rsc advances c2ra20491f paper composite fluorescent vesicles based on ionic and cationic amphiphilic...

RSC Advances c2ra20491f

PAPER

Composite fluorescent vesicles based on ionic and cationicamphiphilic calix[4]arenes

Paul K. Eggers, Thomas Becker, Marissa K. Melvin, RamizBoulos, Eliza James, Natalie Morellini, Alan R. Harvey,Sarah A. Dunlop, Melinda Fitzgerald, Keith A. Stubbs andColin L. Raston*

The surface structure and localisation of charged amphiphiliccalixarenes that self-assembled into vesicles and were wrapped ina peptide–glycol coat for enhanced stability.

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Paper: c2ra20491f

Title: Composite fluorescent vesicles based on ionic and cationic amphiphilic calix[4]arenes

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; Composite fluorescent vesicles based on ionic and cationic amphiphiliccalix[4]arenes{

Paul K. Eggers,a Thomas Becker,b Marissa K. Melvin,a Ramiz Boulos,a Eliza James,a Natalie Morellini,c Alan

R. Harvey,d Sarah A. Dunlop,c Melinda Fitzgerald,c Keith A. Stubbse and Colin L. Raston*a

Received 16th March 2012, Accepted 27th April 2012

DOI: 10.1039/c2ra20491f

Amphiphilic calixarenes bearing ionisable phosphonic acid groups or cationic trimethylamine groups

attached to the upper rim of calix[4]arenes in the cone conformation, with dodecyl moieties attached

to the lower rim, have a high uptake in PC-12 cells, with the calixarenes being localised

cytoplasmically. The calixarenes form tightly packed layers, as established using scanning tunnelling

microscopy (STM) on atomically flat surfaces, and can form vesicles or micelles. The vesicles have

been wrapped in a peptide–glycol coat for enhanced stability.

Introduction

The ability to deliver a multi-component package consisting of

drugs, enzymes and possibly genetic material to a specific

location is the ‘‘Holy Grail’’ in medicine. Such a system would

allow known ratios of enzymes and drugs to be delivered, for

regeneration of injury sites as well as for preventing toxic drugs

from affecting healthy tissue in the treatment of diseases such as

cancer.

One possible approach for packaging functional materials is to

use amphiphilic phospholipids. The human body uses the self-

assembly of amphiphiles to create membranes that separate

various components and provide a barrier against external

environments.1 In principle, these same compounds in the form

of vesicles offer scope to deliver packages2 to specific locations

using biotags.

Vesicles readily form through self-assembly, enclosing the

contents of the surroundings in which they formed, and can be

small enough to be delivered into the body in a variety of ways.2

However, in reality the lipids which the body uses are not stable

enough in their vesicle form for long duration drug delivery. The

cells within our body reinforce the lipids with various complex

protein structures to ensure their structural integrity.3 A range of

strategies have been enacted with greater or lesser success in order

to increase the stability of lipid vesicles within the body. Such

strategies include: (i) high-melting-temperature lipids which result

in wax-like, instead of fluid-like, bilayers at body temperature, (ii)

the addition of cholesterol which stabilises the standard lipids, (iii)

polymerisation of head groups and/or tails to stabilise the vesicles,

and (iv) the PEGylation of the head groups to inhibit fusion and

multilamellar vesicles which use multiple bilayers in order to

reduce the probability of lysis.4 Another strategy which could be

used involves a virus capsid which also uses self-assembly

associated with repeating protein units to form a rigid shell.5

These shells are highly ordered helical or icosahedral structures.6

The capsid of viruses is extremely stable under a variety of

conditions and has proven time and again to be an effective

delivery system.7,8 Furthermore, viruses often use a coat or

envelope on top of their capsid to assist in delivery. However, the

capsid proteins are complex, often immunogenic and are

extremely difficult to synthesise and manipulate, and thus are

not necessarily suitable for routine enzyme/drug delivery.

Our packaging approach herein is to design a hybrid system

that combines the simplicity of the amphiphilic lipids with the

stability of the virus capsid. We use positively and negatively

charged amphiphilic calix[4]arenes to form a robust bilayer. The

symmetry and the opposing charges of the amphiphilic

calixarenes reduce the fluidity of the membrane, hence creating

structural rigidity, while derivatisation of the calixarene head

groups allows the incorporation of a biotag, a fluorescent tag

and/or an extra-vesicular matrix framework to be attached

without disrupting the integrity of the vesicles. The charged

amphiphiles are based on p-phosphonated calix[4]arenes which

readily self assemble via interactions between the polar head

groups and C18 alkyl chains.9,10 We report the formation of

aCentre for Strategic Nano-Fabrication, The University of WesternAustralia, 35 Stirling Hwy, Crawley 6009, Australia.E-mail: [email protected]; Fax: +618 65488 3045;Tel: +618 3045 8683bNanochemistry Research Institute, Curtin University, Kent Street,Bentley, 6102, Australia. E-mail: [email protected];Fax: +618 9266 4699; Tel: +618 9266 7806cSchool of Animal Biology, The University of Western Australia, 35Stirling Hwy, Crawley 6009, Australia. E-mail: [email protected];Fax: +618 6488 7527; Tel: +618 6488 1403dSchool of Anatomy and Human Biology, The University of WesternAustralia, 35 Stirling Hwy, Crawley 6009, Australia.E-mail: [email protected] of Chemistry and Biochemistry, The University of WesternAustralia, 35 Stirling Hwy, Crawley 6009, Australia.E-mail: [email protected]; Fax: +618 6488 7330;Tel: +618 6488 2725{ Electronic Supplementary Information (ESI) available. See DOI:10.1039/c2ra20491f/

