triplet-triplet annihilation upconversion based nanocapsules for bioimaging under excitation by red...

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Triplet–Triplet Annihilation UpconversionBased Nanocapsules for Bioimaging UnderExcitation by Red and Deep-Red Lighta

Christian Wohnhaas, Volker Mail€ander, Melanie Dr€oge, Mikhail A. Filatov,Dmitry Busko, Yuri Avlasevich, Stanislav Baluschev,* Tzenka Miteva,Katharina Landfester, Andrey Turshatov*

Non-toxic and biocompatible triplet–triplet annihilation upconversion based nanocapsules(size less than 225nm) were successfully fabricated by the combination of miniemulsion andsolvent evaporation techniques. A first type of nanocapsules displays an upconversionspectrum characterized by the maximum of emission at lmax¼ 550nm under illumination byred light, lexc¼ 633nm. The second type of nanocapsules fluoresces at lmax¼ 555nm when
excited with deep-red light, lexc¼ 708nm. Conventionalconfocal laser scanning microscopy (CLSM) and flowcytometry were applied to determine uptake andtoxicity of the nanocapsules for various (mesenchymalstem and HeLa) cells. Red light (lexc¼ 633nm) withextremely low optical power (less than 0.3mW) or deep-red light (lexc¼ 708nm) was used in CLSM experimentsto generate green upconversion fluorescence. The cellimages obtained with upconversion excitation demon-strate order of magnitude better signal to backgroundratio than the cell images obtainedwith direct excitationof the same fluorescence marker.
Dr. C. Wohnhaas, Dr. V. Mail€ander, M. Dr€oge, Dr. M. A. Filatov,D. Busko, Dr. Y. Avlasevich, Prof. Dr. S. Baluschev,Prof. K. Landfester, Dr. A. TurshatovMax Planck Institute for Polymer Research, Ackermannweg 10,55128 Mainz, GermanyE-mail: [email protected]

Dr. V. Mail€anThird DepartJohannes GuGermanyProf. Dr. S. BOptics and SUniversity ‘‘SBulgariaDr. T. MitevaMaterials ScHedelfingers

aSupporting Information is available at Wiley Online Library or fromthe author.

� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimMacromol. Biosci. 2013, 13, 1422–1430

wileyonlinelibrary.com

1. Introduction

The process of the generation of photons with a higher

energy under excitation by photons with a lower energy is

known as photon upconversion (UC). There are a number of

upconversion techniques, for example, second harmonic

generation,[1] two-photon absorption,[2] sequential energy

transfer (or excited-state absorption) in rare earth ion-

derment of Medicine, University Medicine of thetenberg University, Langenbeckstr. 1, 55131 Mainz,

aluschevpectroscopy Department, Faculty of Physics, Sofiat. Kliment Ochridski,’’ James Bourchier 5, 1164 Sofia,

ience Laboratory, Sony Deutschland GmbH,tr. 61, 70327 Stuttgart, Germany

DOI: 10.1002/mabi.201300149

Triplet–Triplet Annihilation Upconversion Based Nanocapsules . . .

www.mbs-journal.de

doped inorganic glasses,[3] and triplet–triplet annihilation

assisted upconversion (TTA-UC).[4–13] In comparison with

the mentioned upconversion tools, TTA-UC shows several

outstanding advantages. First, UC emission is observed

under very weak excitation intensity (down to mW cm�2)

and low spectral power density (as low as mW nm�1).

Therefore, the technical requirements for the appropriate

excitation sources could be fulfilled by low-power continu-

ous wave (cw) – diode lasers or other conventional light

sources (such as light emitting diodes). Second, the

excitation wavelength of the TTA-UC process is easily

tunable and can be gradually extended from visible to NIR

region by using sensitizers with red-shifted absorption

bands.[14] By tuning the molar ratio of the sensitizer to the

emitter, one can find a composition where the optical

signals of the UC fluorescence and the residual phospho-

rescence of the sensitizer have a comparable intensity.

The phosphorescence of the sensitizer shows a bath-

ochromic shift relative to the excitation wavelength

(DlPhos> 150nm) whereas the corresponding UC fluores-

cence has a hypsochromic shift relative to the excitation

wavelength (DlUC> 100nm). Consequently, two optical

signals (the residual phosphorescence and the delayed UC

fluorescence) could provide a ratiometric measuring

scheme. Furthermore, phosphorescence and upconversion

fluorescence are examples of delayed emission. Therefore, a

time-resolved registration might provide an additional

improvement of the detection quality: the problems,which

are caused by scattering of the excitation light or by

autofluorescence with short lifetime, can be eliminated by

using a pulsed excitation and time-gated detection.

