In Situ Preparation of Amphibious ZnO Quantum Dots with Blue Fluorescence Based on Hyperbranched Polymers and their Application in Bio-Imaging

A new strategy for preparing amphibious ZnO quantum dots (QDs) with blue fluorescence within hyper-branched poly(ethylenimine)s (HPEI) was proposed in this paper. By changing [Zn2+]/[OH−] molar ratio and heating time, ZnO QDs with a quantum yields (QY) of 30% in ethanol were obtained. Benefiting from the amphibious property of HPEI, the ZnO/HPEI nanocomposites in ethanol could be dissolved in chloroform and water, acquiring a QY of 53%, chloroform and 11% in water. By this strategy, the ZnO/HPEI nano-composites could be applied in not only in optoelectronics, but also biomedical fields (such as bio-imaging and gene transfection). The bio-imaging application of water-soluble ZnO/HPEI nanocomposites was investigated and it was found that they could easily be endocytosed by the COS-7 cells, without transfection reagent, and they exhibited excellent biological imaging behavior.


Synthesis of HPEI/Zn 2+
A totally of 384.0 mg of HPEI was dissolved by 30 mL of ethanol in a 250 mL conical flask. Then, the solution 13.0 mg of Zn(OAC) 2 in 10 mL of ethanol was added. The mixture was vigorously stirred for 2 days.

Synthesis of Amphibious ZnO QDs with Blue Fluorescence Based on HPEI Hyperbranched Polymers
Typically, 10 mL of HPEI/Zn 2+ was deoxygenated and stirred in two-neck flask. Reflux was carried out for 2 h with oil bath heating at 100 • C under ordinary pressure. The heating temperature was then cooled down to 80 • C and 1.4 mg of NaOH ([Zn 2+ ]/[OH − ] = 1:2) in 4 mL ethanol and 1 mL ultrapure water was quickly injected into the reaction flask. Test samples were taken out from the reaction flask at 2, 4, 6, 8, and 10 h, respectively. The sample collected at 10 h was abbreviated as ZnO 1:2 /HPEI-10 nanocomposites. A similar procedure was also applied for other ZnO

Bio-Imaging Application of ZnO/HPEI Nanocomposites with Blue Fluorescence
The original ethanol solution of ZnO/HPEI nanocomposites were firstly concentrated and then dialyzed by dialysis bag with molecular weight cut off (MWCO) of 1 k Da against distilled water to obtain an aqueous solution of ZnO/HPEI nanocomposites. The original culture medium of COS-7 cells was replaced by phosphate buffer solution (PBS) solution of ZnO/HPEI nanocomposites. After culturing for 6 h, the cell imaging of ZnO/HPEI nanocomposites were characterized by fluorescence microscopy (Olympus Optical Co., Tokyo, Japan).

Measurements
Transmission electron microscopy (TEM) and elemental characterization were performed on a FEI Tecnai F20 microscope (Thermo Fisher Scientific, Waltham, MA, USA), with an energy-dispersive X-ray spectrometer (EDS) (Thermo Fisher Scientific, Waltham, MA, USA), at an accelerating voltage of 200 kV. The ethanol solution of ZnO/HPEI nanocomposites was filtered by 220 nm polyethersulfone filter and sonicated for 10 s immediately before TEM sample preparation. TEM samples were made by adding 10 µL ethanol solution of ZnO/HPEI nanocomposites to a copper grid coated with ultrathin carbon film. UV-Vis spectra were characterized on a UV-1780 spectrophotometer with a 1 cm quartz spectrophotometer cell. For the quantum yield (QY) measurement, the solutions of ZnO/HPEI nanocomposites were commonly diluted with the corresponding solvents to make the absorbance at an excitation wavelength (360 nm) in the range of 0.03 to 0.1. Photoluminescence (PL) spectra were investigated by using a Cary Eclipse spectrometer (Agilent, Santa Clara, CA, USA) with an excitation wavelength of 360 nm. Dynamic light scattering (DLS) data were collected on a Zetasizer Nano-ZS (Malvern Instruments, Malvern, United Kingdom). Photographs were taken with a SAMSUNG WB150F digital camera (Samsung, Seoul, South Korea). TGA weight loss curves were acquired by using Netzsch STA-449F3 (Netzsch, Selb, Germany) under nitrogen at a heating rate of 20 • C/min from 30 to 800 • C.

