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Abstract
Core/shell nanoparticles composed of a silica core over which a propargyl methacrylate (PMA) shell was polymerized around were synthesized. To employ the shell coating, the surface of the silica nanoparticles (SiNPs) was modified with an alkene-terminated organometallic silane linker that allowed for the covalent attachment of a poly(propargyl methacrylate) (pPMA) shell. The alkyne groups resulting from the pPMA shell were utilized in copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) reactions to attach azide-modified Förster resonance energy transfer (FRET) pairs of naphthalimide (azNap), rhodamine B (azRhod), and silicon phthalocyanine (azSiPc) derivatives to the shell surface. The luminescence of the system was manipulated by the covalent attachment of one, two, or three of the fluorophores resulting in no energy transfer, one energy transfer, or two energy transfers, respectively. When all three fluorophores were attached to the core/shell particles, an excitation of azNap with a wavelength of 400 nm resulted in the sequential energy transfer between two FRET pairs and the sole emission of azSiPc at 670 nm. These particles may have applications as bioimaging probes as their luminescence is easily detected using fluorescence microscopy.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
The morphology and functionalization of silica-based nanoparticles (SiNPs) have been explored over the past several decades. Most research has focused on internally functionalizing SiNPs by encapsulating dyes, drugs, or proteins in the SiNPs during initial formulation [1–8]. Typically, these encapsulation techniques require difficult and lengthy synthetic procedures [9,10], where moieties are electrostatically conjugated, covalently bound through organosilane bonds, or sterically hindered by the SiNP to afford encapsulation. These techniques possess drawbacks that include undesirable leaching of the moiety from the SiNP, restrictions on the types of moieties that can be used due to a limited number of functional groups, and long durations needed to complete these reactions [11]. Alternatively, there has been limited research on externally functionalizing SiNPs [12–14]. Many SiNP surface functionalization techniques utilize amine group reactions and conjugations, requiring extensive synthetic procedures [15,16]. A copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) reaction has been used to attach organic dyes and maleimide groups to the surface of SiNPs; however, this synthesis required one week to complete [17]. Here, a facile and quick method to synthesize core/shell nanoparticles composed of a silica core and poly(propargyl methacrylate) (pPMA) polymer shell is presented. The pPMA shell of the core/shell nanoparticles decorates the particle surface with readily available alkynes such that the particles can be highly functionalized using CuAAC reactions. In this work, SiNPs coated with a pPMA shell were synthesized and subsequently used to attach up to three azide-modified fluorophores to the particle’s surface using rapid CuAAC reactions. Up to two Förster resonance energy transfers (FRETs) could be induced by attaching up to three fluorophores to the surface of the nanoparticles. The particles exhibited a 670 nm emission upon excitation at 400 nm, a 270 nm difference.
2. Results and discussion
In this work, core/shell nanoparticles composed of a silica core and a poly(propargyl methacrylate) (pPMA) polymer shell were synthesized. In brief, the silica nanoparticles (SiNPs) were produced using a standard Stöber synthesis and surface functionalized with a silane linker (3-methacryloxypropyltrimethoxysilane) that had a terminal, polymerizable double bond using a hydrolysis reaction. These particles were employed in a seeded, free radical emulsion polymerization resulting in SiNPs with a shell of pPMA (SiO$_2$/pPMA) (cf. Fig. 1(a)). The pPMA shell yielded free alkyne functional groups on the shell surface, which were exploited by covalently attaching an organic dye (2-(3-azidopropyl)-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (azNap) (D$_1$)) using a copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) reaction (SiO$_2$/pPMA/D$_1$). This simple system was used to ensure that the dye remained fluorescent once attached to the particle. Additionally, the core/shell nanoparticles were functionalized with multiple fluorophores that form Förster resonance energy transfer (FRET) pairs with one another to manipulate the system’s luminescence. Two or three fluorophores were attached to the nanoparticle’s surface such that one or two energy transfers could be induced, respectively. In the case of the SiO$_2$/pPMA/D$_2$ system, which possesses two fluorophores (D$_2$), azNap is the donor and rhodamine B 3-azido-propyl-1 ester (azRhod) is the acceptor. In the case of the SiO$_2$/pPMA/D$_3$ system, which possesses three fluorophores (D$_3$), azNap and azRhod remain FRET pairs with one another, but azRhod becomes a donor to azide-modified silicon phthalocyanine (azSiPc), which is the acceptor (cf. Fig. 1(b)). Furthermore, these particulate systems may have applications as bioimaging probes, so the pPMA layer also acts as a passivation layer to shield the inorganic particle from a biological environment.
