Non-Traditional Intrinsic Luminescence (NTIL): Dynamic Quenching

Article Cite This: J. Phys. Chem. C 2019, 123, 18007−18016

pubs.acs.org/JPCC

Non-Traditional Intrinsic Luminescence (NTIL): Dynamic Quenching Demonstrates the Presence of Two Distinct Fluorophore Types Associated with NTIL Behavior in Pyrrolidone-Terminated PAMAM Dendrimers Maciej Studzian,†,‡ Łukasz Pułaski,‡,§ Donald A. Tomalia,∥,⊥,# and Barbara Klajnert-Maculewicz*,†

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†

Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/143 Pomorska Street, Lodz 90-236, Poland ‡ Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, Lodz 90-237, Poland § Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS, Lodowa 106, Lodz 93-232, Poland ∥ NanoSynthons LCC, 1200 N. Fancher Avenue, Mt. Pleasant, Michigan 48858, United States ⊥ Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States # Department of Physics, Virginia Commonwealth University, Richmond, Virginia 23284, United States S Supporting Information *

ABSTRACT: Historically, poly(amidoamine) (PAMAM) dendrimers were the first macromolecular structures reported to exhibit “non-traditional intrinsic luminescence” (NTIL) properties. Initially, this unique intrinsic luminescent property suggested the possibility of dendrimer-based biological imaging without the need for conjugating external labels. Unfortunately, low NTIL intensity levels exhibited by most simple surface-modified PAMAMs presented a serious barrier to progress in that area. Unexpectedly, a simple surface modification of amine-terminated PAMAM dendrimers with N-(4-carbomethoxy)pyrrolidone groups (4-CMP) was found to dramatically increase NTIL fluorescence intensity (i.e., >50-fold) while substantially enhancing biocompatibility and reducing cytotoxicity/complement activation properties. This current study focuses on the use of conventional and time-resolved spectroscopic measurements to characterize the NTIL behavior of 4-CMP PAMAM dendrimers over four generation levels (i.e., G2−G5). We describe and discuss the impact of polymer size and composition on NTIL intensity levels and quantum yields. Fluorescence lifetime measurements revealed two discrete major lifetime components, which were similar for all dendrimer generations and remained unaffected by changes in pH. Time-resolved fluorescence quenching studies involving a collisional quencher (methyl red) and a dynamic proximity quencher (nitrobenzoxadiazole dipeptide derivative) provided evidence for two spatially separated NTILtype emission sites within this 4-CMP PAMAM dendrimer series. In summary, these results provide important insights into the molecular-level NTIL mechanism and demonstrate the critical role of pyrrolidone surface modification as well as separate contributions made by interior dendrimer components to the observed enhancement of NTIL fluorescence intensity.

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host interactions4,5 and have been extensively reviewed elsewhere.6 Recently, a new photoluminescence phenomenon, referred to as “non-traditional intrinsic luminescence” (NTIL), has been described and extensively reviewed.7 This NTIL phenomenon has become of great interest and has been recognized as a new emerging property associated with many common dendritic frameworks such as poly(amidoamine) (PAMAM),8−10 poly-

INTRODUCTION

Dendrimers, members of a fourth major architectural class of synthetic macromolecules after classical linear, cross-linked and branched polymers, are distinguished by many unique properties derived from their hyperbranched features. These highly branched macromolecules are monodisperse, nanoscale in size, and adopt a globular shape with higher generations.1,2 It is recognized that despite exhibiting dense peripheries, certain dendrimers possessing symmetrical branch cell junctures (i.e., poly(amidoamine) (PAMAM))3 retain dynamic inner solvent filled cavities that enable encapsulation properties via guest− © 2019 American Chemical Society

Received: March 22, 2019 Revised: June 24, 2019 Published: June 25, 2019 18007

DOI: 10.1021/acs.jpcc.9b02725 J. Phys. Chem. C 2019, 123, 18007−18016

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The Journal of Physical Chemistry C

physicochemical fluorophore properties and their dependence on dendrimer−environment interactions.

(propyl ether imine) (PETIM)11,12 or poly(propylene imine) (PPI) dendrimers.13 Although these dendrimer families lack any traditional chromophore groups in their structure, it was shown that these aliphatic polymers, rich in internal tertiary amino groups, manifest unexpected luminescent properties when excited at near-UV wavelengths (340−380 nm). Although the physicochemical mechanisms underlying this phenomenon are becoming better understood, a current opinion is that NTILtype fluorophores may involve certain interior components of the dendrimer such as tertiary ammonium groups as well as imidic acid groups that may be excited, thus resulting in productive fluorescence due to dendritic architectural confinement within a protected and constrained nanoenvironment.14−16 Dendritic polymers were one of the first well-defined nanoparticles reported to exhibit NTIL-type autofluorescent properties in the absence of traditional fluorophores. However, this NTIL phenomenon, exhibited with variable intensity, has now been observed in many other types of linear and hyperbranched polymers.17−19 More specifically, for PAMAM dendrimers terminated with carboxyl groups, it was shown that both NTIL emission intensities and lifetimes increase with dendrimer generation.20,21 NTIL-type fluorescence for PAMAM dendrimers is also strongly increased at lower pH,22−24 although simple surface group modifications (i.e., amino, carboxyl, hydroxyl) do not seem to affect the luminescence process.23,25 At the same time, chemical oxidation of PAMAM or PPI dendrimers with ammonium persulfate was reported to enhance the observed emission although there is no simple explanation underlying this phenomenon.8,25,26 More recently, we have shown that fluorescence intensity of the nanoparticle is enhanced considerably when the surface amines of the PAMAM dendrimer are converted to N-(4carbomethoxy)pyrrolidone groups (4-CMP).27,28 Linear or branched polymers containing pyrrolidone moieties are well known for their dipolar aprotic properties, high solubility in organic solvents or water, as well as unusual biological compatibility, which allow them to be used in pharmaceutical formulations as excipients.29 Their other applications include use as emulsifiers, stabilizers, dispersing agents, solubility enhancers, or surfactants.30 Similarly, dendritic polymers decorated with pyrrolidone moieties become highly biorthogonal. Modification with pyrrolidone derivatives negates the high inherent toxicity of amino-terminated PAMAM dendrimers. 4CMP PAMAM dendrimers are virtually nontoxic for cell cultures in vitro. As opposed to “naked” PAMAM structures (i.e., amine terminated), they do not induce apoptosis or reactive oxygen species or changes in mitochondrial membrane potentials.31 It has also been shown that they display only minimal dendrimer−protein interactions and low complement activation properties, which are highly valuable for prototypical drug delivery systems.31−36 Thus, the unique biocompatible surface chemistry of 4-CMP PAMAM dendrimers in combination with their intrinsic fluorescence properties postures them up as high potential candidates suitable for exploitation in nanomedical applications. However, a more thorough understanding of the chemistry and physics behind their non-traditional fluorescence is required to comprehend the role of pyrrolidone moieties in defining these unique modified dendrimer properties. Therefore, we have set out to use time-correlated fluorescence spectroscopy and fluorescence quenching to draw inferences concerning the