This journal is � The Royal Society of Chemistry 2012 RSC Adv., 2012, 1–8 | 1

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RSC Advances Dynamic Article Links

Cite this: DOI: 10.1039/c2ra20491f

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vesicles using these amphiphilic calixarenes and the surface

structure resulting from their self-assembly. In addition, we

report the toxicology of these compounds, the location of the

calixarenes after incubation with PC-12 cells via a fluorescent tag

attached to the calixarene, and demonstrate a method to create a

tertiary structure on the surface of the vesicle for enhancing their

stability.

The amphiphilic calix[4]arenes possess four lower rim C12

alkyl chains, with the incorporation of more than one such

surfactant moiety in a single molecule resulting in a greater

viscosity and stability of the self assembled vesicles.11,12 Lower

rim O-alkyl calix[4]arenes in general can form well defined

bilayer structures, including the case where the alkyl moieties are

interdigitated.13 The choice of calix[4]arenes rather than the

larger ring systems which are also readily accessible, especially

for calix[6 and 8]arenes, relates to the ability to lock the

calix[4]arenes in a cone conformation with all the alkyl chains on

the same side of the plane of the lower rim phenolic O-atoms.

This occurs for O-propyl groups and longer alkyl groups with

the smaller groups resulting in a conformation change via

threading through the annulus of the molecules.14

Results and discussion

Amphiphile design and synthesis

The amphiphilic calix[4]arenes used in this study, 1–4, are shown

in Scheme 1.

With calix[4]arenes as the structural unit of the amphiphiles,

one out of the four upper rim head groups may be used for

attaching a biotag, a fluorescent molecule as in compound 3, or

the support point for an extra vesicular matrix. The other head

groups can maintain the ability of the calixarene to assemble into

bilayers without producing significant defects, i.e. an assembly

mechanism similar to the calixarene where all head groups are

the same. Incorporation of the azide group as the functional

group for the attachment of a fluorescent molecule, as in

compound 4, was undertaken because of the bioorthogonal,

biocompatibility15 and high yields associated with click chem-

istry (see below), and the polarity of the azide group maintaining

the amphiphilic nature of the calixarene. The choice of benzylic

head groups in all the compounds in the present study relates to

both the ease of their synthesis and the extra degree of freedom

the methylene unit attached to hetero-atoms allows in packing

the bilayers of calixarenes.

The two different head groups chosen were trimethylamine,

compound 1, and a phosphonate, compound 2, due to their

opposite charge and biocompatibility, and thus the lower

likelihood of imparting significant toxicity. Calix[4]arenes 1

and 2 and the associated analogues with a fluorescent molecule

or an azide group attached, compounds 3 and 4, have O-dodecyl

alkyl chains on the lower rim. Targeting such an intermediate

length C12 alkyl chain relates to our recent studies on the

toxicology of p-phosphonated calix[4]arenes, where the head

groups are attached directly to the phenol rings. Longer alkyl

chains result in higher toxicity16 and shorter alkyl chains can

result in a higher degree of calixarene directed self-assembly; that

is the formation of micelles and vesicles is controlled more by the

calixarene itself rather than through the interplay of the alkyl

chains.

Synthesis of calix[4]arenes 1, 2, 3 and 4

The synthesis of compounds 1–4 is shown in Scheme 1. The

synthetic steps up to and including the alkylation have been

described previously.16 The formylation reaction, which was the

lowest yielding reaction (70% yield), was adapted from the

method of Dondoni et al.17 All subsequent reactions were

completed in close to quantitative yields. Click chemistry was

chosen for in situ modifications as it has been shown to be

biocompatible, is highly selective and consistently achieves high

yields.15,18–20 The click reaction to attach the fluorescent

molecule was monitored by fluorescence spectroscopy showing

a 40 nm shift in the fluorescence peak and a 10 fold increase in

the fluorescence intensity after attachment.21

Effects of calix[4]arenes 1 and 2 on cell viabilities

In order to establish whether compounds 1 and 2 had any

toxicity, cell viability studies using a LIVE/DEAD cell-based

assay were performed on rat PC-12 cells. The concentrations

assessed were 0.001, 0.01, 0.1, 0.3, 1 and 3 mg mL21. As can be

seen from Fig. 1, 1 has a significant effect on PC-12 cell viability

from 0.01 mg mL21, with 74% of the cells still viable at this

concentration; 2 has a noticeable effect from 0.001 mg mL21,

with 78% of the cells still viable at this concentration. Both

compounds had an IC50 (50% of cells still viable) of approxi-

mately 1 mg mL21. This level of toxicity could be beneficial for a

delivery system targeting cancer cells as long as the delivery

system maintains its integrity until it reaches the targeted cells.

