Profluorescent PPV-Based Micellar System as a Versatile Probe for

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Profluorescent PPV-based Micellar System as a Versatile Probe for Bioimaging and Drug Delivery Neomy Zaquen, Hongxu Lu, Teddy Chang, Russel Mamdooh, Laurence Lutsen, Dirk Vanderzande, Martina Heide Stenzel, and Thomas Junkers Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01653 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016

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Profluorescent PPV-based Micellar System as a Versatile Probe for Bioimaging and Drug Delivery Neomy Zaquen†, Hongxu Lu§, Teddy Chang§, Russel Mamdooh§, Laurence Lutsen⊥, Dirk Vanderzande⊥†, Martina Stenzel§,* Thomas Junkers†⊥*. † Institute for Materials Research, Hasselt University, Martelarenlaan 42, 3500 Hasselt, Belgium. § Center for Advanced Macromolecular Design (CAMD), School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia. ⊥ Imec associated lab IMOMEC, Wetenschapspark 1, 3590 Diepenbeek, Belgium. e-mail: [email protected], [email protected]

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ABSTRACT

Although micelles are commonly used for drug delivery purposes, their long-term fate is often unknown due to photo-bleaching of the fluorescent labels or the use of toxic materials. Here, we present a metal-free non-toxic non-bleaching fluorescent micelle that can address these shortcomings. A simple, yet versatile profluorescent micellar system – built from amphiphilic poly(p-phenylene vinylene) (PPV) block copolymers – for use in drug delivery applications is introduced. Polymer micelles made from PPV show excellent stability for up to 1 year and are successfully loaded with anti-cancer drugs (Curcumin or Doxorubicin) without requiring introduction of physical or chemical crosslinks. The micelles are taken up efficiently by the cells, which triggers disassembly, releasing the encapsulated material. Disassembly of the micelles and drug release is conveniently monitored as fluorescence of the single polymer chains appear, which enables not only to monitor the release of the payload, but in principle also the fate of the polymer over longer periods of time.

KEYWORDS Conjugated Polymers, Fluorescent Micelles, PPV, Bioimaging, Drug Delivery


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INTRODUCTION Synthesis and characterization towards conjugated polymers has seen significant improvement over the last years, leading to a variety of complex polymer materials of different chemical nature and architecture.1;2 The most significant area of application is organic electronics3-7 – due to their usually excellent optoelectronic characteristics – with poly(p-phenylene vinylene)s (PPV)s as one of the best studied type of material.8-10 Although mostly replaced nowadays by other polymers in the field of organic electronics,11,12 PPVs still show excellent fluorescent properties, high reproducibility and non-toxic behavior, making them ideal candidates for bioimaging and drug delivery applications.13-15 Over the years, many synthesis routes were developed towards tailor-made PPVs, with the so-called sulfinyl precursor route as one of the most versatile pathways to obtain conjugated PPVs in a two-step mechanism.16-18 Upon carefully chosen reaction conditions, a fully anionic (and hence living) mechanism can be accessed with the sulfinyl route.19 Combined with the use of dual initiator approaches, well-defined PPV block copolymers of various types can be synthesized. The alpha-end group of the initiator is easily functionalized, and the combination with single electron transfer living radical polymerization (SET-LRP)20-23 chain extensions has proven to be a very promising synthesis pathway.24,25 Depending on the chemical structure of the comonomers, amphiphilic block copolymers that form micellar structures may be obtained. Also ring-opening metathesis polymerization (ROMP) can in principle be used to synthesize conjugated PPV block copolymers, requiring however significantly more synthetic effort.26;27 The dual initiator approach is in contrast more versatile and simple to carry out, creating hence a universal platform for the synthesis of PPV block copolymers. To date, the technique has been applied for hydrophobic materials, and while a proof of principle for amphiphilic materials was given before, this route hasn’t been explored yet in depth, especially with regards to micellar self-assembly of such block copolymers.


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In recent decades, tremendous efforts have been taken to improve current fluorescent-based materials in the biomedical field.28-30 Great advances have been made towards the development of imaging tools upon using inorganic semiconductor nanoparticles (NPs) – better known as quantum dots (QDs) – or the use of fluorescent dyes. Although promising at first, the former one still shows signs of cytotoxicity – despite the numerous efforts to reduce this by either encapsulating the QDs or by modifying the surface – while fluorescent dyes show signs of bleaching over time. 31-37 Hence, conjugated polymers seem like a promising candidate for fluorescent nanoprobes, as they show excellent optical properties, no signs of bleaching and there is a variety of nontoxic materials available.38-44 Surprisingly, most of the research is focused on using conjugated polymers as core-shell particle or as encapsulate, but not as self-assembling polymer material itself.45;46 Hence, conjugated polymers are in this respect employed in the same way as quantum dots, while the materials in principle offer many more advantages. Direct incorporation of the conjugated polymers into the polymer amphiphile has the advantage that fluorescent behavior and micelle stability can be directly linked to each other. Further, little is known about the fate of micelles over a longer period of time in many drug delivery applications, as well as their pathway and disassembly mechanism in the cells. Inherently fluorescent polymers enable to trace these materials also after disassembly and can hence be used in principle to study if these materials are efficiently removed from the body or may accumulate. In addition, many classical micellar systems selfassemble spontaneously, yet do not show very high stability with payload encapsulation, hence requiring physical crosslinking of one block segment in order to create a drug carrier.47,48 These crosslinks must then be opened upon an exogenous trigger to open the micelles and to release the payload. An intrinsically metal-free profluorescent stable micellar system would hence be highly favorable to combine the excellent properties of polymeric


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micelles for drug delivery, while the drug carrier can be monitored over an extended period of time without requiring complex core-shell particles to protect the potentially toxic payload.

Figure 1: Schematic overview of the π-π stacking of the PPV-b-P(EGMA) block copolymer. Spontaneous self-assembly of the amphiphilic block copolymers in water leads to the process of micelle formation, in which the hydrophobic PPV is situated at the core of the micelle (blue) and the hydrophilic P(EGMA) at the outside (pink).