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EXPERIMENTAL METHODS Materials. N-(4-Carbomethoxy)pyrrolidone-terminated PAMAM (4-CMP PAMAM) dendrimers with diaminobutane (DAB) cores (generations G2−G5) were synthesized and characterized as described previously.28 Chemical characterization of these compounds by NMR, mass spectrometry, and Fourier transform infrared spectroscopy has been previously published, confirming the structures ascribed to them.4,37,38 The purity of dendrimer preparations was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) with Coomassie staining (Figure S1). All dendrimers were dissolved in sterile phosphate-buffered saline (PBS) (pH = 7.4) to obtain stock solutions of 1 mM and were stored at 4 °C in darkness prior to use. A high-quality sample (fluorescence grade) of quinine used for fluorescence quantum yield (QY) measurement was purchased from Sigma-Aldrich. Its 1 mM stock solution as well as all dilutions were prepared in 0.1 M sulfuric acid and were kept in darkness prior to use. Methyl red sodium salt (ortho isomer of DABCYL: 2-(N,N-dimethyl-4aminophenyl)azobenzenecarboxylic acid sodium salt) and nitrobenzoxadiazole chloride (NBD-Cl; 4-chloro-7-nitro-1,2,3benzoxadiazole) used for time-resolved quenching studies were purchased from Sigma-Aldrich. To evaluate the influence of pH on dendrimer fluorescence, 50 mM buffers of acetic, HEPES, and glycylglycine were used to set its solution to pH equal to 3.2, 6.1, and 11.4, respectively. Spectroscopic Measurements. UV−vis absorption and fluorescence measurements were obtained at room temperature using a V-650 UV−vis spectrophotometer (JASCO) and an LS55 spectrofluorometer (PerkinElmer), respectively. Measurements were performed using a quartz cuvette with a 1 cm path length and low volume (0.7 mL). Fluorescence spectra used for quantum yield measurements were obtained with a scan speed of 600 nm min−1. Excitation and emission slits were kept constant (5.0 nm for excitation; 2.7 nm for emission). To evaluate fluorescence quantum yield of dendrimers, a comparative protocol was used.39 Since absorbance values of standard and test samples measured at the same excitation wavelength are proportional to the amount of the photons absorbed, a simple ratio of the integrated fluorescence intensities of the two samples relative to their molar extinction coefficient (recorded at excitation wavelength) gives the ratio of their fluorescence quantum yield (QY) values. QY of the test sample is then calculated from the equation ji I ·c zy εstandard, λEx QYtest = QY standard·jjj F test standard zzz· j IF standard·ctest z εtest, λ k { Ex

where IF is integrated fluorescence intensity at the given sample concentration, c is the sample concentration, and ελEx is the molar extinction coefficient at the given excitation wavelength λEx. Extinction spectra of dendrimers were collected and deconvoluted into absorption and Rayleigh scattering plots to obtain scatter-free absorption spectra. At higher generations, dendrimers are relatively large nanoparticles (5−6 nm diameters) and can significantly scatter light in the shorter wavelength range. Rayleigh scattering function was tail-fitted to experimental data assuming that the absorption of the 18008


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where Itotal is the total number of photons counted, and the intensity fraction f i equals

dendrimer chromophores starts below 450 nm, and the spectral range from 500 to 700 nm represents only the loss of transmitted light intensity due to out-scattering. This portion of the spectra was fitted to the equation A = log

fi = Ai τi /∑ Ai τi i

1 + A0 1 − kλ−4

Time-Resolved Fluorescence Quenching Measurements. For fluorescence quenching studies, the sodium salt of methyl red was dissolved in PBS at various concentrations (ranging from 0.1 to 1 mM) and mixed with equal volumes of 1 mM 4-CMP PAMAM dendrimer solutions in PBS. A stock solution (i.e., 10 mM) of the glycine dipeptide derivative of nitrobenzoxadiazole (NBD-GlyGly) was prepared by overnight reaction. The water-soluble conjugate was prepared by combining an appropriate aliquot of NBD-Cl with a 100 mM water solution of glycylglycine and adjusting the pH to 7.4. Dilutions of NBD-GlyGly were subsequently prepared in PBS (concentrations between 0.05 and 0.2 mM) and mixed with an equal volume of a 1 mM 4-CMP PAMAM dendrimer solution in PBS. Chemical structures of the quenchers are presented in Figure 1.

