Fluorescent conjugated block copolymer nanoparticles by controlled mixing

Fluorescent conjugated block copolymer nanoparticles by controlled mixingt

Friederike Schiitze, Beate Stempfle, Christian Jiingst, Dominik Woll, Andreas Zumbusch and Stefan Mecking*

001: 1O.1039/c2ccl7066c

Monitoring of the fonnation of stable fluorescent nanoparticles from controlled mixing of a THF solution of poly(fluorene ethynylene)- block-poly( ethylene glycol) in a microfluidic laminar flow cross- junction by spatially resolved fluorescence spectroscopy reveals the time scale of particle fonnation as well as incorporation of small molecule guests and the role of solvent mixing.

Fluorescent nanoparticles are of interest for optoelectronics, live cell imaging and biosensing. Their extraordinary fluorescence brightness and much higher fluorescence emission rates with respect to single dye molecules are beneficial for time resolved observations or intracellular studies. Amongst different classes of fluorescent nanoparticles studied, n-conjugated polymer nano- particles 1.1 combine photoluminescence, and high absorption coefficients, fluorescence quantum yields, as well as non-linear optical absorption. 1(/.3 Thus, they are utilized as probes for cell labelling and bioimaging.lb.d,/~~

In principle, polymer nanoparticles can be obtained either directly by polymerization in disperse heterophase systems, ^1<,4 or by post-polymerization dispersion techniques.la The latter approach has been employed most commonly for the preparation of conjugated polymer nanoparticles in the form of so-called reprecipitation.^1/d A dilute solution of the conjugated polymer in a water-miscible organic solvent (THF) is injected rapidly into an excess of water manually. The colloidal stabilization mechanism in these surfactant free dispersions often remains unclear. Without steric or electrostatic stabilization even for these dilute dispersions (N 10¹⁵ L -I) coagulation should occur on the time scale of seconds.⁶

In view of the strong demand for defined fluorescent organic nanoparticles, enhanced preparative procedures are desirable.

Amphiphilic block copolymers⁷ lend themselves to this purpose.

With regard to the hydrophilic block, poly(ethylene oxide) otTers the advantage of biocompatibility and non-toxicity in vivo or in vitro^B We report on the post-polymerization generation of sterically stabilized conjugated block copolymer nanoparticles

Chair of Chemical Materials Sciellce, Departmellt of Chemistry, Ulliversity of Konstallz, 78464 Konstallz, Germany.

E-mail: stefall.meckillg@lIl1i-kollstallz.de; Fax:

+

Tel: +497531885151

t Electronic supplementary information (ESI) available: Additional synthetic procedures and analytical data. See DOl: 10.1039/c2ccI7066c

2104

polymer inTHF

water

Fig. I Overview and details of mixing zone (channel width: 100 ~un).

by continuous microfluidic mixing and insights on the particle formation process from spatially resolved spectroscopy.

A poly[9,9'-di(2-ethylhexyl)fluorene ethynyleneJ (PFE) with alkyne endgroups was generated by Sonogashira coupling of 2, 7-dibromo-9 ,9'-di(2-ethylhexyl)fluorene and 2,7 -diethynyl- 9,9'-di(2-ethylhexyl)fluorene with a slight excess of the latter.

Quenching of this reaction with a.-( 4-bromo phenol)-oo-methoxy poly(ethylene glycol) (a) (M" = 2000 g mol-I) yielded a diblock copolymer PFE-PEG (1) after workup with an apparent M" ^(apC) 3.2 x 10⁴ g mol-I and a weight ratio of the hydrophobic conjugated vs. the hydrophilic PEG block of ca. IS (ef ESI't).

Hydrodynamic flow focussing⁹ allows for controlled mixing of two solvent streams in the laminar flow regime. In a cross- junction glass device with channels of 100 11m x 20 11m cross section, a stream of THF containing dissolved polymer was focussed by two streams of excess water (Fig. I).

Introduction of THF solutions of 1 at flow rates of 0.5 to 1.2 ilL min-I at a water flow rate of 10 ilL min-I (corres- ponding to a velocity of ca. 83 mm S-I) in all cases yielded stable dispersions of 50 to 80 nm particles. In this range studied, with increasing amount of polymer introduced per time and thus per amount of water introduced, particle sizes slightly increase (ef ESlt). Under given conditions, particle sizes are satisfactorily reproducible.

