Polymer Nanoparticles Microenvironment: Using Photophysical

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C: Physical Processes in Nanomaterials and Nanostructures

Polymer Nanoparticles Microenvironment: Using Photophysical Probes to Investigate Internal Porosity and Polarity Suxiao Wang, Shreyas Phadke, ZIXU ZHAO, Stephanie Reid, Jason Beirne, and Gareth Redmond J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b05960 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 25, 2018

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The Journal of Physical Chemistry

Polymer Nanoparticles Microenvironment: Using Photophysical Probes to Investigate Internal Porosity and Polarity Suxiao Wang†,‡, Shreyas Phadke‡, Zixu Zhao‡, Stephanie Reid‡, Jason Beirne‡, Gareth Redmond‡* †

Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional

Molecules, Hubei Collaborative Innovation Centre for Advanced Organic Chemical Materials, College of Chemistry and Chemical Engineering, Hubei University, Wuhan, 430062, China. ‡School

of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland

ACS Paragon Plus Environment

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Abstract

The objective of this work was to probe the internal morphology, porosity and polarity of conjugated polymer poly[9,9-di-(2-ethylhexyl)-fluorenyl-2,7-diyl] (PF2/6) nanoparticles in order to assess their feasibility as drug-delivery vehicles. To this end, conjugated polymer PF2/6 nanoparticles of varying size were prepared using a reprecipitation method; size was controlled using different injection speed and agitation power. Dynamic light scattering and fluorescencebased MEMFCS measurements indicate the hydrodynamic diameters of nanoparticles increase from Method 1 to Method 3 (40 nm, 60 nm, 80 nm, respectively). However, AFM measurements indicate that nanoparticles shrink to ca. 10 nm upon drying. Further experiments suggest that this occurs due to NP porosity, which increases with increasing hydrodynamic size as progressively more water becomes trapped within the microenvironment of the NP. Single nanoparticle fluorescence images show that fluorescence brightness for NPs of differing hydrodynamic size does not change, indicating that an increase in size may not be due to additional polymer chains. The solvatochromic fluorescent dyes Nile red and DCM were used to precisely probe the internal polarity of NPs. They were separately doped into the hydrophobic core of PF2/6 NPs with different hydrodynamic sizes. The photophysical properties of both of the two dyes agrees that the polarity of the nanoparticles increase with increasing PF2/6 NP size, providing further evidence of trapped water in NPs of larger size. Furthermore, the restricted motion of the dye molecules in the nanoparticles has been investigated by time-resolved anisotropy. The anisotropy of dyes in PF2/6 NPs with different sizes are the same, indicating the randomness, rotational motion restriction and rigidity of the encapsulated dyes doesn’t change as NP size varies. The Lippert-Mataga polarity of PF2/6 NPs were calculated for three different methods by applying the two fluorescent dyes.


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Introduction Conjugated polymer nanoparticles (CPNPs) are emerging as multifunctional nanoscale materials that offer great potential in many application areas due to their high fluorescence brightness, excellent photostability, highly efficient energy transfer and sensing capabilities. 1 Their properties can be easily tuned for desired applications through the choice of conjugated polymers and surface modification.

Additionally,

their

facile

synthesis,

tuneable

properties

and

superior

biocompatibility compared to existing inorganic nanoparticles make these materials highly attractive as smart drug-delivery vehicles. 2-4 For applications such as light harvesting,2, 5-8 photodynamic therapy 9-10 and photoswitching 3-4, 11-19

where multiple components may be encapsulated into the NP core, it is extremely important

to understand the internal structure and photophysical behaviour of the NP microenvironment in physiological conditions. For the purpose of controlling photophysical processes such as charge and energy transfer 20-26, precise understanding of the internal morphology and polarity effects on conjugated polymer nanoparticles emission behaviour was desired.27-28 However, efforts were directed dominantly toward manipulation of their dimensions, shapes and surface functionalities, leaving their interior rarely addressed. 29 Patra et al studied organic dyedoped PVK polymer nanoparticles with varying polymer concentrations and found that microviscosity and rigidity increases with increasing PVK concentration, influenced by the restricted motion of dye molecules in polymer nanoparticles. 30-31 Various researchers: Muller et al.32, Sommerdijk et al.33, and Yuan et al.29, 34 reported the multi-lamellar, onion-like and striped ellipsoid-like morphology of self-assembled CPNPs using cryo-electron microscopy (cryo-TEM) and tomography (3D cryo TEM, cryo-ET). These techniques can image hydrated CPNPs, but are quite costly and require high quality sample preparation.


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Determining the internal polarity of CPNPs is beneficial of designing the smart nanomaterials, but very limited studies have been presented so far. Solvatochromic probe can be a good candidate to perform here. Due to their remarkable sensitivity, fluorescent probes have been used quite extensively to determine the local environment and micropolarity of proteins 35-37, cyclodetrins,3839

micelles,40-41 reversed micelles,42 and zeolites.43-44 Nile red (9-diethylamino-5-benzo[α]phenoxazinone) and DCM (4-dicy-anomethylene-2-

methyl-6(p-dimethylamino-styryl)-4H-pyran)

are

hydrophobic,

highly

fluorescent,

solvatochromic dyes which are ideally suited as microenvironment probes.45-46 The photophysical properties of these dyes can be used to report back information about the microenvironment, where intermolecular and intramolecular interactions strongly influence the emission behaviour Fluorescence

decay

and

anisotropy

measurements

revealed

how

the

structure

47-48.

of

microheterogeneous systems influence the photophysical properties of Nile red. 49-51 In our previous work, we revealed dynamic behaviour of the small organic molecule tetraphenylporphrin (TPP) which was found to wobble inside the core of CPNPs at a semiangle of ca. 70°and laterally diffuse on the CPNP surface with a diffusion coefficient of ca. 10 -4 cm2 s-1 .52 In the present contribution, we focus on the polymer nanoparticle itself and precisely analyse the internal morphology and polarity of the CPNPs. Conjugated polymer poly[9,9-di-(2-ethylhexyl)fluorenyl-2,7-diyl] (PF2/6) nanoparticles were made with different hydrodynamic sizes but which dried to the same size, suggesting the porous morphology formed by trapping more water inside of NPs. And the solvatochromic fluorescent dyes Nile red and DCM are doped into the hydrophobic core of PF2/6 nanoparticles to detect the internal environmental changes among different sizes of NPs. The photophysical properties of both of the two dyes agrees that the polarity of the nanoparticles increase with the increasing of PF2/6 NPs sizes which provide another


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evidence of more water trapped in NPs with bigger sizes. The Lippert-Mataga polarity of PF2/6 NPs were also calculated. Furthermore, the fluorescence anisotropy has been applied to investigate the localization and restricted motion of dye in NPs and wobbling coefficient were calculated. Our work makes the contribution of more accurate understanding the internal morphologies of conjugated polymer NPs helps to achieve an improvement in smart nanomaterial development.