Fig. 1. Schematic representation of (a) silica nanoparticles (SiNPs) functionalized with 3-methacryloxypropyltrimethoxysilane (MPS) (i.e. surface modification). These particles were then used in a seeded emulsion polymerization to attach a shell of poly(propargyl methacrylate) (pPMA) on the particle yielding core/shell SiO$_2$/pPMA particles (i.e. shell formation). (b) The core/shell particles were then modified with azide-modified fluorophores through copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) reactions. The fluorophores utilized were 2-(3-azidopropyl)-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (azNap) (D$_1$), azNap and rhodamine B 3-azido-propyl-1 ester (azRhod) (D$_2$), or azNap, azRhod, and azide-modified silicon phthalocyanine (azSiPc) (D$_3$) yielding particulate systems SiO$_2$/pPMA/D$_1$, SiO$_2$/pPMA/D$_2$, and SiO$_2$/pPMA/D$_3$, respectively.
2.1 Photophysics of SiO$_2$/pPMA/D$_1$ particles
To ensure the azNap dye remained photoluminescent upon covalent attachment to the nanoparticle, the absorbance and emission of the single molecule dye and SiO$_2$/pPMA/D$_1$ particles were assessed. The maximum absorbance of azNap was 402 nm, whereas the maximum absorbance of the SiO$_2$/pPMA/D$_1$ nanoparticles was found to be slightly red-shifted from the dye at 406 nm. The maximum emission of azNap was 518 nm, while the maximum emission of the SiO$_2$/pPMA/D$_1$ nanoparticles was slightly red-shifted from the dye at 521 nm (cf. Fig. 2). These findings are in good agreement with the literature that particles tend to cause a red-shift in the absorbance and photoluminescence of organic dyes [18].
Fig. 2. Absorbance (blue) and photoluminescence (red) spectra of azNap (dashed lines) and SiO$_2$/pPMA/D$_1$ nanoparticles (solid lines) in THF. Excitation wavelength was at 400 nm.
2.2 FRET capability of SiO$_2$/pPMA based nanoparticles
The dye combination of azNap, azRhod, and azSiPc was chosen because these dyes have good spectral overlap and form FRET pairs with each other. AzNap has a maximum absorbance of 402 nm with a broad maximum emission peak at 518 nm, which coincides well with the aggregate and monomeric absorbance of azRhod at 550 nm and 560 nm, respectively. AzRhod emits at 585 nm and overlaps the absorbance of azSiPc, which has aggregate absorbances beginning at ca. 610 nm with a maximum absorbance at 672 nm. AzSiPc has a maximum emission at 680 nm (cf. Fig. 3). The Förster distance between the azNap donor and azRhod acceptor is 4.71 nm and between the azRhod donor and azSiPc acceptor is 5.16 nm.
Fig. 3. Absorbance (solid lines) and photoluminescence (dashed lines) spectra of azNap (blue), azRhod (green), and azSiPc (red) with excitation at 400 nm, 550 nm, and 660 nm, respectively, for the photoluminescence spectrum. Notice the overlap of the emission of azNap with absorbance of azRhod and the overlap of the emission of azRhod with the absorbance of azSiPc. The concentration of each dye was 5 $\mu$g/mL in THF.
AzNap, azRhod, and azSiPc were covalently attached to the core/shell SiO$_2$/pPMA nanoparticle’s surface through multiple CuAAC reactions. When the SiO$_2$/pPMA/D$_3$ nanoparticles were excited at 400 nm (i.e., within the absorbance of azNap), the resulting emission occurred at 670 nm (i.e., the emission of azSiPc) with no other emission observed between 500 nm and 700 nm (cf. Fig. 4(a)). These results indicate that FRET occurs twice: once between azNap and azRhod followed by a second transfer between azRhod and azSiPc. The FRET efficiency (E$_F$) of the two energy transfers occurring in the SiO$_2$/pPMA/D$_3$ nanoparticles was calculated using the integrated intensities of the donor (I$_D$) and acceptor (I$_A$) emissions and Eq. (1).
In the SiO$_2$/pPMA/D$_3$ nanoparticles, the E$_F$ of the energy transfer between azNap (415 - 580 nm) and azRhod (580 - 650 nm) was 35% and the E$_F$ of the energy transfer between azRhod (580 - 650 nm) and azSiPc (650 - 750 nm) was 85%. To further investigate this FRET phenomenon, the particulate system was excited at 550 nm (i.e., within the absorbance of azRhod) and the only resulting emission occurred at 672 nm, confirming FRET between azRhod and azSiPc (cf. Fig. 4(b)). In this case, the E$_F$ of the energy transfer between azRhod (580 - 650 nm) and azSiPc (650 - 750 nm) was 86%. Additionally, the SiO$_2$/pPMA/D$_3$ nanoparticles were excited at 660 nm (i.e., the within the absorbance of azSiPc) and the emission of azSiPc at 670 nm was observed (cf. Fig. 4(c)). For all excitation wavelengths, the emission of the SiO$_2$/pPMA/D$_3$ nanoparticles remains at ca. 670 nm. It should be noted that when SiO$_2$/pPMA/D$_2$ nanoparticles were excited at 400 nm (i.e., within the absorbance of azNap), the particles were not highly luminescent. However, the azRhod serves as a conduit for the energy transfer observed in the SiO$_2$/pPMA/D$_3$ particulate system because a significant energy transfer does not occur without the azRhod being attached to the particle.
