Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Podsumowanie

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Streszczenie

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca²⁺) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

Wprowadzenie

In recent years, lanthanide-doped upconversion nanocrystals (UCNs) have been widely used as an alternative to conventional organic dyes and quantum dots in biomedical applications, which are mainly based on their outstanding chemical and optical properties, including great biocompatibility, high resistance to photobleaching, and narrow-bandwidth emission^1,2,3. More importantly, they can serve as a promising nanotransducer with excellent tissue penetration depth in vivo to convert near-infrared (NIR) excitation into a broad range of emissions from the UV, visible, and the NIR regions through a multi-photon upconversion process^4,5. These unique properties make lanthanide-doped UCNs serve as a particularly promising vector for biological sensing, biomedical imaging, and diseases theranostics^6,7,8.

The general components of UCNs are mainly based on the doped lanthanide ions in the insulating host matrix containing a sensitizer (e.g., Yb³⁺, Nd³⁺) and an activator (e.g., Tm³⁺, Er³⁺, Ho³⁺) within the crystal homogeneously⁹. The different optical emission from the nanocrystals is attributed to the localized electronic transition within the 4f orbitals of the lanthanide dopants due to their ladder-like arranged energy level¹⁰. Therefore, it is critical to precisely control the size and morphology of synthesized UCNs with multicomponent lanthanide dopants. By right, some promising methods have been well established for the preparation of lanthanide-doped UCNs, including thermal decomposition, high-temperature co-precipitation, hydrothermal synthesis, sol-gel processing, etc.^11,12,13 Among these approaches, the high-temperature co-precipitation method is one of the most popular and convenient strategies for UCNs synthesis, which can be strictly controlled to prepare desired high-quality nanocrystals with uniform shape and size distribution in a relatively short reaction time and low-cost¹⁴. However, most nanostructures synthesized by this method are mainly capped with hydrophobic ligands such as oleic acid and oleylamine, which generally hinder their further bioapplication due to the limited of hydrophobic ligand solubility in aqueous solution¹⁵. Therefore, it is necessary to perform suitable surface modification techniques to prepare biocompatible UCNs in biological applications in vitro and in vivo.

Herein, we present the detailed experimental procedure for the synthesis of core-shell UCNs nanostructures through the high-temperature co-precipitation method and a feasible modification technique to functionalize biocompatible polymer on UCNs surface for further cellular applications. This UCNs nanoplatform incorporates three lanthanide ions (Yb³⁺, Nd³⁺, and Tm³⁺) into the nanocrystals to acquire strong blue emission (~480 nm) upon NIR light excitation at 808 nm, which has greater penetration depth in living tissue. It is well known that Nd³⁺-doped UCNs display minimized water absorption and overheating effects at this spectral window (808 nm) as compared to conventional UCNs upon 980 nm irradiation^16,17,18. Moreover, to utilize the UCNs in biological systems, the hydrophobic ligands (oleic acid) on the surface of UCNs are firstly removed by sonication in acid solution¹⁹. Then the ligand-free UCNs are further modified with a biocompatible polymer (polyacrylic acid, PAA) to acquire great solubility in aqueous solutions²⁰. Furthermore, as a proof-of-concept in cellular applications, the hydrophilic UCNs are further functionalized with molecular ligands (dibenzyl cyclooctyne, DBCO) for specific localization on the N₃-tagged cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission at 480 nm can effectively activate a light-gated channel protein, channelrhodopsins-2 (ChR2), on cell the surface and thus facilitate cation (e.g., Ca²⁺ ion) influx across the membrane of living cells.

This video protocol provides a feasible methodology for lanthanide-doped UCNs synthesis, biocompatible surface modification, and UCNs bioapplication in living cells. Any differences in the synthesis techniques and chemical reagents used in nanocrystal growth will influence the size distribution, morphology, and upconversion luminescence (UCL) spectra of final UCNs nanostructures used in cell experiments. This detailed video protocol is prepared to help new researchers in this field to improve the reproducibility of UCNs with the high-temperature co-precipitation method and avoid the most common mistakes in UCNs biocompatible surface modification for further cellular applications.

Protokół

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Please use all appropriate safety practices when performing the synthesis of UCNs at a high temperature (~290 °C), including the use of engineering controls (fume hood) and personal protective equipment (e.g., safety goggles, gloves, lab coat, full length pants, and closed-toe shoes).