This then would give dual-action toxicity with both the

packaging system as well as the contents of the package being

able to kill the cancer cells. Nevertheless, the present study is an

analysis of the structure of the packaging system and its

plausibility, not an attempt to demonstrate efficacy in drug

delivery. Furthermore, we have recently shown that the toxicity

of these compounds is related to chain length and that

p-phosphonated calix[4]arenes with four lower rim O-octyl

groups are less toxic.16 Thus, future work will also be undertaken

using a slightly shorter chain which is less toxic. See below for

toxicity studies on compound 1 and 2.

Scheme 1 The amphiphilic calix[4]arenes used in the present study, with

R = C12H25.

2 | RSC Adv., 2012, 1–8 This journal is � The Royal Society of Chemistry 2012

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Localisation studies

As a follow on to the toxicology, an imaging study on the

localisation of the fluorophore calixarene 3 within cells was

undertaken. Compound 3 has an excitation wavelength of

350 nm and an emission wavelength of 440 nm (Fig. 2, red

squares). The precursor fluorophore to compound 3 has a

fluorescence peak at 404 nm, with the shift to 440 nm indicative

of a successful ‘click’ reaction.21

Compound 3 was added to the media of PC-12 cultures 24 h

prior to imaging. PC-12 cells are originally derived from rat

pheochromocytoma and are used as a model system for primary

neuronal cells.22 These cells are relatively small, have a small

amount of cytoplasm, a large nucleus and their doubling time

exceeds five days.22 As a control to clarify the intracellular

localisation of the calixarene, calcein acetoxymethyl (Calcein-AM)

was added 30 min prior to imaging. Calcein-AM is a

P-glycoprotein substrate, cell permanent and a non-fluorescent

compound.23 Once inside the cell Calcein-AM is rapidly hydro-

lysed by intracellular esterases to form the strongly green

fluorescent calcein anion. The calcein ion is retained in live cells

and has an excitation wavelength of 475 nm and an emission

wavelength of 525 nm (Fig. 2, blue diamonds). The distinctly

different excitation wavelengths of 3 and calcein allow both

fluorophores to be located in the cell and excited separately.

Fig. 3a and d show differential interference contrast images of

the PC-12 cells 24 h after compound 3 was added. In these

images, the cells have extended multiple processes indicating that

they are in a healthy state. In an effort to gain insight into the

cellular localisation of 3, we undertook photon microscopy

experiments. Fig. 3b and e show two photon microscopy images

of the same area as Fig. 3a and d but depict the calcein emission

wavelength. These images show that calcein is retained

predominantly in the cytoplasm. Fig. 3c and f are two photon

microscopy images of the same area as Fig. 3b and e but

depicting the emission wavelength of 3. It can be seen from

Fig. 3c and f that 3 has the same localisation as calcein in that it

penetrates the membrane into the cytoplasm and does not

appear to enter the nucleus. Furthermore there is also very little

evidence of the fluorescence of 3 outside the PC-12 cells

indicating close to 100% uptake of the added calixarene.

Surface organisation

To determine the surface organizational structure and the

capability of these bilayers to produce a physical barrier, the

vesicles (as identified by dynamic light scattering (DLS) and

described later in this paper) were allowed to fuse with a

decanethiol self-assembled monolayer (SAM) on an atomically

smooth Au(111) surface, forming a biomimetic bilayer.24 This

technique is well established in producing consistent reproduci-

ble results. The biomimetic bilayer is produced through a

thermodynamic driving force which results from the increase in

entropy achieved when water is removed from the non-polar

distal head groups of the alkanethiols by the non-polar tails of

the amphiphilic calixarenes.24 Studies on the assembly of a

mixture of compounds 1, 2 and 4 are detailed below.

Fig. 2 Fluorescence spectra for calcein (excitation 475 nm) and

compound 3 (excitation 350 nm).

Fig. 3 The same field of view of PC-12 cultures was imaged by

differential interference contrast (a and d), and two photon fluorescence

for calcein, (b and e) or compound 3 (c and f).

Fig. 1 Toxicology data for compounds 1 and 2. The effects of the

preparations of 1 and 2 on PC-12 cell viabilities (mean ¡ SEM =) after

24 h in culture and representative images of untreated (control) and

treated PC-12 cells (objective 406). Statistically significant reductions in

the viability of the cultures treated with the calixarene preparations

compared to the untreated cells are indicated by * (p ¡ 0.05). Scale bar =

100 mm.

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Impedance

Impedance studies were undertaken as an integrated approach to

understanding the self assembly of compound 2. Impedance

studies give information on the capability of the layer to separate

charge and hence will show if a biomimetic layer is formed and if

that layer is uniform.

If an insulating compound covers a metal surface forming a

uniform layer without defects, then that layer can be represented

solely as a Helmholtz capacitor in the equivalent circuit. Thus,

the equivalent circuit for the electrochemical cell would be a

capacitor, representing the uniform coverage of the surface of a

metal, and a resistor, representing the resistance of the solution.