Fig. 4. Photoluminescence spectrum of SiO$_2$/pPMA/D$_3$ nanoparticles excited at (a) 400 nm, (b) 550 nm, and (c) 660 nm. At all excitations, only the emission from azSiPc is observed.
2.3 in vitro characterization via microscopy
The particulate systems’ cytotoxicity and potential bioapplication was investigated in a human hepatoma (HepG2) cell line and a human embryonic kidney (HEK293T) cell line. Cell viability of SiO$_2$, SiO$_2$/pPMA, and SiO$_2$/pPMA/D$_3$ particles in HepG2 cells were investigated at concentrations of 1.56$\times$10$^5$ and 1.56$\times$10$^4$ particles per cell, where the cells were incubated with particles for 24, 48, and 72 hours (cf. Fig. 5). The SiO$_2$, SiO$_2$/pPMA, and SiO$_2$/pPMA/D$_3$ particles showed no significant toxicity levels at both concentrations during the 24 - 72 hour time period.
Fig. 5. Cell viability of (a) SiO$_2$ particles (b) SiO$_2$/pPMA particles and (c) SiO$_2$/pPMA/D$_3$ particles in a HepG2 cell line at 1.56$\times$10$^5$ (blue) and 1.56$\times$10$^4$ (red) particles/cell for 24, 48, and 72 hours.
SiO$_2$/pPMA/D$_1$ and SiO$_2$/pPMA/D$_3$ particles were resuspended in PBS via sonication and ample vortexing prior to in vitro microscopy. Following serial dilutions, a concentration of 1$\times$10$^7$ particles per well was used for incubation viability analysis and was incubated for 24 hours with HEK293T cells. The SiO$_2$/pPMA/D$_1$ and SiO$_2$/pPMA/D$_3$ particles were excited with a 488 nm and 565 nm laser line, respectively. The light emitted from each system was easily detected by confocal microscopy (cf. Fig. 6). It should be noted that with the excitation at 565 nm, the azNap energy transfer is removed and only an energy transfer between azRhod and azSiPc occurs. Nonetheless, the energy transfer between azRhod and azSiPc occurred and was readily detectable in a biological system. Additionally, a time course was performed to ensure the viability of the cells with the SiO$_2$/pPMA/D$_1$ particles. The particles were incubated for 48 hours (1$\times$10$^8$ particles per well) and 72 hours (1$\times$10$^7$ particles per well) in HEK293T cells and were imaged with fluorescence microscopy. The cells were washed twice with PBS and once with fresh media to ensure the elimination of free floating particles that had not been endocytosed. The remaining particles appeared colocalized within the cells at both incubation periods and particle concentrations (cf. Fig. 7). No fluorescence was detected outside of cellular structures, indicating the particles’ endocytosis. Moreover, no significant toxicity levels were observed during the 24 - 72 hour time period, as all cells had normal morphology and were still proliferating (cf. Fig. 7).
Fig. 6. Fluorescence microscope images (using the ICC protocol described) of 4’,6-diamidino-2-phenylindole (DAPI) stained human embryonic kidney (HEK293T) cells incubated with SiO$_2$/pPMA/D$_1$ at dye concentrations of (a) 100 mg/well or (c) 10 $\mu$g/well excited with the blue (405 nm) channel and green (488 nm) channel. Also presented are fluorescence microscope images of DAPI stained HEK293T cells incubated with SiO$_2$/pPMA/D$_3$ at dye concentrations of (b) 100 mg/well or (d) 10 $\mu$g/well excited with the blue (405 nm) channel and red (565 nm) channel. Both sets of particles were excited with the blue (405 nm) channel to illuminate the DAPI stain, which stains the nucleus of the cell. All scale bars are 10 $\mu$m.
