Multicolored protein nanoparticles: synthesis, characterization and cell

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Multicolored protein nanoparticles: synthesis, characterization and cell uptake Bobbi S. Stromer, Sonali Roy, Melissa R. Limbacher, Bardwi Narzary, Manobjyoti Bordoloi, Julia Waldman, and Challa V. Kumar Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00282 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

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Bioconjugate Chemistry

Multicolored protein nanoparticles: synthesis, characterization and cell uptake Bobbi S. Stromer1, Sonali Roy2, Melissa R. Limbacher1, Bardwi Narzary2, Manobjyoti Bordoloi2, Julia Waldman1, and Challa Vijaya Kumar1,3* 1

University of Connecticut, Department of Chemistry, 55 N. Eagleville Road, Storrs CT 06269-3060

2

Natural Product Chemistry Group, Chemical Sciences & Technology Division, CSIR-North East Institute of Science and Technology, Jorhat, Assam, India, 785006

3

Department of Molecular and Cellular Biology, 91 N. Eagleville Road, U-3125, University of Connecticut, Storrs Connecticut 062693125 *Corresponding Author 55 N Eagleville Road, Storrs, CT 06269-3060 Phone: (860)-486-2012 Fax: (860)-486-3213 [email protected]

KEYWORDS: quantum dots, protein, nanoparticles, fluorescence, photobleaching, dynamic light scattering, gel electrophoresis, imaging ABSTRACT: Synthesis, characterization, and applications of strongly fluorescent, multicolored protein nanoparticles (GlowDots) are reported here. Bovine serum albumin was cross-linked under controlled conditions to form nanoparticles, where particle size was controlled from 20 to 100 ± 10 nm by choosing appropriate reaction conditions. The absorption as well as the emission wavelengths were controlled without changing the particle size, unlike quantum dots. Each GlowDot was loaded with up to 214 ± 50 chromophores and hence, the particles have high molar absorptivities (106 M-1cm-1) as well as high brightness (105 to 106 M-1cm-1). A large number of functional groups cover the particle surface and these are further functionalized to enhance cellular uptake. GlowDots that were labeled with fluorescein and functionalized with taurine, for example, were quickly taken up by HeLa, MDAMB-231, PC3, and L6 myoblast cells, as interrogated by fluorescence imaging studies. GlowDots were biocompatible, size tunable, biodegradable, strongly fluorescent, and stable for months at room temperature, and they may serve as substitutes for quantum dots in a variety of practical applications.

Introduction

ther surface modification of the GlowDots. Earlier reports of the synthesis of protein nanoparticles involved toxic solvents and resulted in a wide range of particles >100 nm in diameter.20 Thus, alternative approaches to making particles 106 M-1 cm-1) expressed in terms of particle concentration, and this high value is due to the large number of dyes present per particle. The number of dyes attached per particle is estimated by dividing the total dye concentration with particle concentration. The dye concentration was obtained from dye extinction coefficients by assuming that the extinction coefficients of the dye do not change upon attachment. The particle concentration was obtained by dividing the protein concentration with the number of BSA molecules present per particle (previously described). Particles had 109 to 215 dyes each, depending on the dye (Table 2). The large dif-

The absolute quantum yields of the labeled GlowDots (Table 2) were measured (SI, Figure S6) by using Rhodamine B (RhB) as a reference sample. The fluorescence quantum yield of GlowDot (Фf) was calculated by equation 2,

(2)

where Φr is the quantum yield of Rhodamine B28, Ir is the fluorescence intensity of the Rhodamine, and If is the fluorescence intensity of the GlowDot sample (10 mM phosphate buffer pH 7.2). Absorbance of the sample and the reference were matched at the absorption maximum, specific to each dye and the corresponding emission intensities were recorded. For example, absorbance of Rhodamine B and GlowDot494 in phosphate buffer pH 7.2 were adjusted to match at 494 nm and their emission intensities recorded at 520 nm. While quantum yields of GlowDots varied, these , , , are in a range that is similar to those of the quantum dots. 29 30 31 32

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Table 2: Photophysical properties of GlowDots Dyes per particle

ε (cm-1 M-1) particle

Relative Fluorescence Intensity

Quantum Yield, Φ

Brightness (M-1cm-1)