At low frequencies the phase angle is dominated by a Helmholtz

capacitor with values of less than 90u representing defects in the

capacitor, at high frequencies the phase angle is dominated by

the solution resistance and thus should be close to zero.25–27 If

the layer is completely uniform and is thick enough not to have

any resistive character, then at 1 Hz the phase angle of the system

will be 90u.25,26 Thus, if there is a resistive component such as

from defects in the uniform coverage or electron tunnelling

through the layer, at 1 Hz the impedance measurement will be

less than 90u. As can be seen in Fig. 4, the decanethiol SAM has

the lowest phase angle at 85u at 1 Hz followed by the bilayer

formed from 2 which has a phase angle of 87u.It is expected that a decanethiol SAM even with uniform

coverage would have a lower phase angle than a bilayer due to

an increase in the thickness created by the coverage of calixarene

lipids. Although an increase in thickness decreases the capaci-

tance, it also decreases the resistance of the SAM (due to electron

tunnelling). This increases the capacitive character of the layer

resulting in a phase angle closer to 90u. As indicated in Fig. 4, the

bilayer made from 2 has an increase in capacitive character

relative to the decanethiol SAM. Since the phase angle is greater

than the decanethiol SAM, the impedance measurements imply

that biomimetic bilayers have formed.24

STM results

The Au(111) on mica substrates were studied using scanning

tunnelling microscopy (STM). Hydrogen flamed gold on mica

substrates show the triangular terracing expected for atomically

flat Au(111), Fig. 5a.28 As shown in both Fig. 5a and b, the

images of the decanethiol SAMs depict the expected pitting

caused by the formation of alkanethiol SAMs. The STM image

in Fig. 5b, as a close up of the decanethiol monolayer shows the

typical hexagonal packing of an alkanethiol SAM on gold.28,29

Fig. 6a and b show STM images of 2 on top of a layer of

decanethiol. The closest packing of the SAM has a separation

between 0.8 to 0.9 nm with the next closest packing between 1 to

1.1 nm. These distances agree with the model in Fig. 6d and the

expected size of a calixarene. The model in Fig. 6d is more

chaotic than the STMs of compound 2, however the packing

distances are approximately the same. This indicates that each

spot in the STMs of compound 2 contain four phosphate groups.

The packing is very close to the hexagonal packing of an

alkanethiol monolayer which suggests that the underlying layer

is influencing the packing.

Molecular simulation studies were undertaken to investigate

the effect of the length and width of the unit cell on the packing

arrangement and energy of the p-phosphonated calix[4]arenes

sitting on decanethiols. The optimized structure corresponding

to the lowest energy had a unit cell containing four calixarenes

with a width and length of 22 A. As shown in Fig. 6c, this

corresponds to the calixarenes being tilted, which maximises

packing, optimises the van der Waals interactions and the

Fig. 4 Impedance spectra for a SAM of decanethiol and a biomimetic

bilayer of 2 formed on top of the decanethiol SAM.

Fig. 5 STM images of a decanethiol SAM on Au(111) >.

Fig. 6 (a and b) STM images of a biomimetic bilayer of 2 formed on top

of a SAM of decanethiol on Au(111), and (c and d) a simulation of the

same system, different colours represent different calixarenes.


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electrostatic forces between the calixarenes, and interactions

between the calixarenes and the decanethiols. The tilt angle is

approximately 30u, which is the same angle of the underlying

alkane layer. However, the hexagonal packing may not strictly

be due to the underlying decanethiol monolayer. In this context

we note that previous studies of crystallised calixarenes with

O-alkyl chains have also shown a hexagonal lattice with a similar

tilt with respect to the plane of the bilayer.30,31

In relation to the structure of the bilayers, the STM images

and impedance results show that 2 can self-assemble into a

highly ordered tight packed layer, completely covering the

substrate.

Vesicles

Compounds 1 and 2 were designed to form vesicles when mixed

and this has been confirmed experimentally using dynamic light

scattering (DLS). Compound 2 and a 1 : 1 mixture of 1 and 2

formed vesicles with a mean hydrodynamic diameter of 107 nm

and standard deviations of 5 nm and 4 nm, respectively. This was

the expected vesicle size after extruding using a 100 nm pore filter

with an Avanti Polar Lipid Mini-Extruder. Thus, these two

calixarene systems are predisposed to self-assembling into

vesicles. However, compound 1 by itself resulted in 5.4 nm

particles with a standard deviation of 0.5 nm. The length of

1 from the tip of the alkane chain to the methyl of the

trimethylamine equates to approximately 2.4 nm. This is

approximately half the diameter of the micelles formed from 1

at 5.2 nm. Thus, compound 1 by itself spontaneously self-

assembles into micelles in preference to vesicles. Interestingly,

shorter chain analogues of compound 2 have also been shown to

form micelles by first solubilising the compounds at high pH,

and then on reducing the pH, the micelles maintained their

integrity to pH , 3.16

Extravesicular matrix

As mentioned in the introduction, vesicles containing lipids alone

do not have long-term stability within the body. In attempting to

increase the stability of the vesicles, we incorporated a pillar

compound where one of the four trimethylamine groups of 1 is

replaced by an azide, compound 4. In this situation it is

envisaged that the three trimethylamine units hold the com-

pound within the bilayer with the azide group available to attach

the extravesicular framework. In demonstrating that it is possible

to form an extravesicular matrix for coating the vesicles, click

chemistry was used to first attach a synthetic peptide consisting

of (glycine-glycine-glyine-propargylglycine)n to the outside of the

vesicle, and then a 1000n ethyleneglycol was attached onto the

peptide coat of the vesicle. The ratio of amphiphilic calixarenes

used to make the vesicles was 8 : 8 : 1 for 1 : 2 : 4, respectively.

