MECHANOCHEMICAL SYNTHESIS

Preparation of functionalized protein materials assisted

by mechanochemistry

Lei Wang1 and Niclas Solin1,*

1Department of Physics, Chemistry, and Biology, Biomolecular and Organic Electronics, Linköping University, 581 83 Linköping,

Sweden

Received: 15 February 2018

Accepted: 15 May 2018

Published online:

18 June 2018

� The Author(s) 2018

ABSTRACT

Herein, we investigate the suitability of hen egg-white lysozyme (HEWL) as a

protein matrix for dispersal of various hydrophobic dyes. Moreover, we

investigate the use of a mixer mill for grinding operation as an alternative to

hand grinding by mortar and pestle. HEWL and various dyes are mixed by

mechanochemistry, and the resulting composite material is dissolved in aque-

ous acid. The samples are then exposed to conditions promoting self-assembly

of HEWL into protein nanofibrils (PNFs). The effect of PNF formation on dye

photophysics is investigated by spectroscopic examination by absorption and

luminescence spectroscopy, and product morphology is examined by scanning

electron microscopy. The self-assembly process results in protein nanofibrils

functionalized with luminescent dyes. Such structures may find future appli-

cations in various devices for light emission. In addition, we demonstrate that

the anticancer drug camptothecin can be incorporated into protein nanofibrils

giving materials that can find application as drug delivery agents.

Introduction

Mechanochemistry has been used since prehistoric

times, and it continues to be important in both

everyday life (e.g., in cooking procedures) and

industrial settings [1, 2]. In contrast, most research

chemists will be highly suspicious of processes not

involving solvent/liquids and may thus not even

consider the option of employing mechanochemical

processes (perhaps with the exception of preparing

samples for IR spectroscopy) [3]. However, recent

years have seen rapid developments in

mechanochemistry both regarding inorganic and

organic systems, including enzymes [2–9].

Mechanochemistry may, for example, enable organic

reactions to be carried out without solvent, thereby

minimizing the environmental impact of synthetic

procedures. In addition, mechanochemistry can be

employed for the formation of composite materials,

e.g., nanofillers, in a polymer matrix [10]. It should

not be overlooked that mechanochemistry may allow

formation of novel metastable materials not accessi-

ble by solution processing [5]. In conjunction with

such processes, it should thus be possible to use

mechanochemistry to prepare starting materials that

Address correspondence to E-mail: niclas.solin@liu.se

https://doi.org/10.1007/s10853-018-2461-7

J Mater Sci (2018) 53:13719–13732

Mechanochemical Synthesis

can be employed to carry out further unique chem-

istry, regardless if occurring in solvent or not. Such

chemistry could be termed mechanochemically

assisted chemistry. Herein, we investigate an exam-

ple of such a process, where mechanochemistry is

employed to prepare a metastable starting material

for a liquid-phase self-assembly process. In short (see

below for a more detailed description), a protein,

capable of self-assembly in aqueous phase, is co-

grinded with a hydrophobic dye molecule/material.

Upon addition of water, the protein dissolves, at the

same time helping to disperse the finely ground dye

in water. The protein is then exposed to conditions

inducing protein self-assembly. As a result of struc-

tural changes in the protein, the dye molecules obtain

new degrees of freedom, and accordingly, both pro-

tein and dye form novel structures. As both

mechanochemical processing of biopolymers [11–13]

and self-assembly processes having a huge number of

possibilities regarding both applicable materials and

exact milling conditions, such processes should

enable facile preparation of novel materials for a

wide range of applications. Self-assembly of proteins

is an efficient process enabling preparation of mate-

rials with potential applications ranging from food

science to light-emitting diodes and organic dye

lasers [14–18]. A very important self-assembly pro-

cess is the conversion of proteins into fibrillar

aggregates, known as amyloid fibrils [19–21]. Such

fibrils are associated with a range of diseases

including Alzheimer’s and Parkinson’s [20]. How-

ever, in recent years a range of amyloid structures

have been discovered, where organisms employ

amyloid for functional purposes [22]. Both in the case

of pathological amyloid and functional amyloid, the

amyloid material consists of protein nanofibrils. The

discovery of functional amyloid hints that assembly

of proteins into nanofibrils can be used to form novel

nanomaterials [23]. Hereafter, we will abbreviate

such protein nanofibrils as PNFs. For PNF formation,

typically water-soluble globular proteins are

employed that upon appropriate stimuli (typically

heating to 65–90 �C in combination with acidic pH)