1. Synthesis of NaYF₄:Yb/Tm/Nd(30/0.5/1%)@NaYF₄:Nd(20%) core-shell nanocrystals

1. Synthesis of NaYF₄:Yb/Tm/Nd(30/0.5/1%) core nanostructure
   1. Preparation of the RE(CH₃CO₂)₃, NaOH, NH₄F methanol stock solution
      NOTE: The rare-earth (RE) lanthanide ions include Yttrium (Y), Ytterbium (Yb), Thulium (Tm), and Neodymium (Nd).
      1. Dissolve 500 mg Yttrium(III) acetate hydrate in 5 mL methanol (100 mg/mL), 250 mg Ytterbium (III) acetate hydrate in 5 mL methanol (50 mg/mL), 10 mg Thulium (III) acetate hydrate in 1 mL methanol (10 mg/mL), and 10 mg Neodymium (III) acetate hydrate in 1 mL methanol (10 mg/mL) in glass vials with an ultrasonic cleaning bath for 2 min.
      2. Combine 400 mg NaOH in a 50 mL centrifuge tube with 20 mL methanol with ultrasonic cleaning bath to prepare NaOH stock solution (20 mg/mL).
      3. Combine 600 mg NH₄F in a 50 mL centrifuge tube with 30 mL methanol with ultrasonic cleaning bath to prepare NH₄F stock solution (20 mg/mL).
         NOTE: Place the stock solution of lanthanide complexes, NaOH, and NH₄F in glass vials, seal with parafilm and store them in a refrigerator at ~4 °C until needed. The prepared methanol stock solutions are replaced once every 2 weeks.
   2. Preparation of NaYF₄:Yb/Tm/Nd core nanostructure
      1. Pipette 3 mL of oleic acid and 7 mL of 1-octadecene into a 50-mL three-neck flask.
      2. Combine 1.089 mL of the Y(CH₃CO₂)₃ stock solution, 0.608 mL of the Yb(CH₃CO₂)₃ stock solution, 83.6 µL of the Tm(CH₃CO₂)₃ stock solution, and 128.5 µL of the Nd(CH₃CO₂)₃ stock solution into the flask.
      3. Fit a thermometer (0 - 360 °C range) in the flask and let its tip touch the solution. Place the flask in a glass container with phenylmethyl silicone oil.
      4. Heat the solution to 100 °C with a hot plate to evaporate off the methanol. Connect the flask to a Schlenk line with a dual vacuum/gas manifold to remove the residual methanol and keep the reaction mixture under vacuum for 2 - 3 min while stirring.
      5. Increase the temperature to 150 °C and keep this temperature for 60 min. Maintain a stirring speed of 700 rpm during the synthesis.
         NOTE: Set a moderate stirring speed to avoid splashing the solution.
      6. Stop the hot plate and keep the flask at room temperature to allow the solution cool down slowly.
         NOTE: The protocol can be paused here.
      7. Combine 2 mL of NaOH-methanol stock solution and 2.965 mL of NH₄F-methanol stock solution into a 15 mL centrifuge tube. Tighten the tube with its cap and mix the solution by powerful vortexing for 5 s.
      8. Add the NaOH-NH₄F mixture slowly into the flask by a glass pipette over 5 min.
      9. Increase the temperature of the mixture to 50 °C and keep this temperature for 30 min.
         NOTE: Set the temperature at no more than 50 °C because the high temperature will promote crystal nucleation and growth.
      10. Increase the temperature (~100 °C) to evaporate off the methanol and connect the flask to a Schlenk line with a dual vacuum/gas manifold to remove the residual methanol and keep the reaction mixture under vacuum for 2 - 3 min.
      11. Switch the position of the stopcock to fill the flask with nitrogen.
      12. Increase the temperature to 290 °C at a heating rate of ~5 °C/min. Keep the reaction mixture at 290 °C for 1.5 h.
      13. Stop the hot plate and remove the flask to allow the reaction mixture to cool down slowly in room temperature while stirring.
          CAUTION: Be careful of the hot plate with high temperature (>400 °C) to avoid severe burns upon skin contact.
      14. Transfer the mixture in the flask into a 50 mL centrifuge tube. Rinse the flask with 30 mL of ethanol and transfer the solution to the centrifuge tube.
