Method Article

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

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DOI:

10.3791/56657

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February 11th, 2018

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1Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, 2Institute of Materials Engineering, College of Engineering and Applied Sciences, Nanjing University, 3Collaborative Innovation Center of Chemistry for Life Sciences, Nanjing University, 4Department of Chemistry, Western Washington University

In This Article

Summary

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Abstract

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

Introduction

Nanocrystals such as semiconductor quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs) have demonstrated great potential for biological detection, imaging, manipulation, and therapy1,2,3,4,5,6. Encapsulating one or more nanocrystals into a micelle has been a widely used method to interface nanocrystals with biological environments3,6. The thus-formed micellar nanocrystals (micelles with nanocrystals encapsulated) have become an emerging class of nanobiomaterials7,8,9,10. Commonly used methods to fabricate micelles that encapsulate various materials (e.g., nanocrystals, small molecule drugs, and dyes) include film hydration, dialysis, and several others7,11.

The present work describes a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-mediated structural control. Compared with other fabrication methods of micellar nanocrystals, our method offers several beneficial features: (1) It is a largely continuous production process. This feature is mainly due to the fact that electrospray is used in our method to form emulsion droplets. In contrast, some other methods use vortexing or sonication to form emulsion droplets, thereby making these methods batch processes in nature12. (2) It results in products with high water-dispersibility, excellent colloidal stability, and intact physical functions of the encapsulated nanocrystals. This process can often give products with superior quality compared with other micelle encapsulation methods, to a large extent because electrospray can form ultrafine and uniform emulsion droplets. (3) The structures of the products, including micelle shape and number of encapsulated nanocrystals, can be controlled by the solvent, which is much more inexpensive compared with other ways of control such as changing the amphiphilic polymers used, and can produce not only the commonly available spherical micelle shape but worm-like micelle shape via micelle fusion13. The thus-formed worm-shaped micellar nanocrystals are found to offer greatly reduced non-specific cellular uptake than the spherical counterparts13. On the other hand, it is worth pointing out that this method requires the setup of an electrospray device, which is somewhat more technically demanding (although far from prohibitive) than the need of instrumentation in the other methods.

The fabrication method involves first generating ultrafine liquid (often oil-in-water emulsion) droplets with uniform sizes by electrospray, followed by evaporation of organic solvent resulting in self-assembly to form micellar nanocrystals (Figure 1).The electrospray setup has a coaxial configuration using concentric needles: the oil phase, which contains amphiphilic block copolymers and hydrophobic nanocrystals dissolved in organic solvent, is delivered to the inner needle (27 G stainless-steel capillary) with a syringe pump; the water phase, which contains a surfactant dissolved in water, is delivered to the outer needle (20 G stainless-steel three-way connector) with a second syringe pump. A high voltage is applied to the coaxial nozzle. Ultrafine droplets with uniform sizes are generated due to electrodynamic force overcoming surface tension and inertial stress in the liquid. Each droplet essentially functions as a 'micro-reactor', in which, upon removal of the organic solvent by evaporation, the self-assembly 'reaction' occurs spontaneously due to hydrophobic interactions. Using different organic solvents leads to different structures of micellar nanocrystals: a water-immiscible organic solvent chloroform leads to spherical micelle shape, while a water-miscible organic solvent THF with a long reaction time leads to worm-like micelle shape along with enhanced nanocrystal encapsulation.

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Protocol

Caution: Due to the use of organic solvents, all operations should be done in a chemical fume hood. Due to the use of high electric voltage, avoid body contact with the apparatus when the power supply is on. Use all appropriate safety practices such as using personal protective equipment (safety glasses, gloves, lab coat, full-length pants, and closed-toe shoes). Consult all relevant material safety data sheets (MSDS).