self-assembles into fibrillar objects with typical

diameters of 5–10 nm and lengths in the lm range

[19, 20]. For many applications, it is desirable to

functionalize such structures with additional mate-

rials [24]. For example, by addition of luminescent

dyes PNFs can be prepared suitable for applications

related to light emission [25–28]; in addition,

interesting nonlinear optical effects have been

observed in dye-doped PNF systems [29]. Moreover,

dye-doped PNFs may be valuable as, e.g., labeled

proteins for amyloidosis studies. Alternatively,

addition of drug molecules enables use of PNFs as

drug delivery systems [30]. There exist a large num-

ber of readily available luminescent dyes that are

highly hydrophobic. When using traditional func-

tionalization approaches, this creates problems as the

employed proteins have a high solubility in water,

whereas dyes are completely insoluble in water.

Standard approaches to functionalize such structures

would be to attach hydrophilic moieties to the

hydrophobic dye; conjugate the hydrophobic dye to

the protein, or in the case of relatively polar

hydrophobic dyes employ an organic co-solvent

miscible with water. We recently reported a novel

method for protein functionalization utilizing a pre-

liminary mechanochemical mixing step [31–36]. By

co-grinding the hydrophobic dye and the protein, a

composite material is formed that can be dissolved in

water. Due to the hydrophobic effect, the dye and the

protein will stay associated in the aqueous environ-

ment, meaning that the dye will be carried along into

whatever protein structures are formed. This means

that functionalized protein materials can be prepared,

avoiding time-consuming synthetic procedures

involving covalent bond formation. An overview of

the process is shown in Fig. 1. The main point of the

mechanochemical treatment is to finely divide and

disperse the hydrophobic material within the water-

soluble material. If during the mechanochemical

treatment fine division is not achieved, upon addition

of water a turbid dispersion is obtained. Hydropho-

bic dyes typically contain a large fraction of aromatic

units and a relatively low degree of polar groups. The

main interactions between dye molecules are thus of

van der Waals type and pi–pi interactions. The most

efficient way of dispersing such dyes within a poly-

mer/protein matrix is to expose the mixture to shear

forces. During the mechanochemical treatment of

dyes within the protein matrix, relatively weak van

der Waals interactions may be broken and the dye

dispersed in the protein matrix. Impact forces should

lead to division of dye particles but is expected to be

less efficient than shear at tearing of thin layers of dye

molecules. With the above point in mind, grinding

with mortar and pestle should be an efficient tech-

nique for dispersing dyes in a protein matrix, and we

have utilized this method in several publications,

13720 J Mater Sci (2018) 53:13719–13732

where the protein bovine insulin has been mixed

with a variety of hydrophobic dyes/materials

[31–36]. However, mechanochemistry by hand

grinding with mortar and pestle suffers from some

drawbacks. As exact grinding conditions may be

difficult to reproduce, there may be undesired vari-

ations between different sample preparations; for

example, it is challenging to control variations in

force and grinding motions. In contrast, the use of a

shaker mill allows for systematic variations of

grinding parameters as well as enhancing repeata-

bility between different sample preparations. In

addition, in our previous studies the employed pro-

tein (bovine insulin) is relatively expensive, and it

would be desirable to investigate lower cost alterna-

tives. Hen egg-white lysozyme (HEWL) is a relatively

low-cost alternative that is well studied with regard

to PNF formation [37]. Herein, we investigate the

suitability of HEWL as a protein matrix for dispersal

of hydrophobic dyes 1–5 (Fig. 2); in addition, we

investigate the use of a shaker mill for grinding

operation as an alternative to hand grinding by

mortar and pestle. HEWL and various dyes are

mixed by mechanochemistry, and the resulting

composite material is dissolved in aqueous acid. The

samples are then exposed to conditions promoting

self-assembly of HEWL PNFs. The effect of PNF

formation on dye photophysics is investigated by

spectroscopic examination by absorption and lumi-

nescence spectroscopy, and product morphology is

examined by SEM.