Summarizing all advantages, we can conclude that the

TTA-UC is a highly promising tool for bioimaging operating

under red or near infrared (NIR) optical excitation,

since only excitation light with wavelength longer than

600 nm[15–17] penetrates deep enough into the tissue and

can locally generate via TTA-UC process blue photons,

which canbeused for FRETassays ormulticolor targetingof

cancer cells.

Despite the demonstrated advantages, a few significant

drawbacks restrict the applicability of TTA-UC in biosci-

ence: the typical TTA-UC system consists of two dyes

(sensitizer and emitter) dissolved in an oxygen-free organic

solvent (toluene, for instance). An imperative requirement

for bio-applications is transferring the TTA-UC process

from the organic solvent into aqueousmedia. Recently, the

TTA-UC was performed in water environment via an

encapsulation of the UC dyes in micelles out of a

biocompatible non-ionic surfactant,[18] polymer nanopar-

ticles with sizes of 1[19] and 322nm,[20] poly(propylene

oxide) core/silica dioxide shell nanoparticles (size nearly

22.5 nm)[21] or microcapsules with the size of 200[22] and

350mm.[23]Most of the fabricatedmaterials have, however,

one similar characteristic: palladium (PdOEP) or platinum

Macromol. Biosci. 20

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octaethyl porphyrin and diphenylanthracene (DPA) were

used as upconverting dyes (UC excitation lexc¼ 532nm,UC

fluorescencewith lmax¼ 435nm).[19–22] Thus, there remain

open questions. First, could the methods suggested

elsewhere[19–22] be expanded with UC sensitizers which

absorb light in red or NIR regions of the spectrum?

Substantial progress was achieved recently when Liu

et al.[24] reported a successful example of red-to-green

upconversion measured in vivo (in living mouse) by using

nanocapsules designed as the soybean oil core and a shell

formed from a bovine serum albumin–dextran conjugate.

Another important question is: are all fabricated nano-

materials able to work efficiently under conditions with

different concentrations ofmolecular oxygen? For instance,

there isasignificantoxygentensionof0.5–2.5 kPa (less than

in air 21 kPa) in a tissue.[25] Thus, TTA-UCmight be strongly

quenched bymolecular oxygen.[26] Monguzzi et al.[19] have

shown that highly crosslinked polymer nanoparticles are

able to protect the TTA-UC process fromoxygen quenching.

Retarding the molecular oxygen by a cross-linked polymer

shell in microcapsules was demonstrated by Kang and

Reichmanis[23] as well. Liu et al.[21] synthesized the poly

(propylene oxide) core/silica dioxide shell nanoparticles

(with PdOEP and DPA as UC dyes) and reported rather high

UC quantum yield of 4% in aerobic conditions. In contrast,

there are two publications[27,28] where the authors

synthesized nanoparticles, which have equivalent proper-

ties with respect to the nanoparticles reported by Liu

etal.[21] (there isonly insignificantdifference in theoriginof

the loaded dyes) and proved the penetration of the

molecular oxygen through the silica dioxide shell. More-

over, nanoparticles (loaded with platinum(II) meso-tetra-

phenyltetrabenzoporphyrin) prepared by Wang at al.[28]

demonstrate strong quenching of the phosphorescence of

the porphyrin by molecular oxygen and are used as an

oxygen sensor. In order to reduce the effect of molecular

oxygen, an alternative method was demonstrated by Kim

and Kim.[22] They used the mixture of hexadecane and

polyisobutylene (PIB) as the inner solvent phase for

upconverting microcapsules and postulated that the

vanishing of the oxygen quenching is related to the oxygen

impermeability of PIB. Indeed, the PIB polymer demon-

strates low permeability to gas molecules. However, this

explanation is unpersuasive in case of 5% PIB solution in

hexadecane.

In our previous paper, we described an efficient method

to incorporate the UC system in polymeric nanocapsules

(NCs) via radical miniemulsion polymerization.[29] The UC

couple of dyes dissolved in a liquid hexadecane core was

embeddedwithin polystyrene-co-polyacrylic acid NCs (size

180nm). However, the radicals generated in the process of

the radical miniemulsion polymerization reacted with the

potential sensitizers for red and deep-red excitation

(tetrabenzoporphyrins and tetranaphthoporphyrins) and

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C. Wohnhaas et al.