      15. Centrifuge the product at 4,000 × g for 8 min at room temperature and discard the supernatant.
      16. Add 10 mL of hexane to the centrifuge tube and re-disperse the product with sonication (60 kHz, 240 W) for 2 min.
      17. Add 30 mL of ethanol to the tube. Spin down the product at 4,000 × g for 8 min and then discard the supernatant.
      18. Re-disperse the solid products in the bottom of centrifuge tube with 5 mL hexane. Store the solution in a refrigerator at ~4 °C for a next step.
          NOTE: The upconversion emission of core-shell UCNs is tested by irradiating the solutions with an 808 nm laser (2 W).
2. Preparation of NaYF₄:Yb/Tm/Nd(30/0.5/1%)@NaYF₄:Nd(20%) core-shell nanocrystals
   1. Combine 3 mL of oleic acid and 7 mL of 1-octadecene into a 50-mL three-neck flask. Add 1.082 mL of the Y(CH₃CO₂)₃ stock solution and 2.87 mL of the Nd(CH₃CO₂)₃ stock solution into the flask.
   2. Add the obtained core nanostructure (80 mg in 5 mL hexane from step 1.1.2.18) into the flask while stirring.
   3. Fit a thermometer (0 - 360 °C range) in the flask and let its tip touch the mixture. Place the flask in a glass container with phenylmethyl silicone oil.
   4. Heat the mixture at 100 °C on the top of hot plate to evaporate off the methanol and hexane. Connect the flask to a Schlenk line with a dual vacuum/gas manifold to remove the residual solvent and keep the reaction mixture under vacuum for 2 - 3 min while stirring.
   5. Increase the temperature to 150 °C and keep it for 60 min. Maintain the stirring speed at 700 rpm in the synthesis.
      NOTE: Set a moderate stirring speed to avoid splashing the solution.
   6. Stop the hot plate and remove the flask to allow the solution to cool down slowly at room temperature.
      NOTE: The protocol can be paused here.
   7. Pipette 2 mL of NaOH-methanol stock solution and 2.965 mL of NH₄F-methanol stock solution into a 15 mL centrifuge tube. Tighten the tube with its cap and mix the solution by powerful vortexing for 5 s.
   8. Add the mixture into the flask by a glass pipette slowly over 5 min.
   9. Increase the temperature of the mixture to 50 °C and keep it at 50 °C for 30 min.
      NOTE: Set the temperature no higher than 50 °C because the high temperature will promote crystal nucleation and growth.
   10. Increase the temperature (~100 °C) to evaporate off the methanol and connect the flask to a Schlenk line with a dual vacuum/gas manifold to remove the residual methanol, keeping the reaction mixture under vacuum for 2 - 3 min.
   11. Switch the position of the stopcock to fill the flask with nitrogen.
   12. Increase the temperature to 290 °C at a heating rate of ~5 °C/min. Keep the reaction mixture at 290 °C for 1.5 h.
   13. Stop the hot plate and remove the flask to allow the reaction mixture to cool down slowly at room temperature while stirring.
       CAUTION: Be careful of the hot plate with high temperature (>400 °C) to avoid severe burns upon skin contact.
   14. Transfer the mixture in the flask into a 50 mL centrifuge tube. Rinse the flask with 30 mL of ethanol and transfer the solution to the centrifuge tube.
   15. Centrifuge the product at 4,000 × g for 8 min at room temperature and discard the supernatant.
   16. Add 10 mL of hexane to the centrifuge tube and re-disperse the product with sonication (60 kHz, 240 W) for 2 min.
   17. Add 30 mL of ethanol to the tube. Spin down the product at 4,000 × g for 8 min and then discard the supernatant.
   18. Re-disperse the solid products in the bottom of centrifuge tube with 5 mL hexane. Store the solution in a refrigerator at ~4 °C until needed.
       NOTE: The upconversion emission of core-shell UCNs is tested by irradiating the solutions with an 808 nm laser (2 W).