In this study, profluorescent non-crosslinked micelles are synthesized based on amphiphilic PPV block copolymers, which spontaneously decompose and release their payload after cell uptake. This is to the best of our knowledge the first time that such PPV-based micelles are used for this purpose. As the micelles are composed of conjugated polymers, no fluorophores are needed to enable cell uptake or drug release visualization. Also, the conjugated segments allow to form highly stable micelles via pi-stacking,49,50 removing the need to introduce chemical crosslinks. To realize this, amphiphilic PPV block copolymers were for the first time synthesized using SET-LRP using either ethylene glycol methyl ether methacrylate (EGMA), 2-hydroxyethyl acrylate (HEA) or 2-hydroxypropyl methacrylate (HPMA) as comonomer, to yield PPV-b-P(EGMA), PPV-b-P(HEA) or PPV-b-P(HPMA) block copolymers respectively. Next, polymer micelles are generated of which the morphological and optical properties are investigated. Although high fluorescence is observed for the free block copolymers, practically all fluorescence is quenched upon self-assembly, making the payload carriers profluorescent. Laser scanning confocal microscopy tracing of the materials hence does not simply allow to monitor where the micelles are located in a specific system, but allow to trace the decomposition and drug release itself. Micelles were loaded with Curcumin


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or Doxorubicin as model drugs and tested towards stability, morphology, optical properties and drug release.


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EXPERIMENTAL

Self-assembly of PPV Block Copolymers 2 mg of block copolymer was dissolved in 0.4 mL of DMF. The solution was placed on a stir plate with a high stirring rate at room temperature. Deionized water (3.6 mL) was added dropwise to the solution with a flow rate of 0.2 mL·h-1, leading to a total polymer concentration of 0.5 mg·mL-1. Afterwards the solution was placed in a dialysis membrane with pore size Mw < 3 500 g·mol-1 and dialyzed against deionized water for 48 hrs.

Encapsulation of Material into PPV Micelles 2 mg of block copolymer and 1 mL of stock solution (concentration = 1 mg·mL-1 in DMF) of the encapsulated material (Nile Red, Curcumin or Doxorubicin) was dissolved in in 0.4 mL of DMF. The solution was placed on a stir plate with a high stirring rate at room temperature. Deionized water (3.6 mL) was added dropwise to the solution with a flow rate of 0.2 mL·h-1, leading to a total polymer concentration of 0.4 mg·mL-1. Afterwards the solution was placed in a dialysis membrane with pore size Mw < 3 500 g·mol-1 and dialyzed against deionized water for 48 hrs.

Critical Micelle Concentration (CMC) The CMC was measured by fluorescence spectroscopy using pyrene as a fluorescent probe.51 Briefly, a stock solution of pyrene was made by dissolving pyrene (1 mg, 5 µmol) in acetone (200 mL) to form a 2.5 × 10−5 M solution. The pyrene solution (48 µL) was dropped into empty vials, and the acetone was evaporated overnight under reduced pressure. A stock solution with either empty or loaded micelles was serially diluted with deionized water starting with concentrations of 0−100 µg/mL. Each polymer solution (2 mL) was transferred


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to a vial containing pyrene and stirred overnight. The final concentration of pyrene in the polymer solutions was 6 × 10−7 M (which is less than the pyrene saturation concentration in water).52 Fluorescence measurements were carried out using an excitation wavelength of λ = 237.96 nm, using a 2.5 nm slit width for excitation and a 2.5 nm slit width for emission. Emission wavelengths were scanned from 350 to 450 nm. The intensity of the I1 (372 nm) and I3 (383 nm) vibronic bands was evaluated for each sample, and the ratio of these values were plotted against the concentration of each sample.53 The CMC was determined at the point where the ratio of I1/I3 starts to increase in the graphs.

Micellar internalization observed with laser scanning confocal microscopy (LSCM) AsPC-1 cells were seeded in 35 mm Fluoro-dishes (0.5 × 105 cells per dish) and incubated for 3 days at 37 °C and 5 % CO2. The micelles were sterilised by passing through a sterile 0.45 µm membrane and loaded to the cells at a concentration of 100 µg/mL. After incubation for 2 h and 18 h, the cells were washed with Hanks' balanced salt solution (HBSS) thrice and stained with 100 nM LysoTracker Red DND-99 (Thermo Fisher Scientific, Australia) for 5 min. After rinsed with HBSS once, the cells were mounted in 1 mL HBSS and observed under a LSM780 laser scanning confocal microscope (Carl Zeiss). Excitation of the micelles was done at 500 nm (PPV), 418 nm (PPV-b-P(EGMA)), 466 nm (PPV-b-P(HPMA)) and 438 nm (PPV-b-P(HEA)) respectively with an Argon ion laser. Emission was detected using a band-pass filter of 565 – 615 nm. An incubation chamber was equipped on the LSM780 to provide the cells with an environment of 5 % CO2 and 37 °C. The observation used a 100 × oil lense (1.4 N.A.), a Diode 405-30 and an argon laser. ZEN2012 software (Zeiss) was used for image acquisition and processing.


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Time lapse observation with laser scanning confocal microscopy (LSCM) AsPC-1 cells were seeded in a 35 mm Fluoro-dishes (0.5 × 105 cells per dish) and incubated overnight. According to the manufacture’s instruction, 15 µL of BacMam 2.0 CellLight® Lysosomes-GFP (Thermo Fisher Scientific, Australia) was added to the dish (approximately 30 particles per cell). The cells were then incubated at 37 °C and 5 % CO2 for another 2 days before treatment with micelles. The micelles were sterilised by passing through a sterile 0.45 µm membrane and loaded to the cells at a concentration of 100 µg/mL. After incubation with micelles for 2h, the cells were washed with Hanks' balanced salt solution (HBSS) thrice and 1 mL phenol-red free RPMI 1640 (supplemented with 10% FBS) was added in the dish. The cells were then observed under a LSM780 laser scanning confocal microscope (Carl Zeiss) equipped with an incubation chamber providing an environment of 37 °C and 5 % CO2. The observation used a 100 × oil lense (1.4 N.A.), a Diode 405-30 and an argon laser. The images were taken every 10 min for duration of 16 h. ZEN2012 software (Zeiss) was used for image acquisition and processing.