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destroyed them chemically. Taking into account this

limitation,we havemodified themethod. Themodification

allowed to incorporate red and deep-red UC sensitizers

into polymer NCs by combination of miniemulsion and

solvent evaporation techniques.[30]

The aim of the current work is a demonstration of the

progress in a development of the TTA-UC NCs for the

excitation with red and deep-red light. Essentially, the NCs

should be nontoxic for living cells and demonstrate UC

performance in vitro. Additionally, the NCs should be

suitable for investigations with well-established biomedi-

cal techniques, namely confocal laser scanningmicroscope

(CLSM), plate reader, or flow cytometry.

2. Experimental Section

2.1. Materials

All solvents were used as receivedwithout additional purification.

Palladium(II) meso-tetraphenyltetrabenzoporphyrin (PdTBP), per-

ylene, and 1-phenyl-heptadecane (PHD), were purchased from

Aldrich. Sodium n-dodecyl sulfate (SDS) was purchased from Alfa

Aesar. Polymethylmethacrylate (pMMA; Mn ¼ 120kDa) was pur-

chased from Merck. For the synthesis of 1,3,5,7-tetramethyl-8-

phenyl-2,6-diethyl dipyrromethane �BF2 (dye 550) we followed

synthetic procedure reported byWagner and Lindsey[31] Palladium

(II) meso-tetraaryltetranaphthoporphyrin (PdTNP) was synthe-

sized in agreement with literature.[32,33] The detailed synthesis

of 3,9(10)-Bis(4-tert-Butylphenylethynyl)perylene (dye 555) is

reported in the Supporting Information.

2.2. Synthesis of the Capsules

The pMMA-capsules NC633 and NC708 were formed via solvent

evaporation process in miniemulsion. The first step of this process

was to mix two phases, the disperse phase and the continuous

phase, and stir them for 1h (1200 rpm) to get an emulsion. The

continuous phase consists of 10mg SDS and 10g demineralized

water. The disperse phase contains 150mg pMMA, 300mg PHD,

0.3mg PdTBP as UC sensitizer, 1.3mg dye 550 as UC emitter,

and 2.5 g chloroform as co-solvent (preparation of NC633).

Alternatively, 0.048mg PdTNP and 0.198mg dye 555 (all other

componentswere taken as for NC633)weremixed in the synthesis

of NC708.

To receive miniemulsion (second step) with a droplet size

between 50 and 500nm, high shear power is necessarywhichwas

reachedwith sonificationbyanultrasound tip (BransonsonifierW-

450D, 1/2 in. Tip, 90% Amplitude, 120 s, 10 s pulse/10 s break, ice

cooling). The last step was the slow evaporation of the solvent

(CHCl3) under stirring and room temperature overnight. Due to the

evaporation of the co-solvent the pMMA precipitated and a phase

separation between the pMMA and PHD took place. As a result of

different surface tensions, the hydrophobic PHD formed the inner

core and the hydrophilic pMMA arranged at the water interphase

as shell, so the core-shell morphology with liquid core and solid

polymer-shell was built up.


� 2013 WILEY-VCH Verlag GmbH

2.3. Characterization of the Nanocapsules

Dynamic light scattering (DLS) was performed with Submicron

Particle Sizer Nicomp 380 (fixed scattering angle of 908, laserwavelengthl¼ 633nm) formeasurements of the size of theNCs in

dispersion (concentration �0.2mgmL�1). The transmission elec-

tron microscopy (TEM) was done with a Zeiss EM-912 (operating

voltage 120kV). For sample preparation, 5mL of diluted dispersion

(3mL of sample in 3mL of demineralized water) were attached

on a carbon coated copper grid and left to dry. The dry sample

was covered by an additionally carbon layer in order to

avoid decomposition of the polymer and to increase the image

contrast.

2.4. TTA-UC Measurements

For upconversion measurements, the dispersion was filled into

a cuvette (1mm thickness) under nitrogen atmosphere (UNIlab

glove box, M.braun GmbH) and then sealed to prevent oxygen

penetration. A supercontinuum laser (repetition rate 20MHz,

mean spectral power density 1mWnm�1, pulse duration 10ps)

SC450-2-PP (Fianium Inc.) was integrated in the optical scheme.