GlowDot340

169 ± 37

7.27 (± 2.2) x 106

37.0

0.14 ± 0.01

7.05 x 105

GlowDot350

175 ± 22

3.52 (± 0.6) x 106

1.2

0.33 ± 0.01

8.05 x 105

GlowDot432

119

4.05 x 106

12.7

1.06 ± 0.04

2.31 x 106

GlowDot494

109 ± 26

8.18 (± 2.8) x 106

0.4

0.45 ± 0.02

2.55 x 106

GlowDot543

110 ± 8

7.13 (± 0.8) x 106

1.6

0.19 ± 0.002

9.39 x 105

GlowDot576

214 ± 50

7.72 (± 2.5) x 106

0.9

0.17 ± 0.06

9.09 x 105

2.3.3 Photostability and Photobleaching Studies Photostability of the nanoparticles is important for their application in imaging studies where the samples are usually subjected to high light intensities for prolonged time periods. For these studies, GlowDots were irradiated at their corresponding peak absorbance wavelengths, with a 500 W mercury vapor lamp fitted with a monochromator (10 nm band pass) and a filter to cut off shorter wavelengths. The decrease in the sample absorbance was monitored as a function of irradiation time. GlowDot543 had the highest photostability, retaining 90 % of its absorbance after irradiation for one hour. This was followed by GlowDot494 and GlowDot576, both of which retained 90 % of their intensities for 30 minutes. GlowDot340, GlowDot350, and GlowDot432 had the lowest photostabilities, requiring only ten minutes to lower their intensity substantially. Thus, some samples were more stable than the others. These properties were quantified by measuring photobleaching quantum yields. Two separate actinometers, Tris(2,2'-bipyridyl)ruthenium (II) (Ru(bpy)3Cl2) and 9,10 diphenylanthracene (DPA)33 were used to calculate the photobleaching quantum yields. GlowDot494 or the free dye was used as the alternate actinometer for samples that did not absorb in the region suitable for the above chemical actinometers. Irradiation was carried out for 10 % loss in absorbance and the actinometer has been used to determine the number of photons absorbed by the samples, The photobleaching quantum yields were calculated (SI Figure S7) from these data. The lowest photobleaching quantum yields were noted with GlowDot576 (4.4 x 104 ) and GlowDot543 (3.1 x 10-3), indicating that they had the highest stability. In contrast, GlowDot340 and GlowDot432 had the highest values (Figure 8), indicating lower stabilities. The photobleaching quantum yield of GlowDot494 (1.9 x 10-2) was about two orders of magnitude higher than that of free FITC in water 1.2x10-4). 34 These photochemical reactions might distort the protein secondary structure and therefore, changes in protein secondary structure were examined by CD as described previously (SI, Figure S8). Less than 50 % of the protein secondary structure was retained for GlowDot340 and GlowDot350 while BSA itself did not lose significant structure, upon irradiation.(SI, Figure S9). To mitigate the photochemical instability of some GlowDot samples, we relabeled the irradiated samples with the same fluorescent dye. Thus, the photosensitive residues on the protein are partially consumed by the first round of irradiation and not available fully for the subsequent irradiations. By doing so, we ob-

served substantial increases in the photostability. For example, GlowDot432 particles were irradiated at 405 nm till 50 % bleaching was observed. Particles were then relabeled with the corresponding dye, purified and characterized by DLS (SI, Figure S10 D & E) and agarose gel electrophoresis (SI, Figure S10 F&G). The d ata showed that the dye used in the second round was covalently attached to the particle and that the particle size did not change significantly. The photostability and photobleaching quantum yields of the relabeled GlowDot432 particles were examined, which indicated a significant increase in the photostability. The photobleaching quantum yield for the relabeled particles was 7.4 ± 1.9 x 10-2 (4x decrease), as opposed to 0.3± 0.2 of the original GlowDot432 particles. Significant improvement was noted when the reactive site on the protein is pre-consumed by the first round of photoexcitation, and this could be a simple but effective strategy to improve the photostabilities of the GlowDot family of particles.

Figure 8: Summary of photobleaching quantum yields for GlowDots. The larger error associated with GlowDot432 is due to particle aggregation during the reaction and this causes significant light scattering with changes in its absorbance. 2.4 Adaptive surface modification of GlowDots One necessary element for a cell imaging agent is a high degree of adaptability and facile surface functionalization for fine-tuning cellular uptake. The GlowDot surface is naturally decorated with numerous amine and carboxyl groups which are amenable to the

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attachment of required ligands without any prior surface modification. The GlowDots were first modified with taurine and tested for uptake by a variety of cell lines. Taurine is an amino acid and it is known to increase the cellular uptake of small peptides.35 Taurine was conjugated with GlowDot280 by EDC coupling. The agarose gel electrophoresis of the modified GlowDot indicated a progressive movement of the modified samples toward the negative electrode as a function of increasing taurine concentration (Figure 9A). Thus, taurine labeling adds positive charges to the particle and eventually reverses their electrical charge (lanes 6 and 7).

