Examining the Roles of Emulsion Droplet Size and Surfactant in the Interfacial Instability-Based Fabrication Process of Micellar Nanocrystals
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The interfacial instability process is an emerging general method to fabricate nanocrystal-encapsulated micelles (also called micellar nanocrystals) for biological detection, imaging, and therapy. The present work utilized fluorescent semiconductor nanocrystals (quantum dots or QDs) as the model nanocrystals to investigate the interfacial instability-based fabrication process of nanocrystal-encapsulated micelles. Our experimental results suggest intricate and intertwined roles of the emulsion droplet size and the surfactant poly (vinyl alcohol) (PVA) used in the fabrication process of QD-encapsulated poly (styrene-b-ethylene glycol) (PS-PEG) micelles. When no PVA is used, no emulsion droplet and thus no micelle is successfully formed; Emulsion droplets with large sizes (~25 μm) result in two types of QD-encapsulated micelles, one of which is colloidally stable QD-encapsulated PS-PEG micelles while the other of which is colloidally unstable QD-encapsulated PVA micelles; In contrast, emulsion droplets with small sizes (~3 μm or smaller) result in only colloidally stable QD-encapsulated PS-PEG micelles. The results obtained in this work not only help to optimize the quality of nanocrystal-encapsulated micelles prepared by the interfacial instability method for biological applications but also offer helpful new knowledge on the interfacial instability process in particular and self-assembly in general.
KeywordsSelf-assembly Nanoparticle Interfacial instability Microencapsulation Quantum dot Magnetic nanoparticle Biological imaging Cell separation
1-Ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride
Poly (styrene-b-ethylene glycol)
Carboxylic acid terminated poly (styrene-b-ethylene glycol)
Poly (vinyl alcohol)
Superparamagnetic iron oxide nanoparticle
Transmission electron microscopy
The potential of applying nanomaterials, such as fluorescent semiconductor nanocrystals (quantum dots, QDs) [1, 2, 3], superparamagnetic iron oxide nanoparticles (SPIONs) [4, 5, 6], and gold nanoparticles [7, 8, 9], for biomedical detection, imaging and therapy has been well established after nearly two decades of research [10, 11]. Thus, in recent years, the focus of nanobiomaterial research has been shifted from proof-of-concept experiments to mechanistic studies, which aim to obtain insights and systematic understanding on nanomaterial fabrication processes, nanomaterial structure-property relationships as well as nanomaterial-biosystem interactions, and translational research, which aims to identify and solve the key problems in translating nanomaterials to industry and the clinic. The present work focuses on gaining new understanding on an emerging fabrication process, which is known as the interfacial instability method, of micellar nanocrystals, which have become a major class of nanobiomaterials.
A main strategy to solubilize hydrophobic nanomaterials (e.g., QDs, SPIONs, and gold nanoparticles synthesized by the commonly used organic solvent-based high temperature synthesis [12, 13, 14]) in water is to use a micelle to encapsulate the hydrophobic nanomaterials [15, 16, 17]. A micelle is a classic self-assembly system, in which amphiphilic molecules spontaneously form a core-shell structure (called a micelle) in an aqueous environment, with the hydrophilic segment of the amphiphilic molecules facing outward as the micelle shell and the hydrophobic segment facing inward as the micelle core, to minimize the total energy of the system. Micelles have a long history of applications as cleaning agents and drug delivery systems [18, 19, 20, 21, 22], mainly based on the fact that hydrophobic molecules (e.g., oils, many anticancer drugs) can be encapsulated into the hydrophobic cores of the micelles driven primarily by hydrophobic interaction . More recently, micelles have been applied to encapsulate single nanocrystals (with each micelle encapsulating a single nanocrystal) for biomedical imaging and detection . Most recently, quite a few research groups have reported the use of a micelle to encapsulate multiple nanocrystals, for multifunctionality or synergistic effects between the different nanocrystals in a micelle [25, 26, 27, 28, 29, 30, 31, 32].