Cellular uptake of the micelles with fluoro spectrometry AsPC-1 cells were seeded in 24 well tissue culture plates at a density of 1.5 × 105 cells per well and incubated for 2 days at 37 °C and 5 % CO2. The micelles were diluted with MilliQ water to 100 µg/mL and sterilised by passing through a sterile 0.45 µm membrane. 500 µL micelles were loaded to each well together with 500 µL 2 × concentrated cell culture medium (3 wells per time point). After incubation for 2 h and 18 h, the cell culture media were collected and freeze-dried. 1 mL dimethylformamide (DMF) was added to the lyophilised powder to dissolve the polymer. The mixture was sonicated for 30 min, shaken for 1h, and filtered through 0.45 µm membranes to remove the precipitations. The fluorescence intensity (FI) was measured with a Cary Eclipse fluorescence spectrophotometer (Agilent). The


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absorption and emission were 416 and 507 nm, respectively. The reading was zeroed with fresh cell culture medium. The micelles mixed with 2 × medium (1:1) was used as the control. The uptake ratio was calculated with the following equation: Uptake ratio (%) =

× 100 %.


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RESULTS AND DISCUSSION

Amphiphilic PPV block copolymers Over the past years a variety of PPV monomers have been applied via the so-called sulfinyl precursor route. Amongst them, 1-chloromethyl-2-methoxy-5-(3,7-dimethyloctyloxy)-4[(octylsulfinyl)methyl] benzene (MDMO) is one of the most commonly used PPV monomer. The sulfinyl precursor route allows to choose in a facile manner between a radical and anionic polymerization mode, simply by changing the solvent and the base that is employed to create the active quinodimethane monomer out of the precursor monomer, Full mechanistic studies towards the polymerization of MDMO-PPV via both the radical as well as anionic route has led to the development of advanced controlled synthesis techniques.19 Best conditions towards living polymerizations were established via the anionic polymerization route. By using a bromine-functionalized anionic initiator, reinitiation of polymerization could be achieved in a second step using single electron transfer living radical polymerization (SET-LRP). 24;25 In this way, complex PPV architectures of different nature became accessible for the first time. In here, we focus on this concept by demonstrating how the technique can be used to synthesize a variety of amphiphilic PPV-containing block copolymers, and their potential application in drug delivery studies. Despite the large synthetic efforts invested in the design of conjugated polymer materials in the recent years, surprisingly such route has not yet been followed before.

Figure 2: Chemical structure of amphiphilic PPV block copolymers used in this work: PPV-b-P(EGMA) (left), PPV-bP(HEA) (middle), PPV-b-PHMA (right).


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Amphiphilic block copolymers with either ethylene glycol methyl ether methacrylate (EGMA), 2-hydroxyethyl acrylate (HEA) or 2-hydroxypropyl methacrylate (HPMA) as comonomer to yield PPV-b-P(EGMA), PPV-b-P(HEA) or PPV-b-P(HPMA) block copolymers, respectively have been synthesized. All block copolymers were characterized with respect to their chain length, optical properties (emission and absorption) as well as quantum yield (φF). (see Table 1 and Figure 2). Table 1: Overview of the different PPV block copolymers synthesized and their characteristics. Mntheory

Mnapp, 1

Mwapp, 1

λmax,abs2

λmax,em3

φF3

g·mol-1

g·mol-1

g·mol-1

nm

nm

%

PPV

5 000

5 100

7 400

1.45

494

584

26.48

PPV-b-P(EGMA)

10 000

9 500

12 400

1.30

418

506

20.56

PPV

5 000

6 400

9 000

1.40

494

584

26.48

PPV-b-P(HEA)

10 000

16 100

22 900

1.40

438

512

19.46

PPV

2 500

2 500

3 800

1.50

494

584

26.48

PPV-b-P(HPMA)

5 000

5 100

6 100

1.20

466

546

21.19

Ð

1

Size exclusion chromatography was used to determine molecular weights of the (block) copolymers. λmax,abs was determined using DMF as solvent. 3 λmax,em and φF were determined using fluorescent spectroscopy and DMF as solvent. 2

The appearance of the C=O vibration band of the acrylic monomers around 1700 cm-1 (Figures S2, S6, S10) in the infrared spectra and the characteristic signal of the CH2COO signal at 3.5 ppm in the 1H NMR spectrum clearly indicate the successful block copolymer formation (Figures S4, S8, S12). In addition, SEC traces show clear shifts towards higher molecular weights for all block copolymers (Figures S1, S5, S9). However, not all theoretical and number average molecular weights (Mn) match up. The main reason is that no full kinetic study was performed for each individual block copolymer, thus conversion values were assumed to be in a range similar for all three second blocks, explaining the offset of theoretical molecular weights. In addition, a blue shift in λmax in the UV-Vis absorption and


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the fluorescence emission spectrum is observed after block copolymerization, indicating a significant effect of the non-conjugated block on the photophysical properties of the PPV block (Figures S3, S7, S11). Possible explanations for this blue shift are that the flexible second block (acrylate, methacrylate or acrylamide in this case) is wrapped around the more rod-like conjugated chain, hence influencing the electronic properties.54 Although influenced, the absorbance / emission wavelengths are still within the range of most commonly used fluorescent dyes. Sufficient quantum yields are obtained for the different PPV block copolymers (taking the high extinction coefficients in mind) and the typical PPV characteristics are mostly retained (Table 1).55;56

Non-loaded PPV micelles The different block copolymers were self-assembled in DMF/water followed by dialysis, see Figure 1, leading to spontaneous micelle formation. Table 2: Overview of different PPV micelles synthesized and their characteristics. Solid content

Addition speed

DLS1 nm

mg·mL-1

mL·h-1

Imean

Vmean

Nmean

TEM nm

λmax 2 nm

Zeta1 potential

PDI

D50

Abs

Em

mV

PPV-b-P(EGMA) 0.5

0.2

119.3

109.2

94.5

0.197

25 ± 5

361

464

-28.9

0.5

0.1

127.8

108.6

82.6

0.096

15 ± 5

361

470

-33.0

183.5

182.5

156.7

0.337

30± 15

389

491

-35.7

346.4

341.7

158.3

0.363

50 ± 5

421

495

-38.7

PPV-b-P(HEA) 0.5

0.2

PPV-b-P(HPMA) 0.5

0.2

1

measurements are performed in water as solvent. 2 measurements are performed in water (90 v/v%) / DMF (10 v/v %) as solvent.

