Titania and Zinc Oxide Nanoparticles: Coating with Polydopamine and Encapsulation within Lecithin Liposomes—Water Treatment Analysis by Gel Filtration Chromatography with Fluorescence Detection

: The interplay of metal oxide nanoparticles, environmental pollution, and health risks is key to all industrial and drinking water treatment processes. In this work we present a study using gel ﬁltration chromatography for the analytical investigation of metal oxide nanoparticles in water, their coating with polydopamine, and their encapsulation within lecithin liposomes. Polydopamine prevents TiO 2 and ZnO nanoparticles from aggregation during chromatographic separation. Lecithin forms liposomes that encapsulate the nanoparticles and carry them through the gel ﬁltration column, producing an increase of peak area for quantitative analysis without any change in retention time to affect qualitative identiﬁcation. To the best of our knowledge, this is the ﬁrst report that demonstrates the potential application of lecithin liposomes for cleaning up metal oxide nanoparticles in water treatment. Encapsulation of graphene quantum dots by liposomes would allow for monitoring of nanoparticle-loaded liposomes to ensure their complete removal by membrane ultraﬁltration from treated water.


Introduction
Nanomaterials exhibit novel physicochemical properties that continue to promote their use in various industrial manufacturing processes. The top applications of nanomaterials in North America includes catalysts, coatings, electronics, food products, paints, rubber tires, and textiles [1]. Three metal oxide nanoparticles having sizes between 1 and 100 nanometers, namely titania (TiO 2 ), zinc oxide (ZnO), and ceria (CeO 2 ), are produced in high tonnage for use as additives in nanomaterials worldwide. TiO 2 nanoparticles are used as a fine whitener in cosmetics and sunscreen products because of their brightness, high refractive index, and resistance to discoloration. However, concerns are increasing recently about environmental pollution and the health risks of widespread exposure to TiO 2 nanoparticles. Long et al. reported that TiO 2 nanoparticles can produce reactive oxygen species that cause many diseases including immortalized brain microglia in mice [2,3]. Trouiller et al. found that TiO 2 nanoparticles are inducers of DNA damage and genetic instability in mice [4]. Chang et al. found a high percentage of biological toxicity when the liver and kidneys were exposed to TiO 2 nanoparticles [5]. Grande and Tucci lately discussed the potential human health risks induced by exposure to TiO 2 nanoparticles [6]. Disdier demonstrated that, despite a lack of brain translocation, exposure to TiO 2 nanoparticles induces blood-brain carrier physiology alteration and the gel filtration column without excessive retention, and to obtain reproducible detection peaks during chromatographic separation. Potential applications for cleaning up metal oxide nanoparticles during industrial wastewater and drinking water treatment are proposed. The idea of using lecithin liposomes for nanoparticle pre-concentration and removal in water treatment analysis is innovative. Its development into an industrial procedure is feasible as the reported critical micellar concentration of lecithin is around 0.4 mg/mL and the assembled lecithin liposomes have a typical size of 100-1000 nm, which is sufficient to accommodate all sizes of nanoparticles.

Materials
Methanol was purchased from VWR (Mississauga, ON, Canada). Soy lecithin in oil form was obtained from Vita Health Products (Winnipeg, MB, Canada). Titanium dioxide nanopowder (21 nm primary particle size), zinc oxide powder (maximum particle size: 45 nm), and Tris base (pK a = 8.2) were obtained from Sigma-Aldrich (Oakville, ON, Canada).

Coating of TiO 2 or ZnO Nanoparticles with Polydopamine
TiO 2 or ZnO nanoparticles (100 mg), dopamine (100 mg), Tris (63 mg), and distilled deionized water (60 mL) were mixed by magnetic stirring for 1 h. The mixture was centrifuged at 4500 rpm for 30 min, the supernatant was discarded, and the TiO 2 -PDA or ZnO-PDA nanoparticles were re-suspended in deionized water (40 mL) for all subsequent experiments.

Lecithin Liposomes Blank
A thick syringe needle was inserted to a capsule of lecithin oil for half an hour to suck lecithin slowly into the syringe. A mixture of lecithin (0.05 mL) and methanol (4.95 mL) was sonicated by an unbranded ultrasonic homogenizer (20 kHz, 108 W, sequentially powered on for 2 s, and powered off for 5 s) for 5 min at room temperature to form a uniform dispersion. This dispersion was mixed with distilled deionized water, obtained from a Millipore Milli-Q water system (Bedford, MA, USA), at a ratio of 1:2 by volume to serve as a lecithin liposomes blank, for comparison with TiO 2 or ZnO nanoparticles encapsulated within lecithin liposomes, in GFC-FD analysis. In a mixture of methanol and water (1:2 ratio), the dispersion of lecithin liposomes is stable for at least an hour, which was enough time the GFC-FD analysis.

Lecithin Liposome-Encapsulated TiO 2 or ZnO Nanoparticles
To prepare lecithin liposome-encapsulated TiO 2 or ZnO nanoparticles, different concentrations of nanoparticles (from 0.01 to 1.0 mg/mL prepared daily in our laboratory using distilled deionized water as the dispersion medium) were mixed with the lecithin liposomes blank at a ratio of 2:1 by volume. The mixtures were homogenized by the ultrasonic homogenizer (20 kHz, 108 W, sequentially powered on for 2 s and powered off for 5 s) for 5 min at room temperature to form uniform suspensions before GFC-FD analysis. Furthermore, different amounts (50-250 µL) of lecithin liposomes were added to either distilled deionized water (500 µL) or 1.0 mg/mL TiO 2 nanoparticles (500 µL), followed by addition of distilled deionized water until the total volume was 750 µL.