Notably, when employing poly[9,9'-di(2-ethylhexyl)fluorene ethynyleneJ without a poly(ethylene glycol) block rather than t, macroscopic precipitation of the polymer occurred and no nanoparticle dispersion was obtained. Complete precipitation was also observed for a physical mixture of the conjugated polymer with a. Covalent binding of the hydrophilic sterically stabilizing moieties appears to be essential for colloidal stability.

For comparison with 'reprecipitation', solutions of t (0.1 mL) were injected into water (1.3 mL) under rapid stirring.

First publ. in: Chemical Communications ; 48 (2012), 15. - pp. 2104-2106

Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-187001

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Fig. 2 (a) Schematic representation of nanoparticle formation from I.

Particle formation causes a bathochromic shift of fluorescence from blue to green. Real·color fluorescence image of the mixing zone at (b) THF flow rate ⁼ 0.6 ilL min-' and (c) THF flow rate ⁼ 1.0 ilL min-I (water flow rate 10 ilL min-', )'e," = 380-400 nm). (d) Emission spectra of 1 in THF (blue), in dispersion (green) and as solid (dashed green) ()'e," = 380 nm). (e) TEM image of nanoparticles of I.

Particles of similar size with higher polydispersities (POI ca. 0.2, cf Table SI, ESIt) resulted.

Due to different conjugation length and energy transfer to lower-energy chromophores,'o fluorescence emission of conjugated polymer nanoparticles is commonly red-shifted vs. polymer solutions in organic solvents. For 1 this results in a change from blue to green fluorescence upon particle formation (Fig. 2).

TEM studies reveal an essential spherical particle shape, and quite uniform particle size distributions (Fig. 2e) with no indication of aggregation. TEM analysis of the particles is in agreement with size distributions from DLS (cf ESIt).

An incorporation of small molecules into polymer particles is of broad interest. A widely studied use of the resulting particles is the delivery and release of drugs and other biologically active substances. In fluorescent nanoparticles, incorporation of a small portion of an appropriate chromophore can enable the tuning of their emission colors by energy transfer and emission from the dye. I I Corresponding particles are studied for live cell imaging.

Perylene dyes are known to be very photostable and to emit with high quantum yields. The diimide 2 (Fig. 3a) possesses an absorption spectrum which overlaps favorably with the emission spectrum of 1 (cf ESIt).

During the particle formation process, 2 is effectively incor- porated into the nanoparticles as evidenced by the batho- chromic shift of the emission (Fig. 3b). Quantum yields were ca. 45% vs. 26% for particles without perylene. Particle sizes were not notably altered by incorporation of the dye (cf ESH).

In order to monitor and better understand the particle formation process, the microfluidic mixing device was inte- grated into a custom-made fluorescence microscope which

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Fig. 3 Particle formation from polymer 1 with incorporation of perylene diimide dye 2 (2 mol% perylene with respect to conjugated polymer repeat units). (a) Schematic representation. (b) Real-color fluorescence image of the mixing channel (J'e," = 380-400 nm).

allows for spatial and spectral resolution (cf ESIt). With increasing distance to the mixing junction the initial solution (THF) fluorescence spectra of 1 converge to solid phase spectra of the dispersion (Fig. 4). The broad emission band of the dispersion at Am"x = 508 nm grows continuously at the expense of the solution emission with Am"x = 420 nm. To exclude a possible contribution of a variation of the emission of the dissolved polymer by changes of the solvent polarity imparted by increasing amounts of water in the THF phase, emission spectra of 1 were recorded in aqueous THF solutions (cf ESIt). The lack of significant changes between spectra underlines that the alteration of emission observed in the mixing device indeed reflects particle formation. As expected, at a given distance to the mixing junction the particle formation process is further progressed at the lower flow rate of the polymer solution of 0.6 ~L min - I (Fig. 4a), than at the flow rate of 0.8 ~L min-I (Fig. 4b). Already after 12 ms, corresponding to

I mm distance from the mixing junction, a substantial particle emission is observed, and after 60 ms (5 mm) emission occurs from the particles nearly exclusively. Note that due to the higher quantum yield of emission from a THF solution (if> = 61 %) vs.

the particles (if> = 26%), the contribution from the former (band with Am"x = 420 nm) is overestimated when simply comparing band intensities in spectra. During the generation of nano- particles with incorporated dye, the ingrowth of the dominating red perylene emission (Am"x = 600 nm) and of the residual emission from the solid polymer occur simultaneously. Dye incorporation and particle formation occur in a concerted fashion, rather than a conceivable uptake of perylene by formed particles (Fig.4c).