1. Setup of Materials

  1. To prepare QD solution, dissolve 10 mg hydrophobic QDs (fluorescent emission peak wavelength = 605 nm, used as the model nanocrystals here) in 20 mL organic solvent (chloroform for producing spherical micelle shape or THF for producing worm-like micelle shape) and vortex for 20 s.
  2. To prepare PS-PEG solution, dissolve 100 mg PS-PEG (amphiphilic block copolymer, with 9.5 kDA PS segment and 18.0 kDA PEG segment) in 10 mL organic solvent (chloroform for producing spherical micelle shape or THF for producing worm-like micelle shape). Mix the solution by vortexing for 1 min (chloroform) or bath sonicate for 2 min (THF).
  3. Mix 1 mL of QD solution and 1 mL PS-PEG solution and vortex for 1 min. Add the mixture to Syringe A. The syringe is made of PTFE.
  4. To prepare PVA solution, dissolve 400 mg PVA (13-23 kDa, 87-89% hydrolyzed) in 10 mL water in a heated water bath at 60–80 °C for 4–5 h. Allow the PVA solution to cool down to room temperature before use.
  5. Add 5 mL of PVA solution to Syringe B. The syringe is made of PTFE.

2. Setup of Equipment

  1. Insert the inner capillary into the outer capillary assembly and gently screw into position. Do not over tighten. Figure 2 shows the overall setup of the coaxial electrospray system. The inner capillary needle is a 27 G (outer diameter 500 µm; inner diameter 300 µm) stainless-steel capillary, and the outer needle is a 20 G (outer diameter 1,000 µm; inner diameter 500 µm) stainless-steel three-way connector. The PTEE tubing used has an inner diameter of 1.8 mm.
  2. Load Syringe A on Syringe Pump A as shown in Figure 2. Connect Syringe A to the inner stainless-steel capillary of the electrospray coaxial nozzle using PTFE tubing.
  3. Load Syringe B on Syringe Pump B as shown in Figure 2. Connect Syringe B to the outer stainless-steel capillary of the electrospray coaxial nozzle using PTFE tubing.
  4. Position the electrospray coaxial nozzle tip approximately 0.8 cm above a grounded steel ring (diameter of 1.5 cm).
  5. Place a glass collection dish approximately 10 cm below the coaxial nozzle.
  6. With the power supply turned off, connect the ground wire (black wire in Figure 2) to the grounded steel ring.
  7. With the power supply turned off, connect the positive terminal (red wire in Figure 2) of the power supply to the inner needle of the coaxial nozzle using a metal alligator clip.

3. Production of Micellar Nanocrystals

  1. Set the speed of Syringe Pump A to 0.6 mL/h.
  2. Set the speed of Syringe Pump B to 1.5 mL/h.
  3. Start both syringe pumps and wait for their respective flow rates to stabilize. Drops forming at the nozzle at a steady rate indicate a stable flow rate. This usually occurs within 60 s after starting syringe pumps.
    NOTE: There should be no bubbles in the tubing and droplets should form at the electrospray coaxial nozzle.
  4. Turn on the power supply to apply a positive high voltage to the electrospray coaxial nozzle. Adjust the applied voltage within the range of 5–9 kV, until a concave cone-jet (i.e., a convergent jet, commonly known as a 'Taylor cone') is observed at the tip of the coaxial nozzle (as shown in the inset of Figure 3a).
    Caution: Be sure not to touch electrospray nozzle when high voltage is applied. Follow appropriate safety precautions.
    NOTE: Insufficient applied voltage will result in droplets forming at the tip of the nozzle (as shown in the inset of Figure 3b), while too high of applied voltage will cause an electrical arc between the nozzle and the grounded steel ring.
  5. After a stable Taylor cone (Figure 3a) has been obtained, add 10 mL deionized water to a clean collection dish and replace the glass collection dish in the setup. The new dish will collect the micellar nanocrystal product.
  6. Run the micellar nanocrystal production process for a certain time duration (for approximately 40 min for producing spherical micelle shape or approximately 90 min for producing worm-like micelle shape). Then remove the collection dish from underneath the electrospray nozzle.
  7. Stop Syringe Pump A and B.
  8. Turn off the high voltage power supply.
  9. Allow the organic solvent to evaporate (in a fume hood) from the uncovered collection dish overnight.
    NOTE: Judging from the characterization results of the micellar nanocrystal products, evaporation overnight is sufficient for removing the organic solvent to obtain products with good quality.
  10. Finally, transfer the micellar nanocrystal product to a 15 mL centrifuge tube for characterization (e.g., fluorescent spectroscopy, dynamic light scattering, transmission electron microscopy, and thermal analysis), application, or storage. Store the final micellar nanocrystal product in a refrigerator at 4 °C.
    NOTE: The product can remain stable under this storage condition for at least one month.