Size distributions of the formed micelles were analyzed by means of dynamic light scattering (DLS) (Table 2, Figures S14, S15, S22, S27). With increasing hydrophilicity, an increasing particle diameter and dispersity (PDI) is observed, leading to observation of aggregates of


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micelles with an average size between 94 nm and 160 nm respectively. As (HEA) and (HPMA) are both capable of forming hydrogen bonds, the resulting micelles will be associated with a somewhat larger diameter as compared to using (EGMA), which behavior is clearly confirmed by the data in Table 2. In addition, size distributions and morphology of the formed micelles were analyzed by means of transmission electron microscopy (TEM) (Table 2, Figure S13, S21, S26). All TEM samples were stained in an equal way to allow for good comparison between the results from the different systems. Average diameters measured by TEM are significantly smaller than those obtained by DLS, which may be explained by aggregation of micelles in water due to hydrodynamic effects, especially with the extremely hydrophilic P(HEA) or P(HPMA) polymers. DLS measurements were performed far above the critical micelle concentration (for CMC see Figure 4) leading to discrepancies between the particle size as observed with DLS and TEM analysis. Lower micelle concentrations should yield DLS results closer to the TEM diameters measured, however, no reliable measurement could be achiveed for such conditions. However, similar trend lines in TEM as DLS were observed – increasing hydrophilicity leads to an increasing particle diameter and dispersity –

resulting in spherical micelles with an average size

between 15 nm and 50 nm respectively (Figure 3). This micelle sizes are also more in line with expectations regarding the molecular weight of the clock copolymers used.

20

Intensity Mean

18 16 14

Intensity / %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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12 10 8 6 4 2 0 0,1

1

10

100

1000

10000

100000

Size / nm

Figure 3: Visualization of size and morphology or PPV-b-P(EGMA) micelles using TEM (left); Hydrodynamic diameter (intensity) measured by DLS of PPV-b-P(EGMA) micelles using a flow rate of 0.2 mL·h-1 (right).


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Next, a closer look at the optical properties – absorbance and emission – of the micelles was taken. Surprisingly at first, the fluorescence of the conjugated block is completely quenched upon micellization in water (Figure S16, S23 and S28), most likely as an effect from stacking of the PPV segments and the formation of supramolecular aggregates. Partly dissolving the micelles into an organic solvent (e.g. DMF) lets the fluorescence of the conjugated polymer (Table 2) reappear while mostly maintaining the morphology of the micelle. The loss of fluorescence is even more pronounced in water as compared to organic solvents and the progressive loss of fluorescence can be observed during micelle formation when organic solvent is partly replaced by water, which promotes also aggregation of the single micelles. In these supramolecular structures the conjugated parts are clustered together in the center of the aggregate, leading to quenched fluorescence of the micelles. Accompanied with the formation of these aggregates is an increase in average size. This behavior is clearly observed by DLS measurements as depicted above. The discrepancy between the TEM – in which the average diameter of a single micelle is measured – and DLS measurements – in which the average size of the supramolecular micelles is measured – can hence be reasoned. Similar behavior was observed for other conjugated polymers in literature as well.57;58

1,4

100

PPV-b-PHPMA PPV-b-PHEA PPV-b-PEGMA

Viability ( % of Control)

1,2 1,0

I1 / I3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0,8 0,6 0,4 0,2 0,0

0,5

1,0

1,5

2,0

2,5

80 60 40 20 0 -1

0

-1

Concentration / µgmL

1

2

3

-1

log ([C]) / µgmL

Figure 4: Determination of the CMC for PPV-b-P(HPMA) (black), PPV-b-P(HEA) (red) and PPV-b-P(EGMA) (blue) by measuring the intensity of the first (I1) and third (I3) vibrational bands of pyrene using fluorescence spectroscopy. Data are presented as the ratio of I1/I3 versus concentration (left); Cell viability of AsPC-1 cells exposed to different concentrations or PPV-b-P(EGMA) micelles for 72 h of exposure determined by the SRB assay (right).


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In the next step, all micelles formed were subjected to a cytotoxicity study (SRB or WST-1 assay). It should be noted that the cellular uptake is dependent on the stability of the micelle. Disassembly of the micelle can prevent the uptake by endocytosis and will also affect the rate of exocytosis.59 Therefore, the stability of the block copolymers was investigated using fluorescence spectroscopy revealing critical micelle concentrations (CMC) of around 1 µg·mL-1 (Figure 4). The proliferation of human pancreatic cancer cell line AsPC-1 upon contact with the micelles was investigated at different concentrations over a period of 72 h (S17, S24, S29). Even at high concentrations of 250 µg·ml-1 (well above the CMC of the micelles), the PPV-b-P(EGMA) micelles showed no sign of significant cytotoxicity as compared to cells exposed to normal growth conditions (Figure 4). Similar results were obtained for PPV-b-P(HEA) micelles, however PPV-b-P(HPMA) micelles did show slight toxicity, leading to an IC50 value of 0.599 µM. The latter is most likely related to insufficient purification of the copper residuals after block copolymer synthesis rather than due to the (HPMA) block, as both PPV as well as (HPMA) separately display no toxicity.


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Micelles

Lysosome

DIC

Merged

Figure 5: Cellular uptake measured by confocal fluorescence microscope. Images of the PPV-b-P(EGMA) micelles in pure water using AsPC-1 cell lines. The micelles were counterstained with lysotracker Red DND-99 upon 2 h of incubation (a d) or 18 h of incubation (e-h). Scale bar represents 10 µm; micelles with a 100x magnification are shown.