This ratio was decided upon to ensure that each molecule of 4

was surrounded by compounds 1 and 2. As can be seen from

Fig. 7a the initial vesicle had a mean diameter of approximately

70 nm. This is smaller than the diameter of the vesicles reported

in the previous section, which is a consequence of using two

filters during the extrusion instead of one. As shown in Fig. 7b,

after the peptide and the glycol was attached to the vesicle, the

peak at 70 nm disappeared and a peak at 150 nm appeared. This

is strongly suggestive that the peptide–glycol coat wraps around

the vesicle. It should be noted that the peak also tailed toward

larger sizes which may indicate that the peptide binds some

vesicles together.

In a phosphate buffered solution coating the vesicles signifi-

cantly enhanced their stability. Without the coating, vesicles made

up of compounds 1 and 2 would start to agglomerate and

precipitate out of solution after 48 h. The solution of coated

vesicles was stable with no observed precipitation over the course

of a month.

Conclusions

Amphiphilic calixarenes bearing ionisable phosphonic acid

groups or cationic trimethylamine groups attached to the upper

rim of calix[4]arenes locked in the cone conformation, and with

dodecyl moieties attached to the lower rim, have an extremely

high uptake in the cytoplasm of PC-12 cells. Also noteworthy is

that the calixarenes form tightly packed layers, and can assemble

into vesicles or micelles. In addition, we have shown that the

vesicles can be wrapped in a peptide–glycol coat which should

increase their stability in vivo.

Experimental

Molecules

The amphiphilic calix[4]arenes used in the self assembly studies,

1–4, are shown in Scheme 1, with their syntheses described

below.

Preparation of 5,11,17,23-tetra-formyl-25,26,27,28-tetra-dode-

cyloxy-calix[4]arene. Under an inert gas atmosphere a mixture of

dry 25,26,27,28-tetra-dodecyloxy-calix[4]arene (3.3 g, 3 mmol)

and hexamethylenetetramine (16.2 g, 120 mmol) in CF3COOH

(100 mL) was stirred for 96 h under reflux. The mixture was

cooled to room temperature and then poured into a stirring

solution of 2 M HCl (200 mL) and CH2Cl2 (200 mL), and

vigorously stirred for 1 h. The mixture was extracted with

CH2Cl2 (2 6 100 mL) and the combined organic layers were

washed with saturated aqueous Na2CO3 (2 6 100 mL) and brine

(2 6 100 mL), dried over NaSO4 and the solvent was then

Fig. 7 DLS data for (a) vesicles prepared from an 8 : 8 : 1 ratio of

1 : 2 : 4, and (b) the same system wrapped in a peptide–ethylene glycol coat.


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removed under reduced pressure. The raw product was further

purified by column chromatography (silica gel 60; hexane : ethyl

acetate, 7 : 3) to give a white solid (2.73 g, 70%).1H NMR (CDCl3, 500 MHz) d: 0.85 (t, 3J = 7.05 Hz, 12H),

1.27 (m, 72H), 1.87 (m, 8H), 3.33, 4.48 (2d, 2J = 13.8 Hz, 2 64H) 3.96 (t, 3J = 7.34 Hz, 8H), 7.13 (s, 8H), 9.53 (s, 4H).

13C NMR (CDCl3, 125.8 MHz) d: 13.9 (CH3), 22.5 (CH2), 26.1

(CH2), 29.3 (CH2), 29.6 (CH2), 29.7 (CH2), 29.8 (CH2), 30.2

(CH2), 30.8 (CH2), 31.8 (CH2), 75.6 (CH2), 130.1 (CH), 131.2

(C), 135.5 (C), 161.8 (C), 191.3 (CH).

Preparation of 5,11,17,23-tetra-methylhydroxy-25,26,27,28-

tetra-dodecyloxy-calix[4]arene. Under an inert gas atmosphere

ethanol (50 mL) was added to a stirring solution of 5,11,17,23-

tetra-formyl-25,26,27,28-tetra-dodecyloxy-calix[4]arene (2.73 g,

2 mmol) in THF (10 mL). NaBH4 (2.6 g, 70 mmol) was then

added and the mixture stirred for 18 h at room temperature. The

mixture was then concentrated under vacuum and the resulting

solid dissolved in CH2Cl2 (100 mL). 2 M HCl (100 mL) was

added slowly and the solution was stirred for 1 h. The reaction

mixture was then extracted with CH2Cl2. The combined organic

fractions were washed with 2 M HCl (3 6 100 mL) and

saturated aqueous Na2CO3 (3 6 100 mL) and dried over

Na2SO4. The solvent was removed in vacuo to obtain a white

solid (1.5 g , 60%).1H NMR (CDCl3, 500 MHz) d: 0.88 (t, 3J = 6.90 Hz, 12H),

1.35 (m, 72H), 1.96 (m, 8H), 3.16, 4.46 (2 d, 2J = 13.1 Hz, 2 64H), 3.91 (t, 3J = 6.95 Hz, 8H), 4.34 (s, 8H), 6.70 (s, 8H).