ing the nucleus (z-axis = 0 at the bottom of the plate). As the top of the cell is approached, the green fluorescence begins to emit from the cell wall. The feasibility of imaging other cell lines by GlowDot494 was also tested by examining three additional lines: Breast cancer (MDA-MB-231) (Figure S12A), prostate cancer (PC3) (Figure S12B), and Rat myoblast (L6) (Figure S12C) cell lines have been tested. Uptake of GlowDot494 labeled with taurine was observed with all cell lines. From the images, it appeared that particles were located in the nucleus of MDA-MB-231 cells while the PC3 and L6 cell lines appeared to have the particles mostly located in the cytoplasm as in the case of HeLa cells. Further work needs to be done to more precisely determine where the particles are located after they are taken up by different cell lines. No morphological changes in cell structure were observed after co-incubation of each of these cell lines with 0.3 mg/mL GlowDot494 for 3 h, suggesting that GlowDot494 has little to no toxicity. To confirm that the particles had low to no toxicity, GlowDot494 treated cells were tested by the standard MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The same four cell lines were studied. The assay was done following the manufacturer's guidelines (Merck Millipore) and the cells examined to determine the number of viable cells in a sample (Table 3).

A.

Table 3: Results of MTT Assay for the cytotoxicity after incubation with GlowDot494, per cent viable.

Control

100

100

100

100

Treated

98.6

97.2

96.9

99.4

A fresh batch of cells were seeded into multiwell plates into complete medium and incubated for 24 h at 37 ⁰C and 5 % CO2. The medium was ten replaced with FBS free medium and GlowDot494 added so that the final concentration in the well was 0.3 mg/mL. After cells were incubated with GlowDot494 for 3 h at 37 ⁰C 5 % CO2, MTT assay was performed according to manufacturer’s instructions. More than 96 % of the cells remained viable in all four cell lines after the 3 h exposure. This concluded the morphological observations from cellular imaging and supported the conclusion that GlowDots are not toxic. The facile synthesis of modified particles taken up by different cell lines and nontoxicity to cells at these levels demonstrates the viability of GlowDots as a suitable nontoxic imaging agents. Table 4. Summary of GlowDots physical properties compared to other types of imaging agents.

Mol

*n.d. – property not determined in literature. GlowDots (This Work)

GlowDot494 particles labeled with taurine (0.3 mg/mL in 10 mM phosphate buffer pH 7.2) were added to HeLa cells grown in Nunc glass slide 8-well microplates in DMEM for 24 h. Cells and particles were co-incubated for 3 h at 37 ˚C in 5 % CO2. Before imaging, the cells were rinsed three times with 10 mM phosphate buffer pH 7.2 to remove any particles that were not taken up by the cells. Green fluorescence could be seen inside the cells (Figure 9B), and at various Z-axis values when imaged by confocal microscopy (SI, Figure S11). Overlaying the transmission channel with the monitoring fluorescence from the 488 nm excitation laser, it appears that the particles are located in the cell walls and cytoplasm. As the Z-axis image progresses from the bottom of the cell to the top, the fluorescence is constant in the space surround-

L6

Small NPs

Control studies were used to test if taurine modification is essential for cellular uptake or if it could be added to the solution with a similar effect. GlowDot494 (0.3 mg/mL in 10 mM phosphate buffer pH 7.2) and 4.5 µM taurine, equivalent concentration of the labeled particles, in 10 mM phosphate buffer pH 7.2 were coincubated with HeLa cells in DMEM for 1 and 3 h at 37 ˚C at 5 % CO2. The cells were washed three times with 10 mM phosphate buffer pH 7.2 to remove unbound particles; no fluorescence was observed from any of the cells. Thus, the presence of free taurine did not induce particle uptake and its attachment to the particles is essential.

PC3

Quantum Dots

Figure 9: A. Agarose gel (150 mM Tris Acetate pH 7.0) of modified GlowDots with taurine. The degree of modification was controlled by increasing EDC concentrations (10, 20, 40, 80, and 160 mM EDC, lanes 3-7, respectively). BSA (lane 1) and unmodified GlowDots (lane 2) shown for comparison. B. Cellular uptake of taurine modified GlowDot494 into HeLa Cells after co-incubation for 3 h. Cells were washed 3X with 10 mM phosphate buffer pH 7.0 to remove particles not taken up by the cells.

MDAMB231

High Quantum Yield

+

-

+

+

+

High tion

+

-

+

+

+

NIR-emission

+

+

+

n.d

+

Non-blinking

+

+

-

+

+

Liposomes

B.