Generally, output radiation of the laser was passed through a

4F-monochromator in order to select the excitation wavelength.

Typically, excitationwithabandwidthofDl�10–15nmwasused.

A series of reflective neutral density filters (Thorlabs Inc.) placed on

a revolving optical holder was used to attenuate smoothly the

beam power. The laser beam was guided by ultrabroadband

mirrors (MaxMirror, Semrock Inc.) and finally focused by an

achromatic lens (NA¼ 0.24) onto the sample. The excitation

spot diameter was permanently controlled by a beam profiler

BP104-VIS (Thorlabs Inc.). The luminescence emissiongeneratedby

the sample was collected by the same achromatic lens, thus the

excitation- and observation-spots were completely spatially over-

lapped. The excitation light (lexc¼633nm) was rejected by a

notch filter NF03-633U-25 (Semrock Inc.). The excitation light

(lexc¼ 708nm) was rejected by a tilted at 458 notch filter NF03-

785E-25 (Semrock Inc.) or a short-pass filter FF01-694/SP-25

(Semrock Inc.). The emission spectra were registered by a

CCD-spectrometer (C10083CA, Hamamatsu Inc.). Detailed exam-

ples of absorption and UC spectra of investigated compounds in

toluene can be found in the literature[34] and in the Supporting

Information.

2.5. Confocal Laser Scanning Microscope (CLSM)

Imaging

The images of NC633 NCs were obtained with a Leica TCS SP5X

microscope. The sample was excited with different wavelengths:

lexc¼488nm of an Arþ laser for direct excitation of dye 550 and

lexc¼633nmofHeNe laser forUCexcitationof PdTBPor excitation

of CellMask Deep Red (Invitrogen). Two detectors were used, a

photomultiplier tube (PMT) and an avalanche photodiode (APD). In

case of theAPD, thedetection channelwas equippedwithayellow-

wavelengthcenteredbandpassfilterHQ525/50M(ChromaInc.) for

fluorescence andUC luminescenceof dye 550. The PMTwasused to

detect the emission from CellMask Deep Red (in the range 650–

700nm).

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www.mbs-journal.de

The images ofNC708NCswereobtainedwith ahomebuilt CLSM

microscope. A supercontinuumfiber laser SC-450HP (Fianium Inc.)

provided both the direct and UC excitation. A wavelength for the

direct excitation of dye 555was extracted from the spectrumof the

supercontinuum laser by a dichroic bandpass filter BP510/20

(Chroma Inc.). A wavelength for excitation of PdTNPwas extracted

byaspeciallydesignedmonochromator (lexc¼708nm,Dl¼5nm).

Bothbeamswere focusedby a1.4NAobjective lens (HCXPLAPOCS

100� oil, Leica Microsystems). The image acquisition was

performed by scanning the sample with a 3D piezo stage

MAX341/M (Thorlabs Inc.). The fluorescence was collected by the

same objective lens, separated from the excitation light by the

dichroic mirrors and an additional bandpass filter BP570/60

(Chroma Inc.), and finally focused into the multimode optical fiber

(diameter 62.5mm) serving as a pinhole to be detected by photon

counting module MP 900 (Perkin Elmer).

2.6. Cell Cultures

HeLa cells were obtained from Deutsche Sammlung von Mikroor-

ganismen und Zellkulturen (DSMZ) and kept in Dubbecco’s

modified essential medium (DMEM), whereas mesenchymal stem

cells (MSC) were generated from bone marrow aspirations or

explanted hips after obtaining informed consent and kept in

a-minimal essential medium (MEM, Lonza). Both media were

supplemented with 10% fetal calf serum (FCS; HeLa) and 20% FSC

(MSC), 100 units penicillin and 100mgmL�1 streptavidin (all

from Invitrogen) and especially for MSC cells, 1mM Na-pyruvate

(Invitrogen) and 0.6% ciprofloxacin (Fluka) were added. For the

incubation, adherent cells were seeded at a density of 5000 cells

cm�2 (HeLa)and4000cells cm�2 (MSC) inam-dish (d¼35mm,high,

Ibidi). On the 2nd day, the NCs were added at a concentration of

3000mgmL�1 to themediawithout using a transfection agent. The

incubation time was 24h in a humidified incubator in order to

facilitate cell uptake (37 8C, 5% CO2). After incubation, themedium

was removed from the m-dish and the sample was washed three

timeswith 3mL of phosphate buffered saline (PBS, Invitrogen). The

additionalmembrane-stainingwas realizedwith 0.25mL CellMask

Deep Red directly added before studies on the CLSM. The treatment

with valinomycin (Aldrich; 1mmol L�1) was proceeded for 10min.