UV-Visible Absorption and Fluorescence Emission Spectroscopy
Fluorescence excitation and emission spectra of samples were measured on a Horiba Jobin Yvon (Edison, NJ, USA) FluoroMax-4 spectrofluorometer using a slit width of 4 nm for both emission and excitation. First under excitation by UV light at a wavelength of 300 nm, the emission wavelength was scanned from 350 to 550 nm to obtain a fluorescence emission spectrum. Next by monitoring the emission intensity at a wavelength of 450 nm, the excitation wavelength was scanned from 200 to 400 nm to obtain a fluorescence excitation spectrum.

Gel Filtration Chromatography
Gel filtration chromatography was performed on a Shimadzu LC-6A system (Columbia, MD, USA), initially using a short column (Shodex SHSB-807G, 50 × 8 mm, 35 µm, MWCO 10,000) for rapid analysis and eventually on a long column (Shodex SB-807HQ, 300 × 8 mm, MWCO 10,000) for extended analysis. Both columns were purchased from Canadian Life Science (Peterborough, ON, Canada). Distilled deionized water was used as the mobile phase at a flow rate ranging from 0.3 mL/min to 1.0 mL/min. The GFC system was coupled to a Jasco FP-2020 Plus intelligent fluorescence detector (Easton, MA, USA). Fluorescence detection (FD) employed λ ex = 365 nm and λ em = 460 nm for TiO 2 -PDA determination, or λ ex,max = 350 nm and λ em,max = 395 nm for TiO 2 determination.

Results and Discussion
Before GFC could be used to investigate the coating of TiO 2 nanoparticles in water with PDA and their encapsulation within lecithin liposomes, their fluorescence properties were studied in order to determine the excitation and emission wavelengths for selective and sensitive detection. Ultrasonic homogenization was evaluated for facilitating the encapsulation of TiO 2 -PDA nanoparticles by lecithin liposomes, by observing if reproducible peaks could be obtained from GFC-FD analysis for the chromatographic characterization of lecithin liposome-encapsulated TiO 2 -PDA nanoparticles. A short GFC column was initially used for rapid analysis and a long column was used for extended analysis to characterize their retention time, retention volume, and peak area at different flow rates of the mobile phase. After the feasibility of determining TiO 2 nanoparticles in the presence of lecithin liposomes was confirmed, the potential application of lecithin liposomes for encapsulating other metal oxide nanoparticles would be demonstrated with ZnO nanoparticles. Optimization of the new method was carried out by adding different amounts of lecithin liposomes to a fixed concentration of TiO 2 nanoparticles. In order to develop a cost effective technology for the removal of metal oxide nanoparticles in water treatment, graphene quantum dots were tested as a fluorescent sensor for monitoring the complete removal of nanoparticle-loaded liposomes from the treated water.

Fluorescence Spectroscopy
The fluorescence property of lecithin liposome-encapsulated TiO 2 nanoparticles was determined first. The maximum excitation and emission wavelengths were measured to be λ ex,max = 350 nm and λ em,max = 395 nm in Figure 1a,a'. Next, λ ex = 365 nm and λ em = 460 nm were determined for lecithin-bound TiO 2 -PDA nanoparticles in Figure 1b,b'. Both excitation spectra look very similar because lecithin liposomes are nearly transparent, the PDA coating is only a couple nanometers thick, and TiO 2 nanoparticles were mainly responsible for absorption of the UV light from 230 nm to 400 nm. The two emission spectra are obviously different due to energy transfer from TiO 2 nanoparticles emission at 395 nm to PDA emission at 460 nm in Figure 1b only. Efficient and facile coating of TiO 2 nanoparticles with PDA had previously been reported in the scientific literature [49,50]. The transmission electron microscopy images of TiO 2 and TiO 2 -PDA nanoparticles in Figure 2 below show how difficult it is to see their difference due to a thin organic PDA coating.

Gel Filtration Chromatography
Gel filtration chromatography was performed on a Shimadzu LC-6A system (Columbia, MD, USA), initially using a short column (Shodex SHSB-807G, 50 × 8 mm, 35 μm, MWCO 10,000) for rapid analysis and eventually on a long column (Shodex SB-807HQ, 300 × 8 mm, MWCO 10,000) for extended analysis. Both columns were purchased from Canadian Life Science (Peterborough, ON, Canada). Distilled deionized water was used as the mobile phase at a flow rate ranging from 0.3 mL/min to 1.0 mL/min. The GFC system was coupled to a Jasco FP-2020 Plus intelligent fluorescence detector (Easton, MA, USA). Fluorescence detection (FD) employed λex = 365 nm and λem = 460 nm for TiO2-PDA determination, or λex,max = 350 nm and λem,max = 395 nm for TiO2 determination.