(CH2), 26.4 (CH2), 29.5 (CH2), 29.8 (CH2), 29.9 (CH2), 30.1

(CH2), 30.4 (CH2), 31.0 (CH2), 32.0 (CH2), 64.5 (CH2), 75.3

(CH2), 127.1 (CH), 134.6 (C), 134.8 (C), 155.9 (C).

Preparation of 5,11,17,23-tetra-methylchloride-25,26,27,28-

tetra-dodecyloxy-calix[4]arene. Under an inert gas atmosphere

thionylchloride (2 mL) was added to 5,11,17,23-tetra-methylhy-

droxy-25,26,27,28-tetra-dodecyloxy-calix[4]arene (0.5 g, 0.4 mmol)

in CH2Cl2 (15 mL) and then stirred at room temperature for 18 h.

The mixture was concentrated under vacuum and the resulting

solid was dissolved in CH2Cl2 (50 mL), washed with saturated

aqueous Na2CO3 (3 6 50 mL) and dried over Na2SO4 .The solvent

was removed under reduced pressure to obtain an off-white solid

(0.436 g, 85%).1H NMR (CDCl3, 500 MHz) d: 0.92 (t, 3J = 7.14 Hz, 12H),

1.34 (m, 72H), 1.93 (m, 8H), 3.17, 4.44 (2 d, 2J = 13.3 Hz, 2 64H), 3.90 (t, 3J = 7.43 Hz, 8H), 4.32 (s, 8H), 6.68 (s, 8H).


(CH2), 29.4 (CH2), 29.5 (CH2), 29.7 (CH2), 29.8 (CH2), 29.9

(CH2), 30.0 (CH2), 30.3 (CH2), 30.9 (CH2), 32.0 (CH2), 46.6

(CH2), 75.4 (CH2), 128.6 (CH), 130.9 (C), 135.2 (C), 156.8 (C).

Preparation of 5,11,17,23-tetra-trimethyammoniumchloride-

methyl-25,26,27,28-tetra-dodecyloxy-calix[4]arene, 1. Under an

inert gas atmosphere trimethylamine (5 mL) was transferred via

cannula to a cooled pressure tube (215 uC) containing 5,11,17,23-

tetra-methylchloride-25,26,27,28-tetra-dodecyloxy-calix[4]arene

(0.30 g, 0.22 mmol) in dry DMF (30 mL). The pressure tube was

sealed, the solution was allowed to warm to room temperature

and was then stirred at 100 uC for 16 h. An off-white solid was

collected by filtration, washed with water (10 mL) and dried in

vacuo. To produce a white solid in quantitative yield.1H NMR (CDCl3, 500 MHz) d: 0.72 (t, 3J = 7.04 Hz, 12H),

1.20 (m, 72H), 1.84 (m, 8H), 2.79 (m, 36 H), 3.17, 4.27 (2 d, 2J =

12.9 Hz, 2 6 4H), 4.01 (8H), 4.39 (s, 8H), 7.00 (s, 8H).13C NMR (CDCl3, 125.8 MHz) d: 13.8 (CH3), 22.4 (CH2), 26.1

(CH2), 29.2 (CH2), 29.6 (CH2), 29.7 (CH2), 29.9 (CH2), 30.3

(CH2), 31.7 (CH2), 51.9 (CH3), 68.1 (CH2), 75.8 (CH2), 121.7

(C), 133.4 (CH), 135.2 (C), 157.8 (C).

TOF MS ES+ (m/z): Expected for C92H160Cl3N4O4+,

1490.1505. Found: 1490.1520.

Preparation of 5,11,17,23-tetra-diethylphosphonomethyl-

25,26,27,28-tetra-dodecyloxy-calix[4]arene. Dry 5,11,17,23-tetra-

methylchloride-25,26,27,28-tetra-dodecyloxy-calix[4]arene (2.1 g,

1.62 mmol) was added with stirring triethylphosphite (150 mL)

and the mixture was then refluxed for 16 h. The solution was

cooled to room temperature and the triethylphosphite removed

under reduced pressure. The raw product was purified by column

chromatography (silica gel 60; chloroform : methanol, 10 : 1) to

give a white solid (2.67 g, 96%).1H NMR (CDCl3, 500 MHz) d: 0.73 (t, 3J = 6.95 Hz, 12H),

1.15 (m, 120H), 1.70 (m, 8H), 2.62 (d, 2J = 21.2 Hz, 8H), 2.91,

4.20 (2 d, 2J = 13.5 Hz, 2 6 4H), 3.66 (t, 3J = 7.25 Hz, 8H), 3.80

(m, 16H), 6.36 (s, 8H).13C NMR (CDCl3, 125.8 MHz) d: 13.8 (CH3), 16.0 (d, CH3,

7.5 Hz), 22.4 (CH2), 26.1 (CH2), 29.2 (CH2), 29.5 (CH2), 29.6

(CH2), 29.7 (CH2), 29.8 (CH2), 29.5 (CH2), 29.7 (CH2), 30.0

(CH2), 31.6 (CH2), 33.0 (CH2, d, 138 Hz), 63.3 (CH2), 74.9

(CH2), 123.8 (C), 129.2 (CH), 134.7 (C), 155.5 (C).