2.7. Flow Cytometry

To analyze the viability and the uptake of the NCs to the different

cell lines, flow cytometry experiments were performed in the

followingway. Firstly, the adherent cells were detached in trypsin

(Gibco) and seeded in FCS supplemented medium at a density of

20 000 (HeLa) or 10 000 cells cm�2 (MSC) in a 6-well plate (Greiner

BioOne). On the next day, the pMMA capsules were added at

different concentrations (from 75mg to 5000mgmL�1) to the

medium.The incubation timewas24h inahumidified incubator in

order to facilitate cell uptake (37 8C, 5% CO2). For analysis the cells

were washed with PBS, trypsinized, centrifuged (3min, 1500 rpm)

and stained with 28.6mgmL�1 7-aminoactinomycin (7-AAD) as

fluorescent apoptosis marker for 15min at room temperature,

before resuspending in PBS. The measurements were performed

with a CyFlowML using FlowMax 3.0 software (Partec, Germany).



The FL1 channel (lexc¼488nm, ldet¼ 527nm)was used to analyze

the uptake of NCs and FL6 channel (lexc¼ 561nm, ldet¼675nm)

for 7-AAD experiments. For the analysis, cells were selected on a

forward scatter/sideward scatter plot, thereby excluding cell

debris. These gated events were then further analyzed for the

two channels. This corresponds to the amount of NCs taken up or

associated with individual cells. For 7-AAD the events in cell gate

were analyzed on a FL1/FL6 dot-plot and three different

populations (viable, apoptotic, and dead) were determined by

using negative controls and the apoptotic and dead cells present in

thecell cultures.All valuesare triplicateswithastandarddeviation.

3. Results and Discussion

In general, capsule formation might occur if the polymer

and the dispersed liquid are immiscible and the interface

‘‘polymer/continuous phase’’ is more favorable than the

interface ‘‘dispersed liquid/continuous phase.’’[35] In the

case of fabrication of polymer NCs by the solvent

evaporation method,[36,37] an additional co-solvent pro-

vides compatibility of hydrophobic components before and

during the process of miniemulsification. Later this co-

solventhas tobe removedbyevaporation. Inorder toobtain

a stable polymeric shell, we chose pMMA. Non-volatile

organic solvents (1-phenyl heptadecane) were used as a

liquid core. All synthesized dispersions (none dialyzed)

were stable during months.

Figure 1a shows the TEM image of slightly deformed

NC633NCs.Aperfect core/shell structure is clearlyobserved

in the image. DLS measurements confirm a narrow and

uniform size distributionwith ahydrodynamic diameter of

dh¼ 225nm. Figure 1b shows a typical luminescence

spectrum (the red line) of NC633 NCs dispersed in water

at room temperature. Strong UC fluorescence of dye 550

withlmax¼ 550nmandresidualphosphorescenceofPdTBP

with lmax¼ 800nm are observed. For comparison, the

luminescence spectrum (the black line) for the same

sensitizer/emitter couple in toluene is presented as well.

It is important to mention, that the TTA-UC process in

bulk organic solvent is more efficient than in confined

environment. The substantial decrease of local mobility of

the interacting molecules species is likely the main reason

for the decrease of UC efficiency in the confined environ-

ment.[38,39] Additionally, we observed a decrease in the UC

intensity by a factor of 5, when measurement were done

under air conditions. Apparently, the 20nm pMMA shell of

the NCs is not able to protect TTA-UC process from oxygen

quenching. However, it will be shown further that the level

of theUC intensity is high enough to detectUCfluorescence

with CLSM in living MSC or HeLa cells.