Results and Discussion
HPEI have a large number of primary, secondary, and tertiary amines. Zn 2+ ions were firstly complexed with the amine groups of HPEI, and then reacted with alkali, which was supplied by HPEI or addition. After refluxing for several hours, fluorescent ZnO QDs with blue emission were obtained based on HPEI, as shown in Scheme 1a-c. After dialysis, the ZnO/HPEI nanocomposites with blue emission were applied on bio-imaging, as shown in Scheme 1d.
The  [20,[24][25][26], zinc salts and alkali were used to prepare size-controlled ZnO QDs without other stabilizers, and the hydroxyl ions and their counter ions actually as a capping layer around the QDs, thus, the size-tunability of ZnO QDs could be realized by [Zn 2+ ]/[OH − ] molar ratios. Here, HPEI, with a three-dimensional architecture and plenty of amines, were used to segregate, stabilize and control the size of ZnO QDs. The size-tunability of ZnO QDs might be tuned by adjusting the molar ratio of [Zn 2+ ]/[HPEI], as reported in our previous work for Fe 3 O 4 nanocrystal synthesis [37]. The hydroxyl ions and their counter ions cannot control the size ZnO QDs anymore, but they could still adsorb on the surface of ZnO QDs and influence the emission property of ZnO QDs.
The quantum yields (QY) of ZnO QDs were measured according to the method described in reference [38]. Cumarin [37]. The hydroxyl ions and their counter ions cannot control the size ZnO QDs anymore, but they could still adsorb on the surface of ZnO QDs and influence the emission property of ZnO QDs. The quantum yields (QY) of ZnO QDs were measured according to the method described in reference [38]. Cumarin        Transmission electronic microscopy (TEM) was used to characterize the morphology and size distribution of ZnO 1:2 /HPEI-10 nanocomposites, which were synthesized with [Zn 2+ ]/[OH − ] molar ratio of 1:2 and 10 h heating, as shown in Figure 3. It can be seen the ZnO QDs are relatively uniform and monodisperse. This is because HPEI could segregate ZnO QDs and prevent their aggregation. The ZnO QDs have a mean diameter of 3.0 nm by measuring hundreds of nanoparticles. The corresponding EDS spectrum, shown in Figure 3b, displays the signals of Zn and O elements, confirming the existence of ZnO QDs.
Polymers 2020, 12, x FOR PEER REVIEW 6 of 11 Transmission electronic microscopy (TEM) was used to characterize the morphology and size distribution of ZnO1:2/HPEI-10 nanocomposites, which were synthesized with [Zn 2+ ]/[OH − ] molar ratio of 1:2 and 10 h heating, as shown in Figure 3. It can be seen the ZnO QDs are relatively uniform and monodisperse. This is because HPEI could segregate ZnO QDs and prevent their aggregation. The ZnO QDs have a mean diameter of 3.0 nm by measuring hundreds of nanoparticles. The corresponding EDS spectrum, shown in Figure 3b, displays the signals of Zn and O elements, confirming the existence of ZnO QDs.  Figure 4 shows the size distribution of neat HPEI and ZnO1:2/HPEI-10 nanocomposites, measured by DLS. The hydrodynamic diameter of neat HPEI is 3.6 nm, while the ZnO1:2/HPEI-10 nanocomposites have a diameter of 4.9 nm. According to TEM data and the hydrodynamic diameter change of HPEI before, and after, quantum dot (QD) synthesis, the ZnO QDs should be encapsulated into the interior cavities of HPEI and one ZnO QD is complexed with each HPEI in average.   Figure 4 shows the size distribution of neat HPEI and ZnO 1:2 /HPEI-10 nanocomposites, measured by DLS. The hydrodynamic diameter of neat HPEI is 3.6 nm, while the ZnO 1:2 /HPEI-10 nanocomposites have a diameter of 4.9 nm. According to TEM data and the hydrodynamic diameter change of HPEI before, and after, quantum dot (QD) synthesis, the ZnO QDs should be encapsulated into the interior cavities of HPEI and one ZnO QD is complexed with each HPEI in average. Transmission electronic microscopy (TEM) was used to characterize the morphology and size distribution of ZnO1:2/HPEI-10 nanocomposites, which were synthesized with [Zn 2+ ]/[OH − ] molar ratio of 1:2 and 10 h heating, as shown in Figure 3. It can be seen the ZnO QDs are relatively uniform and monodisperse. This is because HPEI could segregate ZnO QDs and prevent their aggregation. The ZnO QDs have a mean diameter of 3.0 nm by measuring hundreds of nanoparticles. The corresponding EDS spectrum, shown in Figure 3b, displays the signals of Zn and O elements, confirming the existence of ZnO QDs.  Figure 4 shows the size distribution of neat HPEI and ZnO1:2/HPEI-10 nanocomposites, measured by DLS. The hydrodynamic diameter of neat HPEI is 3.6 nm, while the ZnO1:2/HPEI-10 nanocomposites have a diameter of 4.9 nm. According to TEM data and the hydrodynamic diameter change of HPEI before, and after, quantum dot (QD) synthesis, the ZnO QDs should be encapsulated into the interior cavities of HPEI and one ZnO QD is complexed with each HPEI in average.   Figure 5 shows a comparison of FT-IR spectra between 4000 and 500 cm −1 of neat HPEI and ZnO 1:2 /HPEI-10 nanocomposites. The N-H stretching vibration of primary and secondary amine groups in HPEI locate at 3286 cm −1 in Figure 5a, and it red shift to 3429 cm −1 in Figure 5b for the ZnO 1:2 /HPEI-10 nanocomposites. The bands at 2917 (or 2921) and 2813 (or 2850) cm −1 in all curves correspond to asymmetric -CH 2 -stretching vibration, and symmetric -CH 2 -stretching vibration, respectively. The bending vibrations of primary amines and secondary amines, in HPEI in Figure 5a, appear at 1669, and 1589 cm −1 , respectively. While, the bending vibrations of primary amines and secondary amines in ZnO 1:2 /HPEI-10 nanocomposites shift to 1631, and 1604 cm −1 , respectively. These frequency changes in FT-IR can be attributed to the complexation interactions between amines of HPEI and ZnO QDs.