where λ is light wavelength, A0 is a constant, and k is a constant of proportionality. Subsequently, extinction spectra were corrected for Rayleigh scattering function extended to shorter wavelengths for each dendrimer generation. This deconvolution procedure was necessary to obtain true absorption of dendrimer chromophores at maximum excitation wavelength and hence to properly calculate molar extinction coefficients (ε380 nm) and fluorescence quantum yields (QYs). Fluorescence of 4-CMP PAMAM dendrimers has an excitation maximum at around 380 nm and emission maximum at around 460 nm.28 Quinine sulfate, a well-characterized dye with similar spectral properties, was used as a quantum yield reference (QY = 0.546). Molar extinction coefficients were calculated from the slope of absorbance values at 380 nm (out-scattering corrected for dendrimers) versus concentrations of the compound (concentrations up to 1 mM for 4-CMP dendrimers and up to 0.1 mM for quinine sulfate). Integrated fluorescence intensities of dendrimers and quinine sulfate were calculated from emission spectra recorded between 400 and 600 nm with excitation at 380 nm for various concentrations of dendrimers and quinine sulfate (from 0.05 to 0.5 mM for 4-CMP PAMAM dendrimers and from 2 to 10 μM for quinine sulfate). Time-Resolved Fluorescence Measurements. Fluorescence lifetime as well as fluorescence intensity (photon counts) was measured by the time-correlated single photon counting (TCSPC) method with the PicoHarp 300 module (PicoQuant, Germany) and a laser scanning microscope (LSM 780, Zeiss, Germany) equipped with 20 MHz pulsed 405 nm diode laser. Fluorescence emission intensity of 4-CMP PAMAM dendrimers excited with this wavelength equals around 80% of the maximum value (excited with 380 nm).28 In a single experiment, dendrimer solutions were excited at 21 °C, and the data from 10 scans of the volume of 72.9 pl (135 μm × 135 μm × 4 μm) of the sample was recorded and analyzed using the SymPhoTime 64 program (PicoQuant, Germany). To allow fluorescence intensity comparisons between different dendrimer samples, total photon counts were recorded for each polymer during the same period of time. Prior to time-correlated counting, photons were filtered using a CFP emission filter (475/28 nm). A multiexponential fitting model was used to calculate fluorescence lifetimes and amplitude parameters for fluorescence decay histograms (corrected for instrument response function measured separately) according to the equation I (t ) =

Figure 1. Chemical structures of compounds used to quench the intrinsic fluorescence of 4-CMP PAMAM dendrimers.

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RESULTS Quantum Yield and Brightness of Non-Traditional Intrinsic Luminescence (NTIL) of 4-CMP PAMAM Dendrimers. The emission intensity of non-traditional intrinsic luminescence (NTIL) for 4-CMP PAMAM dendrimers is known to be many-fold higher than their amino-terminated counterparts and to increase with dendrimer generation.28 At the same time, excitation and emission maxima remain unchanged and equal ∼380 and ∼460 nm, respectively. To quantitatively compare fluorescence intensity of four different generations of 4-CMP PAMAM dendrimers (G = 2−5), exact measurements of the molar extinction coefficient, quantum yield, and brightness at the excitation maximum were undertaken. At first, we determined absorption spectra of 4-CMP PAMAM dendrimers. Measured extinction spectra were corrected for Rayleigh scattering, and deconvoluted absorbance spectra are shown in Figure 2. There is no apparent absorption band at 380 nm that can be related to well-defined traditional chromophoric moieties, but as can be expected, with increase in generation, dendrimers gradually absorb more light. Next, we set out to evaluate fluorescence quantum yield (QY) for each dendrimer generation using the comparative method and quinine sulfate as a reference dye. Molar extinction coefficients at 380 nm (ε380 nm) were calculated for each dendrimer generation as well as for quinine sulfate (the value was very similar to that reported in the literature). Subsequently, fluorescence intensities were measured for dendrimers and quinine sulfate for various concentrations, wherein, using these data, quantum yields and brightness were calculated. The obtained results are summarized in Table 1.

∑ Aie−t/τ

i

i

where Ai is the amplitude of the decay of the ith component at time t and τi is the lifetime of the ith component. Increasing numbers of fluorescent components were assumed in the multiexponential fitting model, and the goodness-of-fit was evaluated by the reduced chi-square parameter (χ2). Calculated fluorescence intensity (corresponding to theoretical photon counts) of the respective fluorescence component was obtained using the following equation

Ii = Itotal × fi 18009


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quantum yields of this non-traditional intrinsic fluorescence decrease slightly with increasing generation number. Time-Resolved Fluorescence Characteristics of 4CMP-PAMAM Dendrimers. To further characterize unusual intrinsic fluorescence properties of 4-CMP PAMAM dendrimers, we have measured their fluorescence lifetimes. For a given fluorophore moiety, fluorescence lifetime is defined as the average time it resides in the excited state. Importantly, although this value is not dependent on chromophore concentration, excitation intensity, or absorbance of the sample, it is sensitive to several microenvironmental parameters such as pH, heterogeneity in electron density distribution, molecular binding, or proximity of energy acceptors/quenchers. The time-correlated single photon counting (TCSPC) method was used to determine fluorescence lifetime of different generations of 4-CMP PAMAM dendrimers. We have assumed increasing numbers of fluorescent components in the multiexponential fitting model as summarized in Table 2. Fluorescence decay curves for 4-CMP PAMAM dendrimers solutions are complex, and the best goodness-of-fit (χ2) values were obtained when three different components were assumed. Regardless of dendrimer generation, three fluorescence lifetime components were found to be equal to approximately 0.4, 3, and 8 ns. Furthermore, we evaluated fluorescence intensities of each component for all examined generations of the 4-CMP PAMAM dendrimer. Intensity fractions for each component were obtained from fluorescence lifetimes and fluorescence decay curve amplitudes. Photon counts that correspond to the fluorescence intensity of respective components were then calculated for different 4-CMP PAMAM dendrimer generations. Obtained results are summarized in Table 3. In agreement with the previous data, fluorescence intensity of all fluorescence decay curve components increases almost 2-fold with each generation. Fluorescence intensity of the first component (lifetime ca. 0.4 ns) is always negligibly small (only around 1% of the total photons counted). Furthermore, independent of the generation, intensity fractions of the second and the third component are also constant (ca. 10 and 90%, respectively). Fluorescence Properties of 4-CMP-PAMAM Dendrimers at Different pH Values. Since the intrinsic

Figure 2. Deconvoluted absorbance spectra of 4-CMP PAMAM dendrimers. Extinction spectra of 0.5 mM dendrimers of different generations (G = 2−5) were measured and corrected for Rayleigh scattering as described in the Experimental Methods section.