Figure 1 Schematic drawing

of the methodology (a) and

illustration of the procedure

(b) used to prepare an aqueous

dispersion of hydrophobic

dyes@HEWL fibrils. In b, a-sexithiophene (6T or

compound 1) is used as a

representative of the

hydrophobic dye.

Figure 2 Chemical structure of studied compounds: a-sexithiophene (1 or 6T), a-octithiophene (2 or 8T), Nile red (3), pyrene (4) and (S)-(?)-camptothecin (5).

J Mater Sci (2018) 53:13719–13732 13721

Experimental

Materials

Hen egg-white lysozyme, pyrene, Nile red and (S)-

(?)-camptothecin were obtained from Sigma-Aldrich.

a-sexithiophene and a-octithiophene were obtained

from TCl. All chemicals were used as received

without further purification.

Preparation of HEWL-dye composites

The grinding was done with a shaker mill (Mixer Mill

MM 400, Retsch, Germany). The milling operation

was carried out in a stainless steel milling jar with a

volume of 1.5 mL using grinding balls of the same

material of 3 mm in diameter. Fifty milligrams of

HEWL was ground with 0.5, 1, 1.5, 2 or 3 mg of 6T.

The HEWL and 6T mixture put into the grinding jar

with 1, 10, 20 or 30 balls. Shaking time was 1, 5, 10, 20

or 30 min, and frequency was 10, 20 or 30 Hz. For

other hydrophobic dye–HEWL composites, typically,

50 mg of HEWL was ground with 1 mg of

hydrophobic dye by shaking the grinding jar with 20

balls at 30 Hz for 10 min. For comparison, 50 mg of

HEWL was ground with 1 mg of hydrophobic dye by

a mortar and a pestle. The grinding carried out at

intervals for a total of 10 min.

Self-assembly of hydrophobic dyes:HEWLcomposites

The composite material (prepared by grinding) was

dissolved in 5 mL 25 mM hydrochloric acid followed

by filtration through a 0.2-lm PES filter. The resulting

dispersion was heated at 80 �C with magnetic stirring

at 1000 rpm for 24 h to obtain hydrophobic dye-

s@HEWL fibrils.

Determination of concentration of 6Tin 6T:HEWL composites

Fifty milligrams of HEWL was ground with different

amounts of 6T (0.5, 0.75, 1, 1.25 mg) employing the

shaker mill, shaking the grinding jar at 30 Hz for

10 min with 20 balls (3 mm diameter). The resulting

composite materials were dissolved in 5 mL 25 mM

hydrochloric acid to obtain solutions containing 0.01,

0.015, 0.02, 0.025 and 0.03 mg mL-1 of 6T,

respectively.

Uv–Vis spectroscopy

Absorption data were obtained using a PerkinElmer

Lambda 950 UV–Vis spectrometer. Samples mea-

sured were diluted 20 times from the reaction mix-

ture concentration.

Fluorescence spectroscopy

Fluorescence spectra were collected using a Horiba

Jobin–Yvon Fluoromax-4 spectra fluorometer. Sam-

ples prior to measuring were diluted 20 times from

the reaction mixture concentration.

Atomic force microscopy

AFM measurements were taken using a digital

instruments dimension 3100 atomic force microscope.

Samples were diluted 100 times from the reaction

solution prior to applying to silica substrates and left

to dry for 1 min. Excess fluid was removed by

applying a nitrogen gas flow.

Scanning electron microscopy

SEM images were recorded on a Philips XL30 FEG

SEM microscope. Small amount of samples powder

were attached to metal substrates by double adhesive

carbon tape. The stock solution samples were incu-

bated for 1 min prior to drop casting on Si substrates.

Excess fluid was removed by applying a nitrogen gas

flow. All samples were sputter-coated with a thin

layer of Pt under argon in a sputter coater (Leica EM

SCD 500).

