A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

Summary

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a ‘plug and play’ method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

Abstract

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Introduction

Today there is still an urgent need to develop new types of bio-imaging agents. Many novel fluorescent probes have been well documented.^1-6 However, substantial improvements in the image resolution remains a challenge.⁷ One practical method is to directly modulate the fluorescence probes between a ‘light’ emissive state and a ‘dark’ quenched state.^8-12 This particular method has been applied to develop technologies such as stimulated emission depletion (STED) microscopy¹³ and stochastic optical reconstruction microscopy (STORM).¹⁴

Another approach to modulate fluorescence is to couple photoresponsive chromophores together with fluorescent probes.^15,16 Toggling the photoresponsive chromophore between two isomers where only one of the isomers can act as an efficient energy-transfer acceptor, allows control over quenching of the fluorescence from the probe through Förster Resonance Energy Transfer (FRET) and other mechanisms. The result is the creation of an emissive state and a quenched state that can be alternated by exposure of the photoresponsive chromophore to different wavelengths of light.

Photoresponsive diarylethene chromophores can be reversibly toggled between a colorless ring-open isomer and a colored ring-closed isomer upon irradiation with UV and visible light.^17-19 The thermal stability of the two isomers and tunable absorption spectra of the ring-closed isomer make diarylethenes very good candidates as controllable FRET acceptors.^20-23 Lanthanide-doped NaYF₄ upconverting nanoparticles are useful for bio-imaging.²⁴ These nanoparticles absorb near-infrared light and emit light in several regions of the visible spectrum. Examples of fluorescence modulation by combining photoresponsive diarylethene chromophores and nanoparticles have been previously reported by our group.^25-27 However, the systems described in each example required an additional synthetic modification to attach the diarylethenes to the surface of the nanoparticles, which complicates the development of more diverse systems.

Herein we demonstrate a simple ‘plug-and-play’ method to prepare water-dispersible organic dye molecules and photoresponsive upconverting nanoparticles using a self-assembly strategy. The choice of polymers; poly(styrene-alt-maleic anhydride) and polyether amine 2070 provide both a hydrophobic and hydrophilic environment. The hydrophobic sections of the polymer help to hold the normally water insoluble organic molecules and upconverting nanoparticles together, whereas the hydrophilic region of the polymer is critical for maintaining the water-solubility. We will first demonstrate synthesis of the upconverting nanoparticles by the thermal nucleation method. Then, we will prove how the organic molecules and upconverting nanoparticles are encapsulated within hydrophobic regions of the polymer shell and remain stable in aqueous media by simply co-stirring a solution of the upconverting nanoparticles, polymer and different organic dye molecules, followed by a convenient work-up procedure. We also demonstrate how to modulate fluorescence emission of the assemblies using external light irradiation. We anticipate the scope of using this ‘plug-and-play’ method to make water-dispersible nanoassemblies will continue to expand.

Protocol

1. Synthesis of the NaYF₄/Yb³⁺/Er³⁺ Upconverting Nanoparticles (UCNP)

1. Set up the apparatus as followed:
   1. Place a 250 ml heating mantle on a regular stirring plate and plug the mantle onto the thermal couple.
   2. Place a 250 ml round bottom flask equipped with a magnetic stir bar onto the heating mantle with proper clamping.
   3. Attach an air adapter to the left neck of the round bottom flask and connect this air adapter to the Schlenk line with plastic tubing.
   4. Attach a glass adapter to the right neck of the round bottom flask and fix a thermometer adapter onto the glass adapter. Insert the temperature probe into the flask through the thermometer adapter and plug this into the thermocouple.
   5. Attach a distillation head to the middle neck of the round bottom flask. Place a stopper on top of the distillation head. Connect the head to a condenser, followed by a vacuum distillation adapter and a 50 ml round bottom flask. Connect the vacuum distillation adapter to a bubbler through plastic tubing. 
2. Weigh 1.17 g (3.9 mmol) of yttrium acetate, 0.439 g of ytterbium acetate and 0.0727 g (0.1 mmol) of erbium acetate and place them in the reaction round bottom flask.
3. Add 30 ml of oleic acid and 75 ml of octadecene to the flask using a graduated cylinder.
4. Rinse the side of the reaction round bottom flask using 5 ml of methanol to make sure that no oleic acid and octadecene is stuck to the sides of the reaction flask.
5. Connect the reaction flask to a double manifold Schlenk line and turn the corresponding valve to keep the reaction flask connected to the nitrogen line.
6. Turn on the thermocouple, set the temperature to 80 °C, and gradually heat the system to this temperature. At 80 °C and after all starting materials are dissolved, remove the heating mantle and allow the reaction to cool down to 30 °C.
7. When temperature reaches 30 °C, take off the distillation head, switch the air adapter from the left neck to the middle neck and close off the left neck with a stopper. Slowly introduce vacuum to the reaction flask by turning the valve on the Schlenk line from the nitrogen line to the vacuum line. All of the low boiling point components will be pulled out from the reaction at this point.
8. When the solution stops bubbling, raise up the temperature to 115 °C in a speed of 5 °C/min.
9. Once the temperature reaches 115 °C, maintain this temperature for 15 min, then remove the heating mantle and cool down the reaction to 50 °C. Afterwards, quickly switch the set up back to the original form by reattaching the distillation head to the middle neck and the air adapter to the left head.
10. Weigh out 0.74 g (12.5 mmol) of NaOH and 0.50 g (20.0 mmol) of NH₄F during the cooling process, and dissolve them in 50 ml of methanol by sonication.
11. After sonication, pour the solution into the reaction round bottom flask and rinse the sides of the flask with 5 ml of MeOH.
12. Leave the solution stirring at 50 °C for 30 min.
13. Increase the temperature to 75 °C to distill the methanol.
14. During the distillation, empty the collection flask when necessary. After the distillation is finished, heat up the reaction to 300 °C under nitrogen protection as fast as possible.
15. Once the temperature reaches 300 °C, maintain this temperature for 1 hr. If needed, cover the setup with aluminum foil to help maintain the temperature. Then remove heat source and allow the reaction to cool down to room temperature.
16. Once it is cooled down to room temperature, split the solution evenly into three centrifugation tubes (50 ml tubes, roughly 35 ml solution per each tube), and top up the tube to the 50 ml scale using anhydrous ethanol. Centrifuge all the tubes at 3,400 x g for 15 min. After centrifugation, the UCNPs should be observed on the side of the tubes as a white precipitate.
17. Discard the supernatant and redisperse the UCNPs pellets in hexanes (7.5 ml of hexanes per each tube), then top up the tube with ethanol to the 50 ml scale. Centrifuge tubes again at 3,400 x g for 15 min.
18. Once the centrifugation is complete, discard the supernatant and redisperse the solid UCNPs in 30 ml of CHCl₃ for further use.