Table 1. Selected Fluorescence Properties of 4-CMP PAMAM Dendrimersa generation

molar extinction coefficient (ε380 nm) (M−1 cm−1)

quantum yield (QY380 nm)

brightness (QY ε)

40 ± 6 61 ± 8 134 ± 5 249 ± 11 1152 ± 38

0.211 ± 0.003 0.192 ± 0.002 0.171 ± 0.002 0.159 ± 0.002 0.546

8 12 23 40 629

2 3 4 5 quinine sulfate a

Dendrimers were analyzed at 380 nm wavelength. Molar extinction coefficients (average ± standard error) were calculated from absorbance versus concentration slopes. Quantum yields (average ± standard error of mean (SEM)) were calculated from three independent experiments performed at four different concentrations using quinine sulfate as a reference dye.

In line with the previously reported data,27,28 fluorescence intensity (brightness) for 4-CMP PAMAM dendrimers increases almost 2-fold with each generation. Interestingly,

Table 2. Fitting of Fluorescence Lifetime Parameters to Fluorescence Decay Curves of 4-CMP PAMAM Dendrimers (pH = 7.4) Recorded by TCSPCa generation

exponential components

τ1 (ns)

2

1 2 3 1 2 3 1 2 3 1 2 3

6.92 ± 0.02 1.42 ± 0.01 0.42 ± 0.01 6.98 ± 0.03 1.50 ± 0.02 0.41 ± 0.01 6.66 ± 0.02 1.38 ± 0.01 0.40 ± 0.01 6.95 ± 0.03 1.53 ± 0.01 0.44 ± 0.01

3

4

5

τ2 (ns)

τ3 (ns)

χ2

7.64 ± 0.03 2.65 ± 0.07

7.96 ± 0.01

7.58 ± 0.03 2.97 ± 0.01

7.92 ± 0.03

7.32 ± 0.02 2.82 ± 0.02

7.70 ± 0.02

7.64 ± 0.03 2.76 ± 0.01

7.94 ± 0.04

7.62 ± 0.17 1.27 ± 0.03 1.01 ± 0.02 5.19 ± 0.07 1.23 ± 0.01 1.02 ± 0.01 13.78 ± 0.27 1.72 ± 0.03 1.07 ± 0.01 19.25 ± 0.25 1.76 ± 0.02 1.07 ± 0.02

a Fluorescence decay curve was fitted to mono-, bi-, or triexponential equations. Fluorescence lifetimes (τ) as well as respective reduced chi-square (χ2) parameters were calculated. Values are shown as average ± SEM (n = 4). Bold font indicates highest fit quality (values used for further calculations).

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Table 3. Intensity Fraction (percentage) of Each Fluorescence Decay Curve Component and Its Calculated Fluorescence Intensity for 4-CMP PAMAM Dendrimers (pH = 7.4)a generation

f1 (%)

I1

f 2 (%)

I2

f 3 (%)

I3

2 3 4 5

1.24 ± 0.07 1.03 ± 0.05 1.34 ± 0.04 1.12 ± 0.05

549 ± 31 623 ± 30 1637 ± 49 2332 ± 104

10.63 ± 0.75 10.73 ± 0.34 12.22 ± 0.40 10.70 ± 0.12

4715 ± 333 6517 ± 207 14 915 ± 488 22 243 ± 249

88.14 ± 0.81 88.24 ± 0.29 86.43 ± 0.37 88.17 ± 0.11

38 984 ± 358 53 532 ± 176 105 315 ± 451 183 230 ± 229

Fluorescence decay curves were fitted to the triexponential equation and intensity fractions (f i), as well as respective calculated fluorescence intensities (Ii) were obtained for each lifetime component (i) as described in the Experimental Methods section. Values are shown as average ± SEM (n = 4). a

Table 4. Changes in Fluorescence Lifetimes and Calculated Fluorescence Intensity of Two Major Fluorescence Decay Curve Components of the 4-CMP-PAMAM Dendrimer at Different pHa generation

pH

τ2 (ns)

I2 (×103)

τ3 (ns)

I3 (×103)

2

3.2 6.1 7.4 11.4 3.2 6.1 7.4 11.4 3.2 6.1 7.4 11.4 3.2 6.1 7.4 11.4

2.69 ± 0.06 2.66 ± 0.03 2.65 ± 0.07 2.67 ± 0.07 3.01 ± 0.03 2.89 ± 0.08 2.97 ± 0.01 2.72 ± 0.07 2.95 ± 0.04 2.76 ± 0.05 2.82 ± 0.02 2.65 ± 0.03 2.75 ± 0.02 2.74 ± 0.05 2.76 ± 0.01 2.60 ± 0.04

4.99 ± 0.17 4.94 ± 0.16 4.72 ± 0.30 4.70 ± 0.16 7.30 ± 0.24 6.38 ± 0.15 6.52 ± 0.30 6.02 ± 0.27 17.92 ± 0.98 14.70 ± 0.67 14.92 ± 0.63 14.60 ± 0.51 23.87 ± 0.72 22.42 ± 0.63 22.24 ± 0.36 19.90 ± 0.61

7.78 ± 0.04 7.92 ± 0.04 7.96 ± 0.01 8.01 ± 0.04 7.62 ± 0.04 7.83 ± 0.05 7.92 ± 0.03 7.87 ± 0.02 7.34 ± 0.03 7.60 ± 0.03 7.70 ± 0.02 7.57 ± 0.02 7.61 ± 0.02 7.82 ± 0.04 7.94 ± 0.04 7.98 ± 0.01