Results and discussion

Investigation of milling conditionsfor 6T:HEWL

A variety of parameters will influence the efficiency

of the milling process. We investigated the effect of

number of grinding balls, grinding time and fre-

quency. The experiments were performed using a

Retsch mixer mill MM400. The milling process was

performed in stainless steel jars with an interior

volume of 1.5 mL employing stainless steel grinding

balls with a diameter of 3 mm. For this study, we

focused on combinations of 6T and HEWL. A typical

particle size after milling is 5 lm (information from

13722 J Mater Sci (2018) 53:13719–13732

the Retsch Company Web site). However, in the

present material system, the hydrophobic dye (6T) is

milled in a large excess of HEWL. It can therefore be

expected that 6T will be diluted in the form of smaller

particles in the HEWL matrix. As stated earlier, 6T is

insoluble in water, whereas HEWL readily dissolves

in water. As a consequence, the particle size of HEWL

upon milling is of low importance. In contrast, an

inefficient milling process, resulting in the presence

of large 6T particles within the HEWL matrix, will

result in a dispersion of large-sized particles that will

scatter light, resulting in a turbid sample. This effect

is readily apparent, as can be seen in Fig. 3a, b, where

photographs are shown in different samples, dis-

persed in 25 mM HCl and obtained under different

milling conditions (number of grinding balls being 1,

10, 20 and 30). The sample ground with one ball is

turbid, and upon filtration through a 0.2-lm mem-

brane, the majority of 6T is removed (Fig. 3a). In

contrast, for samples ground with several balls all the

samples look transparent. However, when analyzed

by UV–Vis spectroscopy, after filtration through a

membrane it can be seen that 20 grinding balls give

the most efficient result. Ten and 30 grinding balls

lead to less 6T being incorporated (Fig. 3b). We also

tested the effect of frequency and found that 30 Hz

(the highest frequency available) is more efficient

than 20 or 10 Hz (Fig. 3d). We also investigated the

milling process at different time points. For this test,

we employed 20 grinding balls in combination with a

Figure 3 Photographs of 6T:HEWL composites solution prior to

grinding by different mixing numbers of balls (upper solution

before filtration, down solution after filtration) (a). Absorbance

spectra at 25 mM HCl for samples of 50 mg HEWL ground with

1 mg 6T at different grinding conditions (b–d).

J Mater Sci (2018) 53:13719–13732 13723

frequency of 30 Hz. The milling operation was stop-

ped after the allotted time, the grinding jar was

opened and a small amount of sample powder was

collected. This was then dispersed in 25 mM HCl,

filtered through a 0.2-lmmembrane and analyzed for

6T content (Fig. 3c). After 1-min milling, a small

amount of 6T is dispersed, but after 6 min there is an

significant increase in the amount of dispersed 6T.

Finally, after 10 min only small changes are observed

upon increasing the milling time (for up to 30 min). It

is also interesting to compare SEM images of the

composite material, either as prepared or as drop

casted (after treatment with 25 mM HCl) (Fig. 4). In

order to investigate the as-prepared milled powder,

small amounts of the sample were attached to metal

substrates by double adhesive carbon tape. The

resulting SEM images, obtained for powders exposed

to different milling conditions, are shown in Fig. 4. It

is difficult to see any clear difference in particle size

between different samples (Fig. 4 left section). How-

ever, when zooming in on individual particles,

interesting differences in morphology can be

observed as a function of milling parameters (Fig. 4

middle section). For the sample with one grinding

ball, the majority of examined particles have rela-

tively cleanly cleaved surfaces. On the other hand, for

samples where multiple grinding balls are employed,

the majority of particles are of a more irregular shape,

containing components with a ‘‘flake-like’’ shape.

This is true both for samples consisting of ground

pure HEWL and for co-ground of 6T and HEWL. In

contrast, when examining samples with SEM after

treatment with 25 mM HCl, the observable particles

have a smaller size (Fig. 4, right section). These

results indicate that the particles as prepared after

milling consist of mixtures of 6T and HEWL, where

upon addition of 25 mM HCl the HEWL is dissolved,

and the remaining observable particles consist of 6T.