HeLa

Polymer Dots

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


Absorp-

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Good Stability

+

-

+

+

+

NonPhotobleaching

+

-

+

n.d

-

Cytotoxicity

+

+

-

+

+

Modular

-

+

-

+

+

Adaptive

-

+

-

+

+

Inexpensive

n.d

n.d

-

n.d

+

Finally, the above properties of GlowDots are compared with those of QDs (Table 4). There are significant improvement in the ease of synthesis, lower toxicity, facile synthesis in most common laboratories, as well as a pathway to enhance photochemical stabilities. Furthermore, the particles are naturally soluble in water and nearly non-toxic to a number of cell lines tested here. Their low cost and high quality is noteworthy. Conclusions We have reported a facile synthesis of protein nanoparticles with tunable size (20 to 100 nm) with properties much like those of quantum dots. The size, absorption and emission properties of GlowDots are fully tunable. Furthermore, these properties are independent of the particle size for the most part, which cannot be achieved with conventional quantum dots. The strong molar absorptivities and high brightnesses of these particles are similar to those reported for heavy metal quantum dots and hence, these may be viable alternatives for cell studies as well as biological applications. Additionally, no special steps are required for solubilizing GlowDots in the aqueous media or biological media, and their surfaces are adaptable to chemical modification. Photostability experiments validate the particles’ potential as imaging agents due to their reasonable photostability which is an important requirement for practical applications. In addition, there is a systematic approach delineated to enhance the photochemical stabilities of these particles by pre-irradiation and re-labeling. These particles possess similar properties of QDs while addressing some of the areas that QD technology has yet to address. Because the particles are made of BSA, they may degrade in the cellular environment into the constituent amino acids, and hence, may be viewed as non-toxic for consumption.36 The highly controllable, tunable, and biologically friendly properties of GlowDots could be utilized in cellular imaging, and they are promising for drug delivery and other biological nanotechnologies as well. 4. Experimental Section 4.1 Proteins and chemicals Bovine serum albumin (fatty acid) was purchased from Equitech Bio. (Kerrville, TX). 1-pyrenebutanoic acid (λem= 376 nm), 7methoxycoumarin-3-hydroxy (λem= 410 nm), Diethylaminocouramine-3-carboxylic acid (λem= 472 nm), fluorescein isothiocyanate (λem= 519 nm), tetramethylrhodamine -5-(and-6)isothiocynate (λem= 571 nm), and 5-(and-6)-carboxy-xrhodamine (λem= 601 nm) were all purchased from Anaspec (Fremont, CA). Amicon ultra centrifugal filters were purchased from EMD Millipore (Billerica, MA). 1-(3Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS 25952-53-8) was purchased from TCI America (Portland, OR). Tris(2,2'-bipyridyl)ruthenium(II) Chloride Hexahydrate (CAS 50525-27-4) was purchased from TCI America (Portland OR). 9,

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10 Diphenylanthracene, 98 % was purchased from Thermo fisher Scientific (Bridgewater,NJ). 4.2 Synthesis of GlowDots Protein solution was prepared by stirring 150 mg of BSA in 1 mL of de-ionized water (dH2O). EDC (1M, dH2O) was added in 10 mM aliquots and stirred for 20 m between additions. Particle growth was monitored by dynamic light scattering (DLS). The reaction was quenched by adding 1 mL of 20 mM CO32-/HCO3pH 9.3 buffer. Sample was then diluted into 100 mM Phosphate 500 mM Sodium Chloride buffer pH 7.0 and heated to 85˚C for five minutes. Solutions were allowed to cool back to room temperature. Taurine powder was added to the nanoparticle solution (100x concentration of protein) and 100 mM EDC added dropwise to the solution. This was allowed to stir for 30 m. A solution of 1-pyrenebutanoic acid, (emission wavelength 340 nm, 0.5 % (m/m)) in DMSO was added to the protein solution and stirred for 2 h in the dark at room temperature. Samples were filtered with Amicon 100 kDa cutoff centrifuge filter tubes to remove unreacted dyes and protein. Buffer (10 mM Na2HPO4 pH 7.0) was added to wash the sample until filtrate absorbance spectrum was clear of fluorescent dye and protein. This method was also used for making GlowDots with the following fluorescent labels (0.5 % m/m): 7-Methoxycoumarin-3-hydroxy succinimidyl ester (350), Diethylaminocouramine-3-carboxylic acid (432), fluorescein isothiocyanate (494), tetramethylrhodamine -5-(and-6)-isothiocynate (543), and 5-(and-6)-carboxy-x-rhodamine (576) with appropriate adjustments as needed. All samples were denoted as GlowDotX where X is the emission wavelength of the dye used to label the particles. 4.3 Dynamic Light Scattering (DLS) Hydrodynamic radius of GlowDots was monitored by photon correlation spectroscopy with Precision Detectors (Varian Inc., part of Agilent Technologies), CoolBatch+ dynamic light scattering apparatus with 1 cm x 1 cm plastic cuvette and a 658 nm excitation laser source with a 90 ⁰ geometry. Data collection was done at room temperature, for 1 s, three repetitions with 200 accumulations. All samples were filtered with 0.22 µm filter (PDVF, 13 mm, Restek). Precision Ellucidate v 1.1.0.9 and Precision Deconvolve v 5.5 were used to collect and analyze the data respectively. 4.4 Agarose Gel Electrophoresis Agarose gels were prepared by dissolving agarose (0.5 % w/v, Sigma electrophoresis grade) in heated Tris acetate (40 mM, pH 7.0). The gel was poured on a horizontal electrophoresis apparatus (Gibco model 200, Life Technologies Inc., MD) and Tris acetate (40 mM, pH 7.0) was used as the running buffer. Samples were loaded into the wells at the center of the gel with 50 % (v/v) loading buffer (50 % v/v glycerol, 0.01 % m/m bromophenol blue). Electrophoresis was carried out for 30 min at 100 mV at room temperature. The gel was stained overnight with 0.02 % m/m Coomassie Blue, 10 % v/v acetic acid and then de-stained overnight with 10 % v/v acetic acid. This procedure was repeated with 160 mM Tris acetate buffer pH 9.0 for looking at the modification of GlowDots with taurine. 4.5 Circular Dichroism (CD) CD spectra were measured on a Jasco J-710 CD spectrometer. A concentration of 1.25 µM protein in 10 mM Na2HPO4 buffer pH 7.0 was used. Spectra were obtained using a 0.05 cm path length quartz cuvette in the region of 260 – 190 nm. Other operating parameters were: sensitivity 100 mdeg, data pitch 0.5 nm, continuous scanning mode, 50 nm/min scanning speed, 1 s response, 1.0 nm bandwidth and three accumulations. CD spectra were corrected by subtracting buffer signal from sample signal. Enzyme structure retention was assessed by calculating the change in ellipticity