36.17 ± 1.31 38.55 ± 1.39 38.98 ± 1.09 38.29 ± 0.98 54.15 ± 1.53 53.45 ± 1.71 53.53 ± 1.54 53.47 ± 0.85 115.92 ± 3.48 103.40 ± 2.45 105.32 ± 2.68 102.14 ± 2.61 177.55 ± 3.05 182.87 ± 2.48 183.23 ± 2.36 177.32 ± 1.68

3

4

5

Values are shown as average ± SEM (n = 4).

a

Figure 3. Lifetime Stern−Volmer plots for two major fluorescence decay curve components of 4-CMP PAMAM dendrimers quenched with methyl red. Values are shown as average ± SEM (n = 3).

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Figure 4. Lifetime Stern−Volmer plots for two fluorescent components of 4-CMP PAMAM dendrimers quenched with NBD-GlyGly. Values are shown as average ± SEM (n = 3).

fluorescence of some PAMAM dendrimers was reported to be strongly influenced by changes in the acidity of the medium, we examined whether fluorescence lifetimes of the two major fluorescence components (second and third components), as well as their respective calculated fluorescence intensities, changed with a shift in pH. Fluorescence decay curves were recorded for each dendrimer generation at four different conditions. At pH = 3.2, all internal tertiary amines of the PAMAM dendrimer are fully protonated; at pH = 6.1 (i.e., pKa value of internal tertiary amines of the amino-decorated PAMAM dendrimer), pH = 7.4, and pH = 11.4, amino groups of the 4-CMP PAMAM dendrimer become gradually deprotonated. Unexpectedly, as summarized in Table 4, there are almost no changes in either fluorescence lifetimes or calculated fluorescence intensities for 4-CMP PAMAM dendrimers between various pH conditions. These dendrimers retained characteristic fluorescence lifetime values close to 3 ns (component 2) and 8 ns (component 3), and the relation between their calculated intensities remains virtually constant (ca. 1:8). Time-Resolved Fluorescence Quenching of 4-CMPPAMAM Dendrimers by Methyl Red. Next, to get some insight into spatial location(s) of putative fluorophore(s) corresponding to the two major fluorescence components, we determined how 4-CMP PAMAM dendrimers of different generations responded to fluorescence quenching. Initially, we decided to use methyl red, a nonfluorescent dye showing a strong absorption band overlapping with the fluorescence emission of these nanoparticles. Methyl red is expected not to enter inside the dendrimer shell structure (intercalate between dendrimer branches)we performed a qualitative control experiment by 1H NMR, which confirms this assumption, showing no chemical shift in any of the internal dendrimer protons in its presence (Figure S2). Fluorescence lifetimes of the dendrimers were measured in the presence of increasing

concentrations of methyl red, and lifetime Stern−Volmer plots were drawn separately for the second and the third time-resolved fluorescence decay curve component of each generation of the 4CMP PAMAM dendrimer (Figure 3). Component 3 in each dendrimer generation was found to be dynamically quenched by methyl red judging by the linear relationship between the quencher concentration and the lifetime ratio. Fluorescence lifetime of component 2 was not changed in the presence of methyl red; see Figure 4. Dynamic quenching constants as well as bimolecular quenching rates were calculated for different generations of the 4-CMP PAMAM dendrimer and are shown in Table 5. A Table 5. Lifetime Stern−Volmer Parameters of Quenching of Component 3 with Methyl Reda generation

KD (3) (M−1)

kq (3) (×109 M−1 s−1)

2 3 4 5

301.6 ± 6.6 285.0 ± 20.6 265.8 ± 24.5 270.2 ± 22.5

37.47 ± 0.88 36.11 ± 2.63 34.52 ± 3.05 33.75 ± 2.73

a Dynamic quenching constants (KD) and bimolecular quenching rates (kq) for the interaction of 4-CMP PAMAM dendrimers (0.5 mM) with methyl red. Values are shown as average ± SEM (n = 3).

slight decrease in the dynamic quenching constants was observed with increasing dendrimer generation, but there are no significant differences between bimolecular quenching rates. Time-Resolved Fluorescence Quenching of 4-CMPPAMAM Dendrimers by Glycylglycine Nitrobenzoxadiazole (NBD-GlyGly). Subsequently, we quenched the fluorescence of 4-CMP PAMAM dendrimers with a water-soluble derivative, namely, nitrobenzoxadiazole (NBD-GlyGly) (i.e., a glycine dipeptide conjugate), which is a relatively small, electron-rich, fluorescent dye molecule. As such, NBD-GlyGly 18012


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The Journal of Physical Chemistry C Table 6. Time-Resolved Stern−Volmer Parameters of Quenching of Components 2 and 3 with NBD-GlyGlya generation

KD (2) (M−1)

kq (2) (×109 M−1 s−1)

KD (3) (M−1)

kq (3) (×109 M−1 s−1)

2 3 4 5

1101.0 ± 56.9 1271.9 ± 52.6 665.9 ± 24.4 380.8 ± 14.3

437.7 ± 22.6 438.1 ± 18.1 244.9 ± 9.0 151.8 ± 5.7

416.8 ± 40.8 319.0 ± 16.7 273.0 ± 28.9 257.6 ± 33.3

52.4 ± 5.1 40.3 ± 2.1 35.5 ± 3.8 32.4 ± 4.2

a

Dynamic quenching constants (KD) and bimolecular quenching rates (kq) for the interaction of 4-CMP PAMAM dendrimers (0.5 mM) with NBD-GlyGly. Values are shown as average ± SEM (n = 3).