2. Assembly of Water-dispersible Nanoassemblies Containing Organic Dye Molecules and Upconverting Nanoparticles

1. Dissolve 25 mg (0.0147 mmol) of poly(styrene-alt-maleic anhydride) (PSMA) in 3 ml of CHCl₃ into a scintillation vial equipped with a magnetic stir bar. This quantity is an optimized quantity after multiple trials.
2. Add 250 µl (47 mg/ml) of the upconverting nanoparticles chloroform stock solution to the scintillation vial.
3. Cap the vial and place it on the magnetic stirring plate, and stir the solution at room temperature for 2 hr.
4. Weigh 160 mg (0.0773 mmol) of polyether amine 2070, and dissolve it in 1 ml of CHCl₃. Then add this solution to the scintillation vial in one portion using a pipette. The solution will turn to pale yellow indicating the reaction of polyether amine 2070 with the anhydride groups on the PSMA.
5. Continue to stir the solution overnight at room temperature.
6. Measure the appropriate quantity of organic dye molecules then dispense it into the scintillation vial in one portion, stir the resulting solution for 1 hr.
   1. For the sample TPP-NP (nanoassembly containing polymer shell, tetraphenyl porphyrin and upconverting nanoparticles), directly add 1 mg of tetraphenyl porphyrin to the scintillation vial. For the sample DAE-UCNP (nanoassembly containing polymer shell, diarylethene molecules and upconverting nanoparticles), the quantity of each diarylethene molecules is 2 × 10^-7 mol. Add the two diarylethene molecules into the reaction solution. The volumes for the two diarylethene molecules are: DAE-1o (1.8 mM), 111 µl and DAE-2o (1.6 mM), 125 µl.
7. Remove the CHCl₃ solvent under reduced pressure using a rotary evaporator, then add 3 ml of 0.001 M aqueous NaOH (pH ≈ 11) to the scintillation vial, then sonicate the vial until a milky suspension is formed.
8. Place the vial back on the rotary evaporator, and carefully remove the remaining CHCl₃ until the suspension has turned to a clear solution.
9. Transfer the solution from the scintillation vial to two 1.5 ml conical centrifugation tubes, then centrifuge the solution at 20,600 x g for 25 min.
10. Discard the supernatant, then add a total of 3 ml of deionized H₂O into the two tubes (1.5 ml per tube), sonicate the tubes to redisperse the pellets in the deionized H₂O.
11. Centrifuge the two tubes again at 20,600 x g for 25 min.
12. Discard the supernatant, then add a total of 3 ml of deionized H₂O into the two tubes (1.5 ml per tube). Sonicate the tubes to redisperse the pellets in the deionized H₂O.
13. Filter the aqueous nanoparticles dispersion sample through a 0.2 µm syringe filter to obtain the final sample for further testing.

Results

Absorption spectra and photoluminescence spectra were collected for sample DAE-UCNP. The absorption spectra are used for comparing the spectral overlap between the closed diarylethene chromophores and the upconverting nanoparticles. Photographs of the samples (both TPP-UCNP and DAE-UCNP) were also included to demonstrate successful encapsulation of organic dye molecules and upconverting nanoparticles, which are located within the amphiphilic polymer shells in the aqueous phase. The modulation of photochemistry and fluore...

Discussion

The nanoparticles synthesized according to this protocol have a size distribution from 20 to 25 nm centered at around 22.5 nm.^26,27 They can be classified as spherical particles with a α-NaYF₄ host lattice structure. There are two critical steps in this protocol. In the UCNP synthesis, it is crucial to maintain the heating temperature and time as precise as possible to assure a narrow distribution of particle size. Simultaneous addition of NaOH and NH₄F along with the addition of lan...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Yttrium acetate	Sigma	326046	Yttrium(III) acetate hydrate
Ytterbium acetate	Sigma	544973	Ytterbium(III) acetate hydrate
Erbium acetate	Sigma	325570	Erbium(III) acetate hydrate
Oleic acid	Sigma	75096	analytical standard
Octadecene	Sigma	O806	Technical grade
NaOH	Sigma	S5881	reagent grade
NH4F	Sigma	216011	ACS reagent
Poly(styrene-co-maleic anhydride)	Sigma	442399	Average Mn = 1700
JeffAmine 2070	Huntsman	M-2070
Varian Carry 300	Agilent
JDSU NIR laser	JSDU	L4-9897510-100M	980 nm diode laser