Results and Discussion
Before GFC could be used to investigate the coating of TiO2 nanoparticles in water with PDA and their encapsulation within lecithin liposomes, their fluorescence properties were studied in order to determine the excitation and emission wavelengths for selective and sensitive detection. Ultrasonic homogenization was evaluated for facilitating the encapsulation of TiO2-PDA nanoparticles by lecithin liposomes, by observing if reproducible peaks could be obtained from GFC-FD analysis for the chromatographic characterization of lecithin liposome-encapsulated TiO2-PDA nanoparticles. A short GFC column was initially used for rapid analysis and a long column was used for extended analysis to characterize their retention time, retention volume, and peak area at different flow rates of the mobile phase. After the feasibility of determining TiO2 nanoparticles in the presence of lecithin liposomes was confirmed, the potential application of lecithin liposomes for encapsulating other metal oxide nanoparticles would be demonstrated with ZnO nanoparticles. Optimization of the new method was carried out by adding different amounts of lecithin liposomes to a fixed concentration of TiO2 nanoparticles. In order to develop a cost effective technology for the removal of metal oxide nanoparticles in water treatment, graphene quantum dots were tested as a fluorescent sensor for monitoring the complete removal of nanoparticle-loaded liposomes from the treated water.

Fluorescence Spectroscopy
The fluorescence property of lecithin liposome-encapsulated TiO2 nanoparticles was determined first. The maximum excitation and emission wavelengths were measured to be λex,max = 350 nm and λem,max = 395 nm in Figure 1a,a'. Next, λex = 365 nm and λem = 460 nm were determined for lecithinbound TiO2-PDA nanoparticles in Figure 1b,b'. Both excitation spectra look very similar because lecithin liposomes are nearly transparent, the PDA coating is only a couple nanometers thick, and TiO2 nanoparticles were mainly responsible for absorption of the UV light from 230 nm to 400 nm. The two emission spectra are obviously different due to energy transfer from TiO2 nanoparticles emission at 395 nm to PDA emission at 460 nm in Figure 1b only. Efficient and facile coating of TiO2 nanoparticles with PDA had previously been reported in the scientific literature [49,50]. The transmission electron microscopy images of TiO2 and TiO2-PDA nanoparticles in Figure 2 below show how difficult it is to see their difference due to a thin organic PDA coating.

Rapid GFC-FD Analysis of Lecithin Liposome-Encapsulated TiO 2 -PDA Nanoparticles
A short GFC column (50 × 8 mm) was initially used for rapid analysis of lecithin liposomeencapsulated TiO 2 -PDA nanoparticles. As expected, a PDA coating prevented the TiO 2 nanoparticles from aggregation during chromatographic separation. Ultrasonic homogenization apparently facilitated the encapsulation of TiO 2 nanoparticles by lecithin liposomes, thus yielding reproducible peaks for the chromatographic characterization of lecithin liposome-encapsulated TiO 2 -PDA nanoparticles. The standard calibration curve in Figure 3 shows that the GFC-FD peak area increased as the concentration of lecithin liposome-encapsulated TiO 2 -PDA nanoparticles increased. At the lowest concentrations of lecithin liposome-encapsulated TiO 2 -PDA nanoparticles studied, peak areas were very close to each other around the quantification limit of the GFC-FD analysis method. Next, lecithin liposome-encapsulated PDA nanoparticles and PDA nanoparticles were also analyzed by rapid GFC-FD for a comparison. Their peak areas in Figure 4b,c are significantly smaller than that in Figure 4a despite equivalent concentrations. This difference in peak area can certainly be attributed to the presence versus absence of TiO 2 nanoparticles. A rapid method for the quantitative analysis of TiO 2 -PDA nanoparticles by GFC-FD in 6 min is thus verified for the linear dynamic range from 0.05 mg/mL to 1.00 mg/mL.

Rapid GFC-FD Analysis of Lecithin Liposome-Encapsulated TiO2-PDA Nanoparticles
A short GFC column (50 × 8 mm) was initially used for rapid analysis of lecithin liposomeencapsulated TiO2-PDA nanoparticles. As expected, a PDA coating prevented the TiO2 nanoparticles from aggregation during chromatographic separation. Ultrasonic homogenization apparently facilitated the encapsulation of TiO2 nanoparticles by lecithin liposomes, thus yielding reproducible peaks for the chromatographic characterization of lecithin liposome-encapsulated TiO2-PDA nanoparticles. The standard calibration curve in Figure 3 shows that the GFC-FD peak area increased as the concentration of lecithin liposome-encapsulated TiO2-PDA nanoparticles increased. At the lowest concentrations of lecithin liposome-encapsulated TiO2-PDA nanoparticles studied, peak areas were very close to each other around the quantification limit of the GFC-FD analysis method. Next, lecithin liposome-encapsulated PDA nanoparticles and PDA nanoparticles were also analyzed by rapid GFC-FD for a comparison. Their peak areas in Figure 4b,c are significantly smaller than that in Figure 4a despite equivalent concentrations. This difference in peak area can certainly be attributed to the presence versus absence of TiO2 nanoparticles. A rapid method for the quantitative analysis of TiO2-PDA nanoparticles by GFC-FD in 6 min is thus verified for the linear dynamic range from 0.05 mg/mL to 1.00 mg/mL. from aggregation during chromatographic separation. Ultrasonic homogenization apparently facilitated the encapsulation of TiO2 nanoparticles by lecithin liposomes, thus yielding reproducible peaks for the chromatographic characterization of lecithin liposome-encapsulated TiO2-PDA nanoparticles. The standard calibration curve in Figure 3 shows that the GFC-FD peak area increased as the concentration of lecithin liposome-encapsulated TiO2-PDA nanoparticles increased. At the lowest concentrations of lecithin liposome-encapsulated TiO2-PDA nanoparticles studied, peak areas were very close to each other around the quantification limit of the GFC-FD analysis method. Next, lecithin liposome-encapsulated PDA nanoparticles and PDA nanoparticles were also analyzed by rapid GFC-FD for a comparison. Their peak areas in Figure 4b,c are significantly smaller than that in Figure 4a despite equivalent concentrations. This difference in peak area can certainly be attributed to the presence versus absence of TiO2 nanoparticles. A rapid method for the quantitative analysis of TiO2-PDA nanoparticles by GFC-FD in 6 min is thus verified for the linear dynamic range from 0.05 mg/mL to 1.00 mg/mL.