An emerging method to prepare micellar nanocrystals (nanocrystal-encapsulated micelles) is the interfacial instability method [33, 34, 35]. The interfacial instability process was first reported in 2008 by Zhu and Hayward to prepare iron oxide nanoparticle-encapsulated micelles  and was later used by Ruan and Winter et al. to prepare micelles encapsulating both QDs and SPIONs in 2010 and micelles encapsulating QDs of different fluorescent emission colors in 2011 [25, 26]. The interfacial instability process for preparing QD-encapsulated poly (styrene-b-ethylene glycol) (PS-PEG) micelles involve two main steps: (1) Formation of oil-in-water emulsion droplets. In this emulsion, the oil phase contains hydrophobic QDs and the amphiphilic block copolymer PS-PEG dissolved in a non-polar organic solvent (chloroform in the present work); the aqueous phase contains a surfactant poly (vinyl alcohol) (PVA) dissolved in water; (2) Formation of nanocrystal-encapsulated micelles. Upon evaporation of the organic solvent, the oil/water interface of the emulsion becomes unstable, and hydrophobic interaction drives the system to spontaneously form PS-PEG micelles encapsulating hydrophobic QDs. A simple indicator for successful formation of micelles typically used in the experiments is the dramatic visual transformation of the system from a milky dispersion (emulsion) to a transparent one (micellar nanocrystal dispersion), thanks to the nanometer size (typical diameter 30–40 nm) of the micelles. In Ruan and Winter’s previous experiments with encapsulating QDs into PS-PEG micelles using the interfacial instability process, it was found that, although this process had many positive features, a major problem was the frequently observed great loss of QD fluorescence of the system during the fabrication/storage process, and the cause for the fluorescence loss was unknown. The goals of the present work are twofold: on the one hand, we aim to minimize the fluorescence loss of QD-encapsulated PS-PEG micelles prepared by the interfacial instability process; on the other hand, through the technology optimization process and taking advantage of the fluorescence of QDs as a reporter to follow the fabrication process of QD-containing nanocomposite materials, we aim to gain new understanding on the emerging general process for preparing nanocrystal-encapsulated micelles, i.e., the interfacial instability process. Our results suggest that the emulsion droplet size and the surfactant PVA play key roles in the fabrication process: each emulsion droplet essentially functions as a “micro-reactor” in which interfacial instability and self-assembly “reactions” occur, with the surfactant PVA being required for formation of the “micro-reactors”; Using large “micro-reactor” size (~25 μm) leads to a large portion of colloidally unstable nanocrystal-encapsulated PVA micelles in addition to colloidally stable nanocrystal-encapsulated PS-PEG micelles, while using small “micro-reactor” size (~3 μm or smaller, generated by sonication or electrospray) leads to only colloidally stable nanocrystal-encapsulated PS-PEG micelles.
Core-shell CdSe/ZnS quantum dots (QDs, emission wavelength 600 nm, covered with octadecylamine) were purchased from Ocean Nanotech. Poly (styrene-b-ethylene glycol) (PS-PEG) and carboxylic acid terminated poly (styrene-b-ethylene glycol) (PS-PEG-COOH) (PS 9.5 k Dalton, PEG 18.0 k Dalton) were purchased from Polymer Source. Poly (vinyl alcohol) (PVA) (molecular weight 13–23 kg/mol, 87–89% hydrolyzed) was purchased from Sigma-Aldrich. Tat peptide (sequence YGRKKRRQRRR) and RGD peptide (Arg-Gly-Asp) were purchased from ChinaPeptides. 1-Ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and sulfo-NHS were purchased from Sigma-Aldrich. All other chemicals were of reagent grade. The water used for all experiments was double distilled and purified by a Millipore Milli-Q purification system.
Preparation of Micellar Nanocrystals via the Interfacial Instability Process
In a typical procedure, an oil phase was first formed by mixing QDs (0.1 μM, 0.1 ml) and PS-PEG (10 mg/ml, 20 μl) in the organic solvent chloroform. This was followed by adding an aqueous phase (0.6 ml of water containing 5 mg/ml PVA). An oil-in-water emulsion was formed by either manual shaking (vigorously shaking the mixture by hand for 1 min) or sonication (sonicating the mixture in a ShuMei KQ218 bath sonicator for 30 s). In some experiments, electrospray was used to generate ultrafine emulsion droplets for the interfacial instability process . The different treatments were used to generate emulsion droplets with different sizes for studying the effects of droplet size: ~25 μm (in diameter) droplets were formed by manual shaking, ~3 μm (in diameter) droplets were formed by sonication, and a few hundred nanometers to a few micrometer (in diameter) droplets were formed by electrospray. The emulsion was diluted by an additional factor of four with ultrapure water (2.4 ml). The emulsion was left in a chemical fume hood with magnetic stirring at 100 rpm to allow evaporation of chloroform, leading to formation of micellar QDs. A visible transition in appearance from a milky dispersion to a transparent one was indicative of successful micelle formation.