The reliability of the TTA-UC process in the absence of

molecular oxygen is demonstrated in Figure 2. The

experiment underlines, that NCs were successfully loaded

with sensitizer and emitter dyes. When the UC NCs were

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Figure 1. a) TEM image of theNC633 NCs; b) UC spectra ofmixturePdTBP/dye 550 in toluene (black line, CPdTBP¼ 10�5mol L�1/Cdye550¼ 10�4mol L�1) and water dispersion of NC633 NCs (redline, 4% w/w of the dispersed phase, CPdTBP¼ 10�3mol L�1/Cdye550¼ 10�2mol L�1 in the dispersed phase (PHD)). Excitationlexc¼633 nm. Both samples are sealed in a glove-box withoxygen concentration below 2ppm. Inset: sketch of theprocesses leading to TTA-UC in NCs; c) Simplified diagram ofenergy levels explained TTA-UC in the fabricated NCs (indexes (S),(T), (ISC), (TTT), and (TTA) are related to singlet state, triplet state,intersystem crossing, triplet–triplet transfer, and triplet–tripletannihilation, correspondingly). It should be noted that twomolecules (triplet states) of emitter take part in the TTAprocess. One of the excited emitter triplet returns to theground state, but the other molecule accumulates the totalenergy of the two triplets and the excited singlet state ispopulated.

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C. Wohnhaas et al.


� 2013 WILEY-VCH Verlag GmbH1426

placedontopofamicroscope slideandsealed inoxygenfree

atmosphere, the CLSM images under direct (Figure 2a) and

UC photoexcitation (Figure 2b) reflected ideal co-localiza-

tion of the emitting species. In other words, 1:1 overlap of

the CLSM images is observed (Figure 2c).

The cell toxicity of the synthesized NCs was determined

in a standard test: after 24h of incubationwith theNCs, the

cell death rate was detected by staining with 7-amino-

actinomycin D (7-AAD). The amounts of living, apoptotic

and dead cells for different concentrations of NCs (100–

5000mgmL�1) are shown in Figure 3. The long incubation

timewas chosen in order to achieve amaximum loading of

the cells.

As evident from Figure 3, the toxicity of the NCs is

increased at higher NC concentrations. Please note, that in

UC experiments of other groups, the toxicity was deter-

mined for much lower nanoparticles concentrations

(well below 1000mgmL�1): for instance, water dispersions

of inorganic UC nanoparticles with a concentration of

50mgmL�1 and a diameter of d¼ 50nm,[40] or 200mgmL�1

and a diameter of d¼ 10nm,[41] or 10mgmL�1 and

a diameter of d¼ 28 nm[42] have been employed. The

TTA-UC NCs, presented in this work, are relatively large

(dh¼ 225nm), therefore the same number of NCs per

volume could be achieved only at highmass concentrations.

Therefore, inordertohaveacomparablemolarconcentration

ofUCNCs as inother invitro experiments[40–42] thevalues of

3000 and 5000mgmL�1 were chosen. The exact mechanism

of uptake (clathrin, caveolin, or other pathways like flotillin)

was not determined for the particles described here. The

uptake mechanism should not alter the optical properties.

Also, the pMMA NCs are not biodegradable and therefore

the exact cellular environment should not influence the

stability of the capsule on the discussed time scale.

Efficient UC luminescence in living cells was shown by

CLSM. Two cell types were incubated for 24h with NC633

NCs (concentration of 3000 or 5000mgmL�1). The dishes

with cell cultures in PBS (under air) were placed in a

microscope sample holder and the dyes were excited from

underneathwith light, passing through a LeicaHCX PL APO

CS 1.4/63� oil immersion objective. Figure 4 represents

CLSM images of a living MSC cell treated with UC NCs.

Figure 4a,c demonstrate the direct photoexcitation of the

dye 550with lexc¼ 488nm,whereas Figure 4b andd shows

photoexcitation in the UC regime with lexc¼ 633nm. The

two types of photoexcitation (direct and UC regime) are

necessary in order to identify possible artifacts. For a better

visualization, the cell membrane was stained with

CellMask Deep Red. As evident from Figures 4,5 the UC

NCs arewell observable inside of livingMSC andHeLa cells.

The experimental conditions for acquiring of the CLSM

images in UC regime deserve special attention: the cell

images, demonstrated in Figures 4b,d,5c, were collected by

using a cw HeNe laser (l¼ 633nm) and pixel dwell time

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Figure 2. CLSM images of NC633 NCs displaced on a top of a microscope slide: a) direct excitation (lexc¼488nm, ldet¼ 500–550nm); b) UCexcitation (lexc¼633 nm, lde t¼ 500–550nm); c) overlay of images a) and b).