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Experimental Methods Materials. Poly[9,9-di-(2-ethylhexyl)-fluorenyl-2,7-diyl] (PF2/6) (ADS131BE) with a weightaverage molecular weight of 10,000 g/mol was purchased from American Dye Source, Inc. The organic dye 9-diethylamino-5-benzo[α] phenoxazinone (Nile red, NR) with a molecular weight of 318.37 g/mol, was purchased from Sigma Aldrich. 4-dicy-anomethylene-2-methyl-6(pdimethylamino-styryl)-4H-pyran (DCM) with a weight-average molecular weight of 303.37 g/mol was purchased from Exciton®. Tetrahydrofuran (THF) (CHROMASOLV® for HPLC, 99.9%) was purchased from Sigma Aldrich, Inc. De-ionized water (> 16.1 MΩcm, Milli-Q®, Millipore Corp.) was used for all aqueous solutions. The molecular structures of PF2/6, Nile red and DCM are depicted in Scheme 1. Preparation of nanoparticles. The Nile red and DCM doped conjugated polymer nanoparticles with difference sizes were prepared using a reprecipitation method as seen in Scheme 1. PF2/6 powder was first dissolved in anhydrous tetrahydrofuran (THF) with 1 h continuous stirring (1000 rpm; IKA RCT basic IKAMAG hot plate) to make polymer / THF (P/THF) solutions (0.1 mg mL−1; 100 ppm). The fluorescence dye, Nile red and DCM, were separately dissolved in anhydrous THF with 1 h continuous stirring to make dye/THF solutions (0.002 mg mL −1; 2 ppm). The pure PF2/6 nanoparticles were synthesized by rapidly injecting 2 mL of PF2/6/THF solution (20 µg of PF2/6) into 8 mL of deionized water in a round bottomed flask while applying sonication for 3 min. Then a rotary evaporator was used to evaporate THF for 1 h at 10 mbar.7 mL of nanoparticle solution was left in the flask after rotary evaporation. Dye-doped PF2/6 nanoparticles were synthesized as follows: 0.2 mL of 100 ppm PF2/6 / THF stock solution (20 µg PF2/6) was separately mixed with varying volumes of 2 ppm dye / THF stock solution: 0.10 mL Nile red (0.2 µg, 1.0 wt.%), 0.15 mL Nile red (0.3 µg, 1.5 wt. %) and 0.20 mL Nile red (0.4 µg, 2.0 wt. %); or


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0.10 mL DCM (0.2 µg, 1.0 wt.%), 0.20 mL DCM (0.4 µg, 2.0 wt.%) and 0.30 mL DCM (0.6 µg, 3.0 wt.%). THF was added to control the total volume for each sample at 2 mL. For method 1, the preparation involved the rapid injection (2 mL in 1 sec) of the prepared mixture solution into 8 mL de-ionized water in a round-bottomed flask while applying sonication over 3 minutes in order to improve mixing and increase the rate of polymer chain collapse. Rapid mixing with water resulted in a sudden decrease in solvent quality, which drove the formation of a suspension of polymer nanoparticles. For methods 2 and 3, the preparation involved the relatively slow injection (method 2: 2 mL in 12 sec; method 3: 2 mL in 20 sec) of the prepared mixture solution into 8 mL de-ionised water in a round-bottomed flask while applying gentle stirring over 4 minutes. The decreasing injection speed and agitation intensity results in slower polymer chain aggregation and larger nanoparticle formation. Following this, the resulting polymer/dye/THF/water mixture was placed into a rotory evaporator for 60 mins in order to remove the THF under reduced pressure (10 mbar) at room temperature. After removal of THF, approximately 7 mL solution remained in the roundbottomed flask. Physical Characterization. Dynamic light scattering (DLS) and zeta potential measurements were carried out using a Zetasizer Nano ZS system, (Malvern Instuments, Ltd., UK). DTS Application 5.10 software was employed to analyze the data obtained. Atomic force microscopy (AFM) images of nanoparticles were acquired in non-contact mode using an Innova AFM system (Bruker Corp.). To prepare samples for imaging, glass microscope cover slips were first acid-cleaned. Then, a 10-fold dilute suspension (200 µL) of nanoparticles was flowed onto the cleaned cover slip, held in upright position, which was then dried with nitrogen flow. For image analysis, Nanoscope Analysis Software, v1.40 (Bruker Corp.) was


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employed. To construct histograms of nanoparticle heights, topographies of around 150 – 250 nanoparticles were analyzed. Optical Characterization. To minimize artifacts, such as optical scattering, saturation or reabsorption, dilute samples (< 0.1 A.U.) were used to measure absorption and photoluminescence spectra. UV-vis absorption spectra were acquired using a double-beam spectrophotometer (V-650; Jasco, Inc.). Water or solvent was used as a reference. Photoluminescence spectra were acquired using a QuantaMaster™ 40, (PTI, Inc.), equipped with a Xe short arc discharge lamp and CzernyTurner monochromators. A black light illuminator (Gel Logic 200; Kodak, Ltd.) was used to record

true-color

emission

photographs

(Lumix

DMC-TZ30;

Panasonic,

Corp.).