When tetrahydrofuran (THF) was used as the organic solvent, an oil phase was first formed by mixing QDs (0.1 μM, 1 ml) and PS-PEG (10 mg/ml, 0.2 ml) in THF. Deionized water was added to the solution in a drop-wise manner (1 drop/20 s) until the water content reached 50% v/v. The solution was then mixed by votexing for 10–15 min and was then dialyzed against deionized water for 2 days to remove THF (molecular weight cutoff 100,000 Dalton).
When electrospray was used to generate droplets for the interfacial instability process, the operation was as follows . A coaxial electrospray configuration was used. The inner capillary needle was a 27 gauge (outer diameter 500 μm; inner diameter 300 μm) stainless steel capillary, and the outer needle was a 20 gauge (outer diameter 1000 μm; inner diameter 500 μm) stainless steel three-way connector. The nozzle tip was positioned 0.8 cm above a grounded steel ring and 10 cm above a glass collection dish. An oil phase was formed by mixing QDs and PS-PEG and was then delivered to the inner stainless steel capillary at a flow rate of 0.6 ml/h using a syringe pump (SPLab01, Shenzhen, China). The concentrations of PS-PEG and QDs in the oil phase were 5 mg/ml and 0.2 μM, respectively. An aqueous phase was prepared by dissolving PVA in deionized H2O at 40 mg/ml. The aqueous solution was delivered to the outer annulus of the coaxial needle at a flow rate of 1.5 ml/h using a second syringe pump (SPLab01, Shenzhen, China). Typically at a voltage in the range of 6–7 kV, a concave cone-jet (Taylor cone) was observed at the tip of the coaxial nozzle. A glass collection dish containing 10 ml deionized water was placed below the nozzle tip to collect droplets. The electrospray time (after a stable Taylor cone was formed) was typically 30–90 min. This was followed by further evaporation in the chemical fume hood overnight. Finally, the dispersion in the glass collection dish was transferred to a 15 ml centrifuge tube for characterizations.
Characterizations of the Physical Properties of Micellar QDs
The morphology of micellar QDs was characterized by transmission electron microscopy (TEM, JEOL JEM-2100 (HR)), and all samples investigated by TEM in the present work were negatively stained by 1% of phosphotungstic acid (PTA). Particle size was characterized by TEM or dynamic light scattering (DLS). Fluorescent spectra were obtained by a Hitachi F-4600 Fluorescent spectrophotometer.
Cytotoxicity of Nanomaterials
Cytotoxicity study was performed on three well-characterized human cancer cell lines, namely, A549 (alveolar basal epithelial), MCF-7 (breast), and HeLa (cervix) cells (purchased from KeyGen Biotech, China). The cells were maintained with cultured DMEM with 10% fetal bovine serum and antibiotics (penicillin/streptomycin) in a humid incubator (37 °C and 5% CO2). For cytotoxicity evaluation, cells were seeded onto 96-well plates in 200 μl of medium for 24 h. Then, the cells were incubated with different concentrations of micellar QDs in fresh culture medium at 37 °C in 5% CO2 atmosphere. After 24 h incubation, the culture media with dispersed micellar QDs were removed and the MTT assay was applied according to the manufacturer’s protocol. Finally, the optical absorbance in each well was measured at 570 nm in a microtiter plate reader.
Conjugation of QD-Encapsulated PS-PEG-COOH Micelles with Peptides
PS-PEG-COOH micelles were prepared with the above-described interfacial instability procedure, with PS-PEG-COOH molecules being used instead of PS-PEG ones. Conjugation with Tat peptide or RGD peptide was then conducted via the EDC/sulfo-NHS method. To activate the carboxyl groups of micelles, 0.3 ml of 0.1 M MES buffer solution containing 2 mg/ml EDC and 5 mg/ml sulfo-NHS was added to the micelle dispersion (3 ml) and reacted without stirring for 30 min at room temperature. The extra EDC and sulfo-NHS were then removed by using a 30-kD ultrafiltration tube (centrifugation at 10 krpm for 5 min), and the obtained dispersion was re-suspended in PBS (1 ml). Subsequently, 50 μl of Tat peptide (2 mg/ml in PBS) or 50 μl of RGD peptide (0.5 mg/ml in PBS) was added and reacted for 12 h at 4 °C, respectively. The obtained peptide-conjugated PS-PEG micellar QD dispersion was purified by using a 50-kD ultrafiltration tube (centrifugation at 10 krpm for 5 min) for three times to remove the extra peptide molecules and re-suspended in PBS (1 ml).