Fig. 7. Fluorescence microscope images of human embryonic kidney (HEK293T) cells after incubation with SiO$_2$/pPMA/D$_1$ nanoparticles for (a-c) 72 h with 1$\times$10$^7$ particles per well and (d-f) 48 h with 1$\times$10$^8$ particles per well. Presented are (a) & (d) brightfield images, (b) & (e) fluorescence images excited under the green (488 nm) channel, and (c) & (f) overlay of brightfield images with fluorescence images where the fluorescence images are pseudo-colored green. All scale bars are 50 $\mu$m.
3. Conclusion
Silica nanoparticles (SiNPs) were synthesized using a Stöber synthesis followed by a surface coating with a covalently bound poly(propargyl methacrylate) (pPMA) polymer shell. The pPMA coating yielded free surface-active alkyne groups such that the particles were candidates for CuAAC reactions and up to three fluorophores (azide-modified naphthalimide (azNap), rhodamine b (azRhod), and silicon phthalocyanine (azSiPc) derivatives) were attached to the particle’s surface. AzNap forms a Förster resonance energy transfer (FRET) pair with azRhod, where azNap is the donor and azRhod is the acceptor. Additionally, azRhod forms a FRET pair with azSiPc, where, in this case, azRhod is the donor and azSiPc is the acceptor. By using this system of fluorophores and upon excitation of azNap, two sequential energy transfers are induced and the only observable emission is that of azSiPc. The light emitted from these luminescent particles is easily detected in vitro using fluorescence microscopy, suggesting that this particulate system may have applications as bioimaging probes.
4. Experimental
4.1 Reagents and solvents
All chemicals and solvents used to prepare monomers and polymers were purchased from commercial suppliers, such as Alfa Aesar and TCI America, and were used without further purification. All solvents used for reactions were distilled under nitrogen after drying over an appropriate drying reagent. All manipulations involving air- and/or moisture-sensitive compounds were performed with standard Schlenk techniques under nitrogen. Analytical thin-layer chromatography was performed on glass plates coated with 0.25-mm 230-400 mesh silica gel containing a fluorescent indicator. Column chromatography was performed using silica gel (Grade 60A, particle size 63-210 $\mu$m).
4.2 Characterization methods
$^1$H NMR spectra of monomers and polymers were collected on a JEOL Delta 2 300 MHz spectrometer. All chemical shifts are reported against TMS. Absorbance spectra were taken using a Perkin Elmer Lambda 950 spectrophotometer. Photoluminescence spectra were collected using a Jobin-Yvon Fluorolog 3-222 Tau spectrometer. Thermogravimetric analysis (TGA) was performed on a TA Instruments Hi-Res TGA 2950 Thermogravimetric Analyzer equipped with Universal Analysis software. All TGAs were performed from room temperature to 1000$^\circ$C at 10$^\circ$C/min in a nitrogen atmosphere with a switch to air at 700$^\circ$C. Before each TGA run, the sample was purged in the furnace with nitrogen for 15 min. A Hitachi HT7830 STEM was used to acquire transmission electron microscopy (TEM) images. Samples were drop casted onto a Formvar/Carbon 200 mesh TEM grid.
4.3 Synthesis of 1-azido-3-iodopropane
1-Azido-3-iodopropane was synthesized according to a previously reported method [19].
4.4 Synthesis of 2-(3-azidopropyl)-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (azNap)
2-(3-Azidopropyl)-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (azNap) was synthesized according to a previously reported method [20].
4.5 Synthesis of rhodamine B 3-azido-propyl-1 ester (azRhod)
Rhodamine B (0.3 g, 0.626 mmol) was dissolved in dry dimethylformamide (4 mL). Then potassium carbonate (0.22 g, 1.56 mmol) and 1-azido-3-iodopropane (0.198 g, 0.939 mmol) were added and the obtained mixture was stirred at 85$^\circ$C for 20 h (cf. Fig. 8). After cooling, the mixture was extracted with dichloromethane and washed with water. The organic layer was separated, washed with water one more time, dried with Na$_2$SO$_4$, filtered, and evaporated under reduced pressure. The crude residue was purified by flash column chromatography on silica with an eluent of dichloromethane:acetone (5:1), R$_f$=0.3. Yield 0.28 g, 76%, m.p= 86-87$^\circ$C. $^1$H NMR (CDCl$_3$) $\delta$ 8.28 (d, 1H, $\it {J}$=7.6 Hz), 7.83 (m, 1H, $\it {J}$=7.6 Hz), 7.75 (m, 1H, $\it {J}$=7.6 Hz), 7.33 (d, 1H, $\it {J}$=7.6 Hz), 7.08 (d, 2H, $\it {J}$=9.3 Hz), 6.93 (d.d, 2H, $\it {J}$=9.3 Hz, $\it {J}$=2.4 Hz), 6.82 (d, 2H, $\it {J}$=2.4 Hz), 4.12 (t, 2H, $\it {J}$=6.5 Hz), 3.65 (q, 8H, $\it {J}$=7.2 Hz), 3.20 (t, 2H, $\it {J}$=6.5 Hz), 1.75 (m, 2H, $\it {J}$=6.5 Hz), 1.33 (t, 12H, $\it {J}$=7.2 Hz).