Polymers 2020, 12, x FOR PEER REVIEW 7 of 11 Figure 5 shows a comparison of FT-IR spectra between 4000 and 500 cm −1 of neat HPEI and ZnO1:2/HPEI-10 nanocomposites. The N-H stretching vibration of primary and secondary amine groups in HPEI locate at 3286 cm −1 in Figure 5a, and it red shift to 3429 cm −1 in Figure 5b for the ZnO1:2/HPEI-10 nanocomposites. The bands at 2917 (or 2921) and 2813 (or 2850) cm −1 in all curves correspond to asymmetric -CH2-stretching vibration, and symmetric -CH2-stretching vibration, respectively. The bending vibrations of primary amines and secondary amines, in HPEI in Figure 5a, appear at 1669, and 1589 cm −1 , respectively. While, the bending vibrations of primary amines and secondary amines in ZnO1:2/HPEI-10 nanocomposites shift to 1631, and 1604 cm −1 , respectively. These frequency changes in FT-IR can be attributed to the complexation interactions between amines of HPEI and ZnO QDs. The composition of ZnO1:2/HPEI-10 nanocomposites was measured by thermogravimetric analysis (TGA), as shown in Figure 6. The decomposition temperature range of neat HPEI was 295-420 °C, which can be seen in Figure 6a. At 420 °C, the weight loss of HPEI is 100 wt%, indicating that HPEI have completely decomposed and volatilized. For the TGA of ZnO1:2/HPEI-10 nanocomposites, shown in Figure 6b, there is a certain amount of weightlessness of absorbed physics and chemistry water in the temperature range of 100-200 °C. Figure 6b also shows a large weight loss from 326 to 420 °C, which can be assigned to the decomposition of HPEI. After 420 °C, the weight loss is unchanged and the remained mass content, assigned to ZnO QDs, is about 3.2%. The content of ZnO QDs could be adjusted by changing the amount of Zn(OAc)2 and NaOH during ZnO QD synthesis. We have increased the amount of Zn(OAc)2 and NaOH to 196.6 mg (1.075 mmol), and 85.7 mg (2.150 mmol), respectively. While, the amount of HPEI (384 mg, 0.0154 mmol) is unchanged. The content of ZnO QDs could be increased to more than 30% under such synthesis condition.
Benefiting from the excellent solubility of HPEI in many solvents, such as ethanol, chloroform, water, dimethylsulfoxide (DMSO), N-methylformamide (NMF), the ZnO1:2/HPEI-10 nanocomposites should also be soluble in these solvents. Here, the original ZnO1:2/HPEI-10 ethanol solution was dried under vacuum, and then dissolved in ethanol and chloroform with a concentration of 3 mg/mL, respectively. The original ZnO1:2/HPEI-10 ethanol solution was also dialyzed by dialysis bag with molecular weight cut off (MWCO) of 1 k Da against distilled water to get water-soluble ZnO1:2/HPEI-10 nanocomposites. Figure 7 shows a comparison of UV-Vis and PL spectra of ZnO1:2/HPEI-10 nanocomposites in ethanol, chloroform, and water, respectively. The aqueous ZnO1:2/HPEI-10 The composition of ZnO 1:2 /HPEI-10 nanocomposites was measured by thermogravimetric analysis (TGA), as shown in Figure 6. The decomposition temperature range of neat HPEI was 295-420 • C, which can be seen in Figure 6a. At 420 • C, the weight loss of HPEI is 100 wt%, indicating that HPEI have completely decomposed and volatilized. For the TGA of ZnO 1:2 /HPEI-10 nanocomposites, shown in Figure 6b, there is a certain amount of weightlessness of absorbed physics and chemistry water in the temperature range of 100-200 • C. Figure 6b also shows a large weight loss from 326 to 420 • C, which can be assigned to the decomposition of HPEI. After 420 • C, the weight loss is unchanged and the remained mass content, assigned to ZnO QDs, is about 3.2%. The content of ZnO QDs could be adjusted by changing the amount of Zn(OAc) 2 and NaOH during ZnO QD synthesis. We have increased the amount of Zn(OAc) 2 and NaOH to 196.6 mg (1.075 mmol), and 85.7 mg (2.150 mmol), respectively. While, the amount of HPEI (384 mg, 0.0154 mmol) is unchanged. The content of ZnO QDs could be increased to more than 30% under such synthesis condition.
Benefiting from the excellent solubility of HPEI in many solvents, such as ethanol, chloroform, water, dimethylsulfoxide (DMSO), N-methylformamide (NMF), the ZnO 1:2 /HPEI-10 nanocomposites should also be soluble in these solvents. Here, the original ZnO 1:2 /HPEI-10 ethanol solution was dried under vacuum, and then dissolved in ethanol and chloroform with a concentration of 3 mg/mL, respectively. The original ZnO 1:2 /HPEI-10 ethanol solution was also dialyzed by dialysis bag with molecular weight cut off (MWCO) of 1 k Da against distilled water to get water-soluble ZnO 1:2 /HPEI-10 nanocomposites. Figure 7 shows a comparison of UV-Vis and PL spectra of ZnO 1:2 /HPEI-10 nanocomposites in ethanol, chloroform, and water, respectively. The aqueous ZnO 1:2 /HPEI-10 nanocomposites have an obvious absorption peak at 365 nm, which is distinct from ZnO 1:2 /HPEI-10 nanocomposites in ethanol and chloroform phase, due to dialysis and solvent polarity differences. The corresponding emission peak has a red shift from 408 to 423 nm, compared with that of ZnO 1:2 /HPEI-10 nanocomposites in an ethanol and chloroform phase. For the fluorescence property, the QY of ZnO 1:2 /HPEI-10 nanocomposites in ethanol, chloroform, and water is 30%, 53%, and 11%, respectively.