The sample corresponding to 20 grinding balls

(Fig. 4i) shows the least amount of particles, which

matches the result from the absorption spectroscopy

of the corresponding filtered sample (Fig. 3b). For

comparison, SEM images of powders obtained by

grinding HEWL and 6T by hand with mortar and

pestle are shown in Fig. S1. There is a large variety of

particles sizes (see Fig. S1a), and when zooming in an

individual particle (Fig. S1b), the surface of the par-

ticle shows flaky shapes, similar to the particle shown

in Fig. 4h. However, after addition of 25 mM HCl

and deposition on the substrate, the hand ground

sample contains large particles of a size up to 2 lm,

in contrast to the samples ground by the shaker mill

(c.f. Fig. 4i and Fig. S1c). This result indicates that the

grinding with mortar and pestle gives a more

heterogenous sample than the sample ground by the

shaker mill. We also tested combinations of different

amounts of 6T in combination with a constant

amount of HEWL (Fig. 5). Fifty milligrams of HEWL

was combined with 0.5, 1.0, 1.5, 2.0 and 3.0 mg of 6T,

respectively. The weight ratio of HEWL:6T that gives

the highest amount of 6T dispersed in water after

filtration is 50:1. Increasing the amount of 6T does not

lead to a higher concentration of 6T in the liquid

phase. Instead, it was found that a higher 6T ratio

leads to less efficient dispersion of 6T. When

inspecting the 50:2 and 50:3 ratio samples dispersed

in 25 mM HCl, it is readily apparent that large 6T

particles are present, resulting in turbid samples.

Consequently, upon filtration through a 0.2-lmmembrane, it is apparent that the concentrations of

6T in these samples are lower than in the sample with

a 50:1 ratio. In order to quantify the amount of 6T

dispersed in the liquid phase after filtration, we

prepared a calibration curve in the following manner:

Samples were prepared by co-grinding HEWL and

6T in ratios of 50:0.5, 50:0.75, 50:1.0 and 50:1.25. The

resulting composite materials were dissolved in

25 mM HCl and were inspected to make certain that

no non-dissolved material remained. The 6T absorp-

tion maximum is located at 412 nm. The absorbance

at 412 nm versus the concentration of 6T was then

plotted, and a calibration curve was constructed

(Fig. S2). This curve can then be used to calculate the

concentration of 6T in the aqueous phase. Three

independent samples were prepared by grinding

HEWL:6T (with 20 grinding balls at a 30 Hz fre-

quency) in a 50:1 mg ratio (dissolved in 5 mL 25 mM

HCl). According to the calibration curve, the con-

centration of 6T is 0.168 ± 0.008 mg mL-1 in the

6T:HEWL samples (with the maximum possible

amount being 0.2 mg mL-1). This shows good

reproducibility of the shaker mill grinding method.

When comparing results from grinding with one

and many grinding balls, we see that one grinding

ball gives poor incorporation of 6T. In fact, grinding

with one ball gives a poorer result than hand grind-

ing by mortar and pestle. These results indicate that

shear forces are very important in order to efficiently

disperse 6T in the protein matrix. The organic dyes

contain strong intramolecular covalent bonds. In

13724 J Mater Sci (2018) 53:13719–13732

contrast, intermolecular bonds are much weaker and

we assume that such intermolecular bonds are read-

ily cleaved upon exposure to shear forces, leading to

mixing of protein and dye. However, it should be

noted that after grinding the majority of dye mole-

cules are present in aggregates (not as individual

molecules) as judged by absorption and fluorescence

spectroscopy. However, during/after fiber formation

(see below, the next section), the dyes are able to

redistribute within the PNF matrix, resulting in iso-

lated molecules dispersed within the protein fibrils.

Figure 4 SEM images of 6T:HEWL composites powder after

milling with different numbers of grinding balls: 1 ball (a, b); 10

balls (d, e); 20 balls (g, h); 30 balls (j, k). SEM images of the

resulting composites dissolved by 25 mM HCl and then drop

casted onto substrates: 1 ball (c); 10 balls (f); 20 balls (i); 30 balls

(l).