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Results

Figure 1 shows a schematic summarizing the control of structures (shape and encapsulation) of micellar nanocrystals by the organic solvent used in the production process. Briefly, dichloromethane leads to spherical micelles with no encapsulation of nanocrystals; chloroform leads to spherical micelles with a low encapsulation number of nanocrystals; THF leads to spherical micelles with a high encapsulation number of nanocrystals at a short reaction time and wo...

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Discussion

The fabrication method of micellar nanocrystals described in the present work combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. An effective and convenient quality control method is to use the Taylor cone formed at the coaxial nozzle tip. This is because a properly formed Taylor cone indicates balance (or near balance) between electric force and surface tension, which in turn indicates successful formation of micro-reactors (uniform ultrafine droplets) for the self-assembly rea...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors gratefully acknowledge the financial support of a "Thousand Young Global Talents" award from the Chinese Central Government, a "Shuang Chuang" award from the Jiangsu Provincial Government, start-up fund from College of Engineering and Applied Sciences, Nanjing University, China, award from the "Tian-Di" Foundation, grant from the Priority Academic Program Development Fund of Jiangsu Higher Education Institutions (PAPD), grant from the Jiangsu Province Natural Science Fund.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Hydrophobic quantum dotsOcean NanotechQSPSolid hydrophobic CdSe/ZnS quantum dots. Peak fluorescence emission wavelength is 605 nm.
Poly(styrene)-b-poly(ethylene glycol) (PS-PEG)Sigma-Aldrich666476-500MGMolecular weight of PS segment is 9.5 kDa and that of PEG segment is 18.0 kDa.
Poly(vinyl alcohol) (PVA)Sigma-Aldrich363170-500GMolecular weight 13–23 kDa, 87–89% hydrolyzed.
Tetrahydrofuran (THF)Sinopharma Chemical Reagent80124418
ChloroformSinopharma Chemical Reagent40007960
Syringe pumpsBao Ding Shen ChenSPLab01
TubingShanghei Lai Xing2 mm outer diameter and 1.8 mm inner diameter PTFE tubing.
SyringesYi Ming5.CC5 mL disposable syringe made of PTFE.
High voltage power supplyDong WenDW SeriesDirect current power supply (0–50 kV range).
Electrospray coaxial nozzleHunan Chang Sha Na YiStainless steel assembly. Inner capillary needle was a 27 gauge (outer diameter 500 μm; inner diameter 300 μm). Outer capillary was a 20 gauge (outer diameter 1,000 μm; inner diameter 500 μm).
VortexerXi'an HEB Biotechnology Co., Ltd. ChinaMX-SMX-S with wide speed range of 0–2,500 rpm, stepless speed regulation, touch and continuous operations.
Steel ringYiwu Wan TuRings with a range of diameters (0.8–1.8 cm) can be constructued. For example, a 1.3 cm diameter ring was constructed by curling an approximately 25 cm (length) of 0.5-mm diamter (24 gauge, AWG) steel wire.
Glass collecting dishGrainger1u508425-mm height and 120-mm diameter glass dish.
15 mL centrifuge tubeJiangsu Xinkang Medical Instrument Co., Ltd.X-407Centrifuge tube is made of transparent polypropylene (PP).

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Tags

Electrospray TechniqueSelf assembly ProcessSolvent based ControlTetrahydrofuran THFWorm shaped MicellesSpherical MicellesNanocrystal EncapsulationCoaxial NozzleTransmission Electron Microscopy

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