Photoluminescence lifetime measurements were performed on a time-correlated single photon counting (TCSPC) technique. The measurement system (FluoroCube-01-NL; Jobin Yvon, Horiba Ltd.) was equipped with two semiconductor pulsed light emitting diodes, with emission wavelengths of 635 nm. Fluorescence signal from the samples was collected perpendicularly to the excitation path, passed through a 32 nm band pass filter and detected by a single photon counting module. Time resolved anisotropy measurements were measured using FluoroCube-01NL; Jobin Yvon, Horiba Ltd. with two polarizers in the excitation path and emission path. Single nanoparticle photoluminescence imaging was undertaken using a home-built wide-field epi-fluorescence microscopy system consisting of inverted microscope (IX71 ®, Olympus, Inc.) equipped with a 404 nm diode laser (iBeamsmart 405-S, TOPTICA Photonics AG), an electron multiplying charge-coupled device (EMCCD) camera (EvolveTM 512, Photometrics), and a 100 × oil immersion objective lens (1.45 NA; UPLSAPO 100XO, Olympus, Inc.) in combination with a 1.6 × tube lens. Samples were prepared for imaging using the AFM-related method described above and imaged immediately. To acquire photoluminescence images, each sample was


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illuminated at 404 nm at an excitation intensity of 2..12 W cm-2 and 20 frames were acquired with an exposure time of 20 ms at an electron multiplication gain of 200. Metamorph® Microscopy Automation & Image Analysis Software (Molecular Devices, Inc.) was used to control the imaging system. Acquired image stacks were subsequently analyzed using Image-Pro® Plus (Media Cybernetics, Inc.) in combination with a custom-built MATLAB program (MATLAB R2012a, The MathWorks, Inc.), and plotted using Origin® 8.1 (OriginLab Corp.). Fluorescence correlation spectroscopy (FCS) measurements were performed based on a home built on point-illumination confocal microscopy system utilising an inverted microscope (IX71 ®, Olympus, Inc.) mounted on an isolated optical table (RS2000-410-12 Optical Table, I-2000-400 Stabilizers™, Newport ®). Samples were excited at a wavelength of 399 nm. Calibration standards, which consisted of aqueous dilutions of molecular dye (ATTO 425) were measured prior to sample measurements to ensure the size and shape of the confocal volume was determined correctly. Dilute dispersions of PF2/6 NPs were deposited into samples wells on the same chambered coverglass plates (Lab–Tek™ II, Nunc, Inc.) that were used for calibration. Burst acquisitions were recorded in TTTR mode over 2 mins using a TimeHarp 200 electronic and software suite. Autocorrelation and cross–correlation analysis was performed in SymPhoTime 32 software (v. 5.1.3.1, PicoQuant, GmbH).


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Results and Discussions PF2/6 Nanoparticles. PF2/6 nanoparticles were prepared by exposing polymer chains to water in the absence of any surfactant stabilizers. Dynamic light scattering (DLS) was employed to measure the hydrodynamic size distributions of the nanoparticle dispersions in water. The measured data (number mean) indicated that the average hydrodynamic diameter of the PF2/6 nanoparticles synthesized by applying method 1 was ca. 40 nm (PDI: 0.2), method 2 was ca. 60 nm (PDI: 0.2) and method 3 was ca. 80 nm (PDI: 0.2); see Figure 1 (a). From method 1 to method 3, the decreasing injection speed and agitation intensity results in slower polymer chain aggregation and formation of nanoparticles with larger size. The zeta potential of PF2/6 nanoparticles for methods 1 to 3 were ca. -30 mV, as shown in Figure S1 and Figure S2. These negative surface charges which may occur due to the formation of chemical defects at the NP surface during the reprecipitation process, maintain colloidal stability of the NP suspension and inhibit further aggregation.53 The PF2/6 in THF and undoped PF2/6 NPs absorption spectrum exhibited an intense π-π* absorption band centered at 380 nm, as shown in Figure 1 (b). The absorption spectrum broadening with bigger tails on the high wavelength range from method 1 to method 3 might be due to the increased light scattering by the increasing of NPs sizes. The excitation spectrum of PF2/6 nanoparticles for all three synthesis methods exhibit a maximum peak at 380 nm with spectral broadening from method 1 to 3, as seen in Figure 1 (c) inset, top. The emission spectrum of PF2/6 in THF exhibited an emission peak at 413 nm while the nanoparticles from all three synthesis methods exhibited an emission peak at 418 nm due to the vibronic progression of the inhomogeneously broadened S0 → S1 transition, as shown in Figure 1 (c).54-55 The slight red-shift and sharpening observed in emission of the PF2/6 NPs compared to the corresponding spectrum


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measured for the polymers in THF solution are attributed to changing the polymer chain conformation which present more ordered chain segments with higher conjugation length. 56 Therefore, PF2/6 NPs have the combinations of planarized / elongated coil chains and collapsed / aggregated polymer chains. However, there is no additional apparent absorption peak at a lower energy wavelength, indicating no planarized aggregated phase or cystallized aggregates existing. 56

Also, the low-energy emission band (~ 530 nm) starts to appear after PF2/6 NPs formation which

may be assigned to chain defects (e.g. photo-oxidation).57 These emission color changes from PF2/6 in THF to NPs were observed under black light, as shown in Figure 1 (c) inset, bottom. Steady state fluorescence emission anisotropy data (λexc. = 380 nm) were measured for the PF2/6 polymer in solvent solution and in nanoparticle dispersions, as shown in Figure 1 (d). For the PF2/6 solution in THF (black line), an anisotropy value (r) was estimated as 0.10, while for PF2/6 nanoparticles method 1 to method 3, the estimated values were approximately 0.15, 0.18 and 0.20, respectively. The similar r value of PF2/6 in solution has been reported before as 0.096. 58 From method 1 to 3, with increasing particle size, the observation that particles exhibited increasing emission anisotropy values,higher than that of free polymer in solvent solution suggests less freedom of the torsional degree of the conjugated polymer chains with larger particle sizes. 59 Photoluminescence decay data were also measured using the time-correlated single photon counting technique (under pulsed 380 nm excitation); see Figure 1 (e). Excited state lifetimes were extracted from the decay traces using commercial software by fitting the measured photoluminescence decay traces with multi-exponential functions; the data is summarized in Figure 1 (f), while the residuals of the fitting are shown in Figure S3. The fast component estimated for PF2/6 solutions in THF was 0.56 ns and for NPs made from method 1 to 3 these were 0.29 ns, 0.49 ns and 0.23 ns, respectively while the slow component for PF2/6 NPs from methods 1 to 3