J Mater Sci (2018) 53:13719–13732 13725

Self-assembly of HEWL:thiophenecomposites

Whereas 6T can be viewed as the ‘‘fruit fly’’ of organic

electronics, the corresponding compound containing

two additional thiophene moieties is much more

rarely used. A large reason is the low solubility of

these compounds. Already 6T is hard to dissolve in

organic solvents, and with 8T, the solubility limit is

reached. Accordingly, spectral data for 8T in solution

phase are rare. It would be desirable to have a pro-

cessable form of 8T available, and we tested whether

preparation of such materials could be assisted by

mechanochemistry. We compared both grinding by

mortar and pestle and milling by shaker mill with 20

grinding balls at 30 Hz for 10 min. Both methods

work well, and dispersions processable from aqueous

solvent can be obtained (Fig. S3 0 h and Fig. S4a 0 h).

However, it should be noted that the absorption

spectrum of 6T ground with HEWL is narrower

when ground with the shaker mill compared to the

hand ground sample, indicating a more homogenous

sample when ground with the shaker mill. We then

investigated whether the 6T:HEWL and 8T:HEWL

composites were capable of self-assembly into pro-

tein fibrils. We have earlier reported that composite

materials between bovine insulin and hydrophobic

dyes can form PNFs [32]. Oligothiophenes are con-

venient compounds to study as the absorption and

emission spectra of oligothiophenes are sensitive to

the packing of thiophene molecules [32, 38–42].

Typically, 6T or 8T in an aggregated state will give

rise to a redshift of emission with a low intensity.

Conversely, 6T or 8T in a monomeric state will give

rise to a blueshifted emission of high intensity.

Moreover, there will be corresponding changes in

absorption spectra. This means that the development

of structural change can be monitored through vari-

ations in emission and absorption spectra. When

6T:HEWL and 8T:HEWL composites (prepared by a

shaker mill with 20 grinding balls at 30 Hz for

10 min) are exposed to heating in acidic water (80 �Cfor 24 h with stirring in 25 mM HCl), and spectral

changes are monitored, we observe typical changes

as observed earlier in the 6T:insulin system [32]. For

6T, we observe strong emission with a maximum at

508 nm and a typical vibronic structure (Fig. 6a). For

8T, we observe strong emission with a maximum at

548 nm and a typical vibronic structure (Fig. 6b).

Conversely, in the 0 h samples, emission is weak and

typical of aggregated oligothiophenes. Moreover,

when samples are observed by SEM or AFM after the

fibrillation process, typical fibrillar object can be

observed, confirming the presence of PNFs (Fig. 6c,

d). We also observed similar results when fibrillated

6T and 8T samples were observed by AFM (Fig. S7).

For comparison, samples prepared by grinding with

mortar and pestle give similar result for both 6T and

8T (Figs. S4 and S6).

Figure 5 Photograph of at 25 mM HCl for samples of 50 mg HEWL ground with different amounts of 6T (a), absorbance spectra of at

25 mM HCl for samples of 50 mg HEWL ground with different amounts of 6T (b).

13726 J Mater Sci (2018) 53:13719–13732

Other dye:HEWL materials

We investigated three other dyes in combination with

HEWL: Nile red (3), pyrene (4) and campthotecin (5).

Nile red is a famous dye molecule with applications

as a laser dye as well as an optical probe for sensing

applications [29]. For example, Nile red has been

applied as a probe for the presence of amyloid fibrils

[35, 43]. We tested co-grinding of HEWL and Nile red

and exposed the resulting composite materials to

fibrillation conditions (80 �C for 24 h with stirring in

25 mM HCl). When comparing samples before fib-

rillation (0 h) and after fibrillation (24 h), it can be

seen there is a large increase in fluorescence intensity

after 24 h, with a maximum at 620 nm. The high

fluorescence intensity in the 24-h sample is typical of

the presence of PNFs (Fig. 7a). In addition, fibrillar

objects can be observed by SEM in the 24-h sample

(Fig. 7b). For comparison, samples prepared by

grinding with mortar and pestle give similar result

(Fig. S9).