Rapid GFC-FD Analysis of Lecithin Liposome-Encapsulated TiO2 Nanoparticles
Lecithin liposome-encapsulated TiO2 nanoparticles (without PDA coating) were analyzed by rapid GFC for elucidating the analytical merits of PDA. The peak area in Figure 5a was larger than that for lecithin liposome-encapsulated TiO2-PDA nanoparticles in Figure 4a where the fluorescence from TiO2 nanoparticles was quenched by the PDA coating. Using next the maximum excitation and emission wavelengths λex,max = 350 nm and λem,max = 395 nm for FD, a stronger peak was obtained for lecithin liposome-encapsulated TiO2 nanoparticles in Figure 5b. A new standard calibration curve was constructed in Figure 6 to validate the linear dynamic range from 0.05 mg/mL to 1.00 mg/mL. The maximum wavelengths (λex,max = 350 nm and λem,max = 395 nm) generated a better calibration curve than the initial wavelengths (λex = 365 nm and λem = 460 nm) in terms of sensitivity and linearity. These results indicate the real possibility of determining lecithin liposome-encapsulated TiO2 nanoparticles down to 0.10 mg/mL. Unfortunately, the blank (a mixture of lecithin liposomes and distilled deionized water in a ratio of 1:2 by volume) produced a peak area as large as those obtained from all standard concentrations below 0.10 mg/mL. This could probably be attributed to stronger fluorescence from the phosphatidylcholine and phosphatidyl-ethanolamine contents of lecithin at these maximum wavelengths [51]. It became important that the blank peak from lecithin liposomes be separated from the lecithin liposome-encapsulated TiO2 peaks by using a longer GFC column for extended analysis.

Rapid GFC-FD Analysis of Lecithin Liposome-Encapsulated TiO 2 Nanoparticles
Lecithin liposome-encapsulated TiO 2 nanoparticles (without PDA coating) were analyzed by rapid GFC for elucidating the analytical merits of PDA. The peak area in Figure 5a was larger than that for lecithin liposome-encapsulated TiO 2 -PDA nanoparticles in Figure 4a where the fluorescence from TiO 2 nanoparticles was quenched by the PDA coating. Using next the maximum excitation and emission wavelengths λ ex,max = 350 nm and λ em,max = 395 nm for FD, a stronger peak was obtained for lecithin liposome-encapsulated TiO 2 nanoparticles in Figure 5b. A new standard calibration curve was constructed in Figure 6 to validate the linear dynamic range from 0.05 mg/mL to 1.00 mg/mL. The maximum wavelengths (λ ex,max = 350 nm and λ em,max = 395 nm) generated a better calibration curve than the initial wavelengths (λ ex = 365 nm and λ em = 460 nm) in terms of sensitivity and linearity. These results indicate the real possibility of determining lecithin liposome-encapsulated TiO 2 nanoparticles down to 0.10 mg/mL. Unfortunately, the blank (a mixture of lecithin liposomes and distilled deionized water in a ratio of 1:2 by volume) produced a peak area as large as those obtained from all standard concentrations below 0.10 mg/mL. This could probably be attributed to stronger fluorescence from the phosphatidylcholine and phosphatidyl-ethanolamine contents of lecithin at these maximum wavelengths [51]. It became important that the blank peak from lecithin liposomes be separated from the lecithin liposome-encapsulated TiO 2 peaks by using a longer GFC column for extended analysis.

Extended GFC-FD Analysis of Lecithin Liposome-Encapsulated TiO 2 Nanoparticles
A long GFC column (300 × 8 mm) was next used for extended analysis of lecithin liposomeencapsulated TiO 2 nanoparticles, TiO 2 nanoparticles, and lecithin liposomes separately. It was evidenced from the extended GFC-FD chromatograms (not shown here) that TiO 2 nanoparticles (injected at a low concentration of 0.01 mg/mL) were retained excessively on the long column as no peak appeared using a mobile phase flow rate increasing from 0.5 mL/min up to 1.5 mL/min. These results can be explained based on the time needed for water to wash the TiO 2 nanoparticles through the entire length of gel filtration matrix in the column. Fortunately, encapsulation of TiO 2 nanoparticles within lecithin allowed them to go through the column together quickly to appear as one combo peak at a retention time of 4.15 min. It was confirmed that the peak area of lecithin liposome-encapsulated TiO 2 nanoparticles was larger than that for lecithin liposomes alone, to our great delight.
The influence of flow rate (from 0.5 to 1.0 mL/min) on the retention time, retention volume, and peak area of lecithin liposome-encapsulated TiO 2 nanoparticles (injected at a low concentration of 0.01 mg/mL) was systematically investigated. An increase of flow rate obviously decreased their retention time in Figure 7a, while the retention volume stayed constant at 4.03 ± 0.05 mL with a nearly horizontal trend line in Figure 7b, and the peak area decreased in Figure 7c because the peak became narrower as expected. The unidirectional trends of these results, within experimental errors, confirmed consistently the feasibility of determining TiO 2 nanoparticles in the presence of lecithin liposomes.