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were 1.47 ns, 5.00 ns and 1.55 ns, respectively. PF2/6 solutions in THF exhibits single exponential decay behavior without the slow component. 60 The extra slow component of the PF2/6 NP lifetime is likely due to conformational changes of the polymer chain segments,61 increased polymer chain aggregation, 8, 62-63 chain defects quenching (e.g.photo-oxidation)64 and / or excimers65. Fluorescence correlation spectroscopy (FCS) for PF2/6 NP were measured using a home-built FCS optical system. FCS is a correlation analysis of fluctuations in the fluorescence intensity signal. The fluorescence intensity is fluctuating due to Brownian motion of particles within the optical excitation volume. Fluorophores traversing a focal volume will emit fluorescence, either as a burst for a strongly emissive fluorescent nanoparticle or a narrower spike for a molecular dye. The amplitude of this emission burst is dependent on the brightness of the species while the width is dependent on the size; larger objects will take longer to diffuse through the focus giving a broader fluorescence peak.66 Figure 2 (a) shows a typical autocorrelation FCS curve for a dilute dispersion of PF2/6 NPs synthesized by methods 1 to 3. From these curves, the translational diffusion time increases from method 1 to 3 which suggests the sizes of the nanoparticles increase from method 1 to method 3. The single component fitting fits the correlation data assuming a monodisperse, homogenous population of nanoparticles undergoing Fickian diffusion. In contrast, maximum entropy method fluorescence correlation spectroscopy (MEMFCS) assumes a quasicontinuous distribution of diffusing components (αi) according to67: G (𝜏 ) =

∑𝑛𝑖=1 𝛼𝑖

𝜏 −1

(1 + ) 𝜏𝑖

1

(1 +

𝜏 𝜅2 𝜏𝑖

−2

)

(1)

where αi is the amplitude of the ith component with characteristic residence time τi. Convergence of the fitting algorithm occurs when both the entropy has been maximized according to: S = − ∑𝑖 𝑝𝑖 𝑙𝑜𝑔𝑝𝑖

(2)

𝛼𝑖

where 𝑝𝑖 = ∑

𝑗 𝛼𝑗


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As shown in Figure 2 (b)-(d), the intensity means of the hydrodynamic diameter from light scattering-based DLS for PF2/6 NPs made from method 1 to method 3 were ca. 58 nm, ca. 94 nm and ca. 120 nm respectively, and the DLS size distributions (pink) overlap well with the results derived from the fluorescence based MEMFCS (gray) which confirms the different hydrodynamic sizes of NPs formation from method 1-3. Atomic force microscopy (AFM) was used to determine the dry sizes of NP deposited on cleaned glass substrates. AFM images of NPs made from method 1-3 are shown in Figure 3 (a)-(c) insets, respectively. Analysis of measured particle heights provided histogram data for NPs which were employed to estimate average dry nanoparticle diameters. The number mean hydrodynamic diameter of PF2/6 NPs measured by DLS of method 1-3 were ca. 40 nm, ca. 60 nm and ca. 80 nm, respectively, as seen in Figure 3 (a)-(c) (blue). After drying the samples on glass cover slips, the average PF2/6 NPs sizes measured by AFM of method 1-3 were 8.2 nm ± 3.1 nm, 8.3 ± 3.0 nm and 9.7 ± 2.6 nm, respectively, as seen in Figure 3 (a)-(c) (red). From wet-to-dry, the significant size decrease could be due to the loss of entrapped solvent molecules and to further polymer chain aggregation on drying of the NPs in air. The phenomenon observed here suggests that NP porosity increases from method 1 to method 3 by progressive trapping more water with subsequent increase in polarity of NP internal environment. To eliminate the factor that different numbers of polymer chains give different hydrodynamic sizes, single nanoparticle PL imaging (nanoparticles deposited onto glass cover slips and imaged in air) were performed using a home-built wide-field epi-fluorescence microscopy system (λexc. = 404 nm; 2.12 W cm-2 ). Near-diffraction-limited emission spots, consistent with emission from individual PF2/6 nanoparticles, were apparent in the emission images; see Figures 3 (d-f) for NPs made from method 1 to method 3, respectively. The average single particle emission intensity


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values (photons per particle per frame) were estimated by statistical analysis of the image data which exhibited the per particle brightness of the PF2/6 NPs were ca. 1.05 × 104 photons / 0.02s, 1.20 × 104 photons / 0.02s and 1.11 × 10 4 photons / 0.02s for NPs made from method 1 to method 3, respectively, as shown in Figure 3 (g-i). The histogram data were compiled from approximately 50-80 nanoparticles for each sample type. These brightness values provided excellent signal-tobackground characteristics in the emission images. PF2/6 nanoparticles exhibited very similar initial brightness for all three methods. It may be possible to estimate the number of polymer chains from brightness data as proposed by Tian et al. and Reisch et al. 68-69 As we are operating at higher excitation intensity than these authors, we cannot eliminate the effects of non-radiative processes occurring because this multi-chromophoric system can generate fluorescence quenchers and lead to further brightness decrease with increasing power density.

68-69

Instead, we employ average

particle size and molecular weight to estimate the number of polymer chains per NPs, N, using 70 4

𝐷 3 𝜌𝑁𝐴

3

2

𝑁 = 𝜋( )

𝑀𝑤

,

(3)

where D is the particle diameter determined by AFM methods, ρ is the particle density (taken as 1 g / cm3), NA is Avogadro’s number and Mw is the weight-average molecular weight of polymer. The estimated per particle polymer chain number is 32. To compare with other nanoparticle system, the per-particle brightness are very similar with APFO-3 NPs and PCDTBT NPs under the same detection condition.8 In order to understand the internal morphology changes with vary sizes precisely, spectroscopic studies of the NP interior were performed using an environmentally sensitive probe dye. Nile red as Probe Doped into PF2/6 Nanoparticles. The hydrophobic, highly fluorescent, solvatochromic dye Nile red (NR) was used as microenvironment probe to detect photophysical


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properties of the nanoparticle environment. The intensity-normalized absorption, emission and excitation spectrum of NR in THF and in water are shown in Figure S4 (a) and (b), respectively. NR in THF exhibits an absorption maximum at 527 nm and an emission maximum peak at 590 nm under 527 nm excitation, while Nile red in water exhibits an absorption maximum at 560 nm and an emission maximum at 650 nm under 560 nm excitation. The absorption band of the Nile red in visible region corresponds to a transition moment largely parallel to the long axis of the molecule (π to π* transition).71 The large red shift of the absorption and emission spectrum of Nile red with the environment changing from THF to water is due to its strong solvent dependence; with increasing polarity, the spectrum of Nile red red shifts.72 The emission spectrum of Nile red in water is more noisy with lower intensity than in THF due to its solvatochromic properties with emission intensity decreasing as the solvent polarity increases. In addition, we have dialyzed all the samples by using a dialysis membrane having molecular cutoff of 1000 Da. We have observed no emission intensity of the dyes outside of the dialysis bag. This indicates that a negligible amount of free dye leakage from NPs into solution. Based on this, the number of dyes per particles can be estimated by assuming that all of the dyes were uniformly encapsulated into NPs. The concentrations of prepared NPs were measured by nanoparticle tracking analysis (NTA) as ca. 0.17 nM. The 1 wt. % and 2 wt. % Nile red doping concentration can be calculated as ca. 90 nM and 180 nM, which yields the number of dyes per nanoparticles 529 and 1058, respectively. Similarly, the 1 wt. %, 2 wt. % and 3 wt. % of DCM doping concentration can be calculated as ca. 94 nM, 188 nM and 282 nM, respectively, which yields the number of dyes per nanoparticles 553, 1106 and 1659, respectively. In order to properly understand the internal polarity of PF2/6 NPs, 1.0, 1.5, 2.0 wt. % Nile red doped PF2/6 NPs were made. The strategies of dye encapsulation in NPs can be achieved either