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at 222 nm where BSA in 10 mM Na2HPO4 buffer pH 7.0 was taken as 100 % ellipticity. 4.6 Polyacrylamide Gel Electrophoresis A 7 % separating with 5 % stacking gel was used. Samples were prepared by adding loading buffer (10 µL, 2 % SDS, 10 % BME) to the sample then boiled for 2 minutes. Samples were loaded into the gel so that each well contained 6 µg of protein. The gel was run in SDS running buffer at 200 V constant in Bio-Rad Mini Protean Electrophoresis apparatus until the dye front was 1 cm from the bottom of gel plate. The gel was stained in Stain I (10 % v/v acetic acid, 10 % v/v isopropanol, 0.02 % coomassie blue) for 1 h. Gel was then placed in Stain II (10 % v/v acetic acid, 0.02 % coomassie blue) overnight. The gel was de-stained in 10 % v/v acetic acid until bands were clearly distinguished from clear background of gel. 4.7 Transmission Electron Microscopy (TEM) GlowDots solution (30 µg/mL) was applied to a carbon-coated Cu grid (400-mesh). An aliquot of 12 µL was incubated on the grid for 60 s, blotted with filter paper (Whatman #4), and stained with 5 µL of 1 % uranyl acetate for 10 s followed by blotting. The grid was dried overnight and then imaged using an FEI Tecnai T-12 TEM with an operating voltage of 120 kV. 4.8 Absorption Spectroscopy Absorption spectra were measured on an HP 8450 diode array spectrophotometer (Varian Inc., Santa Clara, CA). Samples were diluted to 0.412 mg/mL in 10 mM Na2HPO4 pH 7.0 and the baseline averaged from 700-800 nm was subtracted. 4.9 Fluorescence Spectroscopy Fluorescence Spectra were recorded on Cary Eclipse Fluorimeter. GlowDots were diluted to approximately 0.4 mg/mL in 10 mM Na2HPO4 pH 7. All spectral measurements were done in a 1 cm x 1 cm quartz cuvette. Extinction coefficients for the particles at the peak wavelengths were calculated by first estimating the particle concentrations using the total mass of protein present in 1 L of the suspension and the mass of one particle, assuming that the density of the particle is similar to that of the BSA. Extinction coefficients at specific wavelengths for particular particles were then calculated from the above absorbance data and the particle concentration Relative fluorescent quantum yields with respect to the corresponding free dye were calculated by comparing the peak emission intensity of a specific GlowDot to the peak intensity of the corresponding dye. The absorbencies of the two samples were matched at the same excitation wavelength and their emission intensities measured under the same conditions of buffer, pH and slit widths. 4.10 Photostability/Photobleaching Studies Photostability experiments were completed by monitoring loss in absorbance using UV/Vis spectroscopy at specific wavelengths (associated with confocal microscopy). In a small volume, 1 cm path length quartz cuvette, dilutions of each GlowDot were done using 10 mM phosphate buffer (pH 7.2). Samples were irradiated using a Bausch and Lomb high intensity monochrometer (Cat. Number: 33-86-76) with entrance slit set to four and the exit slit set to three. 4.11 Relabeling of GlowDots for increased photostability GlowDot particle were irradiated and monitored using UV/vis spectroscopy until the dye intensity decreased to less than 50 % of its original absorbance value. Upon reaching 50 % irradiation, particles were mixed with 0.5 % (m/m) fluorescent dye solution in DMSO and stirred for 2 hours at pH 9. After labeling, particles