Extended GFC-FD Analysis of Lecithin Liposome-Encapsulated TiO2 Nanoparticles
A long GFC column (300 × 8 mm) was next used for extended analysis of lecithin liposomeencapsulated TiO2 nanoparticles, TiO2 nanoparticles, and lecithin liposomes separately. It was evidenced from the extended GFC-FD chromatograms (not shown here) that TiO2 nanoparticles (injected at a low concentration of 0.01 mg/mL) were retained excessively on the long column as no peak appeared using a mobile phase flow rate increasing from 0.5 mL/min up to 1.5 mL/min. These results can be explained based on the time needed for water to wash the TiO2 nanoparticles through the entire length of gel filtration matrix in the column. Fortunately, encapsulation of TiO2 nanoparticles within lecithin allowed them to go through the column together quickly to appear as one combo peak at a retention time of 4.15 min. It was confirmed that the peak area of lecithin liposome-encapsulated TiO2 nanoparticles was larger than that for lecithin liposomes alone, to our great delight.
The influence of flow rate (from 0.5 to 1.0 mL/min) on the retention time, retention volume, and peak area of lecithin liposome-encapsulated TiO2 nanoparticles (injected at a low concentration of 0.01 mg/mL) was systematically investigated. An increase of flow rate obviously decreased their retention time in Figure 7a, while the retention volume stayed constant at 4.03 ± 0.05 mL with a nearly horizontal trend line in Figure 7b, and the peak area decreased in Figure 7c because the peak became narrower as expected. The unidirectional trends of these results, within experimental errors, confirmed consistently the feasibility of determining TiO2 nanoparticles in the presence of lecithin liposomes. The influence of flow rate (from 0.3 to 1.5 mL/min) on the retention time, retention volume, and peak area of lecithin liposome-encapsulated TiO2 nanoparticles (injected at a high concentration of 1.0 mg/mL) was also systematically investigated. An increase of flow rate again decreased their retention time in Figure 8a while their retention volume remained constant at 3.95 ± 0.07 mL with a nearly horizontal trend line in Figure 8b. Interestingly, the retention volume for lecithin liposomes was actually higher, indicating a smaller size than lecithin liposome-encapsulated TiO2 nanoparticles. This makes sense because the high concentration of encapsulated TiO2 nanoparticles could enlarge the size of each and every lecithin liposome. As smaller molecules have greater access and larger molecules are excluded from the gel filtration matrix, they would be eluted from the GFC column in decreasing order of size. The difference in peak area between lecithin liposomes and lecithin liposome-encapsulated TiO2 nanoparticles allowed easily for quantitative determination of TiO2 nanoparticles at this high concentration. Again, the peak area decreased in Figure 8c because the peak became narrower as the flow rate was increased. The influence of flow rate (from 0.3 to 1.5 mL/min) on the retention time, retention volume, and peak area of lecithin liposome-encapsulated TiO 2 nanoparticles (injected at a high concentration of 1.0 mg/mL) was also systematically investigated. An increase of flow rate again decreased their retention time in Figure 8a while their retention volume remained constant at 3.95 ± 0.07 mL with a nearly horizontal trend line in Figure 8b. Interestingly, the retention volume for lecithin liposomes was actually higher, indicating a smaller size than lecithin liposome-encapsulated TiO 2 nanoparticles. This makes sense because the high concentration of encapsulated TiO 2 nanoparticles could enlarge the size of each and every lecithin liposome. As smaller molecules have greater access and larger molecules are excluded from the gel filtration matrix, they would be eluted from the GFC column in decreasing order of size. The difference in peak area between lecithin liposomes and lecithin liposome-encapsulated TiO 2 nanoparticles allowed easily for quantitative determination of TiO 2 nanoparticles at this high concentration. Again, the peak area decreased in Figure 8c because the peak became narrower as the flow rate was increased. The influence of flow rate (from 0.3 to 1.5 mL/min) on the retention time, retention volume, and peak area of lecithin liposome-encapsulated TiO2 nanoparticles (injected at a high concentration of 1.0 mg/mL) was also systematically investigated. An increase of flow rate again decreased their retention time in Figure 8a while their retention volume remained constant at 3.95 ± 0.07 mL with a nearly horizontal trend line in Figure 8b. Interestingly, the retention volume for lecithin liposomes was actually higher, indicating a smaller size than lecithin liposome-encapsulated TiO2 nanoparticles. This makes sense because the high concentration of encapsulated TiO2 nanoparticles could enlarge the size of each and every lecithin liposome. As smaller molecules have greater access and larger molecules are excluded from the gel filtration matrix, they would be eluted from the GFC column in decreasing order of size. The difference in peak area between lecithin liposomes and lecithin liposome-encapsulated TiO2 nanoparticles allowed easily for quantitative determination of TiO2 nanoparticles at this high concentration. Again, the peak area decreased in Figure 8c because the peak became narrower as the flow rate was increased. The flow rate of 1.0 mL/min was deemed optimal for all subsequent extended GFC-FD analyses of TiO2 (and other metal oxide) nanoparticles. Encapsulation within lecithin liposomes improved their stability when travelling through the entire length of the gel filtration matrix in the column. Lecithin liposomes apparently carried the TiO2 nanoparticles through the gel column, as evidenced by a larger peak area, without increasing its original retention time-which indicated a relatively large size corresponding to liposomes. Vemuri and Rhodes had previously explored the use of size exclusion chromatography, as a large-scale process, to separate liposomes from free drug in a liposome