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of 1.2ms. The sample was irradiated with a laser power

less than 0.3mW, consequently, the average light intensity

at the focal spot of the microscope was less than

100Wcm�2. In order to prove the detection of UC

fluorescence we tested NCs loaded only with the dye 550

(Figure S9, Supporting Information). HeLa cells incubated

with such NCs demonstrate bright fluorescence when the

laser with lexc¼ 488nm is used. In contrast, irradiation

with red light (lexc¼ 633nm) is not accompanied by the

Figure 3. Toxicity of the NC633 NCs for HeLa cells a) and MSCs b).



detection of any fluorescent photons within the detection

interval Dldet¼ 500–550nm.

The additional advantage of the UC excitation scheme

can be persuasively demonstrated: the required excitation

intensity is extremely low, therefore, not only the scattered

laser light is reduced, also the possibility of unwanted (not

controlled) nonlinear processes (such as two-photon

absorption) is completely avoided. Since the optical

registration region has a substantial hypsochromic shift

relative to the excitation wavelength, the cell autofluor-

escence is also entirely rejected. As a result, an order of

magnitude higher signal to background (S/B) ratio for the

cell images, demonstrated in Figure 4, is observed. For

Figure 4. CLSM images of living MSCs incubated with NC633 NCs(5 000mgmL�1): a) direct excitation (lexc¼488nm, Dldet¼ 500–550nm); b) UC excitation (lexc¼633 nm, Dldet¼ 500–550nm).CLSM images of living MSCs incubated with the NC633 NCs (3000mgmL�1): c) direct excitation (lexc¼488nm, Dldet¼ 500–550nm); d) UC excitation (lexc¼633 nm, Dldet¼ 500–550nm),cell membrane was additionally stained with CellMask Deep Red.

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Figure 5. CLSM images of living HeLa cells incubated with NC633 NCs (3000mgmL�1): a) bright-field transmission b) direct excitation(lexc¼ 488nm, Dldet¼ 500–550nm); c) UC excitation (lexc¼633nm, Dldet¼ 500–550nm).

Figure 6. CLSM images of living MSC incubated with the NC633NCs (3 000mgmL�1); a) bright-field transmission; b) directexcitation (lexc¼488nm, Dldet¼ 500–550nm); c) UC excitation(lexc¼633nm, Dldet¼ 500–550nm); d) UC excitation after 10mintreatment with valinomycin. Blue and orange ovals are given forbetter visualization of points of interest.

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C. Wohnhaas et al.

1428

instance, the value for the regime of direct excitation

(lexc¼ 488nm) is (S/B)direct¼ 4.5 and for the TTA-UC regime

(lexc¼ 633nm) is (S/B)UC¼ 40. Additionally, the extremely

mild conditions of optical excitation guaranty a low

collateral phototoxicity and a minor probability for cell

heating.

It is a well-known fact, that different cell compartments

might be characterized by different oxygen concentrations.

Therefore, when the concentration of O2 is high, the UC

fluorescence is quenched (since the polymer shell of NCs

cannotprovidea full oxygenprotection). Thus, theUCsignal

canbe considered as an additional tool for the investigation

of oxygen distribution within a living cell. It is clearly

observed (Figure 4c,d or Figure 5b,c) that there is no UC

fluorescence within the large spherical species strongly

loaded with the upconversion NCs (marked with

yellow arrows for better visualization). This might corre-

spond to a high local oxygen content. In the case of

NCs relatively homogenously distributed in the cytoplasm

the oxygen level is low enough to allow strong UC

fluorescence.

A direct proof of the hypothesis of the local oxygen

concentration impact is shown in Figure 6, where

valinomycin was applied as an oxygen concentration

regulator. Valinomycin is an ionophore that has been used

to increase the oxygen consumption by mitochondria of

HeLa cells. Thereby, a decrease of the local oxygen

concentration inside the cells is achieved.[43] A treatment

of 10min with valinomycin (1mmol L�1; Figure 6d) in

comparison to Figure 6c leads to an evident increase of

detected brightness and number of the detected UC NCs.

Since cell compartments demonstrate different pH

values, for instance, in case of cytoplasm (pH 7–7.4),[44]

late endosomes (pH 5.5–6), or lysosomes (pH 5–5.5),[45] we

investigated theUCfluorescence at physiological pHvalues

to exclude effect of pH on the UC intensity. A minor

dependence of the TTA-UC process in NC633 NCs on pH is

observed. Practically, the UC fluorescence signal stays



constant in the range of 1–11 of pH units (Figure S10,

Supporting Information).