Polymers 2020, 12, x FOR PEER REVIEW 8 of 11 nanocomposites have an obvious absorption peak at 365 nm, which is distinct from ZnO1:2/HPEI-10 nanocomposites in ethanol and chloroform phase, due to dialysis and solvent polarity differences. The corresponding emission peak has a red shift from 408 to 423 nm, compared with that of ZnO1:2/HPEI-10 nanocomposites in an ethanol and chloroform phase. For the fluorescence property, the QY of ZnO1:2/HPEI-10 nanocomposites in ethanol, chloroform, and water is 30%, 53%, and 11%, respectively.  HPEI, with a hyperbranched structure and many terminal amines, have been regarded as one of the most promising non-viral gene vectors. They are also widely used in uses, including gene transfection and drug delivery. While, ZnO QDs have excellent fluorescence and are non-toxic compared with cadmium-series QDs, the fluorescent ZnO/HPEI nanocomposites combine the advantages of HPEI and ZnO QDs together, and have great prospects on bio-imaging, gene transfection, and drug release with ZnO QDs as fluorescence probes. Here, the application of ZnO1:2/HPEI-10 nanocomposites on bio-imaging was investigated. The COS-7 cells showed no nanocomposites have an obvious absorption peak at 365 nm, which is distinct from ZnO1:2/HPEI-10 nanocomposites in ethanol and chloroform phase, due to dialysis and solvent polarity differences. The corresponding emission peak has a red shift from 408 to 423 nm, compared with that of ZnO1:2/HPEI-10 nanocomposites in an ethanol and chloroform phase. For the fluorescence property, the QY of ZnO1:2/HPEI-10 nanocomposites in ethanol, chloroform, and water is 30%, 53%, and 11%, respectively.  HPEI, with a hyperbranched structure and many terminal amines, have been regarded as one of the most promising non-viral gene vectors. They are also widely used in uses, including gene transfection and drug delivery. While, ZnO QDs have excellent fluorescence and are non-toxic compared with cadmium-series QDs, the fluorescent ZnO/HPEI nanocomposites combine the advantages of HPEI and ZnO QDs together, and have great prospects on bio-imaging, gene transfection, and drug release with ZnO QDs as fluorescence probes. Here, the application of ZnO1:2/HPEI-10 nanocomposites on bio-imaging was investigated. The COS-7 cells showed no HPEI, with a hyperbranched structure and many terminal amines, have been regarded as one of the most promising non-viral gene vectors. They are also widely used in uses, including gene transfection and drug delivery. While, ZnO QDs have excellent fluorescence and are non-toxic compared with cadmium-series QDs, the fluorescent ZnO/HPEI nanocomposites combine the advantages of HPEI and ZnO QDs together, and have great prospects on bio-imaging, gene transfection, and drug release with ZnO QDs as fluorescence probes. Here, the application of ZnO 1:2 /HPEI-10 nanocomposites on bio-imaging was investigated. The COS-7 cells showed no fluorescence under a fluorescence microscope. However, after the COS-7 cells were cultured for 6 h in the PBS solution of ZnO 1:2 /HPEI-10 nanocomposites, it can be clearly seen that blue fluorescence was observed under a fluorescence microscope, as shown in Figure 8. This phenomenon indicates that the ZnO 1:2 /HPEI-10 nanocomposites could be internalized by COS-7 cells via endocytosis. Green et al. reported that bare cysteine-capped particles could not be endocytosed by the cells and cationic liposome treated QDs can be internalized into human breast cancer cells [39]. It can be seen that the transfection reagent is essential for cell imaging applications. Benefiting from the gene transfection property of HPEI, the ZnO 1:2 /HPEI-10 nanocomposites could be easily endocytosed by cells.
Polymers 2020, 12, x FOR PEER REVIEW 9 of 11 fluorescence under a fluorescence microscope. However, after the COS-7 cells were cultured for 6 h in the PBS solution of ZnO1:2/HPEI-10 nanocomposites, it can be clearly seen that blue fluorescence was observed under a fluorescence microscope, as shown in Figure 8. This phenomenon indicates that the ZnO1:2/HPEI-10 nanocomposites could be internalized by COS-7 cells via endocytosis. Green et al. reported that bare cysteine-capped particles could not be endocytosed by the cells and cationic liposome treated QDs can be internalized into human breast cancer cells [39]. It can be seen that the transfection reagent is essential for cell imaging applications. Benefiting from the gene transfection property of HPEI, the ZnO1:2/HPEI-10 nanocomposites could be easily endocytosed by cells.