Fig. 8. Synthetic scheme to yield rhodamine B 3-azido-propyl-1 ester referred to as azide-modified rhodamine B (azRhod).
4.6 Synthesis of azide-modified silicon phthalocyanine (azSiPc)
Azide-modified silicon phthalocyanine (azSiPc) was synthesized according to a previously reported method [21].
4.7 Synthesis of silica nanoparticles (SiNPs)
SiNPs with a diameter of 289 $\pm$ 22 nm were prepared by the hydrolysis and condensation of tetraethyl orthosilicate (TEOS) in ethanol in the presence of ammonium hydroxide (i.e. Stöber process). TEOS (3 mL) was added into a mixture of ethanol (85 mL) and deionized (DI) water (10 mL). The mixture was stirred for 1 min followed by the addition of an aqueous ammonium hydroxide solution (29%, 6 mL). The obtained mixture was stirred at room temperature for 24 h. The SiNP suspension was used without further purification. Transmission electron microscopy (TEM) images of SiNPs are shown in Fig. 9(a,c).
Fig. 9. Transmission electron microscopy (TEM) images of (a,c) uncoated SiO$_2$ particles and (b,d) pPMA coated SiO$_2$ particles (SiO$_2$/pPMA).
4.8 Synthesis of MPS surface-modified SiNPs
3-Methacryloxypropyltrimethoxysilane (MPS) (0.6 mL) was added to the suspension from the previous step and was stirred for 20 h at room temperature. Subsequently, the suspension was stirred at 60$^\circ$C for 3 h. After cooling, the particles were separated via centrifugation and were washed three times with methanol. The particles were dried under vacuum at 40$^\circ$C. Yield 0.8 g. By mass, the MPS covered 1.8% of the nanoparticles.
4.9 Synthesis of core/shell silica/poly(propargyl methacrylate) nanoparticles (SiO$_2$/pPMA)
SiNPs modified with MPS (0.72 g) were dispersed in DI water (16 mL) followed by the addition of an aqueous solution of SDS (29%, 0.2 mL). Potassium persulfate (0.16 g) was added to the mixture and stirred for 1 minute and the obtained suspension was degassed with nitrogen. Propargyl methacrylate (0.6 mL) was added and the mixture was placed in a preheated oil bath at 65$^\circ$C with stirring under a nitrogen atmosphere for 4 h (cf. Fig. 10). After cooling, the particles were separated via centrifugation and were washed with DI water three times with subsequent centrifugation and re-dispersement in water. The obtained core/shell particles were dried and used in the next step. By mass, the pPMA composed 5.4% of the total mass of the nanoparticles. Transmission electron microscopy (TEM) images SiO$_2$/pPMA particles are shown in Fig. 9(b,d).
Fig. 10. Synthetic scheme to yield core/shell silica/poly(propargyl methacrylate) (SiO$_2$/pPMA) particles.
4.10 Synthesis of naphthalimide-modified SiO$_2$/pPMA nanoparticles (SiO$_2$/pPMA/D$_1$)
Synthesis of SiO$_2$/pPMA/D$_1$ nanoparticles was performed by utilizing a standard copper(I)-catalyzed azide/alkyne cycloaddition click reaction. Core/shell nanoparticles (100 mg dispersed in 1.25 mL DI water) were added to a reaction vessel equipped with a stir bar. Then a solution of azNap (1.72 mg) in tetrahydrofuran (THF) (34 $\mu$L) was added to the reaction mixture. Additional THF (2.715 mL) was added to the reaction mixture. Finally, aqueous solutions of copper(II) sulfate (1.72 mg in 0.5 mL DI water) and sodium ascorbate (3.41 mg in 0.5 mL DI water) were added to the reaction vessel. The reaction vessel was placed in a J-KEM mini-reactor with stirring at 28$^\circ$C in the dark with a nitrogen purge. The reaction was allowed to proceed for 24 h (cf. Fig. 11). Upon reaction completion, the product was washed once with THF followed by a wash with a DI water/EDTA solution to remove the copper catalyst. The product was washed five more times with THF. Washes were performed to remove any unattached dye from the particles. All washes were performed via centrifugation at 15,000 rpm for 15 min. The particles were said to be free of unattached dye when the absorbance of the supernatant no longer contained dye peaks. By mass, the total organic content accounted for 18.3% of the total mass of the particulate system.