may be postulated to be able to penetrate the dendrimer shell structure (and potentially intercalate between dendrimer branches and interact with them)we performed a qualitative control experiment by 1H NMR, which indeed showed significant changes in chemical shifts in the presence of the dye specifically for signals from internal protons, even for those in the dendrimer core, confirming the presence of dye molecules inside the dendrimer (Figure S2). NBD derivatives are frequently used as a fluorescence donor in Förster resonance energy transfer (FRET)-like interactions, but NBD-GlyGly was used here as an energy acceptor (quencher) since its absorption spectrum overlaps significantly with the emission spectrum of 4CMP PAMAM dendrimers. Fluorescence lifetimes of 4-CMP PAMAM dendrimers were measured in the presence of increasing concentrations of NBDGlyGly; and again, lifetime Stern−Volmer plots were obtained separately for the second and the third time-resolved fluorescence decay curve component of each generation (Figure 4). Interestingly, both components were found to be dynamically quenched by nitrobenzoxadiazole but with varied intensity depending on dendrimer generation. Dynamic quenching constants as well as bimolecular quenching rates were calculated for the data presented in Figure 4 and are summarized in Table 6. For both major fluorescence decay curve components of 4-CMP PAMAM dendrimers, bimolecular quenching rates with NBD-GlyGly decrease with increasing dendrimer generation (i.e., the only exception being between G2 and G3 for the second component). Discussion. Although the phenomenon of non-traditional intrinsic luminescence (NTIL) was initially reported for PAMAM dendrimers almost 20 years ago,20,21 the chemical identity of the fluorophore(s) responsible for this process (not necessarily identical in all relevant types of compounds) has remained elusive until recently.7 The main issue encountered by researchers when investigating this type of luminescence is that traditional techniques and theories are not easily adaptable to properly or fully explain the data obtained for non-traditional fluorophores. As such, we decided to attempt a more thorough physical characterization of this phenomenon in a subfamily of PAMAM dendrimers, namely, pyrrolidone-terminated dendrimers, where this surface modification was shown to significantly enhance fluorescence.27,28 In addition to the exact quantification of the NTIL quantum yield, it was necessary to use time-resolved techniques to gain deeper insights into the molecular mechanism of this fluorescence. For dendrimers as nonhomogenous macromolecules, many confounding physical parameters (i.e., including optical parametershigher scatteringand diffusional characteristics) had to be taken into account. Fortunately, dendrimers have two important advantages that allow a more meaningful analysis:

erations); therefore, it is possible to infer the importance of the surface-to-volume ratio and the distance between the external environment and potential internal fluorophores;40 they can be surface-modified, introducing an insulating layer, which is in itself demonstrably inert optically, but can influence internal fluorophores both by separating them from the environment and by shifting the electron distribution along dendrimer branches.41 Previously, pyrrolidone derivatization of surface amino groups in PAMAM dendrimers has only been shown to enhance fluorescence intensity.28 As described above, we quantified the contribution of increased excitation wavelength absorbance vs. increased fluorescence yield. On the other hand, amineterminated PAMAM dendrimers, while weakly fluorescent, were reported to have unmeasurably low fluorescence quantum yields.20 Similarly, other dendritic scaffolds (PETIM, PPI) showed very low quantum yield values.12,13 In our case, pyrrolidone modification ensured a relatively high, measurable value of quantum yield (ca. 15−20%), which compares favorably with many other traditional macromolecular fluorophores. Similar and even higher values have been reported for “oxygenated” or “aged” PAMAM; however, a direct comparison is made difficult by differing experimental procedures and unclear chemical characteristics and homogeneity of the oxidant-modified molecules.8,25,42 As such, it is clear that pyrrolidone termination is the first well-defined type of PAMAM dendrimer surface modification that produces enhanced fluorescence intensities and quantum yields to values acceptable for use as labels in biological systems. Interestingly, the literature reports that a correlation exists between larger, higher generation dendrimers and brighter fluorescence (e.g., PETIM dendrimers12 or triazine dendrimers40). On the other hand, our analysis showed that for 4CMP-PAMAM dendrimers, the quantum yield unexpectedly decreases slightly with an increase in dendrimer generation, wherein the increase in brightness appears to be mediated exclusively by a substantial increase in the absorption coefficient. As such, in spite of the fact that the number of active fluorophores increases with generation, it appears that some of them are environmentally modified in a way that decreases their excitation and/or emission efficiency. Moreover, if one assumes that every new dendrimer branch contained an active fluorophore, a quadratic increase in both absorbance and brightness would be expected. However, our results point to a crucial fluorescence role involving surface-to-volume relations. This suggests that only the outer, subsurface layer of potential fluorophores may be active in the described phenomenon, accompanied by an additional subtle quenching effect due to the increasingly bulky interior, which is especially visible in higher (4−5) generation dendrimers. Since intensity analysis can only yield limited information on the molecular mechanism of fluorescence, we applied TCSPC

it is possible to compare molecules with an analogous structure, differing in size (successive dendrimer gen18013