Yet, all three micellar systems were subjected to cell uptake studies. AsPC-1 cell lines were incubated for 72 h, after which the micelles were loaded into the cells and incubated for 2 h. Although the fluorescence of the micelles was quenched in water, upon cell uptake into the cytosols the micelles become visible again and the amount of micelles taken up by the cells is directly linked to the appearing fluorescence intensity. This actually means that for the first time, a profluorescent system is created which allows visualization of the fate of the micelles over a longer period of time, as well as their pathway and disassembly mechanism in the cells. Consequently, accumulation of the material over time can easily be followed as compared to currently used metal-containing systems as a differentiation can be made between the loaded particles and the free block copolymers. The absence of photobleaching allows for following materials over a longer period of time, even if in-vivo observation is mostly limited by the UV absorbance of the polymer. Fluorescence of the micelles is also seen overlapping with the lysosomes, which is an indication that the micelles entered the lysosomes after endocytosis. The larger PPV-b-P(EGMA) micelles show an easier and better


17

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uptake as compared to the smaller ones, which is in line with previous studies, indicating best cell uptake by particles with a size of 40 – 50 nm (Figure S18, S19).60 PPV-b-P(HEA) and PPV-b-P(HPMA) micelles are in a similar size range and thus should show similar uptake as the large PPV-b-P(EGMA) micelles (Figure S25, S30). The slower uptake of these micelles might be related to the design – hydrophobic / hydrophilic part – of the block copolymer61 Yet, fluorescent polymer is present in all cases indicating the uptake and subsequent break down of the micelles in the cells.

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20

10

0 2h

18 h

Figure 6: Uptake ratios of PPV-b-P(EGMA) micelles by AsPC-1 cells after incubation for 2h and 18h. Data represent means ± standard deviation, n =3. *, significant difference, P ˂ 0.05 revealed by a two-tailed unpaired t-test. Measurements were performed in water and an uptake value of 10.7 % after 2 h (blue) and 18.7 % after 18 h (red) was observed.

PPV-b-P(EGMA) micelles were subjected to additional cellular uptake tests in which the incubation time was extended from 2 h to 18 h and a colored lysotracker (LysoTracker Red DND-99) was used to counterstain the lysosomes (Figure 5; A CellLight® lysosome-GFP was also used to stain the lysosomes, the results were shown in Figure S20). After incubation with micelles for 18 h, more micelles were internalized into the cells – 18.7 % after 18 h as compared to 10.7 % for an incubation time of 2 h – and a full transport of the micelles from the endosomes into the lysosomes is seen (Figure 6). The higher fluorescence intensity over time indicates that more micelles were taken up and disassembled within the cell. Even after


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18 h most of the micelles are still present in the lysosomes, showing living stable cells. A slight increase in the size of the cells as compared to the size of the lysosomes is indicating that some micelles or block copolymers are released from the lysosomes. Various trials to mimic cell conditions on lab scale – surrounding the micelles in an acidified PBS solution for up to 24 h – did not lead to the desired disassembly and hence fluorescence signal as observed when the micelles are taken up by the cell. Alternatively, it may be speculated that enzymatic processes could trigger the disassembly and thus stability of the micelles once taken up by the cell. A closer look into the exact mechanistic pathway as well as disassembly of the micelles upon cell uptake are currently underway as a follow-up study.

In-situ loading of the PPV micelles After the general non-toxicity of the micelles (and their block copolymers) was proven and their long term fate over 18 h in the cells was shown, the next step was to test the micelles for release of a payload within the cells (note that at this stage the exact entry mechanism and reason for micelle breakdown is not fully clear, but irrespective for the herein described application). Therefore, the different PPV micelles were successfully loaded (in-situ) with a fluorescent drug (Curcumin (Cur) or Doxorubicin (Dox)) allowing both the monitoring of the drug carrier as well as release and disassembly at a later stage (Table 3).

Table 3. Overview of different PPV micelles and their loading (Nile Red (NR), Curcumin (Cur) or Doxorubicin (Dox)) synthesized and their characteristics. Encapsulation mg PPV-b-P(EGMA)

DLS nm

λmax1 nm

TEM nm

Zeta potential

η2

Imean

Vmean

Nmean

PDI

D50

Abs

Em

mV

wt%

119.3

109.2

94.5

0,197

22±2.0

357

466

-28.9

0

NR

0.5

300.7

450.4

116

0.335

n.a.

543

650

-37.3

28.9

Cur

0.5

672.4

812.3

109.4

0.485

30±2.5

428

538

-43.2

27.8

Dox

0.5

260.5

262.2

259.2

0.943

30±2.5

480

593

-35.6

9.1

PPV-b-P(HEA)

183.5

182.5

156.7

0.337

30±1.5

389

491

-25.3

0


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NR

0.5

148.5

149.1

147.5

1

n.a.

543

625

-32.9

36.3

Cur

0.5

163.3

163.9

161.4

0.985

n.a.

428

525

-28.0

28.4

Dox

0.5

399.3

404.9

390.7

0.765

n.a.

480

575

-27.9

14.9

364.4

341.7

128.3

0.363

50±5

421

495

-38.7

0

PPV-b-P(HPMA) NR

0.5

120.5

120.7

119.4

1

n.a.

543

650

-32.8

25.5

Cur

0.5

71.38

71.31

70.26

0.874

n.a.

428

515

-22.4

11.1

Dox

0.5

700.3

714.3

699.4

0.743

n.a.

480

575

-1.58

10.1

1

measurements are performed in water (90v/v%) / DMF (10 v/v%). 2 loading of the micelles was determined using UV-Vis analysis. Measurements are performed in water (90v/v%) / DMF (10 v/v%).