were purified using dialysis in 10 mM sodium phosphate buffer, pH 7.4. Photostability experiments were carried out using the same methods listed in 4.10. 4.11 Microscopy studies For Z-Stack Imaging Studies HeLa cells were grown in DMEM at 37 ⁰C, 5 % CO2 for 24 hours in an 8 well chamber with cover plate bottoms. To each well of cells, 0.3 mg/mL GlowDot494+Taurine or GlowDot494 and 4.5 µM taurine was added. Samples were incubated at 37 ⁰C and 5 % CO2 for one or three h. Imaging was done on a Nikon A1R confocal microscope. The green channel was excited with 488 nm argon laser and monitored at 525 nm. All images were processed with FIJI (Fiji Is Just ImageJ). For Single-depth Imaging Studies L6 (Rat muscle cell line), HeLa (Human cervical cancer cell line), PC3 (Human prostate cancer cell line) and MDA-MB-231 (Human breast adenocarcinoma cell line) Cell Line procured from NCCS, Pune and cultured in respective complete media (DMEM for L6 and MDA-MB-231, MEM for HeLa, and Ham's F12k for PC3) supplemented with 10 % Fetus Bovine Serum (FBS), 10 % Penstrep, 1 % Gentamycin and incubated under standard conditions in 37 °C humidified 5 % CO2 atmosphere. After reaching confluence cells (1 x 106 per ml) were seeded in tissue culture grade 4 well Millicell EZ Slide (Millipore) in complete medium and incubated. After 24 h, the complete medium was replaced with FBS free medium and incubated overnight. The cells were then treated with GlowDot494 in a concentration of 0.3 mg/ml of media into each well and incubated for 3 h. Cell maintained in sample free medium served as control. After the treatment the cells washed three times with phosphate buffer to remove any particles not taken up by the cell. The slide removed from the holder and mounted with fluoroshield (Sigma). The mounted slide checked under fluorescent microscope (Motic AE31) and image of the cells captured with excitation at 495 nm.

4.12 MTT Assay

Cytotoxicity was evaluated by in vitro assay described by Roy et al 2016. After reaching confluence, cells (1 x 106 per ml) were seeded in tissue culture grade multiwell plates (Nunc) in complete medium and incubated at 37 ⁰C in 5 % CO2. After 24 h, the complete medium was replaced with FBS free medium and incubated at the same conditions previously mentioned overnight. The cells were then treated with samples (GlowDot 0.3 mg/mL) for 3 h. Well containing medium alone (untreated cells) serves as a control. The MTT assay was performed according to manufacturer’s guideline provided with the MTT assay kit (Merck Millipore). Briefly, after the treatment period (3 h), 10µl of MTT (5 mg/ml) was added into each well, mixed gently and again incubated for 4 hours. After incubation period, cells were viewed under an inverted microscope for the presence of dark purple crystals of formazan at the bottom of the wells. 0.1 mL isopropanol with 0.04 N HCl was added to each well and mixed thoroughly by repeated pipetting with a multichannel pipettor. The HCl converts the phenol red in tissue culture medium to a yellow color that does not interfere with MTT formazan measurement. The isopropanol dissolves the formazan to give a homogeneous blue solution suitable for absorbance measurement. The absorbance was measured on an ELISA plate reader (FilterMax F3 MultiMode Microplate Readers, Molecular Devices) with a test wavelength of 570 nm and a reference wavelength of 630 nm. All experiments were performed at least in triplicate. The effect of the samples on the proliferation of cells was expressed


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as the % cell viability, using the following formula (Khonkarn et al., 2010). % cell viability = Absorbance of treated cells /

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Absorbance of control cells × 100.

SUPPORTING INFORMATION AVAILABLE: Information about the fluorescent dyes used and more detailed data showing particle sizes determined by dynamic light scattering, agarose control studies, absorbance and fluorescence plots of GlowDots compared to their respective free dye, photostability and photobleaching studies, z-stack cell imaging, and cell imaging of MDAMB breast cancer, PC3 human prostate, and L6 rat myoblast uptake of GlowDot494. This material is available free of charge via the internet at http://pubs.acs.org.