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The Journal of Physical Chemistry C for an in-depth analysis of putative fluorophores and their spatial distribution. Various NTIL-dendrimer types were previously reported to have 2 or 3 components within their time-resolved fluorescence decay curve. Similar to amino-terminated PAMAM10 or carboxyl-terminated PAMAM20 and PETIM,12 4-CMP PAMAM dendrimers exhibit 3 components. The first (shortest lifetime) component has negligible intensity (always around 1%), and for other dendrimers in the literature, it was also always the weakest component. Rather than ascribe it to a solvent, as Larson and Tucker did, we think it may be a synthetic impurity, an experimental artifact associated with the photon counting mode or potentially a very dim independent fluorophore center, since its intensity changes proportionally to dendrimer generation. Measured lifetimes of remaining components (i.e., described here as components 2 and 3) are comparable to those reported in the literature for other PAMAM-type dendrimers, especially those of higher generations (i.e., including those that have been treated with oxidants). This suggests a similar photoluminescent mechanism involving two distinct fluorophore centers.13−15,20 This distinction need not necessarily be considered structural or spatial. It could relate to different modes of excitation and/or different nanoenvironments involving the same compositional substructures within the interior of the dendrimeric macromolecule. Interestingly, while the ratio of fluorescence intensity between these components in unmodified, amine-terminated PAMAM dendrimer is close to 1:1, in 4-CMP PAMAM, component 3 is substantially (ca. 8-fold) brighter. This suggests that the increase of fluorescence intensity upon pyrrolidone modification of PAMAM is virtually exclusively due to an increase in brightness of the third, long-lived fluorescence component. Moreover, this ratio remains unchanged in pyrrolidone-modified dendrimers of varying sizes (generation), showing that addition of another dendrimer generation layer adds both types of fluorophore, thus strengthening the conclusion about a tight link between them. Contrary to unmodified, amine-terminated PAMAM,10 we show that for 4-CMP PAMAM there is no pH-dependent fluorescence behavior (i.e., either lifetimes of emission components or their intensity fractions). Thus, both fluorophore types/modes are not dependent on the protonation level. Since 4-CMP PAMAM has no primary amino groups remaining, and their presence has previously been shown to be dispensable for fluorescence,20,25 this demonstrates that the most probable spatial location of fluorophores is on the distal (subsurface) branches of the dendrimer, wherein they may require stabilization by electrostatic repulsion between charged branches (i.e., especially in the case of amino-terminated PAMAM)22 or by spatial order enforced by bulky terminal modifications, such as pyrrolidone moieties, involving possible zwitterionic imidic acid forms. We suggest that the resulting stiffening of the PAMAM branch extremities caused by total surface decoration with 4-CMP may selectively enhance the probability of a molecular state corresponding to the third (i.e., long-lived) fluorescence decay curve component, which can no longer be influenced by charge shift at lower pH. On the other hand, in unmodified, amine-terminated PAMAM dendrimers, this pH-based charge shift may only lead to partial stabilization and a fractional increase in fluorescence intensity compared with that in 4-CMP PAMAM.8,42 Therefore, terminal pyrrolidone modification is the preferred (i.e., covalent chemically welldefined) mode of enhancing PAMAM fluorescence for practical

applications since it does not significantly alter the NTIL mechanism as a function of pH. Since these experimental data allow limited insight into the spatial location and relationship between the two fluorophore centers/modes, we applied chemical quenching together with fluorescence lifetime analysis to gain deeper insights for this reasoning. The prototypical (DABCYL-like) collisional quencher, methyl red, which does not penetrate to the interior of the dendrimer molecule, demonstrated that the two fluorescent components indeed represent distinct fluorophore types rather than excitation modes of a single fluorophore: one (longer-lived, third component) accessible to dynamic collisional quenching and one (shorter-lived, second component) inaccessible. The location of the “third component” fluorophore can be further inferred from the lack of dependence of the collisional secondorder kinetic constant (bimolecular quenching rate constant) on dendrimer generation. Thus, the fluorophore moieties must all be located exclusively at a constant (low) distance from the external dendrimer surface. On the other hand, the smaller (and more dendrimer-soluble) NBD-GlyGly, which is a dynamic proximity (nonbinding, FRET-like) quencher,43 was able to enter inside the dendrimer molecule and quench both fluorophore types. However, in this case, the bimolecular quenching rate constants for both fluorophores significantly decrease with increasing dendrimer generation, reflecting the more restricted access to the interior (where NBD-GlyGly has to enter to efficiently quench both fluorophores due to its lower quenching radius in comparison with methyl red44) due to tighter pyrrolidone shell packing inherent in higher generations. This is even more obvious taking into account that for generations 2 and 3, where the terminal pyrrolidone shell is still relatively open, the quenching constant for the second component does not decrease significantly.

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CONCLUSIONS

In conclusion, 4-CMP PAMAM appears to exhibit NTIL behavior by involving two distinct fluorophoric emission types (i.e., sites), which are distinguished by a difference in spatial accessibility, as well as fluorescence lifetimes. Especially in higher generation dendrimers, the terminal pyrrolidone shell has at least three important roles in this phenomenon: (a) it sterically stabilizes dendrimer branches, allowing for more efficient formation of the fluorophore; (b) it restricts interior access by external solutes, thus preventing accidental quenching of the long-lived exciplex; and (c) it negates the importance of the protonation state (i.e., which is crucial for enhanced NTIL in unmodified, amine-terminated PAMAM dendrimers) by replacing external primary amines and stabilizing the location of internal ternary amines and amides independent of pH. Moreover, results on non-traditional fluorescence in dendrimers in our studies and in the bulk of the literature suggest that electron density effects are as crucial as steric stabilization.11,45,46 We suggest that a disruption of regular electron density gradient along dendrimer branches (in their distal parts) leading to electron resonance (e.g., by electron−hole recombination) is necessary for formation of a stable fluorophorethis may be achieved by noncovalent interactions with molecular oxygen,8,26,42 by introduction of carbonyl or ether groups within the branches,12,19,46 or by surface modification with an electronrich moiety such as 4-carbomethoxypyrrolidone. Thus, 4-CMP PAMAM is uniquely suited for further studies on application in bioimaging and labeling. 18014