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by staining already synthesized blank NPs or by encapsulating the dyes directly during the NP preparation.73 The way used here is the most straightforward approach which uses mixtures of dyes and polymers in the organic phase to achieve physical trapping of the dyes in the formed polymer NPs. During the injection of THF solution of polymer / dye into water (a poor solvent for the polymer and dyes) under stirring conditions, the solution is divided into many droplets by strong shearing force while the dyes are captured into the droplets with polymers. Simultaneously, THF molecules quickly diffuse into water to expose the polymer and dyes to bulk water. Hydrophobic chain collapse together with entrapped dyes to form NPs. At the time of the chain coiling, there should be some confined / caged water molecules inside the polymer NP. Hence, a certain fraction of encapsulated dyes should reside with the direct contact to water and undergo Hbonding. On the other hand, the rest of the fraction resides in a more hydrophobic pocket due to direct contact with the hydrophobic polymer chain.74 As shown in Figure S5, the hydrodynamic diameters from DLS of Nile red-doped PF2/6 NPs made from method 1, 2 and 3 were ca. 40 nm, 60 nm and 80 nm, respectively. This low Nile red doping concentration has thus negligible effect on the NP size. After drying the sample on glass cover slips, the average PF2/6 / 2 wt. % NR NP sizes measured by AFM of method 1 to 3 were 11.5 nm ± 5.8 nm, 12.3 ± 5.6 nm and 7.9 ± 3.3 nm, respectively, as seen in Figure S6. Similar to undoped P2/6 NPs, the sizes of the Nile red doped NPs significantly decrease from wet-to-dry which could also be explained by the loss of entrapped solvent molecules. The zeta potential of PF2/6 NPs of different NR doping concentration made by different synthesis methods were all similar to undoped PF2/6 NPs (ca. -30 mV), see Figure S2(d)-(i). The absorption spectra of 0.0 wt. % (black line), 1.0 wt. % (red line), and 2.0 wt. % (blue line) of Nile red-doped PF2/6 NPs for different methods are shown in Figure 4 (a)-(c). For each method,


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the two absorption peaks at ca. 380 nm and ca. 555 nm suggest the presence of both PF2/6 and Nile red in the doped NPs. The Nile red absorbance slightly increased with increasing of Nile red concentrations, however the light scattering contribution became more apparent (bigger absorption tails shown) from Method 1 to Method 3, as shown in the higher magnification of Nile red absorption peak in the insets of Figure 4 (a)-(c). The light scattering background subtracted absorption spectra of 1 wt. % NR doped PF2/6 NPs exhibited the same Nile red absorbance from method 1 to method 3 which illustrates that encapsulation of 1 wt.% Nile red across the different methods has been accurately controlled, as shown in Figure 4 (d). The emission spectra of Nile red-doped PF2/6 NPs synthesized by different methods and excited directly at Nile red, are shown in Figure 5 (a), (c) and (e). The intensity-normalized excitation spectra of these NPs collected at 635 nm (directly collect at Nile red) are shown in Figure 5 (b), (d) and (f). For each method, the emission intensities increase with increasing Nile red concentration. To directly compare the emission intensities of Nile red in different sized NPs, the emission spectrum overlaps of PF2/6 / NR (1 wt. %) NPs under directly excitation at Nile red is shown in Figure 5 (g). From method 1 to 3, the emission intensity of 1 wt.% Nile red decreases in a very pronounced manner, and the emission maxima determined by Gaussian fit are at ca. 633 nm, ca. 635 nm and ca. 639 nm, respectively while NR in water (black line in Figure 5 (g)) exhibited an emission maximum at 660 nm. The excitation spectra of Nile red in NPs maxima determined by Gaussian fit from method 1 to 3 are at 552 nm, 553 nm and 562 nm, respectively. Therefore, the emission and excitation spectra of Nile red in NPs exhibit gradual red shift from method 1 to 3 and all blue shift compared to Nile red in water. According to the solvatochromic properties of Nile red, the emission and excitation spectra exhibits an appreciable red shift with increase in solvent polarity 36, which indicates that the excited


17


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state of the Nile red molecule experiences a more polar environment than the ground state. 72 One possible cause could be the geometrical distortion of the molecule in the excited state. 72 In our case, the emission spectra of Nile red in nanoparticles blue shift compared with Nile red in water suggest the internal polarity of nanoparticles is lower than pure water and the gradual red shift of emission and excitation spectra from method 1-3 illustrated the internal polarity of NPs increasing from method 1 to 3. Also, the emission intensity of Nile red decreases with increasing solvent polarity

43

as the

activation barrier of TICT (twisted intramolecular charge transfer) process decreases. This results in an increase in the rate of non-radiative processes to the triplet state, which manifests as a decrease in the quantum yield of such molecules in a highly polar medium.36, 72 Herein, with PF2/6 NPs of increasing size, the emission intensity of Nile red decreases, also indicating that the polarity of the internal nanoparticles environment gradually increases from method 1 to 3 and the polarity of the nanoparticles are all smaller than water. In addition, the photoluminescence decay lifetime of Nile red decreases with increasing polarity of the solvent and TICT is the principle process responsible for the changes.72 The fluorescence lifetime data of PF2/6 / NR NPs (from left to right, method 1 to method 3) were measured upon exciting at 635 nm and collecting at 670 nm (exciting and collecting at Nile red); as seen in Figure 6 (a). Excited state lifetimes were extracted from the decay traces by fitting the measured photoluminescence decay traces with double exponential functions; see Figure 6 (c). Within each method, the lifetime remained unchanged with increasing Nile red concentration. The lifetime overlapping of 1 wt. % NR doped PF2/6 NPs made by different methods is shown in Figure 6 (b). The fast component decays are 0.84 ns (Method 1), 0.62 ns (Method 2) and 0.41 ns (Method 3). The slow component decays are 3.50 ns (Method 1), 2.97 ns (Method 2) and 2.37 ns (Method 3).


