Development of Biocompatible Quantum Dots using Zwitterionic Copolymer for Biological

Biocompatible Inorganic Nanomaterial Polymer Coating for Biomedical Applications, 2013, Kicheol Yoon, Graduate Program in Chemistry, Ulsan National Institute of Science and Technology (UNIST). In recent years, zwitterionic polymers have been extensively studied for their unique water resistance and highly effective antifouling properties. This work describes a new class of polymeric ligands for quantum dots (QDs), a QD-coated short-chain zwitterionic ligand stabilized against aggregation from proteins.

The zwitterionic polymers are synthesized by reversible addition-fragmentation-chain-transfer-mediated polymerization to control the uniform molecular weight of random copolymer with histidine group for the binding of QD surface, and (3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl )ammonium hydroxide (MPDSAH) group for water solubility, and primary amine group as binding site for derivatization. By then carefully handling the surface primary amine, folate-conjugated QDs (QD-FAs) with high photostability are prepared. We demonstrate receptor-mediated conjugation of folic acid-functionalized QDs on living cell lines such as Hela cells.

AIBN Azobisisobutyronitrile CTA Chain Transfer Agent DCM Dichloromethane DLS Dynamic Light Scattering DMF N,N'-Dimethylformamide FA Folic Acid. MW Molecular Weight NHS N-Hydroxysuccinimide NMR Nuclear Magnetic Resonance OEG Oilgo Ethylene Glycol PC Phosphorylcholine PEG Poly(ethylene glycol) PBS Phosphate Buffered Saline PS Penicillin/Streptomycin QD Quantum Dot.

Introduction

Results and Discussion

The conditions of ligand exchange were relatively mild and, as a result, the promotion of a mixture of QDs and polyzwitterionic ligand in a mixture solvent of chloroform and methanol at 50 ℃ (Scheme 3). QD-PZLs were dissolved in water or PBS and then further purified by filtration. The absorption point of QD-PZL, QD-PZ(aminoPEG3)10% and QD-PZ(aminoPEG3)25% exhibited a slight red shift (Table 2).

And we measured the DLS of QD-PZ(aminoPEG3)10% and QD-PZ(aminoPEG3)25% once again after folic acid conjugation. As a result, folic acid-conjugated QD-PZLs were shown to reduce the particle size less than QD-PZ(aminoPEG3) without folic acid. TEM analysis of the QDs after ligand exchange with the polyzwitterionic ligand shows that the QDs are well dispersed.

We measured absorbance about three times, QD-PZ(aminoPEG3) before derivatization and QD-FITC and QD-FITC mixture after filter. The number of working FITC was calculated by comparing the increased absorbance intensity of the filtered QD-FITC to the pure QD-PZ (aminoPEG3) absorbance. We confirmed that 38, 47 amine functional groups per particle were attached for each QD-PZ(aminoPEG3) 10% and QD-PZ(aminoPEG3) 25% respectively.

Before adding the amine, the QD-PZLs had a zeta potential of -24.8 mV, indicating that they are well dispersed in water. In order to find a suitable pH for the stability of the dispersed QD-PZL in water, we measured the DLS of the particles compared to QD-PIL2. As shown in Figure 5, QD-PIL increased in size at pH lower than 5, while QD-PZL had a linear size without variation over the entire pH range.

To demonstrate the nonspecific binding of polyzwitterionic ligand-coated QDs, we incubated Hela cells with various amine functional group-modified QD-PZLs and then washed the Ferri cells with phosphate-buffered saline (PBS). In case QD-PZL without functional group, which was not conjugated with folic acid, did not bind to the folate receptor of Hela cells (Figure 6A). Alternatively, folic acid-conjugated QD-PZL showed binding folate receptor of Hela cells, consistent with an increase in the amount of amines on the QD surface.

Folate was conjugated to the functional amine on the surface of QD-PZL using EDC/NHS coupling chemistry. QD-PZ(aminoPEG3) 10%-FAs targeted folate receptor and (C) QD-PZ(aminoPEG3) 25%-FAs targeted folate receptor, Fluorescence imaging of Hela cells by nm emission scanning with 488 nm excitation.

Table 1 Nomenclature of polyzwitterionic ligand used compound.

Summary

Experimental

Ditert-butyl dicarbonate was added dropwise with stirring at 4°C and the reaction was left overnight at room temperature. The appropriate amount of water was added to separate DMF from the crude solution, and the solution was extracted with CHCl3 (4 x 20 mL). The pure product was obtained by means of a silica column (change the ratio of the ethyl acetate/hexanes gradient).

Synthesis of 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid (TC) 41 TC was purchased from Aldrich and also synthesized according to the literature method. Synthesis of TC-terminated poly(ethylene glycol) (PEG-CTA)42 TC (7.2 g, 20 mmol) and oxalyl chloride (8.6 mL, 100 mmol) were added to 40 mL of dry CH2Cl2 and stirred at room temperature, until gas evolution has stopped. Excess reagents were then removed under vacuum and the residue redissolved in 80 mL dry CH2Cl2 followed by the addition of methoxy poly(ethylene glycol) (Mn = 2000 g/mol) (8 g, 4 mmol in 100 mL dry CH2Cl2).

The reaction was stirred at room temperature for 24 hours and the mixture was concentrated before precipitating from excess cold Et2O. The crude polymer obtained by filtration was purified by redissolving in CH 2 Cl 2 , precipitated in Et 2 O and dried in vacuo overnight. The deprotected polymer was dissolved in water and a solution of NaOH in MeOH (1 M) was added dropwise to adjust the pH close to 7-8.

The solvent was removed in vacuo and then the product was dissolved in water and the solution was filtered through a 0.2 µm cellulose syringe filter. The QD stock solution was mixed with polymer of PZL (100 mg) and stirred for 30 min at room temperature, after which 100 μL of water was added followed by stirring for another 120 min at 50 ℃ in oil bath. QD-PZLs in solvent were precipitated by adding ethanol (100 μL), CHCl3 (100 μL) and excess hexanes for washing.

The clear supernatant was discarded, and the pellet was dried in vacuo, followed by the addition of PBS (500 μL, pH 7.4). We used SLS to measure molecular weight of polymer rather than GPC because the zwitterionic polymer was dissolved in only water. Hela cells were propagated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with FBS (Fetal Bovine Serum 10%) and PS (Penicillin/Streptomycin 1%).

Cultured cells were then trypsinized and resuspended in DMEM at a concentration of about 2 × 104 cells/ml. To each chamber, DMEM medium (500 uL) and folic acid-conjugated QDs (400 ug/mL) were added and incubated for 12 h.