Conclusions
In summary, water-soluble and oil-soluble HPEI were used as nanoreactors to synthesize uniform and amphibious ZnO QDs with blue fluorescence in ethanol. By changing [Zn 2+ ]/[OH − ] molar ratio and heating time, ZnO QDs with a QY of 30% were gained. The resulting ZnO/HPEI nanocomposites in ethanol could be dissolved in chloroform and water after vacuum drying, or dialysis, respectively. The QY of ZnO/HPEI nanocomposites can reach to 53% in chloroform and 11% in water. Thus, the application of the resulting ZnO/HPEI nanocomposites could be extended, not only to optoelectronics, but also biomedical field (such as bio-imaging, gene transfection). The resulting water-soluble ZnO/HPEI nanocomposites could easily be endocytosed by the COS-7 cells without transfection reagent and exhibited excellent biological imaging behavior.

Conflicts of Interest:
The authors declare no conflict of interest.

Conclusions
In summary, water-soluble and oil-soluble HPEI were used as nanoreactors to synthesize uniform and amphibious ZnO QDs with blue fluorescence in ethanol. By changing [Zn 2+ ]/[OH − ] molar ratio and heating time, ZnO QDs with a QY of 30% were gained. The resulting ZnO/HPEI nanocomposites in ethanol could be dissolved in chloroform and water after vacuum drying, or dialysis, respectively. The QY of ZnO/HPEI nanocomposites can reach to 53% in chloroform and 11% in water. Thus, the application of the resulting ZnO/HPEI nanocomposites could be extended, not only to optoelectronics, but also biomedical field (such as bio-imaging, gene transfection). The resulting water-soluble ZnO/HPEI nanocomposites could easily be endocytosed by the COS-7 cells without transfection reagent and exhibited excellent biological imaging behavior.