2. Synthesis of Biocompatible UCNs Nanostructures

1. Preparation of ligand-free UCNs nanoparticle
   1. Combine 30 mL ethanol with the as-prepared oleate-capped UCNs solution (step 1.2.18) in a 50 mL centrifuge tube. Centrifuge the mixture at 4,000 × g for 8 min at room temperature and discard the supernatant.
   2. Combine 10 mL acid aqueous solution (pH = 4) adjusted by HCl (0.1 M) with the precipitate in 50 mL centrifuge tube and dissolve the precipitate by sonication (60 kHz, 240 W) for 30 min.
   3. Transfer the solution in a glass vial with vigorous stirring for 2 h.
   4. Extract the aqueous solution with 30 mL diethyl ether to remove the oleic acid, repeat three times.
   5. Add 10 mL water in the combined ether layers with gentle shaking.
   6. Collect the aqueous phase together (20 mL) and add 20 mL acetone with vortexing for 5 s.
   7. Spin down the product at 35,000 × g for 10 min and then discard the supernatant. Dissolve the precipitate in 2 mL water.
2. Preparation of polymer modified UCNs (PAA-UCNs)
   1. Combine 200 mg of polyacrylic acid (PAA, Mw = 1,800) with 20 mL water by sonication (60 kHz, 240 W) for 20 min. Add the aforementioned ligand-free UCNs in the PAA solution with vigorous stirring.
   2. Adjust the pH value to 7.4 by NaOH solution (1 M) with sonication (60 kHz, 240 W) for 30 min. Keep the mixture for 24 h at room temperature while stirring.
   3. Collect the precipitate by centrifugation at 35,000 × g for 10 min. Re-suspend the product in 10 mL water by sonication (60 kHz, 240 W) for 5 min and centrifuge again at 35,000 × g for 10 min. Repeat this step three times.
   4. Re-disperse the product in 8 mL water by sonication (60 kHz, 240 W) for 5 min. Store the solution in a refrigerator at ~4 °C until needed.
3. Preparation of functional DBCO-UCNs nanoparticle
   1. Spin down the as-prepared PAA@UCNs (1 mg) by centrifugation at 35,000 × g for 10 min. Re-suspend the precipitate in 1 mL dry DMF by sonication (60 kHz, 240 W) for 1 min and centrifuge again at 35,000 × g for 10 min. Repeat this step twice.
   2. Dissolve the precipitate in 200 µL dry DMF in a glass vial. Add HOBT (12.2 mg), EDC (14 mg), DBCO-NH₂ (5 mg), and DIPEA (16 µL) in the vial with magnetic stirring for 24 h.
   3. Collect the product by centrifugation at 35,000 × g for 10 min. Remove the supernatant and re-suspend the precipitate in 1 mL DMSO and centrifuge again at 35,000 × g for 10 min. Repeat this step three times.
   4. Disperse the product in 0.2 mL DMSO and store in a refrigerator at ~ 4 °C before use.

3. Bioapplications of DBCO-UCNs in the Regulation of Membrane Channels in Living Cells

1. Culture the HEK293 cells in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin and maintain in a humidified incubator with 5% CO₂ at 37 °C. Seed 1×10⁵ cells in 1 mL DMEM/well in a 12-well plate and keep it in the incubator for 24 h.
2. Combine the plasmid (pCAGGS-ChR2-Venus, 1 µg) with P3000 transfection reagent (2 µL) in Eagle's Minimum Essential Media (MEM) (100 µL) in microcentrifuge tube A, and add transfection reagent (1.5 µL) in MEM (100 µL) in tube B.
3. Incubate the mixture of solutions in tubes A and B for 10 min at room temperature.
4. Combine the solution in tube A and B with 400 µL MEM. Wash the cells with 1 mL serum-free DMEM twice.
5. Add the 600 µL transfection mixture into each well of a 12-well plate and incubate the cells at 37 °C incubator for 4 h. Remove the medium and wash twice with 1 mL DMEM.
6. Add 1 mL DMEM containing 1 µL Ac₄ManNAz (50 mM in DMSO) in the well and keep it in the incubator for 2 days.
7. Remove the medium and wash once with 1 mL PBS. Add 1 mL Trypsin-EDTA (0.25%) solution in each well of the 12-well plate and incubate at 37 °C until cells have detached (~2 min). Re-culture the cells in a confocal dish at 1×10⁵ cells/well in 1 mL DMEM for overnight.
8. Remove the medium and add 1 mL fresh DMEM with 2 µL DBCO-UCNs (50 mg/mL) in the dish for 2 h at 37 °C. For confocal imaging, wash cells twice with DMEM and add 1 µL Rhod-N₃ (10 mM) for 30 min.
9. For intracellular calcium analysis, incubate the cells with the mixture of 1 µL Rhod-3 AM (10 mM), 10 µL Probenecid (250 mM), and 10 µL loading buffer (Pluronic surfactant polyols,0.1% w/v)) in 1 mL DMEM medium for 30 min in the dark.
10. Wash the cells with serum-free DMEM and irradiate with 808 nm NIR light at the dosage of 0.8 W/cm² for 20 min (5 min break after 5 min illumination).
11. Record the imaging results on the confocal microscope (laser source: 561 nm laser; detector range: 610/75 nm; lens: 100x 1.4 NA oil, exposure time: 100 ms). Add 1 mL Trypsin-EDTA (0.25%) solution in each well and incubate at 37 °C until cells have detached (~2 min). Re-suspend the cells in 1 mL PBS for flow cytometry (FCM) analysis (Ex: 561 nm, Em: 582/15 nm).

