1.10).

Functionalization of Lipid Bilayer Membranes

23

Fig. 1.10 Superparamagnetic iron oxide nanoparticles can be guided to the site of action using

an externally applied magnetic field.77 (This figure was adapted from reference 77)

In the beginning, liposomes were studied only for their physicochemical

properties as models of membrane morphology. Today, they are used as

delivery devices to encapsulate cosmetics, drugs, fluorescent detection

reagents, and as vehicles to transport nucleic acids, peptides, and proteins

to specific cellular sites in vivo. Advances in therapeutic applications of

liposomes have been achieved through surface modifications. With these

surface modifications, their biological stability could be increased, which

includes reduced constituent exchange and leakage as well as reduced

unwanted uptake by cells of the mononuclear phagocytic system.78

Targeting components such as antibodies can be attached to liposomal

surfaces and were used to create large antigen-specific complexes. In this

sense, liposomal derivatives are being used to target cancer cells in vivo, to

enhance detectability in immunoassay systems.

Chapter 1

24

1.4 Motivation and Thesis Overview

The overall goal of the work described in this thesis was to use DNA

nanotechnology as a tool to manipulate lipid bilayer surfaces. Our group

synthesized and characterized a new family of DNA amphiphiles containing

modified nucleobases. The modification is introduced in uracil and consists

of hydrophobic moieties. Through solid phase synthesis, the modified

nucleotides can be incorporated in any desired position and several

modifications per DNA strands can be introduced.79 The resulting DNA

sequences still undergo specific Watson-Crick base pairing. This property

combined with the amphiphilic nature of this lipid-DNA qualifies the

material as appealing candidate to interact with and manipulate biological

membrane structures.

In chapter 2, a powerful new approach was introduced by modifying DNA

with lipid chains at four nucleobases to tightly anchor the nucleotide to the

lipid membrane. This strategy allows highly stable incorporation of DNA

into the liposomal bilayer, thereby limiting dissociation. Several assays

were employed proving the incorporation and stable anchoring in the

phospholipid bilayer. These measurements involve small vesicles and

fluorescence energy transfer. These experiments allow to measure how

long the DNA amphiphiles remain in the bilayer.

In chapter 3, efficient fusion of liposomes was studied using lipid-DNA

introduced in the chapter before. While the orientation of DNA

hybridization played a significant role in the efficacy of full fusion of DNA-

grafted vesicles, the number of anchoring units was found to be a crucial

factor as well. As compared to vesicles functionalized with single-anchored

or double-anchored DNA, liposomes containing quadruple-anchored

oligonucleotides were found to be highly fusogenic, achieving considerable

full fusion of up to 29% without notable leakage. This study demonstrates

the importance of the DNA-anchoring strategy in hybridization-induced

vesicle fusion, as not only the structural properties of the unit itself, but

also the number of anchoring units determines its favorable fusion-

inducing properties. Several fluorescence assays, dynamic light scattering

and cryogenic transmission electron microscopy were utilized to prove

these results.

Functionalization of Lipid Bilayer Membranes

25

In chapter 4, we expand the functionality of DNA encoded vesicles

significantly. It was demonstrated that strand replacement can be carried

out. In this chapter it will be outlined what sequences and what DNA

amphiphiles are needed to reach this goal, i.e. changing the surface

functionalities of liposomes by the simple addition of oligonucleotides.

Moreover, it will be detailed how such a surface modification can be

amplified by a simple DNA-triggered supramolecular polymerization.

In chapter 5, we investigated whether it is possible to insert the lipid-

modified DNA sequences into the membrane of live zebrafish to function as

artificial receptor. We demonstrate that oligonucleotides functionalized

with a membrane anchor can be immobilized on a zebrafish. Protruding

single-stranded DNA atop the fish was functionalized by Watson-Crick base

pairing employing complementary DNA sequences. In this way, small

molecules and liposomes were guided and attached to the fish surface. The

anchoring process can be designed to be reversible allowing exchange of

surface functionalities by simple addition of DNA sequences. To achieve

this on a fish surface, the strand exchange experiments established in

chapter 4 on simple vesicles as model were crucial. Finally, a DNA based

amplification process was performed atop of the zebrafish enabling the

multiplication of surface functionalities from a single DNA anchoring unit.

Chapter 1

26

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