UV-Vis and fluorescent analysis indicate the successful loading of the materials in the different PPV micelles (Table 3, Figure S33, S38, S40). The expected absorbance and emission values of the loading (NR, Cur, Dox) was clearly observed at all three micellar systems. Next, UV-Vis analysis was used to determine the loading efficiency (η) of the individual micelles for each of the different loadings. Table 3 clearly shows that the synthesized micelles can be used as drug delivery vesicle. Without any chemical of physical crosslinks, loading efficiencies of up to 36.3 wt% – depending on the type of loading and micellar system – are observed. In addition, zeta potential measurements show the expected negative value, due to the net positive charge of the (meth)acrylates / acrylamide situated at the surface. Upon loading the micelles, a more negative value is obtained in most cases, indicating an increased stability of the micelle upon loading with encapsulate. In case of PPV-b-P(HPMA) micelles loaded with Doxorubicin however, the zeta potential seems to increase to a value of -1.58 mV as compared to the much lower value of -38.7 mV for the non-loaded micelles. This is most likely related to incomplete micelle formation, as precipitate was present in the vial after micelle synthesis (Figure S32, S37, S39). Loading of the PPV-b-P(EGMA) micelles was studied in more detail.


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Nile Red Curcumin Doxorubicin

2.0x10-4

[C] / molL-1

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1.5x10

-4

1.0x10

-4

λmax,em PPV

λmax,em Doxorubicin

non-loaded micelles loaded micelles

loaded micelles

5.0x10-5

0.0 0

1000

2000

3000

400

450

t / min.

500

550

600

650

700

Wavelength / nm

Figure 7: Release of loaded dye (Nile Red, black) or drugs (Curcumin (red) or Doxorubicin (blue)) using PPV-b-P(EGMA) micelles (prepared a flow rate of 0.2 mL·h-1) as measured by UV-Vis in water (90v/v%) / DMF (10 v/v%) as solvent (left); Fluorescence of PPV-b-P(EGMA) micelles in water (90v/v%) / DMF (10 v/v%) (black) non loaded λmax,em = 443 nm, (red) loaded with Doxorubicin λmax,em = 443 nm, (blue) loaded with Doxorubicin λmax,em = 505 nm (right).

Drug release studies on loaded PPV-b-P(EGMA) micelles performed via UV-Vis indicate the expected release of the different payloads within the first hour (Figure 7). Even when dissolved in water (90v/v%) / DMF (10 v/v%) and excited at both the PPV-b-P(EGMA) as well as the Dox wavelength, both materials become visible in the fluorescence spectra (Figure 7), confirming the successful loading of the material.

Figure 8: Visualization of size and morphology or Doxorubicin loaded PPV-b-P(EGMA) micelles using TEM (left) and cell viability of AsPC-1 cells exposed to different concentrations of Doxorubicin loaded PPV-b-P(EGMA) micelles for 72 h of exposure determined by the SBR assay. An IC50 value of 1.51µM was observed (right).

Next, size and morphology was confirmed by TEM measurements (Figure 8, Figure S31). Spherical micelles with increased particle size from 22 nm to 30 nm were obtained when


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using Cur or Dox as payload. Although non-toxic by nature, once loaded with Cur or Dox, the micelles show the expected toxicity with a IC50 value of 1.18 µM or 1.51 µM respectively (Figure 8, S34) and indicate the successful loading of drug into the PPV-b-P(EGMA) micelles. As a last step, cell uptake by means of confocal microscopy on the Dox loaded micelles was performed (Cur could not be used in this case, as the emission of Cur overlaps with the emission of the PPV micelles – this in comparison to the use of Dox). Incubation of the micelles for 2 h (using AsPC-1 cell lines incubated for 72 h before micelle loading) shows a clear uptake of the loaded micelles in the cell and subsequent release of the payload is confirmed by confocal microscopy results (Figure S35, S36).

Figure 9: Dox-encapsulated micelles (blue) were taken up by the cells and located in the lysosomes after 2 hr. Green area partially separated from the blue dots indicates that Dox was released from the micelles. Scale bar represents 50 µm.

The micelles are able to internalize into the lysosomes and spontaneously release the drug upon cell uptake, without the need for an exogenous trigger. After 2 h of incubation, some of the cells are starting to decompose, clearly confirming the release of the toxic Dox into the cells. A new micellar system without the use of crosslinks to stabilize the micelles, a trigger to break them down or to release the payload has been developed, opening new possibilities for the use of conjugated polymers in biomedical applications. CONCLUSIONS


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In conclusion, a unique profluorescent non-crosslinked nanosized micellar system to be used for drug delivery applications has been synthesized. The fate of the micelles, as well as their transport, disassembly and payload release in the cell can directly be visualized for the first time by high-contrast fluorescent imaging methods. In addition, an easy way to load the micelles – and their subsequent release without the use of a trigger – indicates the versatility of these materials. As a result, a novel pathway for an intrinsically fluorescent stable micellar system of highly added value for targeting concomitant bioimaging and drug delivery has been developed. Current work on the uptake and disassembly mechanism of the micelles into the cells and the use of different PPV monomers as starting component is ongoing in our laboratories and will be reported in due course.

ASSOCIATED CONTENT

Supporting Information. Experimental procedures and reaction schemes for the polymer synthesis, SEC, FT-IR, UV-Vis and NMR data of all polymers, DLS and TEM of micelles, cytotoxicity analysis of polymers and cellular uptake measured by fluorescence microscopy. This material is available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION

Corresponding Author * E-mail: [email protected] * E-mail: [email protected]


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources The Authors thank the Agency for Innovation by Science and Technology in Flanders (IWT) and Fund for Scientific Research (FWO) for funding.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT All authors are grateful for funding from the Belgian Science Policy (BELSPO) in the framework of the Inter University Attraction Pole program P7/05 - Functional Supramolecular Systems (FS2). N. Z. is grateful for the funding from the ‘Agency for Innovation by Science and Technology’ in Flanders (IWT) and for a travel grant from the ‘Fund for Scientific Research” Flanders (FWO). T. J. is grateful for funding from the FWO in the framework of the Odysseus scheme. Furthermore, preliminary work on PPV nanoparticles by Martijn Peters is kindly acknowledged.