18

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The progressive decrease of fluorescence lifetime from method 1 to 3 is consistent with emission and excitation spectral observations, supporting the suggestion that increased polarity of internal nanoparticle environment is observed from method 1 to method 3. DCM as an Internal Probe Doped into PF2/6 Nanoparticles. In order to further check the internal polarity of PF2/6 NPs with different sizes, 4-dicyanomethylene-2-methyl-6(p-dimethylamino-styryl)-4H-pyran

(DCM)

another

hydrophobic,

highly fluorescent, solvatochromic dye was used. Intensity-normalized absorption, emission and excitation spectra of DCM in THF are shown in Figure S7. DCM in THF exhibit the maximum absorbance at 465 nm while the emission maximum is observed at 605 nm without any vibronic shoulder (under 465 nm excitation). Only one fluorescence vibrionic peak of DCM in polar solvents is clearly related to the degree of solute-solvent interaction.75 Similar with Nile red-doped PF2/6 NPs, DCM has been doped into conjugated polymer PF2/6 NPs of different sizes (45 nm, 55 nm and 75 nm for Method 1, 2,3, respectively). PF2/6 NPs with varying DCM concentration (1, 2, 3 wt. %) were made, as shown in Figure S8. The zeta potentials were all ca. -30 mV, see Figure S9. The absorption spectra of 0 wt. % (black line), 1.0 wt. % (red line), 2.0 wt. % (blue line) and 3.0 wt. % (pink line) DCM-doped PF2/6 NPs made by different methods are shown in Figure S10. For each method, the two absorption peaks (ca. 380 nm and ca. 475 nm) indicate the presence of both PF2/6 and DCM in the doped NPs. With increasing DCM concentration, the absorbance of DCM at ca. 475 nm slightly increases. In contrast to Nile red, DCM undergoes several ultrafast processes, such as intramolecular charge transfer, ICT, twisting about the double bond by photoisomerization and solvation dynamics. The formation of TICT conformers "twisted" around single bonds (connected


19


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dimethylamino group and pyran ring with two cyano groups) are stabilized in a polar solvent and are characterized by a high quantum yield of fluorescence. In a nonpolar solvent, TICT conformers are not stabilized by the solvent, and then the processes of rotation around double bond (connected dimethylamino group and pyran ring with two cyano groups) occur. Stereoisomers formed through rotation around a double bond are nonpolar or low-polar.76 A strong lowering of the energy level of stereoisomers in DCM "twisted" around a double bond, resulting in an effective nonradiative deactivation of the energy of electronic excitation has been demonstrated before. 48, 77-78 Therefore, the emission intensity of DCM is expected to increase in response to increasing solvent polarity. Herein, the PF2/6 NPs doped with different concentrations of DCM were excited directly at DCM and the emission spectra are shown in Figure 7 (a), Figure 7 (c) and Figure 7 (e). The intensity normalized excitation spectra collected directly at DCM were shown in Figure 7 (b), Figure 7 (d) and Figure 7 (f). In each method, DCM exhibits an emission maximum at 620 nm, while the intensities increase with increasing concentration of encapsulated DCM. To directly compare the emission intensity, the PF2/6 / DCM (3 wt. %) NPs emission spectra overlapping of three methods is shown in Figure 7 (g). From method 1 to 3, with the sizes increasing, the emission intensity increases in a very pronounced manner, and the spectra of DCM in NPs exhibited an emission maximum at ca. 625 nm, ca. 630 nm, and ca. 631 nm, respectively, while DCM in THF has the emission maximum at 605 nm which shows the emission of the DCM in the nanoparticles red shift gradually from method 1 to 3 and blue shift from the emission of DCM in THF. All of these phenomena indicate that as the size of the PF2/6 NPs increases, the polarity of the internal nanoparticles environment gradually increases to values higher than in THF, which is consistent with the observations of PF2/6 / Nile red NPs.


20

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For the purpose of checking the concentration of DCM, 0.5 mL of THF was added into 0.5 mL of 1 wt. % DCM doping NPs for all three sizes NPs. The hydrophobic NPs fell apart and CP chains re-dissolved back into THF resulting in DCM been released into THF/water environment. The emission spectra of the re-dissolved CP THF/ Water solution were measured under 470 nm excitation (directly on DCM), shown in Figure S11. For the three types of NPs, the emission intensities were all around 3.8 × 10 5 cps suggesting that an equivalent amount of DCM has been doped into different-sized PF2/6 NPs, and the increased emission intensity from method 1 to method 3 is due to an internal polarity increase and not experimental variation. Also, these water/THF samples were diluted by half compared to the original concentration of DCM-doped PF2/6 NPs in aqueous suspension. Thus, the emission intensity of DCM in THF/water solution at its original concentration should be 7.6 × 105 cps which is higher than any 1 wt. % DCM in PF2/6 NPs, indicating the polarity of NPs is lower than in a 50:50 (THF: water) environment. In addition, the emission spectra maxima of DCM in re-dissolved water/THF solutions (640 nm) shows a 20 nm red shift compared to DCM in NPs (620 nm). As we know from literature, dipole-dipole interactions between solvent and solute stabilize polar excited-states, lowering their energy and therefore red-shifting the fluorescence band of DCM 79-80 Hence, the polarity of the internal PF2/6 NPs is higher than THF and smaller than 50 % water/ 50 % THF. Localization and restricted motion of dyes in NPs. Another important aspect of the encapsulation of dye molecules inside the polymer nanoparticles is their restricted motion which directly controls the photophysics of encapsulated dye molecules. 31 To elucidate the relation between photophysics of encapsulated dye molecules and their rotational behaviors in the abovementioned nanoparticles from the three synthesis methods, time-resolved fluorescence anisotropy measurements were performed, as shown in Figure 8. Single exponential decay is used to fit the


21


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anisotropy of PF2/6 / NR NPs. The r(0) values for Nile red in PF2/6 / NR (2 wt. %) NPs were 0.12 (method 1), 0.10 (method 2) and 0.10 (method 3), respectively, and the rotational correlation time were summarized in Figure 8 (c). The anisotropy decay of PF2/6 / DCM NPs exhibited double exponential decay behaviour. The r(0) values for DCM in PF2/6 / DCM (2 wt. %) NPs were 0.28 (method 1), 0.28 (method 2) and 0.25 (method 3), respectively, and the rotational correlation time for DCM in water were summarized in Figure 8 (c). Following the method in our previous published paper52 , the “wobbling-in-cone” model was applied here. The diffusion coefficient of wobbling motion Dw of Nile red in NPs for method 1-3 are calculated as 7.5 × 108 s-1 , 8.3 × 108 s1,

and 7.6 × 108 s-1 , respectively. The semiangle of wobbling θ for all synthesis methods are 90º.