Preparation of 5,11,17,23-tetra-phosphonomethyl-25,26,27,28-

tetra-dodecyloxy-calix[4]arene, 2. Under an inert gas atmosphere

dry 5,11,17,23-tetra-methylchloride-25,26,27,28-tetra-dodecyloxy-

calix[4]arene (200 mg, 0.117 mmol) was added to bromotrimethyl-

silane (3 mL) and stirred at room temperature for 5 h. The

bromotrimethylsilane was removed under reduced pressure. The

resulting solid was dissolved in water (5 mL) and stirred for 2 h.

The resultant white solid that formed was collected via vacuum

filtration (163 mg, 94%).1H NMR (MeOD/CDCl3, 600.1 MHz) d: 0.79 (t, 3J = 6.90 Hz,

12H), 1.25 (m, 72H), 1.87 (m, 8H), 2.82 (m, 8H), 3.03, 4.30 (2 d,2J = 12.7 Hz, 2 6 4H), 3.72 (t, 3J = 7.55 Hz, 8H), 6.67 (s, 8H).

13C NMR (MeOD/CDCl3, 150.9 MHz) d: 13.9 (CH3), 22.6

(CH2), 26.2 (CH2), 29.3 (CH2), 29.6 (CH2), 29.7 (CH2), 29.8

(CH2), 29.9 (CH2), 30.3 (CH2), 31.8 (CH2), 32.6 (CH2), 33.5

(CH2), 75.4 (CH2), 125.5 (C), 129.6 (CH), 134.7 (C), 154.9 (C).

TOF MS ES2 (m/z): Expected for C80H130O16P422, 735.4155.

Found: 735.4182.

Preparation of 5,11,17,23-tetra-(trimethyammoniumchloride/

azido)methyl-25,26,27,28-tetra-dodecyloxy-calix[4]arene, 4.

Under an inert gas atmosphere sodium azide (100 mg, 1.53 mmol)

was added to a pressure tube containing 5,11,17,23-tetra-

methylchloride-25,26,27,28-tetra-dodecyloxy-calix[4]arene (1.94 g,

1.39 mmol) in dry DMF (50 mL). The pressure tube was heated to

100 uC for 18 h and then cooled to room temperature. Under an

inert gas atmosphere trimethylamine (10 mL) was transferred via

cannula to the cooled pressure tube (215 uC). The pressure tube


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was then sealed, the solution allowed to warm to room

temperature, then heated to 100 uC and stirred for 16 h. An off-

white solid was collected by vacuum filtration, washed with water

(10 mL) and dried in vacuo to produce a white solid in quantitative

yield.13C NMR (CDCl3, 125.8 MHz) d: 13.8 (CH3), 22.4 (CH2), 29.2

(CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 29.8 (CH2), 29.9

(CH2), 30.0 (CH2), 30.1 (CH2), 30.2 (CH2), 30.3 (CH2), 30.7

(CH2), 31.6 (CH2), 52.0 (m, CH3), 54.2 (m, CH2), 68.3 (m, CH2),

75.8 (m, CH2), 121.7 (m, C), 129.3 (m, CH), 133.4 (m, CH), 135.2

(m, C), 157.8 (m, C).

Preparation of 5,11,17,23-tetra-(trimethyammoniumchloride/

naphthalimide)methyl-25,26,27,28-tetra-dodecyloxy-calix[4]arene,

3. 5,11,17,23-Tetra-(trimethyammoniumchloride/azide)methyl-25,26,

27,28-tetra-dodecyloxy-calix[4]arene (19 mg, 0.015 mmol) was added

to the naphthalimide ligation probe (2.2 mg, 0.01 mmol) in ethanol

(3 mL) and stirred for ten minutes. CuSO4?5H2O (155 mg,

0.001 moles) in ethanol (10 mL) and ascorbic acid (175 mg, 1 mmol)

was then added and the solution and stirred for 2 h. The organic layer

was extracted with chloroform (40 mL) and washed with water

(2 6 20 mL), dried over Na2SO4 and then evaporated under reduced

pressure to obtain a red solid (22 mg).

Fluorescence

Excitation: 350 nm. Emission: 450 nm (fluorescence at 400 nm

prior to click reaction)21

Vesicle preparation

Three different vesicle compositions were prepared. They were 1

alone, 2 alone and a 1 : 1 mixture of 1 and 2. The vesicles were

formed by dissolving 4 mg, of the amphiphilic calixarenes in

10 mL of a methanol–chloroform mixture. This solution was

transferred to a 50 mL round bottom flask (RBF) and dried

under vacuum in a rotary evaporator such that the compound

was spread evenly on the sides of the RBF. The RBF was then

placed under high vacuum for 8 h. 5 mL of 10 mM pH 7.2

phosphate buffer made up to an ionic strength of 154 mM with

NaCl was added to the RBF. The RBF was then connected to

the rotary evaporator, the water bath was set to 37 uC and the

flask was rotated at ambient pressure for 3 h. The resulting

dispersion was then extruded 11 times using an Avanti Polar

Lipids Mini-Extruder with 100 nm filters.