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(11) Bagul, R. S.; Rajesh, Y. B. R. D.; Jayamurugan, G.; Bera, A.; Sood, A. K.; Jayaraman, N. Photophysical Behavior of Poly(Propyl Ether Imine) Dendrimer in the Presence of Nitroaromatic Compounds. J. Photochem. Photobiol., A 2013, 253, 1−6. (12) Jayamurugan, G.; Umesh, C. P.; Jayaraman, N. Inherent Photoluminescence Properties of Poly(Propyl Ether Imine) Dendrimers. Org. Lett. 2008, 10, 9−12. (13) Antharjanam, P. K. S.; Jaseer, M.; Ragi, K. N.; Prasad, E. Intrinsic Luminescence Properties of Ionic Liquid Crystals Based on PAMAM and PPI Dendrimers. J. Photochem. Photobiol., A 2009, 203, 50−55. (14) Jasmine, M. J.; Kavitha, M.; Prasad, E. Effect of SolventControlled Aggregation on the Intrinsic Emission Properties of PAMAM Dendrimers. J. Lumin. 2009, 129, 506−513. (15) Jasmine, M. J.; Prasad, E. Fractal Growth of PAMAM Dendrimer Aggregates and Its Impact on the Intrinsic Emission Properties. J. Phys. Chem. B 2010, 114, 7735−7742. (16) Ji, Y.; Qian, Y. A Study Using Quantum Chemical Theory Methods on the Intrinsic Fluorescence Emission and the Possible Emission Mechanisms of PAMAM. RSC Adv. 2014, 4, 58788−58794. (17) Pastor-Pérez, L.; Chen, Y.; Shen, Z.; Lahoz, A.; Stiriba, S.-E. Unprecedented Blue Intrinsic Photoluminescence from Hyperbranched and Linear Polyethylenimines: Polymer Architectures and pH-Effects. Macromol. Rapid Commun. 2007, 28, 1404−1409. (18) Wu; Liu, Y.; He; Goh, S. H. Blue Photoluminescence from Hyperbranched Poly(Amino Ester)s. Macromolecules 2005, 38, 9906− 9909. (19) Yang, W.; Pan, C.-Y. Synthesis and Fluorescent Properties of Biodegradable Hyperbranched Poly(Amido Amine)s. Macromol. Rapid Commun. 2009, 30, 2096−2101. (20) Larson, C. L.; Tucker, S. A. Intrinsic Fluorescence of Carboxylate-Terminated Polyamido Amine Dendrimers. Appl. Spectrosc. 2001, 55, 679−683. (21) Varnavski, O.; Ispasoiu, R. G.; Balogh, L.; Tomalia, D.; Goodson, T. Ultrafast Time-Resolved Photoluminescence from Novel Metal− Dendrimer Nanocomposites. J. Chem. Phys. 2001, 114, 1962−1965. (22) Huang, J.-F.; Luo, H.; Liang, C.; Sun, I.; Baker, G. A.; Dai, S. Hydrophobic Brønsted Acid−Base Ionic Liquids Based on PAMAM Dendrimers with High Proton Conductivity and Blue Photoluminescence. J. Am. Chem. Soc. 2005, 127, 12784−12785. (23) Wang, D.; Imae, T. Fluorescence Emission from Dendrimers and Its PH Dependence. J. Am. Chem. Soc. 2004, 126, 13204−13205. (24) Wang, Y.; Niu, S.; Zhang, Z.; Xie, Y.; Yuan, C.; Wang, H.; Fu, D. Reversible PH Manipulation of the Fluorescence Emission from Sectorial Poly(Amido Amine) Dendrimers. J. Nanosci. Nanotechnol. 2010, 10, 4227−4233. (25) Lee, W. I.; Bae, Y.; Bard, A. J. Strong Blue Photoluminescence and ECL from OH-Terminated PAMAM Dendrimers in the Absence of Gold Nanoparticles. J. Am. Chem. Soc. 2004, 126, 8358−8359. (26) Haupert, L. M.; Simpson, G. J.; Slipchenko, L. V. Computational Investigation of Amine−Oxygen Exciplex Formation. J. Phys. Chem. A 2011, 115, 10159−10165. (27) Janaszewska, A.; Studzian, M.; Petersen, J. F.; Ficker, M.; Paolucci, V.; Christensen, J. B.; Tomalia, D. A.; Klajnert-Maculewicz, B. Modified PAMAM Dendrimer with 4-Carbomethoxypyrrolidone Surface Groups-Its Uptake, Efflux, and Location in a Cell. Colloids Surf., B 2017, 159, 211−216. (28) Konopka, M.; Janaszewska, A.; Johnson, K. A. M.; Hedstrand, D.; Tomalia, D. A.; Klajnert-Maculewicz, B. Determination of NonTraditional Intrinsic Fluorescence (NTIF) Emission Sites in 1-(4Carbomethoxypyrrolidone)-PAMAM Dendrimers Using CNDPBased Quenching Studies. J. Nanopart. Res. 2018, 20, No. 220. (29) Haaf, F.; Sanner, A.; Straub, F. Polymers of N-Vinylpyrrolidone: Synthesis, Characterization and Uses. Polym. J. 1985, 17, 143−152. (30) Teodorescu, M.; Bercea, M. Poly(Vinylpyrrolidone) − A Versatile Polymer for Biomedical and Beyond Medical Applications. Polym.-Plast. Technol. Eng. 2015, 54, 923−943. (31) Janaszewska, A.; Ciolkowski, M.; Wróbel, D.; Petersen, J. F.; Ficker, M.; Christensen, J. B.; Bryszewska, M.; Klajnert, B. Modified PAMAM Dendrimer with 4-Carbomethoxypyrrolidone Surface Groups

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.9b02725. SDS-PAGE analysis for PAMAM dendrimers with N-(4carbomethoxy)pyrrolidone group (4-CMP) generations: G2−G5 was performed (Figure S1), interactions between the dendrimer and the quenchers (methyl red and NBDGlyGly) were studied by analysis of 1H NMR spectra of dendrimers; no changes in the presence of the nonintercalating methyl red (DABCYL) were observed, whereas the interaction-related change in chemical shifts for the intercalating NBD-GlyGly was shown (Figure S2) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Barbara Klajnert-Maculewicz: 0000-0003-3459-8947 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Polish National Science Centre (Project Harmonia “Intrinsically fluorescent dendrimers spectrofluorimetric and cell biology studies” UMO-2014/14/ M/NZ3/00498).

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REFERENCES

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Non-Traditional Intrinsic Luminescence (NTIL): Dynamic Quenching

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