However, the Dw of DCM in NPs from method 1-3 are 5.1 × 106 s-1 , 4.5 × 106 s-1, and 9.2 × 106 s, respectively, and the θ of DCM in NPs are 12.6º, 14.3º, 15.8º, respectively. Therefore, Nile red

1

appears to diffuse relatively more freely within the polymer NPs for three different methods than DCM. The more freely diffusing Nile red in NPs may be due to its better water solubility than DCM. Internal polarity of nanoparticles. Lippert-Mataga polarity calculations have been applied here to calculate the polarity of the internal CPNPs,. The Lippert-Mataga polarity parameter Δf can be deduced using the following equation: ∆𝑓 =

𝜀−1 2𝜀+1

−

𝑛2−1 2𝑛2+1

(4)

where ε is the dielectric constant of the solvent and n is the refractive index of the solvent. In this equation, the first term (ε-1)/(2ε+1) represents the low-frequency polarizability of the solvent and the second term (n2 -1)/ (2n2 +1) represents the high-frequency polarizability of the solvent. According to Eq. 4 and reference data, different Lippert-Mataga polarity parameters (Δf) for Nile red and DCM in different solvents were calculated in S12. 59


22

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The following equation can be used to describe the energy difference between the ground and excited states of dyes as a property of Δf, called the Lippert-Mataga equation. 81-82 ∆𝜐 = 𝜐𝐴 − 𝜐𝐹 =

𝜆𝐹 −𝜆𝐴 𝜆𝐴 ×𝜆𝐹

=

2 ℎ𝑐

Δ𝑓

(𝜇𝐸−𝜇𝐺 )2 𝑎3

+𝑘

(5)

where λA and λF are the wavelengths of the absorption and emission maxima of the dye, respectively, h is Planck’s constant and c is the speed of the light; µE and µG are the dipole moments of the excited state and ground state of the dye; a is the radius of the cavity in which the dye resides. The values of the slope, 2(µE - µG2/hca3) and the intercept were obtained by linear fitting the plot of Stokes shift of NR in a series of solvents with solvent Lippert-Mataga polarity. Nile red and DCM was dissolved in a series of solvents and the absorption and emission spectra were measured. The absorption wavelength at maxima (λA) and emission wavelength at maxima (λF) were used to calculate Δν. By linear fitting Δν against Δf, the linear function y=4911.90x +1065.01 (for Nile red) and y=6810.96 x +3302.92 (for DCM) was achieved, as shown in Figure S12. By substituting stokes shift Δν for Nile in PF2/6 NPs achieved from method 1 to 3 into this linear function, the polarity of the PF2/6 NPs were calculated as 0.235, 0.240 and 0.252, respectively. The same calculation was applied into PF2/6 / DCM NPs and the polarity for method 1-3 were calculated as 0.251, 0.269 and 0.272, respectively, as shown in Figure 9 and Figure S12. These results indicate the internal polarity of PF2/6 NPs gradually increase from method 1 to 3 which is consistence with the spectra and lifetime changes. To compare the polarity calculated here with the polarity estimated from the result in Figure S12, the polarity of 50 % water / 50 % THF needs to be worked out. The dielectric constant ε MS and refractive index nMS of a water/THF mixture can be calculated using the following equations 83

𝜀𝑀𝑆 = 𝑓𝑤 𝜖𝑤 + 𝑓𝑠 𝜀𝑠


23


2 2 + 𝑓 𝑛2 𝑛𝑀𝑆 = 𝑓𝑤 𝑛𝑤 𝑠 𝑠

Page 24 of 48

(6)

where the subscripts “w” and “s” represent water and THF, respectively, and f represents their volume fractions. So the polarity of 50 % water / 50 % THF was calculated as 0.30. The nanoparticle polarity (0.27) is smaller than 50 % water / 50 % THF (0.30) and higher than THF (0.21) which is consistent with the conclusion made from Figure S11. To explain the swelling properties, conjugated polymer nanoparticles from reprecipitation method here were formed by rapidly injection the polymer / good solvent (THF) into large amount of bad solvent (water) under agitation. Mixing in this manner would result in a gradual decrease in solvent quality giving rise to the formation of a dispersion of polymer particles via collapse and coil of the polymer chains. Therefore, at the time of the chain coiling, there should be some confined/ caged water molecules inside the polymer NP. 31 In this case, the concentration of polymer / THF solution is extremely low (10 µg / mL) which could give rise to a relatively large volume of caged water being present inside the polymer NPs as the NPs form from THF droplets in water. Conclusion In this work, the internal morphology and polarity of conjugated polymer nanoparticles were fully investigated. Conjugated polymer PF2/6 nanoparticles of varying size were prepared using a reprecipitation method and the sizes were controlled using different injection speed and agitation power. Both light scattering-based DLS and fluorescence-based MEMFCS measurements corroborate that nanoparticles synthesized by method 1 to 3 increase in size from ca. 70 nm, ca. 95 nm to ca. 120 nm respectively. Single nanoparticle fluorescence imaging taken on a home build epi-fluorescence microscopy system shows the same fluorescence brightness for all the synthesis methods in single NP level suggesting very similar numbers of polymer chains in each type of


24

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NPs. The nanoparticles shrink down to the similar sizes ca. 10 nm after drying measured by AFM suggesting that the NPs are porous and porosity increases from method 1 to method 3 by progressive trapping of more water giving rise to an increased polarity of the NP internal environment. The solvatochromic fluorescent dyes Nile red and DCM were separately doped into the hydrophobic core of PF2/6 NPs. The photophysical properties of both of the two dyes agrees that the polarity of the nanoparticles increase with increasing PF2/6 NPs size which provide further evidence of more water trapped in NPs of larger size. The Lippert-Mataga polarity of PF2/6 NPs were calculated for both dyes and the gradual increasing of polarity form method 1 to 3 were observed. Furthermore, the restricted motion of the dye molecules in the nanoparticles has been investigated by the time-resolved anisotropy measurement. The anisotropy of the dye in differentsized PF2/6 NPs are the same indicating the randomness and rotational motion of the encapsulated dye doesn’t change among the three different synthesis methods and the motion restriction inside the nanoparticles doesn’t change with the size variation. Also, the wobbling diffusion coefficient were derived and the more freely diffusion for Nile red in NPs than DCM might be due to its better water solubility. Overall, this work helps give a more precise understanding of the internal morphologies of conjugated polymer NPs made by the reprecipitation method and it helps elucidate how further control of these formation processes may lead towards improvements in smart nanomaterial development. Associated Content Supporting Information. The Supporting Information is available free of charge on the ACS Publications website.