ABBREVIATIONS ° C, Degrees Celsius; ∆T, Elevated temperature; λ, Wavelength; λmax, Wavelength at maximum absorbance; η, loading efficiency; φ, Quantum yield; AsPC-1, Human pancreas cell culture; -b-,Block; CDCl3, Deuterated chloroform; Cu, Copper; Cur, Curcumin; Ð,


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Polydispersity index; D50, average particle size;

DLS, Dynamic light scattering; DMAc,

Dimethylacetamide; DMF, Dimethylformamide; Dox, Doxorubicin; DPn, Degree of polymerization; (EGMA), Ethylene glycol methyl ether methacrylate; GFP, Green fluorescent protein; h, Hour; (HEA), 2-hydroxyethyl acrylate; (HPMA), 2-hydroxypropyl methacrylate; IC 50, Half maximum inhibitory concentration; Imean, Intensity mean diameter; In, Initiator; LHMDS, Lithium hexamethyldisilazide; MDMO-PPV, Poly[2-methoxy-5-(3,7dimethyloctyloxy)-p-phenylene vinylene]; Me6TREN, Tris[2-(dimethylamino)ethyl]amine; Mn, Number-average molecular weight; Mnapp, Apparent number-average molecular weight; Mwapp, Apparent weight-average molecular weight; Nmean, Number mean diameter; NMR, Nuclear magnetic resonance; NR, Nile Red; PPV, Poly(p-phenylene vinylene); ROMP, Ring opening metathesis polymerization; SEC, Size exclusion chromatography; SET-LRP, Single electron transfer – living radical polymerization; SRB, Sulforhodamine B; TEM, Transmission electron microscopy; THF, Tetrahydrofuran; UV-Vis, Ultraviolet-visible.


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REFERENCES

1

Letian, D.; Yongsheng, L.; Ziruo, H.; Gang, L.; Yang, Y. Chem. Rev. 2015, 115, 12633.

2

Robb, M.J.; Montarnal, D.; Eisenmenger, N.D; Ku, S.-Y.; Chabinyc, M.L.; Hawker, C.J.

Macromolecules 2013, 46, 6431. 3

Braun, D.; Heeger, A.J. Appl. Phys. Lett. 1991, 58, 1982.

4

Grimsdale, A.C.; Leok Chan, K.; Martin, R.E.; Jokisz, P.G.; Holmes, A.B. Chem. Rev.

2009, 109, 897. 5

Friend, R.H.; Gymer, R.W.; Holmes, A.B.; Burroughes, J.H.; Marks, R.N.; Taliani, C.;

Bradley, D.D.C.; Dos Santos, D.A. Bredas, J.L.; Lőgdlund, M.; Salaneck, W. R. Nature

1999, 397, 121. 6

Horowitz, G. Adv. Mater. 1998, 10, 365.

7

Gűnes, S.; Neugebauer, H.; Sariciftci, N.S. Chem. Rev. 2007, 107, 1324.

8

Burroughes, J.H.; Bradley, D.D.C.; Brown, A.R.; Marks, R.N.; Friend, R.H.; Holmes, A.B.;

Mackay, K.; Burns, P.L. Nature 1990, 347, 539. 9

Kulkarni, A.P.; Tonzola, C.J.; Babel, A.; Jenekhe, S.A. Chem. Mater. 2004, 16, 4556.

10

Gomez, D.E.; Lee, S.S.; Kim, C.S.; Loo, Y.-L. Mol. Org. Electron. Devices 2010, 109.

11

Yue, W.; Larsen-Olsen, T.T.; Hu, X.; Shi, M.; Chen, H.; Hinge, M.; Fojan, P.; Krebs, F.C.;

Yu, D. J. Mater. Chem. A 2013, 1, 1785. 12

Geng, Y.; Cong, J.; Tajima, K.; Zeng, C.; Zhou, E. Polym. Chem. 2014, 5, 6797.

13

Dini, D. Chem. of Mater. 2005, 17, 1933.

14

Schwartz, B.J. Ann. Rev. of Phys. Chem. 2003, 54, 141.

15

Heeger, A.J. Chem. Soc. Rev. 2010, 39, 2354.

16

Junkers, T.; Vandenbergh, J.; Adriaensens, P.; Lutsen, L.; Vanderzande, D. Polym. Chem.

2012, 3, 275.


26

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

17

Van Breemen, A.; Issaris, A.; de Kok, M.; van Der Borght, M.; Adriaensens, P.; Gelan, J.;

Vanderzande, D. Macromolecules 1999, 32, 5728. 18

Kesters, E.; Gilissen, S.; Motmans, F.; Lutsen, L.; Vanderzande, D. Macromolecules 2002,

35, 7902. 19

Zaquen, N.; Lutsen, L.; Vanderzande, D.; Junkers, T. Polym. Chem. 2016, 7, 1355.

20

Percec, V.; Guliashvili, T.; Ladislaw, J. Wistrand, A.; Stjerndahl, A.; Sienkowska, M.;

Montiero, M.; Sahoo, S. J. Am. Chem. Soc. 2006, 128, 14156. 21

Nguyen, N.H.; Percec, V. J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 5109.

22

Nguyen, N.H.; Rosen, B.M.; Ligadas, G.; Percec, V. Macromolecules 2009, 42, 2379.

23

Anastasaki, A.; Waldron, C.; Wilson, P.; McHale, R.; Haddleton, D.M. Polym. Chem.

2013, 4, 2672. 24

Cosemans, I.; Vandenbergh, J.; Voet, V.S.D. Loos, K. Lutsen, L.; Vanderzande, D.;

Junkers, T. Polymer 2013, 54, 1298. 25

Cosemans, I.; Vandenbergh, J.; Lutsen, L.; Vanderzande, D.; Junkers, T. Polym. Chem.

2013, 4, 3471. 26

Yu, C.-Y.; Horie, M.; Spring, A.M.; Tremel, K.; Turner, M. L. Macromolecules, 2010, 43,

222. 27

Lidster, B.J.; Behrendt J. M.; Turner, M.L. Chem. Commun. 2014, 50, 11867.

28

Su, F.; Alam, R.; Mei, Q.; Tian, Y.; Meldrum, D.R. Plosone 2011, 9, 24425.