Pyrene is polyaromatic hydrocarbon that can pack

efficiently into crystals. The pyrene molecule can be

excited by UV light and fluoresces in the blue region

of the spectrum. Due to the tight packing, the pyrene

crystal should be relatively challenging to fragment

by milling. This is readily observed when mixing

pyrene with HEWL. When pyrene is co-ground with

HEWL, a white-colored composite material is

obtained. Upon addition of aqueous 25 mM HCl, a

turbid white-colored dispersion is obtained, demon-

strating the presence insoluble pyrene particles

(Fig. S10a). Still the resulting dispersion can be fil-

tered, and the sample retains a strong absorption

Figure 6 Fluorescence spectroscopy spectra at 25 mM HCl for

samples of 6T:HEWL composites (excited at 465 nm) (a) and

8T:HEWL composites (excited at 495 nm) (b) before (0 h) and

after fibril formation (24 h); SEM image of HEWL ground with 6T

(c) and 8T (d) after fibrils formation (24 h of heating and stirring).

J Mater Sci (2018) 53:13719–13732 13727

from pyrene (Fig. S10b, c). Pyrene photophysics is

sensitive to the concentration of pyrene molecules

[44, 45]. It is known that pyrene readily forms exci-

mers (an excited state dimer) which show a red-

shifted emission without vibronic fine structure. In

contrast, the monomer shows vibronic structure and

a relatively blueshifted emission. Spectral properties

were compared for as-prepared (0-h) and fibrillated

(24-h) samples (Fig. 8a). The 0- and 24-h samples

show markedly different spectral properties. Before

formation of PNFs, the emission spectrum is domi-

nated by a broad peak typical of excimer emission

with a maximum at 465 nm. This indicates that

before fiber formation pyrene molecules are tightly

packed together. In contrast, upon PNF formation the

emission spectrum shows typical characteristics of

monomer emission, with and emission maxima at 375

and 395 nm and featuring vibronic structure. In

addition, when the 24-h sample is examined by SEM,

fibrillar objects can be observed, confirming PNF

formation (Fig. 8b). As the emission spectra are typ-

ical of monomer emission, this indicates that in the

fibrillated sample the pyrene molecules are relatively

spread out within the fibril. For comparison, samples

prepared by grinding with mortar and pestle gave

similar result; however, less pyrene is incorporated

into PNFs using hand grinding compared to use of a

mixer mill (c.f. Fig. S10c and Fig. S11a). Interestingly,

Figure 7 Fluorescence spectroscopy spectra at 25 mM HCl for samples HEWL ground with Nile red (excited at 555 nm) (a) and SEM

image of HEWL ground with Nile red (b) after fibrils formation (24 h of heating and stirring).

Figure 8 Fluorescence spectroscopy spectra at 25 mM HCl for samples HEWL ground with pyrene (excited at 345 nm) (a) and SEM

image of HEWL ground with pyrene (b) after fibrils formation (24 h of heating and stirring).

13728 J Mater Sci (2018) 53:13719–13732

pyrene fluorescence is known to be sensitive to the

polarity of the environment. Pyrene fluorescence has

been measured in organic solvents of varying polar-

ity, and it has been found that the ratio of the inten-

sity of the I to III emission bands varies from * 0.6 in

hydrocarbon media to * 2 in DMSO. In our sample,

the I peak occurs at 375 nm and the III peak occurs at

395 nm. For our samples prepared with a shaker mill

(Fig. 8a), the III/I ratio is 1.038. For our sample pre-

pared by hand grinding (Fig. S11b), the III/I ratio is

1.097. These results indicate that the pyrene mole-

cules in PNFs are located in hydrophobic domains

[45, 46].

Camptothecine is a fluorescent natural product

with anticancer activity [47]. It has been demon-

strated that PNFs can act as drug delivery agents [30];

it is thus of interest to test whether the

mechanochemistry approach can be used to prepare

protein–drug complexes. Camptothecine has dye-like

properties and is fluorescent, which enables conve-

nient monitoring of the dye by spectroscopy [48].