preparation [52]. Interestingly, lecithin presented no distinct critical micelle concentration in either alcohol solution or water suspension, as Hara et al. had measured using electric conductivity and fluorescence [53]. Hypothetically, in the present work, TiO2 nanoparticles were first stripped of all adsorbed anions by the methanol inside lecithin liposomes. Methanol had previously been used for desorption of picrate anions from magnetic nanoparticles that were coated with cetyltrimethylammonium bromide [54]. Presumably, lecithin molecules dispersed around the bare non-ionic TiO2 nanoparticles that interacted favorably with the hydrophobic lipid moiety of each lecithin molecule. Under strong ultrasonication, they merged to form nanoliposomes, each containing several TiO2 nanoparticles. Being metastable aggregates of lipids [55], coalescence between lecithin nanoliposomes occurred when they were compressed by ultrasonic homogenization [56]. A calibration curve was constructed for the extended GFC analysis of lecithin-bound TiO2 nanoparticles at a flow rate of 1.0 mL/min, using λex,max = 350 nm and λem,max = 395 nm for FD. As shown in Figure 9, initially the GFC-FD peak area increased with increasing uptake of TiO2 nanoparticles by the lecithin liposomes, up to 0.03 mg/mL. However, no significant increase in peak area could be observed from 0.03 to 0.06 mg/mL, apparently indicating no further uptake of TiO2 nanoparticles by the lecithin liposomes. Above 0.06 mg/mL, the peak area continued to increase only moderately to suggest a possible restructuring of the TiO2 nanoparticle-loaded lecithin liposomes. Further research needs to elucidate the mechanism of action behind this irregular shape of the calibration curve.
Optimization of the GFC-FD method was carried out by adding different amounts of lecithin liposomes into either distilled deionized water or TiO2 nanoparticles (1.0 mg/mL). The peak area at 4.15 min for lecithin liposomes exhibited a linear correlation with the amount of lecithin liposomes as shown in Figure 10. However, a significantly steeper slope was obtained for TiO2 nanoparticles (than that for water). Apparently, the more lecithin liposomes were added, the lower the concentration of TiO2 nanoparticles that would be encapsulated within each and every liposome. Thus, the excitation of TiO2 fluorescence became more efficient and the emission of TiO2 fluorescence became less self-quenched. This strongly suggested that more lecithin liposomes can be used to obtain a larger peak area for any given concentration of TiO2 nanoparticles after subtraction of the lecithin liposomes blank, thereby improving the analytical sensitivity of this extended GFC method. The flow rate of 1.0 mL/min was deemed optimal for all subsequent extended GFC-FD analyses of TiO 2 (and other metal oxide) nanoparticles. Encapsulation within lecithin liposomes improved their stability when travelling through the entire length of the gel filtration matrix in the column. Lecithin liposomes apparently carried the TiO 2 nanoparticles through the gel column, as evidenced by a larger peak area, without increasing its original retention time-which indicated a relatively large size corresponding to liposomes. Vemuri and Rhodes had previously explored the use of size exclusion chromatography, as a large-scale process, to separate liposomes from free drug in a liposome preparation [52]. Interestingly, lecithin presented no distinct critical micelle concentration in either alcohol solution or water suspension, as Hara et al. had measured using electric conductivity and fluorescence [53]. Hypothetically, in the present work, TiO 2 nanoparticles were first stripped of all adsorbed anions by the methanol inside lecithin liposomes. Methanol had previously been used for desorption of picrate anions from magnetic nanoparticles that were coated with cetyltrimethyl-ammonium bromide [54]. Presumably, lecithin molecules dispersed around the bare non-ionic TiO 2 nanoparticles that interacted favorably with the hydrophobic lipid moiety of each lecithin molecule. Under strong ultrasonication, they merged to form nanoliposomes, each containing several TiO 2 nanoparticles. Being metastable aggregates of lipids [55], coalescence between lecithin nanoliposomes occurred when they were compressed by ultrasonic homogenization [56]. A calibration curve was constructed for the extended GFC analysis of lecithin-bound TiO 2 nanoparticles at a flow rate of 1.0 mL/min, using λ ex,max = 350 nm and λ em,max = 395 nm for FD. As shown in Figure 9, initially the GFC-FD peak area increased with increasing uptake of TiO 2 nanoparticles by the lecithin liposomes, up to 0.03 mg/mL. However, no significant increase in peak area could be observed from 0.03 to 0.06 mg/mL, apparently indicating no further uptake of TiO 2 nanoparticles by the lecithin liposomes. Above 0.06 mg/mL, the peak area continued to increase only moderately to suggest a possible restructuring of the TiO 2 nanoparticle-loaded lecithin liposomes. Further research needs to elucidate the mechanism of action behind this irregular shape of the calibration curve.
Optimization of the GFC-FD method was carried out by adding different amounts of lecithin liposomes into either distilled deionized water or TiO 2 nanoparticles (1.0 mg/mL). The peak area at 4.15 min for lecithin liposomes exhibited a linear correlation with the amount of lecithin liposomes as shown in Figure 10. However, a significantly steeper slope was obtained for TiO 2 nanoparticles (than that for water). Apparently, the more lecithin liposomes were added, the lower the concentration of TiO 2 nanoparticles that would be encapsulated within each and every liposome. Thus, the excitation of TiO 2 fluorescence became more efficient and the emission of TiO 2 fluorescence became less self-quenched. This strongly suggested that more lecithin liposomes can be used to obtain a larger peak area for any given concentration of TiO 2 nanoparticles after subtraction of the lecithin liposomes blank, thereby improving the analytical sensitivity of this extended GFC method.