In order to show the universal character of the method

developed here, we fabricated pMMA NCs with an UC dye

couple PdTNP/dye 555, dissolved in a liquid PHD core. DLS

measurements give the value of dh¼ 238nm. When the

capsules are irradiated by a laser with l¼ 708nm, the

strong UC fluorescence of dye 555 with lmax¼ 555nm is

observed (Figure 7). The residual phosphorescence of PdTNP

isdetectedatl> 900nm.Thus, theencapsulationof thedye

couple PdTNP/dye 555might be considered as further, very

promising imaging agent, because the excitation spectrum

perfectly fits the tissue transparency window and a low

3, 13, 1422–1430


Figure 7. a) TEM images of the NC708 NCs; b) UC spectra of water dispersion of NC708 NCs (4w/w% of the dispersed phase,CPdTNP¼ 1� 10�4mol L�1 and Cdye555¼ 1� 10�3mol L�1 in the dispersed phase (PHD)). Excitation l¼ 708 nm. The sample is sealed in aglove-box with oxygen concentration below 2 ppm. Inset: sketch of the processes leading to TTA-UC in NC708 NCs; c) Simplified diagram ofenergy levels explained TTA-UC in the fabricated NCs (indexes (S), (T), (ISC), (TTT), and (TTA) are related to singlet state, triplet state,intersystem crossing, triplet–triplet transfer, and triplet–triplet annihilation, correspondingly). It should be noted that two molecules(triplet states) of emitter take part in the TTA process. One of the excited emitter triplet returns to the ground state, but the other moleculeaccumulates the total energy of the two triplets and the excited singlet state is populated; CLSM images of fixed HeLa cell incubatedwith the NC708NCs (3 000mgmL�1): d) direct excitation (lexc¼ 510 nm,Dldet¼ 540–600nm); e) UC excitation (lexc¼ 708nm,Dldet¼ 540–600nm). Before CLSM imaging the sample was sealed in oxygen free atmosphere.


www.mbs-journal.de

autofluorescence under excitation with lexc � 700nm is

expected. To demonstrate theUCfluorescence of theNC708

NCs in cells, we designed a homebuilt CLSM microscope.

Spectrally separated output (lexc¼ 708nm) of a super-

continuum laser was used for excitation of the NCs. The

UC fluorescence (lmax¼ 555nm) inside fixed HeLa cells

was detected and results are presented in Figure 7e

(together with CLSM images obtained under direct excita-

tion (lexc¼ 510nm) of the dye 555 (Figure 7d).

4. Conclusion

In summary, we developed an universal methodology for

fabrication of polymer NCs with TTA-UC capability by a

combination of miniemulsion and solvent evaporation

techniques. The preparation method allows straightfor-

ward variation of the sensitizer/emitter UC couple. Such

NCs show low cell toxicity together with effective

internalization by different cell lines. Red excitation light



(lexc¼ 633nm)withextremely lowoptical power (<0.3mW

in CLSM) generates TTA-UC fluorescence with lmax at

550nm.We demonstrated that CLSM could be also used for

detection of UC excited by deep-red light (lexc¼ 708nm).

The cell images obtainedbya conventional CLSMtechnique

with an UC excitation regimes show order of magnitude

better quality than the cell images obtained in the direct

excitation of the same fluorescence marker. The simple

synthesis of the TTA-UC NCs, together with the high

reproducibility make them a reliable alternative for

imaging applications, local generation of blue shifted

photons for fluorescence resonance energy transfer assays

or multicolor targeting of cancer cells.

Acknowledgements: C.W. and V. M. contributed equally to thiswork. A.T. acknowledges the EU-founded FP-7 project EphoCell(N 227127) for the financial support, and S.B. acknowledges theReintegration Grant RG-09-0002(DRG-02/2) Bulgarian NationalScience Fund for the financial support.

13, 13, 1422–1430


www.mbs-journal.de

C. Wohnhaas et al.

1430

Received: March 21, 2013; Revised: June 6, 2013; Published online:July 19, 2013; DOI: 10.1002/mabi.201300149

Keywords: confocal laser scanning microscopy; living cell imag-ing; polymer nanocapsules; triplet–triplet annihilationupconversion

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