29

Tan, H.; Zhang, Y.; Wang, M.; Zhang, Z.; Zhang, X.; Yong, A.M.; Wong, S.Y.; Chang,

A.Y.; Chen, Z.-K.; Lu, X.; Coolani, M.; Wang, J. Biomaterials 2012, 33, 237. 30

Tian, Y.; Wu, W.-C.; Chen, C.-Y.; Strovas, T.; Li, Y.; Jin, Y.; Su, F.; Meldrum, R.; Jen,

A.K.-Y. J. Mater. Chem. 2010, 20, 1728. 31

Wu, W.-C.; Chang, H.-H. Coll and Polym. Sci. 2015, 293, 453.


27

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

32

Page 28 of 30

Wang, H.; Wang, H.H.; Urban, V.S.; Littrell, K.C.; Thiyagarajn, P.; Yu, L. J. Am. Chem.

Soc. 2000, 122, 6855. 33

Jung, Y.; Hickley, R.J.; Park, S.-J. Langmuir 2010, 26, 7540.

34

Wu, W.-C.; Cheng, C.-Y.; Lee, W.-Y.; Chen, W.-C. Polymer 2015, 65, A1.

35

Walling, M.; Novak, J.; Shepard, J.R.E. Int. J. Mol. Sci. 2009, 10, 441.

36

Tiwari, D.K.; Jin T.; Behari, J.; Int. J. Nanomed. 2011, 6, 463.

37

Behrendt, M.; Sandros, M.G.; McKinney, R.A.; McDonald, K.; Przybytkowski, E.;

Tabrizian, M.; Maysinger, D. Nanomedicine 2009, 4, 747. 38 39

Feng, L.; Zhu, C.; Yuan, H.; Lv, F.; Wang, S.; Chem. Soc. Rev., 2013, 42, 6620. Peters, M.; Zaquen, N.; D’Olieslaeger, L.; Bove,́ H.; Vanderzande, D.; Hellings, N.;

Junkers, T.; Ethirajan, A. Biomacromolecules 2016, 17, 2562. 40

Yuan, Y.; Lui, B. ACS Appl. Mater. Interfaces 2014, 6, 14903.

41

Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Chem. Rev. 2012, 112, 4687.

42

Jiang, H.; Taranekar, P.; Reynolds, J. R.; Schanze, K. S. Angew. Chem., Int. Ed. 2009, 48,

4300. 43

Yuan, Y.; Ding, D.; Li, K.; Liu, J.; Liu, B. Small 2014, 10, 1967.

44

Feng, G.; Ding, D.; Liu, B. Nanoscale 2012, 4, 6150.

45

Lee, E.S.; Deepagan, V.G.; You, D.G.; Jeon, J.; Yi, G.; Lee, J.Y.; Lee, D.S.; Suh, Y.D.;

Park, J.H. Chem. Commun. 2016, 52, 4132. 46

Patra, S.K.; Ahmed, R.; Whittel, G.R.; Lunn, D.J.; Dumphy, E.L.; Winnik. M.A.; Manners,

I. J. Am. Chem. Soc. 2011, 133, 8842. 47

Xu, Y.; Meng, F.; Cheng, R.; Zhong, Z. Macromol. Biosci 2009, 9, 1254.

48

Yue, J.; Wang, R.; Liu, S.; Wu, S.; Xie, Z.; Huang, Y.; Jing, X. Soft Matter 2012, 8, 7426.


28

Page 29 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

49

Zhao, Y.; Sakai, F.; Su, L.; Lui, Y.; Wei, K.; Chen, G.; Jiang, M. Adv. Mater. 2013, 25,

5215. 50

Paira, T.K.; Saha, A.; Banerjee, S.; Das, T.; Das, P.; Jana, N.R.; Mandal, T.K. Macromol.

Biosci. 2014, 14, 929. 51

Wang, L.; Zeng, R.; Li, C.; Qiao, R. Colloids Surf. B 2009, 74, 284.

52

Colombani, O.; Ruppel, M.; Schubert, F.; Zettl, H.; Pergushov, D. V.; Müller, A. H. E.

Macromolecules 2007, 40, 4338. 53

Huynh, V. T.; Quek, J. Y.; de Souza, P. L.; Stenzel, M. H. Biomacromolecules 2012, 13,

1010. 54

Xu, B.; Holdcroft, S. Macromolecules, 1993, 17, 4457.

55

Wolfbeis, O.S. Chem. Soc. Rev. 2015, 44, 4743.

56

Michalet, X.; Pinaud, F.F.; Bentolila, L.A.; Tsay, J.M.; Doose, S.; Li, J.J.; Sundaresan, G.;

Wu, A.M.; Ghambir, S.S.; Weiss, S.Science, 2005, 307, 538. 57

Tu, G.; Li, H.; Forster, M.; Heiderhoff, R.; Balk, L. J.; Sigel, R.; Scherf, U. Small 2007, 3,

1001. 58

Patra, S. K.; Ahmed, R.; Whittell, G. R.; Lunn,D. J.; Dunphy, E. L.; Winnik, M. A.;

Manners, I. J. Am. Chem. Soc. 2011, 133, 8842. 59

Kim, Y.; Pourgholami, M. H.; Morris, D. L.; Stenzel, M. H. Biomacromolecules, 2012, 13,

814-825. 60

Jiang, W.; Kim, B.Y.S.; Rutka, J.T.; Chan, W.C.W. Nature Nanotech. 2008, 3, 145.

61

Chang, T.; Lord, M.S.; Bergmann, B.; Macmillan, A.; Stenzel, M.H. J. Mater. Chem. B

2014, 2, 2883.


29

Biomacromolecules

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TOC to

Non-crosslinked Profluorescent PPV-based Micellar System as a Versatile Probe for Bioimaging and Drug Delivery

A profluorescent, metal-free non-photobleaching non-crosslinked micellar system is used for drug delivery applications. PPV-containing block copolymer micelles decompose upon cell uptake, releasing the encapsulated material, at the same time directly allowing for confocal microscopy visualization.


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Profluorescent PPV-Based Micellar System as a Versatile Probe for

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