Preliminary studies of a poly(ethylene glycol)-block-poly[2,5- di(2-ethylhexyl)oxyphenylene ethynylene]-b/ock-poly(ethylene glycol) triblock copolymer 3 (PEG blocks Mil = 2000 g mol-I;

conjugated polymer block Mil (ope) = 6000 g mol-I) underlined the applicability of the procedure studied here to other similar polymer architectures and conjugated backbones. With the same mixing geometry, concentrations and flow rates as given above for 1, stable dispersions of 30 to 60 nm particles resulted (cf ESIt).

In order to further interpret these observations on nanoparticle formation, knowledge of the mixing rates of the two fluid streams and the resulting fluid phase compositions is required.

The formation of nanoparticles by hydrodynamic flow focussing of an acetonitrile solution of poly(lactide-co-glycolide)- block-poly(ethylene glycol) diblock copolymer with a water stream has previously been studied under similar conditions,

i.e. channel geometry and flow rates reported by Farokhzad

et a/. 12.13 By analogy to a previous study of Prud'homme with an impinging jet mixer,9h Farokhzad assumed a solvent mixing time 'mix of < 0.4 ms. This is much faster than particle formation (r"ggr), which led to the conclusion that particles form from a very rapidly generated highly supersaturated aqueous block copolymer solution.

Coherent anti-Stokes Raman scattering (CARS) microscopy shows the fluid composition over the mixing device (Fig. 5).14 A central stream richer in THF vs. the surrounding laminar aqueous flow layers persists throughout the channel. In the center of the channel, the THF solvent is diluted e.g. I: I with water after ca. 40 ms (i.e. 3.5 mm from the junction, 2105

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Fig.4 Spatially resolved fluorescence spectra of I. Spectra are normalized to the emission maximum of 1 in THF solution at 423 nm. Water flow rate = 10 ilL min-I (12 ms mm-I). (a) THF flow rate = 0.6 ilL min-I, CI = 0.03 wt%. (b) THF flow rate = 0.8 ilL min-I, CI = 0.03 wt%.

(c) Mixture of 1 and 2 with 0,6 ilL min-I, CI = 0.05 wt% (J.cxe = 350-400 nm) (0) excitation lamp artifact.

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Fig. 5 CARS image of microfluidic mixing of THF and water (left: close-up, upper right: overview) and decay of the CARS-signal ofTHF at the center of the focussed stream at 0.6, 0.8 and 1.0 ilL min I (lower right). Imaging at the CI-1₂ resonance at 2860 em-I. Water flow rate = 10 ilL min-I. This corresponds to a flow rate of the focussed stream of 0.083 m S-I.

at 0.8 ilL min-I THF). The aforementioned studies of the effect of variable proportions of water on fluorescence spectra ofTHF solutions of 1 indicated that up to 15 vol% of water the nature of these mixtures is that of true sol utions, and no aggregation to particles occurs; this was also confirmed by parallel DLS measurements. Above 15 vol% water THF-swollen particles

> 200 nm had been observed.

The .combined results of spectral monitoring of particle formation and of fluid mixing by CARS microscopy reveal that the time scale of mixing approaches the time scale of particle formation.

In conclusion, from linear block copolymers with a conjugated polymer block and hydrophilic blocks, highly fluorescent nano- particles with a well defined surface chemistry that provides steric stabilization can be generated in a controlled and reproducible fashion via microfluidic mixing. The typical enhanced emission from lower energy chromophores due to energy funneling upon aggregation allows for monitoring of particle formation processes via fluorescence microscopy. Under the conditions studied, in the laminar flow regime, both particle formation and the relative mixing process occur on a. timescale of several ms to several tens of ms. Both processes occur concurrently, rather than in two distinguishable steps of a rapid formation of a supersaturated solution followed by a much slower particle formation ('tm;x « 'tagg,.). Possibly, fluid mixing is the rate determining step. This agrees with the observation that the solvent flow rate impacts the particle size. The incorporation

2106

of small molecules in the particle formation process can be monitored by energy transfer to dyes. Perylene diimide as a model of a small molecule payload of the particles is incorpo- rated gradually directly into the particles simultaneously to their formation, rather than being taken up into preformed particles.

Financial support by the OFG (MeI388J7-l), the Baden- Wilrttemberg Stiftung (CJ, AZ) and the Zukunftskolleg of the University of Konstanz (BS, OW) is gratefully acknowledged.

Notes and references

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