Wyniki

The schematic synthesis process of core-shell lanthanide-doped UCNs are shown in Figure 1 and Figure 2. The transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images of core and core-shell UCNs nanostructures were collected respectively (Figure 1). The ligand-free UCNs are prepared by removing the hydrophobic oleic acid on the surface of UCNs in acid s...

Dyskusje

This article has presented a method for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their surface modification with functional moieties for cellular applications. This novel nanomaterial possesses outstanding optical properties, which can emit UV and visible light upon NIR light excitation through a multi-photon upconversion process. In this protocol, the core-shell UCNs nanostructures (NaYF₄:Yb/Tm/Nd (30/0.5/1%)@NaYF₄:Nd (20%)) are prepared by a high-temperatur...

Materiały

Name	Company	Catalog Number	Comments
1-Octadecene	Sigma Aldrich	O806	Technical grade
oleic acid	Sigma Aldrich	364525	Technical grade
Methanol	Fisher Scientific	A412	Technical grade
Ethanol	Fisher Scientific	A405	Technical grade
Acetone	Fisher Scientific	A18	Technical grade
Hexane	Sigma Aldrich	H292	Technical grade
Thulium (III) acetate hydrate (Tm(CH3CO2)3)	Sigma Aldrich	367702	99.9% trace metals basis
Neodymium (III) acetate hydrate (Nd(CH3CO2)3)	Sigma Aldrich	325805	99.9% trace metals basis
Ytterbium (III) acetate hydrate (Yb(CH3CO2)3)	Sigma Aldrich	326011	99.9% trace metals basis
Yttrium(III) acetate hydrate (Y(CH3CO2)3)	Sigma Aldrich	326046	99.9% trace metals basis
Sodium hydroxide (NaOH)	Sigma Aldrich	S5881	reagent grade
Ammonium fluoride (NH4F)	Sigma Aldrich	338869	ACS reagent
Hydrogen chloride (HCl)	Fisher Scientific	A144	reagent grade
polyacrylic acid (PAA)	Sigma Aldrich	323667	average Mw 1800
1-Hydroxybenzotriazole hydrate (HOBT)	Sigma Aldrich	54802	ACS reagent
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)	Sigma Aldrich	E7750	commercial grade
Dibenzocyclooctyne-amine (DBCO-NH2)	Sigma Aldrich	761540	ACS reagent
N,N-Diisopropylethylamine (DIPEA)	Sigma Aldrich	D125806	ACS reagent
Dimethyl sulfoxide (DMSO)	Fisher Scientific	BP231	Technical grade
HEK293 cell line	ATCC	CRL-1573	human embryonic kidney
Fetal Bovine Serum (FBS)	Sigma Aldrich	F1051	ACS reagent
Penicillin-Streptomycin	Thermo Fisher	15140122	10,000 U/mL
plasmid (pCAGGS-ChR2-Venus)	Addgene	15753	Plasmid sent as bacteria in agar stab
Dulbecco's Modified Eagle Medium (DMEM)	Thermo Fisher	11965092	High glucose
opti-Modified Eagle Medium (MEM)	Thermo Fisher	51985034	Reduced Serum Media
Lipofectamine 3000 Transfection Reagent	Thermo Fisher	L3000015	Lipid-Based Transfection
N-Azidoacetylmannosamine, Acetylated (Ac4ManNAz)	Sigma Aldrich	A7605	ACS reagent
Trypsin-EDTA (0.25%)	Thermo Fisher	25200056	Phenol red
Rhod-3 AM Calcium Imaging Kit	Thermo Fisher	R10145	Fluorescence dye
5-carboxytetramethylrhodamine-azide (Rhod-N3)	Sigma Aldrich	760757	Azide-fluor 545
Confical dish	ibidi GmbH	81158	Glass Bottom, 35 mm
50 ml conical centrifuge tubes	Greiner Bio-One	227261	Polypropylene
15 ml conical centrifuge tubes	Greiner Bio-One	188271	Polypropylene
1.5 ml conical microcentrifuge tubes	Greiner Bio-One	616201	Polypropylene
Phenylmethyl silicone oil	Clearco Products	63148-52-7	Less than 320 degrees Celsius
Glass thermometer	GH Zeal	L0111/10	From -10 to 360 degrees Celsius
12-well plate	Sigma Aldrich	Z707775	Polystyrene