Cell culture

Rat pheochromocytoma cells (PC-12) were obtained from the

Mississippi Medical Centre (Jackson, MS) and grown in a RPMI

medium supplemented with 10% horse serum (HS), 5% foetal

bovine serum (FBS), 2 mM L-glutamine, 2 mM penicillin-

streptomycin, 1 mM MEM sodium pyruvate and 0.1 mM MEM

non-essential amino acids (GIBCO1, Invitrogen, Carlsbad,

California, USA). Cells were grown on flasks coated with

poly-L-lysine (PLL; 10 mg mL21; Sigma, St Louis, Missouri,

USA) and housed in an incubator at 37 uC, 5% CO2. PC-12 cells

were seeded at 2 6 105 cells mL21 in 96 well plates or chamber

slides coated with PLL as above. Compounds were assessed by

dissolving each calixarene preparation (3 mg mL21) in DMSO?

(final maximum concentration of DMSO in solution 1%) by

heating in a thermomixer (Eppendorf, Crown Scientific Pty Ltd.

Minto, NSW, Australia) at 70 uC, 750 rpm and serially diluted to

give final concentrations of 1, 0.3, 0.1, 0.01 and 0.001 mg mL21.

This procedure typically results in micelles forming in solution2

prior to the addition to cells, for example a micelle diameter of

6.4 nm with a standard deviation of 0.9 nm was measured by

DLS for compound 1. The effects of each concentration of

calixarene on cell viabilities were assessed in triplicate as

described.16 Control cultures were incubated in complete media

+ 1% DMSO.

Electrode preparation

The gold ball electrodes were prepared by melting 1 mm

diameter 99.99+% gold wire to a 0.4 mm drop using a hydrogen

flame. Melting of the wire was done according to the procedure

published by Darwish et al.32 Both the gold on mica substrates

and the gold ball electrodes were hydrogen flamed immediately

prior to use.

SAM formation

Immediately after hydrogen flaming the gold ball electrodes and

the gold on mica substrates were placed in a 1 mM decanethiol

ethanolic solution. After 18 h the substrates were rinsed with

ethanol and MilliQ water then immersed in the vesicle containing

solution for a further 18 h. The gold ball electrodes and the gold

on mica substrates with the same biomimetric bilayers were

prepared at the same time and in the same flasks/solutions.

Dynamic light scattering (DLS)

The DLS measurements were carried out on a Malvern

Instruments Zetasizer Nano S (ZEN1600). The measurements

were taken for 30 s and 7 serial 30 s measurements were

combined to form a subset, and 100 subsets were used to

calculate the vesicle size and standard deviation.

Electrochemistry

The impedance studies were done on a Gamry Reference

3000 Potentiostat/Galvanostat/ZRA, using a gold ball working

electrode, a platinum counter electrode and a Thermo Orion

900200 Ag/AgCl double junction reference half-cell with epoxy

body (sure-flow junction) with filling solutions 900002 (inner)

and 900003 (outer) for the reference electrode. The electrolyte for

the impedance was 10 mM KCl and the potential was held at

0 V vs. the reference electrode. Impedance measurements were

performed with an amplitude of 10 mV.

STM Measurements

The STM measurements were performed at room temperature

with a PicoPlus scanning probe system (Agilent, California,

USA) equipped with a 2 mm STM scanner (model N9501A).

STM tips were cut from a 0.25 mm Pt/Ir (80/20) wire

immediately prior to the experiment. Typically, a bias voltage

of 20.7 V and tunnelling current of 0.9 nA was used. The

feedback parameters of the system were adjusted for operation in

constant current mode. After installation of the probe and


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sample the instrument was left for 30 min to equilibrate before

images were recorded.

Molecular modelling

The starting geometry of decanethiols sitting on gold nanopar-

ticles were obtained from published literature data.33 A

hexagonal geometry base with sulphur atoms 4.97 A apart was

built. Decanethiols were attached to the sulphur atoms on one

side and gold particles attached on the other side. Following

construction, the structure was cleaned to correct for unortho-

dox bond lengths and angles. The angle between the decanethiols

chains and the normal was then reduced to 30u to match the

experimental value.

The crystal structure of p-phosphonated calix[4]arene was

imported from CCDC and used as the basis for the calix[4]arene

structure used in the modelling. Dodecyl chains were attached to

the lower rim of the calix[4]arene structure. The unit cell was set

to a A by a A by 59.7 A where a took on values from 20–28 to

ascertain the effect of the calix[4]arene density on the total

energy and morphology of the system post minimisation.

Acknowledgements

Support of this study by the Australian Research Council is

acknowledged, along with the facilities, scientific, and technical

assistance of the Australian Microscopy & Microanalysis Research

Facility at the Centre for Microscopy, Characterisation &

Analysis, which is funded by The University of Western

Australia, State and Commonwealth Governments. M.F. is

supported by the Neurotrauma Research Program of Western

Australia and S.A.D. by the NHMRC (APP1002347). We would

like to thank Anja Werner and The Corrosion Centre for

Education, Research & Technology at Curtin University for their

help with the impedance measurements and Cameron Evans who

assisted in obtaining the confocal microscope images.

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