Live Cell Imaging
Live cell imaging was used to study the cellular internalization and intracellular transport of Tat peptide-conjugated PS-PEG micellar QDs. HeLa cells (purchased from KeyGen Biotech, China) were seeded on glass-bottom tissue culture plates at an initial confluency of 20% (seeding density 1 × 105 cells/ml) in 600 μl of medium (DMEM + 10% fetal bovine serum) and were cultured for 40 h in 5% CO2 at 37 °C. Tat peptide-conjugated PS-PEG micellar QDs (10 nM of QDs in cell culture medium) were then added. After being incubated with the micellar QDs for 1 h, the cells were washed twice with fresh culture medium to remove free micellar QDs (the washing step was done in order that the starting time of the intracellular transport of the internalized micellar QDs could be roughly the same for all the nanoparticles added). After 6 h, each plate of cells was imaged by a live cell imaging system, which consists of a cell incubation chamber (IX3W, Tokai Hit), an epi-fluorescent microscope (IX-83, Olympus, with halogen lamp as the light source), a spinning disk confocal system (Andor) and an electron multiplying charge-coupled device (EMCCD) camera (Evolve 512, Photometrics). The live cell confocal imaging system used here permits spinning-disk confocal imaging of live cells cultured on the microscope stage, which is particularly useful for studying the cellular transport process. By keeping the live cells cultured on the microscope stage, one could ensure that the natural biological process is monitored with minimal disturbance. To counter-stain the cell nucleus, right before imaging (at a particular time point of cellular transport), the fluorescent dye Hoechst 33342 (5 μM in cell culture medium) was incubated with live cells for 20 min.
Live cell imaging was also applied to study the specific binding of RGD peptide-conjugated PS-PEG micellar QDs with α v β3-integrin molecules, using an α v β3-integrin over-expressed cell line (U87 MG cells, purchased from KeyGen Biotech, China) versus a cell line without α v β3-integrin over-expression (MCF-7 cells, purchased from KeyGen Biotech, China). The above cellular imaging protocol used for Tat peptide-conjugated PS-PEG micellar QDs was adopted, with the main modification being that the concentration used for RGD peptide-conjugated micellar QDs was 100 nM (QDs in cell culture medium).
Results and Discussion
We and others recently introduced the interfacial instability method to encapsulate nanocrystals to form composite nanoparticles for biological applications. However, we frequently encountered irreproducible and sometimes conflicting results on fluorescence intensity of QDs (QDs were used as the model of nanocrystals here). This issue needs to be addressed for translation to industry and the clinic. Many factors (e.g., solvent, polymer, temperature, “micro-reactor” size) involved throughout the fabrication process could lead to fluorescence loss and irreproducible results. We have investigated the various factors involved and have found that the “micro-reactor” (emulsion droplet) size is a key factor in this regard, with the use of surfactant PVA being a closely related factor. Below, we primarily describe the results on the effects of emulsion droplet size as well as the surfactant PVA.
Furthermore, we also conducted emulsification treatment in the absence of the surfactant PVA and found that virtually no emulsion droplets were successfully formed, judging from the light microscopy result (Fig. 1a, top), and virtually, no micelles were successfully formed, judging from the observation of nearly complete phase separation (QD precipitation) in the final product, i.e., failure to form micelle product (Fig. 1a, bottom). The results of Fig. 1a suggested that the surfactant PVA is required in the interfacial instability process for successful formation of emulsion droplets (as the “micro-reactors”) and of micelles (as the final products). This is non-trivial because it suggests that, although PS-PEG is also amphiphilic in nature, the presence of PS-PEG alone (without the presence of PVA) in the system cannot give the emulsion droplets needed for the interfacial instability process.
In addition, it should be mentioned that, for a well-dispersed oil-in-water emulsion to form, a surfactant is often required to lower the surface tension between the oil phase and water phase, and PVA was selected here as the surfactant because it was applied in nearly all the previous works on using the interfacial instability method to fabricate micelles [25, 26, 33, 34, 35]. We cannot rule out the possibility that other surfactants could give different results. Examining the effects of different types of surfactant would be part of the future studies.
In conclusion, we have used QDs as the model nanocrystals to follow the interfacial instability process, an emerging general method to fabricate nanocrystal-encapsulated micelles. Our results reveal the key roles of emulsion droplet size and the surfactant PVA in the interfacial instability process. These results not only help to optimize the quality of nanocrystal-encapsulated micelles for biological applications such as biological detection, imaging and therapy, but offer helpful new knowledge on the interfacial instability process in particular and self-assembly in general.
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).
YXS performed the experiments, analyzed the data, and co-wrote the manuscript. LM, NH, XYD, CHY, and WJY assisted in the experiments and data analysis. GR supervised the research and co-wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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