Fig. 11. Synthesis of naphthalimide-modified core/shell SiO$_2$/pPMA nanoparticles via a copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) reaction (SiO$_2$/pPMA/D$_1$).
4.11 Synthesis of naphthalimide- and rhodamine B-modified SiO$_2$/pPMA nanoparticles (SiO$_2$/pPMA/D$_2$)
Synthesis of SiO$_2$/pPMA/D$_2$ nanoparticles was performed by utilizing a standard copper(I)-catalyzed azide/alkyne cycloaddition click reaction. SiO$_2$/pPMA nanoparticles (100 mg dispersed in 1.25 mL DI water) were added to a reaction vessel equipped with a stir bar. Then a solution of azNap (1.25 mg) and azRhod (8.75 mg) in THF (2.715 mL) was added to the reaction mixture. Finally, aqueous solutions of copper(II) sulfate (9.17 mg in 0.5 mL DI water) and sodium ascorbate (18.18 mg in 0.5 mL DI water) were added to the reaction vessel. The reaction vessel was placed in a J-KEM mini-reactor with stirring at 28$^\circ$C in the dark with a nitrogen purge. The reaction was allowed to proceed for 24 h. Upon reaction completion, the product was washed once with THF followed by a wash with a DI water:EDTA solution to remove the copper catalyst. The product was washed five more times with THF. Washes were performed to remove any unattached dye from the particles. All washes were performed via centrifugation at 15,000 rpm for 15 min. The particles were said to be free of unattached dye when the absorbance of the supernatant no longer contained dye peaks. By mass, the organic content on the particles comprised 18.2% of the total mass of the particulate system.
4.12 Synthesis of naphthalimide-, rhodamine B-, and silicone phthalocyanine-modified SiO$_2$/pPMA nanoparticles (SiO$_2$/pPMA/D$_3$)
Synthesis of SiO$_2$/pPMA/D$_3$ nanoparticles was performed by utilizing a standard copper(I)-catalyzed azide/alkyne cycloaddition click reaction. SiO$_2$/pPMA nanoparticles (100 mg dispersed in 1.25 mL DI water) were added to a reaction vessel equipped with a stir bar. Then a solution of azNap (1.25 mg), azRhod (2.5 mg), and azSiPc (6.25 mg) in THF (2.715 mL) was added to the reaction mixture. Finally, aqueous solutions of copper(II) sulfate (7.36 mg in 0.5 mL DI water) and sodium ascorbate (14.59 mg in 0.5 mL DI water) were added to the reaction vessel. The reaction vessel was placed in a J-KEM mini-reactor with stirring at 28$^\circ$C in the dark with a nitrogen purge. The reaction was allowed to proceed for 24 h. Upon reaction completion, the product was washed once with THF followed by a wash with a DI water:EDTA solution to remove the copper catalyst. The product was washed fifteen more times with THF. Washes were performed to remove any unattached dye from the particles. All washes were performed via centrifugation at 15,000 rpm for 15 min. The particles were said to be free of unattached dye when the absorbance of the supernatant no longer contained dye peaks. By mass, the organic content on the particles comprised 20.5% of the total mass of the particulate system.
4.13 Cell culture
For the cell viability studies, human hepatoma cells (HEPG2 cell line (ATCC # HB-8065)) were maintained at 37$^\circ$C in 5% CO$_2$ in Dulbecco’s modied eagle’s media (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were then plated in 96-well plates at a cell density of 2000 cells per well. After the cells were plated for 24 hours, the cells were incubated with SiO$_2$, SiO$_2$/pPMA, and SiO$_2$/pPMA/D$_3$ particles at concentrations of 1.56$\times$10$^5$ and 1.56$\times$10$^4$ particles/cell. At each concentration the cells were incubated for 24, 48, and 72 hours. At the end of each time point, the cell viability was measured using a [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] inner salt (MTS) assay. The media was removed and the plates were washed several times with phosphate buffer solution (PBS) before adding a solution containing 200 $\mu$L of DMEM media and 40 $\mu$L of MTS assay reagent. After 3 hours, the absorbance was measured using a plate reader at OD = 490 nm.
For microscopy, human embryonic kidney cells (HEK293T cell line (ATCC # CRL-3216)) were maintained at 37$^\circ$C in 5% CO$_2$ in DMEM supplemented with 10% FBS and 5% penicillin/streptomycin. The cells were grown in 24-well plates. For transient incubation of nanoparticles, cells were split the day before ca. 1 - 4$\times$10$^5$ cells/well. Cells were then incubated with SiO$_2$/pPMA/D$_1$ and SiO$_2$/pPMA/D$_3$ for 24 -72 hours and then analyzed.