ACKNOLEDGEMENTS SR is thankful to DST-SERB (SR/FT/LS-298/2012) and BN and MJB is thankful to CSIR (CSC-207 and CSC-130) for the financial support and the director of CSIR-NEIST, Jorhat, Assam for providing the facility and administrative support. 6YT.

ASSOCIATED CONTENT This material is available free of charge via the Internet at http://pubs.acs.org Figures show properties of reactive fluorescent dyes, DLS data, changes in Absorbance/fluorescence data

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Wegner, K.D., Hildebrant, N. (2015) Quantum Dots: bright and versatile in vitro and in vivo fluorescence imaging biosensor. Chem. Soc. Rev., 44, 4792-4834. 2 Vu, T. Q., Lam, W. Y., Hatch, E. W., Lidke, D. S., (2015) Quantum dots for quantitative imaging from single molecules to tissue. Cell and Tissue Research, 360, 71 – 86. 3 , T. Q., Lam, W. Y., Hatch, E. W., Lidke, D. S., (2015) Quantum dots for quantitative imaging from single molecules to tissue. Cell and Tissue Research, 360, 71 – 86. 4 Yoo, J. M., Kang, J. H., Hong, B. H., (2015) Graphene-based nanomaterials for versatile imaging studies. Chem Soc. Rev., 44, 4835 – 4852. 5 Mustafa, G., Komatsu, S. (2016) Toxicity of heavy metals and metal-containing nanoparticles on plants. Biochim. Biophys. Acta. 1864: 932-944. 6 Zhang, S., Guo, W., Wei, J., Li, C., Liang, X., Yin, M. (2017) Trrylenediimide-based intrinsic theranostic nanomedicines with high photothermal conversion efficiencty for photoacoustic Imaging-Guided Cancer Therapy. Che. 11, 3797 – 3805. 7 Massey, M., Wu, M., Conroy, E. M., Algar, W. R. (2015) Mind your P’s and Q’s: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications. Current Opinion in Biotechnology, 34, 30 – 40. 8 Cheng, W., Cheng, H., Wan, S., Zhang, X., and Yin, M. (2017) Dual-stimulus-responsive fluorescent supramolecular prodrug for antitumor drug delivery. Chem. Mater., 29, 4218 – 4226. ACS Paragon Plus Environment