However, in contrast to the other dyes used herein,

camptothecin emission does not appear to be sensi-

tive toward the presence of PNFs, as both as-pre-

pared (0-h) and fibrillated (24-h) samples show the

same spectral features, typical of camptothecine at

low pH (Fig. 9a) [48]. Investigation of the 24-h sample

by SEM clearly shows fibrillar objects confirming

PNF formation (Fig. 9b). For camptothecin, it appears

that grinding with mortar and pestle somewhat more

efficient than grinding by the shaker mill, as hand

ground samples give higher absorption than samples

prepared by shaker milling (c.f. Figs. S12, S13

and 9a).

Herein, we have ground HEWL with various dyes.

Co-grinding of dyes with other powder materials is a

well-known technique for dispersion of dyes. For

example, mechanical dry milling of organic pigments

(aggregate of dye molecules) has been performed in

the presence of silica nanoparticles, resulting in effi-

cient dispersion [49]. Such organic pigments can be

dispersed in a liquid and solid solutions and

employed as inks, coatings and plastics. Dynamic

processes of dyes in polymer matrices are known. For

example, in so-called latent pigment technology a

soluble pigment precursor is employed, that upon

chemical or physical treatment is converted to the

(insoluble) pigment in situ [50]. In contrast, in the

approach reported herein, the mechanochemical step

results in composite material consisting mostly of

small dye molecule aggregates dispersed in a HEWL

matrix. However, the HEWL matrix is under appro-

priate conditions capable of self-assembly resulting in

PNFs and these structures are in turn able to promote

disruption of dye aggregates into monomeric species.

These effects are clear for a variety of dyes (6T, 8T,

Nile red, pyrene) employed herein.

Conclusions

We have shown that a shaker mill can be used for

materials preparation, in addition to hand grinding

by mortar and pestle. The shaker mill shows better

Figure 9 Fluorescence spectroscopy spectra at 25 mM HCl for samples HEWL ground with camptothecin (excited at 370 nm) (a) and

SEM image of HEWL ground with camptothecin (b) after fibrils formation (24 h of heating and stirring).

J Mater Sci (2018) 53:13719–13732 13729

efficiency and reproducibility than hand grinding.

The samples ground by shaker mill shows a more

homogenous particle distribution as judged by SEM

images. Moreover, the absorption spectrum of 6T

ground with HEWL is narrower when ground with

the shaker mill, again indicating a more homogenous

sample. In addition, we report that HEWL can be

employed for preparation of functionalized PNFs.

Moreover, as one of the components is insoluble in

the solvent, a cohesive force is generated by the sol-

vent rather than any specific favorable interactions

between the reagents. To the best of our knowledge,

this novel approach has a possibility for creativity

from both the mechanochemistry and self-assembly

sides. For example, by variations in mechanochemi-

cal conditions it may be possible to prepare different

metastable materials that may evolve into different

structures in a sequential self-assembly step.

Depending on the selected functionalization agent

(dye), materials can be prepared aiming toward

various applications, for example light-emitting

diodes (for luminescent molecules) or drug delivery

systems (for drug molecules).

Supplementary information

Calibration of 6T concentration by absorbance spec-

tra; absorbance spectra for samples of HEWL ground

with 6T, 8T, Nile red, pyrene and camptothecin by

shaking mixer mill and absorbance and fluorescence

spectroscopy spectra for samples prior to grinding by

hand grinding with mortar and pestle before and

after fibril formation; AFM images of 6T:HEWL and

8T:HEWL fibrils.

Acknowledgements

L. W. acknowledges financial support from the China

Scholarship Council. We acknowledge financial sup-

port from the Swedish Government Strategic

Research Area in Materials Science on Functional

Materials at Linkoping University (Faculty Grant

SFO-Mat-Liu # 2009-00971).

Electronic supplementary material: The online

version of this article (https://doi.org/10.1007/

s10853-018-2461-7) contains supplementary material,

which is available to authorized users.

Open Access This article is distributed under the

terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/

licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, pro-

vided you give appropriate credit to the original

author(s) and the source, provide a link to the Crea-

tive Commons license, and indicate if changes were

made.

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