4.14 Statistical analysis of cell viability
Cell viability of SiO$_2$, SiO$_2$/pPMA, and SiO$_2$/pPMA/D$_3$ particles in a HepG2 cell line at 1.56$\times$10$^5$ and 1.56$\times$10$^4$ particles/cell for 24, 48, and 72 hours showed no statistically significant difference from the control for each concentration and time period. A statistically significant difference from the control was determined by a post-hoc Tukey’s HSD test ($\alpha$ = 0.05). SiO$_2$ particles, 24 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 0.021, p = 0.98. SiO$_2$ particles, 48 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 0.069, p = 0.93. SiO$_2$ particles, 72 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 0.0004, p = 0.99. SiO$_2$/pPMA particles, 24 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 0.20, p = 0.83. SiO$_2$/pPMA particles, 48 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 4.26, p = 0.07. SiO$_2$/pPMA particles, 72 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 0.001, p = 0.99. SiO$_2$/pPMA/D$_3$ particles, 24 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 0.24, p = 0.79. SiO$_2$/pPMA/D$_3$ particles, 48 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 0.12, p = 0.90. SiO$_2$/pPMA/D$_3$ particles, 72 hrs: n = 3, 3, 3; one-way ANOVA, F(2,6) = 0.007, p = 0.99.
4.15 Immunofluorescent protocol: immunocytochemistry (ICC)
For all microscopy techniques, particles were subjected to sonication before dilutions using an ultrasonic processor (Model # GEX 130) for 20 secs at 30%. The sonication was repeated twice. When the nanoparticles were seeded onto the cells, the particles were vortexed for 10 secs before titrating/pipetting onto cells. For immunocytochemistry (ICC), cells were plated on coverslips coated with laminin. Once the cells were ready for fixation, they were fixed in 4% paraformaldehyde (PFA), blocked with 10% donkey serum, and subsequently incubated with 4’,6-diamidino-2-phenylindole (DAPI) for 10 mins. Then the fixed cells were mounted to slides using a fluoromount. Immunofluorescence images were performed on a Nikon Eclipse TE2000-U Inverted Microscope using a 60x lens (Nikon Plan Apo 60x (oil) DIC H (N.A. 1.4) microscope objective). Cells were excited with the blue (405 nm) channel, green (488 nm) channel, and/or red (565 nm) channel.
4.16 Cellular fluorescence microscopy
Live cells (i.e. before fixation) were imaged with a Nikon Eclipse TS100 Inverted Microscope using a 100% LED intensity, 490 nm CoolLED pE-100 excitation light source and 20x magnification (Nikon CFI ACHRO LWD NAMC 20XF (N.A. 0.4) microscope objective).
Funding
National Science Foundation (OIA-1632881); Gregg-Graniteville Foundation.
Acknowledgements
The authors thank the Gregg-Graniteville Foundation and the National Science Foundation (OIA-1632881) for financial support.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
References
1. A. Auger, J. Samuel, O. Poncelet, and O. Raccurt, “A comparative study of non-covalent encapsulation methods for organic dyes into silica nanoparticles,” Nanoscale Res. Lett. 6(1), 328 (2011). [CrossRef]
2. C. M. Carbonaro, F. Orru, P. C. Ricci, A. Ardu, R. Corpino, D. Chiriu, F. Angius, A. Mura, and C. Cannas, “High efficient fluorescent stable colloidal sealed dye-doped mesostructured silica nanoparticles,” Microporous Mesoporous Mater. 225, 432–439 (2016). [CrossRef]
3. A. Kurtz-Chalot, J.-P. Klein, J. Pourchez, D. Boudard, V. Bin, O. Sabido, L. Marmuse, M. Cottier, and V. Forest, “Quantification of nanoparticle endocytosis based on double fluorescent ph-sensitive nanoparticles,” Biomed. Microdevices 17(2), 42 (2015). [CrossRef]
4. A. Noureddine, L. Lichon, M. Maynadier, M. Garcia, M. Gary-Bobo, J. I. Zink, X. Cattoen, and M. W. C. Man, “Controlled multiple functionalization of mesoporous silica nanoparticles: Homogeneous implementation of pairs of functionalities communicating through energy or proton transfers,” Nanoscale 7(26), 11444–11452 (2015). [CrossRef]