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Yuan, H., Miao, J., Du, Y.-Z., You, J., Hu, F.-Q., and Zeng, S. (2008) Cellular uptake of solid lipid nanoparticles and cytotoxicity of encapsulated paclitaxel in A549 cancer cells. Int. J. Pharm. 348, 137-145. 10 Lian, W., Litherland, S. A., Badrane, H., Tan, W., Wu, D., Baker, H. V., Gulig, P. A., Lim, D. V., Jin, S. (2004) Ultrasensitivie detection of biomolecules with fluorescent dye doped nanoparticles. Anal. Biochem., 334, 135 – 144. 11 Agdeppa, E.D. and Spilker, M.E., (2009) A review of imaging agent development. AAPS. J. 11(2), 286-299. 12 Bilan, R., Fleury, F., Nabiev, I., Sukhanova, A., (2015) Quantum dot surface chemistry and functionalization for cell targeting and imaging. Bioconj. Chem., 26, 609 – 624. 13 Yong, K., Law, W., Hu, R., Ye, L., Liu, L., Swihart, M. T., Prasad, P., N. (2013) Nanotoxicity assessment of quantum dots: from cellular to primate studies. Chem. Soc. Rev. 42, 1236 – 1250. 14 Bradburne, C. E., Delehanty, J. B., Boeneman Gemmill, K., Mei, B. C., Mattoussi, H., Susumu, K., Blanco-Canosa, J. B., Dawson, P. E., and Medintz, I. L. (2013) Cytotoxicity of Quantum Dots Used for In Vitro Cellular Labeling: Role of QD Surface Ligand, Delivery Modality, Cell Type, and Direct Comparison to Organic Fluorophores. Bioconjugate Chem. 24, 1570-1583. 15 Ahmad, F., Pandey, A. K., Herzon, A. B., Rose, J. B., Gerba, C. P., Hashsham, S. A. (2012) Environmental applications and potential health implications of quantum dots. J. Nanoparticle Research, 14, 1038. 16 Raybould, A., Burns, A., and Hamer, M. (2014) High concentrations of protein test substances may have nontoxic effects on Daphnia magna: Implications for regulatory study designs and ecological risk assessments for GM crops. GM crops & food, 5(4), 296-301. 17 Nunn, B. L., Norbeck, A. Keil, R. G. (2003) Hydrolysis patterns and the production of peptide intermediates during protein degradation in marine systems. Marine Chemistry 83, 59. 18 Lill, J. R. , Ingles, E. S., Liu, P. S., Pham, V., Sandoval, W. N., (2007) Microwave assisted proteomics. Mass Spectrom Rev, 26, 657 - 671 19 Lohcharoenkal, W., Wang, L Chen, Y.C., Rojanasakul, Y. (2014) Protein nanoparticles as drug delivery carriers for cancer therapy. BioMed Res. Int. 2014. 20 Jun, J. Y., Nguyen, H. H., Paik, S., Chun, H. S., Kang, B., Ko, S. (2011) Preparation of size-controlled bovine serum albumin (BSA) nanoparticles by a modified dissolvation method. Food Chem., 127, 1892 – 1898. 21 Jumabekov, A.N., Cordes, N., Siegler, T.D., Docampo, P., Ivanova, A., Forminykh, K., Medina, D.D., Peter, L.M. and Bein, T. (2016) Passivation of PbS quantum dot surface with L – Gluthione in solid state quantum-dotsensitized solar cells. ACS Appl. Mater. Interfaces. 8, 4600-4607. 22 Gallard, C., Ghosh, Y., Steinbruck, A., Skyora, M. Hollingsworth, J.A., Klimov, V.I., and Htoon, H. (2011) Two types of luminescence blinking revealed by spectroelectrochemistry of single quantum dots. Nature. 479(7372), 203-207. 23 Peter T. Jr (1958) Serum Albumin. Adv. Protein. Chem. 37, 161-245. 24 Axelsson, I, J., (1978) Characterization of proteins and other macromolecules by agarose gel chromatography. J. Chromatog. 152(1): 21-32 25 Mahadevan, S., Erfle, J. D., Sauer, F. D. (1980) Degradation of soluble and insoluble proteins by Bacteroides Amylophilus protease and by rumen microorganisms. J. Animal Sci., 50(4),723 – 728. 26 Qin, W., Ding, D., Liu, J., Yuan, W. Z., Hu, Y., Liu, B., and Tang, B. Z. (2012) Biocompatible nanoparticles with aggregation-induced emission characteristics as far-red/near-infrared fluorescen bioprobes for In Vitro and In Vivo imaging applications. Adv. Funct. Mater., 22, 771 – 779. 27 Lim, S. J., Zahid, M. U., Le, P., Ma, L., Entenberd, D., Harney, A. A., Condeelis, J., Smith, A. M., (2014) Brightness-equalized quantum dots. Nat. Commun. 6: 8210 doi:10.1038/ncomms9210. 28 Magde, D., Rojas, G.E., and Seybold, P.R. (1999) Solvent dependence of the fluorescence lifetimes of xanthene dyes. Photochem. Photobio. 70(5): 737-744. 29 Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S., Nitschke, R., Nann, T., (2008) Quantum dots versus organic dyes as fluorescent labels. Nature Meth, 5, 763 – 775. 30 Bruns, O., Bischof, T. S., Harris, D. K., et al. (2017) Next-generation in vivo optical imaging with short-wave infrared quantum dots. Nat. Biomed. Eng., 1, 0056. ACS Paragon Plus Environment

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Orfield, N. J., McBride, J. R., Wang, F., et al., (2016) Quantum Yield Heterogenieity among Single Nonblinking quantum Dots Reviealed by Atomic Structure-Quantum Optics Correlation. ACS Nano, 10, 1960 – 1968. 32 Pu, C., Qin, H., Gao, Y., Zhou, J., Wang, P., Peng, X., (2017) Synthetic control of Excitation Behavior in Colloidal Quantum Dots. J. Am. Chem. Soc., 139, 3302 – 3311. 33 Pitre, S. P., McTiernan, C.D., Vine, W., DiPucchio, R., Grenier, M., Scaiano, J.C. (2015) Visible-light actinometry and intermittent illumination as convenient tools to study Ru(bpy)3Cl2 mediated photoredox transformations. Sci. Rep., 5. 16397 34 Widengren, J., and Rigler, R. (1996) Mechanism of photobleaching investigated by fluorescence correlation spectroscopy. Bioimaging. 4, 149-157. 35 Zhou, J. Du, X., Li, J., Yamagata, N., Xu, B. (2015) Taurine Boosts Cellular Uptake of Small D-Peptides for Enzyme Instructed Intracellular Molecular Self-Assembly. J. Am. Chem. Soc., 137, 10040 – 10043. 36 Stromer, B., Kumar, C.V. (2017) White-Emitting Protein Nanoparticles for Cell-Entry and pH Sensing. Adv. Funct. Mater., 27, 1603874.


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Multicolored protein nanoparticles: synthesis, characterization and cell

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