Extended GFC-FD Analysis of Lecithin Liposome-Encapsulated ZnO Nanoparticles
In order to demonstrate the potential application of lecithin liposomes for encapsulating metal oxide nanoparticles other than just TiO2 in water treatment, extended GFC-FD analysis was next

Extended GFC-FD Analysis of Lecithin Liposome-Encapsulated ZnO Nanoparticles
In order to demonstrate the potential application of lecithin liposomes for encapsulating metal oxide nanoparticles other than just TiO2 in water treatment, extended GFC-FD analysis was next

Extended GFC-FD Analysis of Lecithin Liposome-Encapsulated ZnO Nanoparticles
In order to demonstrate the potential application of lecithin liposomes for encapsulating metal oxide nanoparticles other than just TiO 2 in water treatment, extended GFC-FD analysis was next performed on lecithin liposome-encapsulated ZnO nanoparticles, ZnO nanoparticles, and lecithin liposomes. Just like TiO 2 , encapsulation of ZnO nanoparticles within lecithin liposomes allowed them to go through the column together to appear as a combination peak at a retention time of 4.34 min in Figure 11a. It was next evidenced that ZnO nanoparticles were strongly retained on the long column until its peak appeared at 11.47 min in Figure 11b. The chromatogram in Figure 11c shows a peak at 4.15 min with an area linearly proportional to the concentration of lecithin liposomes (R 2 = 0.98, data not shown). In hindsight, for the sake of contrasting, TiO 2 nanoparticles had not shown any peak and lecithin liposome-encapsulated TiO 2 nanoparticles had exhibited a retention time of 4.42 min. performed on lecithin liposome-encapsulated ZnO nanoparticles, ZnO nanoparticles, and lecithin liposomes. Just like TiO2, encapsulation of ZnO nanoparticles within lecithin liposomes allowed them to go through the column together to appear as a combination peak at a retention time of 4.34 min in Figure 11a. It was next evidenced that ZnO nanoparticles were strongly retained on the long column until its peak appeared at 11.47 min in Figure 11b. The chromatogram in Figure 11c shows a peak at 4.15 min with an area linearly proportional to the concentration of lecithin liposomes (R 2 = 0.98, data not shown). In hindsight, for the sake of contrasting, TiO2 nanoparticles had not shown any peak and lecithin liposome-encapsulated TiO2 nanoparticles had exhibited a retention time of 4.42 min. The peak area of 1.0 mg/mL lecithin liposome-encapsulated ZnO nanoparticles was significantly stronger than that of lecithin liposomes alone. A standard calibration curve has been constructed in Figure 12 for the extended GFC analysis at a flow rate of 1.0 mL/min, using λex = 350 nm and λem = 395 nm for FD. Binding of lecithin liposomes with ZnO nanoparticles is confirmed by the larger peak areas for increasing concentrations from 0.01 mg/mL to 0.10 mg/mL. For lecithin liposomeencapsulated ZnO nanoparticles, a detection limit of 0.05 mg/mL is attained by this GFC-FD method. The peak area of 1.0 mg/mL lecithin liposome-encapsulated ZnO nanoparticles was significantly stronger than that of lecithin liposomes alone. A standard calibration curve has been constructed in Figure 12 for the extended GFC analysis at a flow rate of 1.0 mL/min, using λ ex = 350 nm and λ em = 395 nm for FD. Binding of lecithin liposomes with ZnO nanoparticles is confirmed by the larger peak areas for increasing concentrations from 0.01 mg/mL to 0.10 mg/mL. For lecithin liposome-encapsulated ZnO nanoparticles, a detection limit of 0.05 mg/mL is attained by this GFC-FD method.

Extended GFC-FD Analysis of Lecithin Liposome-Encapsulated Graphene Quantum Dots
It is important to develop a cost effective technology for the removal of TiO2 nanoparticles ranging from industrial wastewater to contaminated drinking water treatment. A limiting factor in drinking water treatment is the ability to collect all the waterborne nanoparticles for removal. Industrial filtration technologies are currently used on a very large scale but they are often plagued by structural variation and surface fouling of membrane filters [57][58][59]. The results discussed so far show the possibility of using lecithin liposomes for simple water treatment. One monitoring requirement is that the nanoparticle-loaded lecithin liposomes must subsequently be removed from the treated water before it is released to local waterways. Fluorescent GQDs have attracted tremendous attention for a wide range of sensing applications because of their good water dispersibility, high photostability, excellent biocompatibility, and low toxicity [60][61][62]. They can potentially serve as a sensor probe for monitoring the complete removal of nanoparticle-loaded liposomes from the treated water. Extended GFC analysis of a small aliquot of GQDs that had previously been synthesized and characterized in our lab [19] produced a sharp FD peak at 21.2 min in Figure 13a, as expected for their small size of 5-6 nm. Extended GFC analysis of GQDs in mixture with lecithin liposome-encapsulated TiO2 nanoparticles showed a significant reduction in the GQDs peak area at 20.8 min and the appearance of a small peak at 9.4 min in Figure 13b. This new peak could be attributed to the encapsulation of GQDs by lecithin liposomes (loaded with TiO2 nanoparticles). This result is consistent with a previous work by Chen et al. on the encapsulation of quantum dots in liposomes and the separation of nanoparticle-loaded liposomes from unencapsulated nanoparticles by size exclusion chromatography using a Sepharose gel [63]. They used fluorescence correlation spectroscopy to determine that each liposome encapsulated an average of three quantum dots. Hence, both studies have indicated GQDs can potentially serve the purpose of monitoring the nanoparticle-loaded liposomes to track their complete removal from treated water by ultrafiltration using the membrane separation technology [19,64,65].