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Data sets presenting , ζ-potential distributions, lifetime fitting residuals, hydrodynamic diameters distributions measured for dye-doped and undoped PF2/6 NPs, photophysical characterization of Nile red and DCM in THF solution, PF2/6 in THF solution, dye doped PF2/6 NPs redissolving in THF, AFM images and height histograms of dye doped PF2/6 NPs, Lippert-Mataga polarity parameter of Nile red and DCM in different solvent. Author Information Corresponding Author *(G.R.) Telephone: + 353 1 716 2881. E-mail: [email protected] ORCID Suxiao Wang: 0000-0001-8048-1454 Gareth Redmond: 0000-0002-0507-7707 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. Acknowledgement S.W gratefully acknowledges the financial support by China Scholarship Council. Abbreviations ca.,circa; DLS,dynamic light scattering; CPNPs,conjugated polymer nanoparticles; Nile red, 9diethylamino-5-benzo[α]phenoxazinone;

DCM,4-dicy-anomethylene-2-methyl-6(p-


26

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dimethylamino-styryl)-4H-pyran; TPP,tetraphenylporphrin; PF2/6,poly[9,9-di-(2-ethylhexyl)fluorenyl-2,7-diyl]; THF,tetrahydrofuran; TCSPC,time-correlated single photon counting; EMCCD,electron multiplying charge-coupled device; FCS,fluorescence correlation spectroscopy; PDI,polydispersity index; MEMFCS,maximum entropy method fluorescence correlation spectroscopy


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Scheme 1. Schematic representation of the reprecipitation procedure of method 1, 2 and 3 for the synthesis of the different sizes of water-dispersed PF2/6 ±Dye NPs.


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Figure 1. (a) Number-averaged hydrodynamic size distributions of nanoparticles using different synthesis methods for PF2/6 nanoparticles. (b) Absorption spectra and (c) emission spectra (λexc. = 380 nm) of PF2/6 in THF and PF2/6 NPs from different synthesis methods. Inset of (c); excitation spectra of corresponding samples (λcoll = 420 nm) and photos of PF2/6 in THF (left) and PF2/6 NPs (right) under black light illumination. (d) Photoluminescence emission anisotropy spectra measured for a dilute PF2/6 / THF solution (black line), and aqueous dispersions of PF2/6 nanoparticles made by different methods. (e) Photoluminescence emission decays (λexc. = 380 nm, λcoll = 420 nm) measured for PF2/6 in THF and PF2/6 nanoparticles from different synthesis methods. The black lines indicate instrument response functions (IRF). (f) Summary of photoluminescence emission decay data of PF2/6 in THF and in nanoparticles.


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Figure 2. (a) Normalized auto correlation curves of PF2/6 NPs made from three different methods. (b)-(d) Overlay of extracted hydrodynamic diameters distribution of PF2/6 NPs from DLS (red) and MEMFCS fitting (grey) for PF2/6 NPs synthesized using method 1 to 3. Excitation power for FCS experiments was 400 nW.


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Figure 3. AFM images (inset, scale bar: 1 µm), height histograms from AFM images acquired on glass substrates (red), and number-averaged hydrodynamic size distribution from DLS (blue) of PF2/6 NPs synthesized using (a) method 1, (b) method 2 and (c) method 3. (d-f) Typical single nanoparticle photoluminescence intensity images and (g-i) single nanoparticle emission intensity (brightness) distribution of PF2/6 NPs for method 1-3, respectively.


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Figure 4. Absorption spectra of 0.5 wt. % (black line), 1.0 wt. % (red line) and 2.0 wt. % (blue line) of Nile red doped PF2/6 NPs made using (a) method 1(b) method 2 and (c) method 3. Insets: higher magnification of Nile red absorption peak in PF2/6 NPs. (d) Light scattering background contributions subtracted absorption spectra of 1 wt. % Nile red doped PF2/6 NPs.


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Figure 5. The photoluminescence spectra excited at Nile red (left) and excitation spectra collected at Nile red (right) of PF2/6 ± NR NPs with different Nile red concentration prepared using method 1: (a) & (b), method 2: (c) & (d) and method 3: (e) & (f). (g) and (h) are emission and excitation spectra overlapping of 1 wt. % NR doped PF2/6 NPs for each method.


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Figure 6. (a) Photoluminescence decays (λexc. = 635 nm, λcoll. = 670 nm) of PF2/6 / NPs measured for each synthesis method with varying Nile red dopant level. (b) Photoluminescence decay of PF2/6 / NR (1.0 wt. %) NPs for each preparative method. (c) Summary of the photoluminescence decay component data extracted by double exponential fitting of the decay traces.


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Figure 7. Photoluminescence spectra excited at DCM (left) and excitation spectra collected at DCM (right) of PF2/6 ±DCM NPs with different DCM doping concentrations of method 1: (a) & (b), method 2: (c) & (d) and method 3: (e) & (f). (g) and (h) are emission and excitation spectra of 3 wt. % DCM doped PF2/6 NPs for each method.


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Figure 8. Time-resolved anisotropy decay curves of (a) Nile red in PF2/6 / NR (2.0 wt. %) NPs (λexc. = 635 nm, λcoll. = 670 nm) for each synthesis method and (b) DCM in PF2/6 / NR (2.0 wt. %) NPs (λexc. = 488 nm, λcoll. = 620 nm) for each synthesis method. (c) Summary of time-resolved anisotropy decay component with data extracted by exponential fitting of the time-resolved anisotropy decay traces.


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Figure 9. Linear fitting of the Lippert-Mataga polarity parameter with the Stokes shift of (a) Nile red and (b) DCM in different solvent.


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Polymer Nanoparticles Microenvironment: Using Photophysical

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