5. C.-C. Huang, W. Huang, and C.-S. Yeh, “Shell-by-shell synthesis of multi-shelled mesoporous silica nanospheres for optical imaging and drug delivery,” Biomaterials 32(2), 556–564 (2011). [CrossRef]
6. C. Rosu, S. Selcuk, E. Soto-Cantu, and P. S. Russo, “Progress in silica polypeptide composite colloidal hybrids: from silica cores to fuzzy shells,” Colloid Polym. Sci. 292(5), 1009–1040 (2014). [CrossRef]
7. W.-B. Wu, C. Liu, M.-L. Wang, W. Huang, S.-R. Zhou, W. Jiang, Y.-M. Sun, Y.-P. Cui, and C.-X. Xu, “Uniform silica nanoparticles encapsulating two-photon absorbing fluorescent dye,” J. Solid State Chem. 182(4), 862–868 (2009). [CrossRef]
8. C. Tissandier, N. Diop, M. Martini, S. Roux, O. Tillernent, and T. Hamaide, “One-pot synthesis of hybrid multifunctional silica nanoparticles with tunable coating by click chemistry in reverse w/o microemulsion,” Langmuir 28(1), 209–218 (2012). [CrossRef]
9. F. May, M. Peter, A. Huetten, L. Prodi, and J. Mattay, “Synthesis and characterization of photoswitchable fluorescent SiO2 nanoparticles,” Chem. - Eur. J. 18(3), 814–821 (2012). [CrossRef]
10. M. S. Kim and J. Y. Chang, “Preparation of multifunctional mesoporous silica particles: The use of an amphiphilic silica precursor with latent amine functionality in selective functionalization of the inner surface,” J. Mater. Chem. 21(24), 8766–8771 (2011). [CrossRef]
11. D. Ma, A. J. Kell, S. Tan, Z. J. Jakubek, and B. Simard, “Photophysical properties of dye-doped silica nanoparticles bearing different types of dye-silica interactions,” J. Phys. Chem. C 113(36), 15974–15981 (2009). [CrossRef]
12. D. Estupinan, M. B. Bannwarth, S. E. Mylon, K. Landfester, R. Munoz-Espi, and D. Crespy, “Multifunctional clickable and protein-repellent magnetic silica nanoparticles,” Nanoscale 8(5), 3019–3030 (2016). [CrossRef]
13. F. Hagemans, E. B. van der Wee, A. van Blaaderen, and A. Imhof, “Synthesis of cone-shaped colloids from rod-like silica colloids with a gradient in the etching rate,” Langmuir 32(16), 3970–3976 (2016). [CrossRef]
14. M. Kar, P. S. Vijayakumar, B. L. V. Prasad, and S. Sen Gupta, “Synthesis and characterization of poly-l-lysine-grafted silica nanoparticles synthesized via nca polymerization and click chemistry,” Langmuir 26(8), 5772–5781 (2010). [CrossRef]
15. S. M. Saleh, R. Mueller, H. S. Mader, A. Duerkop, and O. S. Wolfbeis, “Novel multicolor fluorescently labeled silica nanoparticles for interface fluorescence resonance energy transfer to and from labeled avidin,” Anal. Bioanal. Chem. 398(4), 1615–1623 (2010). [CrossRef]
16. J. Geng, P. Liu, B. Liu, G. Guan, Z. Zhang, and M.-Y. Han, “A reversible dual-response fluorescence switch for the detection of multiple analytes,” Chem. - Eur. J. 16(12), 3720–3727 (2010). [CrossRef]
17. D. E. Achatz, F. J. Heiligtag, X. Li, M. Link, and O. S. Wolfbeis, “Colloidal silica nanoparticles for use in click chemistry-based conjugations and fluorescent affinity assays,” Sens. Actuators, B 150(1), 211–219 (2010). [CrossRef]
18. N. Subramanian, R. Kesavamoorthy, K. G. Rajan, M. Yousuf, S. Bera, and S. V. Narasimhan, “X-ray excited optical luminescence, photoluminescence, photostimulated luminescence and X-ray photoemission spectroscopy studies on BaFBr:Eu,” J. Phys.: Condens. Matter 9(22), 4769–4780 (1997). [CrossRef]
19. L. Yao, B. T. Smith, and J. Aube, “Base-promoted reactions of bridged ketones and 1, 3- and 1, 4-haloalkyl azides: Competitive alkylation vs azidation reactions of ketone enolates,” J. Org. Chem. 69(5), 1720–1722 (2004). [CrossRef]
20. M. K. Burdette, H. W. Jones, Y. Bandera, and S. H. Foulger, “X-ray radioluminescent hydrogel stabilized crystalline colloidal arrays,” Opt. Mater. Express 9(3), 1416–1429 (2019). [CrossRef]
21. Y. Bandera, M. K. Burdette, J. A. Shetzline, R. Jenkins, S. E. Creager, and S. H. Foulger, “Synthesis of water soluble axially disubstituted silicon (iv) phthalocyanines with alkyne & azide functionality,” Dyes Pigm. 125, 72–79 (2016). [CrossRef]