Extended GFC-FD Analysis of Lecithin Liposome-Encapsulated Graphene Quantum Dots
It is important to develop a cost effective technology for the removal of TiO 2 nanoparticles ranging from industrial wastewater to contaminated drinking water treatment. A limiting factor in drinking water treatment is the ability to collect all the waterborne nanoparticles for removal. Industrial filtration technologies are currently used on a very large scale but they are often plagued by structural variation and surface fouling of membrane filters [57][58][59]. The results discussed so far show the possibility of using lecithin liposomes for simple water treatment. One monitoring requirement is that the nanoparticle-loaded lecithin liposomes must subsequently be removed from the treated water before it is released to local waterways. Fluorescent GQDs have attracted tremendous attention for a wide range of sensing applications because of their good water dispersibility, high photostability, excellent biocompatibility, and low toxicity [60][61][62]. They can potentially serve as a sensor probe for monitoring the complete removal of nanoparticle-loaded liposomes from the treated water. Extended GFC analysis of a small aliquot of GQDs that had previously been synthesized and characterized in our lab [19] produced a sharp FD peak at 21.2 min in Figure 13a, as expected for their small size of 5-6 nm. Extended GFC analysis of GQDs in mixture with lecithin liposome-encapsulated TiO 2 nanoparticles showed a significant reduction in the GQDs peak area at 20.8 min and the appearance of a small peak at 9.4 min in Figure 13b. This new peak could be attributed to the encapsulation of GQDs by lecithin liposomes (loaded with TiO 2 nanoparticles). This result is consistent with a previous work by Chen et al. on the encapsulation of quantum dots in liposomes and the separation of nanoparticle-loaded liposomes from unencapsulated nanoparticles by size exclusion chromatography using a Sepharose gel [63]. They used fluorescence correlation spectroscopy to determine that each liposome encapsulated an average of three quantum dots. Hence, both studies have indicated GQDs can potentially serve the purpose of monitoring the nanoparticle-loaded liposomes to track their complete removal from treated water by ultrafiltration using the membrane separation technology [19,64,65].

Conclusions
Coating with polydopamine and encapsulation within lecithin liposomes prevent TiO2 and ZnO nanoparticles from aggregation during extended chromatographic separation through the gel filtration matrix, even in a long column. To the best of our knowledge, this is the first report on the gel filtration chromatographic analysis of lecithin liposomes that demonstrates their potential application for cleaning up two different types of metal oxide nanoparticles in water treatment, at a high efficiency but with a low cost. The colloidal stability of TiO2-PDA nanoparticles is important but not crucial for this new method because every sample is homogenized ultrasonically both during encapsulation by lecithin liposomes and before each GFC-FD analysis. In water treatment, unstable nanoparticles form aggregates, undergo sedimentation, and thus do not need liposome encapsulation to clean them up. Considering the importance of industrial wastewater and drinking water treatment technologies, liposomes can be completely removed by membrane ultrafiltration but metal oxide nanoparticles cannot. Further research will need to test whether this method is applicable to environmental matrices with a complex composition. Encapsulation of graphene quantum dots by liposomes will potentially allow monitoring of the nanoparticle-loaded liposomes during their subsequent removal from the treated water before it is released to local waterways or delivered to homes for drinking.

Conclusions
Coating with polydopamine and encapsulation within lecithin liposomes prevent TiO 2 and ZnO nanoparticles from aggregation during extended chromatographic separation through the gel filtration matrix, even in a long column. To the best of our knowledge, this is the first report on the gel filtration chromatographic analysis of lecithin liposomes that demonstrates their potential application for cleaning up two different types of metal oxide nanoparticles in water treatment, at a high efficiency but with a low cost. The colloidal stability of TiO 2 -PDA nanoparticles is important but not crucial for this new method because every sample is homogenized ultrasonically both during encapsulation by lecithin liposomes and before each GFC-FD analysis. In water treatment, unstable nanoparticles form aggregates, undergo sedimentation, and thus do not need liposome encapsulation to clean them up. Considering the importance of industrial wastewater and drinking water treatment technologies, liposomes can be completely removed by membrane ultrafiltration but metal oxide nanoparticles cannot. Further research will need to test whether this method is applicable to environmental matrices with a complex composition. Encapsulation of graphene quantum dots by liposomes will potentially allow monitoring of the nanoparticle-loaded liposomes during their subsequent removal from the treated water before it is released to local waterways or delivered to homes for drinking.