RAFT polymerization of 13a showing (A) tunable polymer DP as a function of [Monomer] to [RAFT] ratio. (B) GPC of polyPEG in DMF, showing narrow polydispersity with a [Monomer]:[RAFT] ratio of 30:1 and [AIBN]:[RAFT] ratio of 1:1 (—, black), and poor MW distribution without RAFT agent (, red).

Size analysis of poly(PEG) QDs. (A) TEM of QDs ligand exchanged with polyPEG, showing non-aggregated mono-disperse samples. (B) Dynamic light scattering measurement of QDs ligand exchanged with hydroxyPEG (—, black) and QDs ligand exchanged with polyPEG (, red), both showing an HD of ~11.5 nm.

Non-specific binding of QDs on HeLa cells as a function of ligand coating, with incubation at 500 nM QD concentration for 5 min at 4 °C , followed by 4x wash with PBS buffer before imaging. Top: QD fluorescence at 565 nm with excitation at 488 nm. All images are scaled to the same contrast with the exception of (B) and (C), for which the left section has the same contrast as the other images, while the contrast has been boosted in the right section to highlight the difference between (B) and (C). Bottom: corresponding DIC image. QDs were ligand exchanged with (A) DHLA, (B) hydroxyPEG, (C) poly(PEG), (D) poly(aminoPEG₃)_10%, and (E) poly(aminoPEG₁₁)_25%.

Non-specific binding of QDs with serum proteins after incubation with 95% mouse bovine serum for 4 h at 37 °C. QDs before incubation (, red) and after incubation (—, black) for (A) hydroxyPEG QDs, (B) poly(PEG) QDs, and (C) poly(aminoPEG₃)_10% QDs. Insets show fluorescence spectrum of eluent at time indicated by the black arrow, showing QD emission.

Scheme 1 presents the synthetic strategy that was pursued to deliver the monomers for polymerization. Acrylic acid is coupled to primary amine bearing moieties via an amide bond forming reaction. Conjugate addition to the vinyl group was minimized by first preparing the NHS-ester of acrylic acid (2), and allowing the coupling reaction to proceed at 4 °C for only 30 min, upon which complete consumption of the starting materials was confirmed by TLC. Monomer 3 containing the imidazole group for QD binding was obtained from the reaction of histamine with 2. Since the trithiocarbonate RAFT agent 12 used in subsequent polymerization reactions34 is highly sensitive towards degradation by aminolysis, the imidazole nitrogen was BOC protected to yield the final monomer 4. Likewise, monomer 6 containing a PEG₁₁ group for water solubility was obtained first via the conversion of the terminal hydroxyl group of mono-methoxy PEG to a primary amine 5, followed by reaction with 2. Monomers 8 and 9 with BOC-protected terminal amines were also synthesized in order to afford polymers bearing primary amine groups for derivatization with FRET dyes or proteins. Finally, to demonstrate the scope of monomer incorporation in the polymerization reaction scheme, monomer 11 was synthesized to give polymers functionalized with biotin for binding assays. For each of the monomers, formation of an amide bond, as opposed to an ester bond, was necessary in order to circumvent the possibility of ester hydrolysis under the acidic BOC deprotection conditions following polymerization, and also to afford higher stability in the presence of hydrolytic enzymes for in-vivo and in-vitro applications.

Instability of DHLA-PEG derived ligands over time. Gel electrophoresis of DHLA-carboxyPEG QDs (A) after initial ligand exchange and (B) after 1 week storage at 4 °C in the dark, with increasing titration of His₆-Tagged monovalent streptavidin from left to right showing sharp and discrete bands in the initially prepared sample, but loss of fidelity after storage. QDs in (B) also exhibit increased non-specific binding to cells (data not shown).

(A) Absorption spectra (—, black) and emission spectra of QDs before ligand exchange in octane (, blue), and after ligand exchange in PBS (, red), showing a slight decrease in fluorescence intensity with a final QY in water of >60%. (B) Stability of polyPEG QDs in various pH buffers after incubation at RT for 4 h. pH 10.5* refers to emission intensity after 5 minutes irradiation under 365nm UV light, showing ~20% photobrightening.

Covalent derivatization of poly(aminoPEG₃)_10% with ROX dye molecules. (A) Absorption spectrum of purified conjugate (—, black), least-squares fit of conjugate spectrum (, blue), as sum of QD (, green) and free ROX (, red) contributions. (B) Photoluminescence spectrum of conjugate (—, black) and of control QD (, green) and free dye (, red), normalized to reflect the QD and ROX concentrations, respectively, present in the conjugate sample. Contributions of QD (, green) and free ROX (, red) to conjugate emission spectrum as obtained from a least-squares fit are also shown. All samples are excited at 450 nm, with dye emission showing 31 fold enhancement from the free dye vs. the conjugate.

Targeting of poly(aminoPEG₁₁)_25% QD-SA conjugates to live HeLa cells transfected with AP-YFP-TM. (A) Ensemble labeling with 100 nM QDs. (B) Same as in (A), but with QDs pre-incubated with biotin. (C) Low-density labeling with 10 nM QD-SA reveals single QDs.

(A) Time lapsed live QDs imaging of P008 tumor vasculature in Tie2-GFP/FVB mice. Red fluorescence corresponds to the signal from QDs within the vessel lumen (0 and 3h), or extravascular space in tumors (3 and 6h), while the green fluorescence is from the GFP in vascular endothelial cells that line the vessel wall. B, Mean QD (red channel) intensities within and outside of the blood vessels over time. Vessel regions are assigned by thresholding of the T=0h image. The QDs are initially confined within the vessels at T= 0. Images at later times reveal decreased vascular and increased extravascular fluorescence, indicative of clearance from the vessels and extravasation into tumor tissue, respectively.

The reaction of monomers 4, 6, and 8 shown in Scheme 2 is representative of all polymerization reactions used in this study. The monomer mixture typically consists of 50% mole fraction of monomer 4 to ensure that there are enough imidazole groups for effective binding to the QD surface, with the remaining 50% mole fraction consisting of some mixture of monomer 6 (for water solubility), along with one of monomers 8, 9, or 11 (for derivatizability). In order to minimize the potential of long polymer chains cross-linking and aggregating QDs during ligand exchange, the polymer MW was kept low, with a targeted degree of polymerization (DP) below 30. RAFT polymerization can be carried out with any number of conventional radical initiators. These are typically thermally activated and include AIBN, azobis(2-cyanopentanoic acid), and K₂S₂O₈.35 In our case, AIBN was used as the initiator in the presence of the RAFT agent 12 to afford a controlled living polymerization.34 Using an [AIBN] : [RAFT]:[Monomer] ratio of 0.25:1:28 and an equimolar mixture of monomers 4 and 6 (polymer 13a), the overall polymer conversion over time as monitored by ¹H-NMR spectroscopy (Figure S2) followed a linear relationship, achieving >80% conversion after 10 h (Figure S3A). Increasing the [AIBN] : [RAFT] ratio to 1:1 resulted in a non-linear conversion efficiency versus time, yielding >90% conversion after 2 h (Figure S3B); interestingly, a low PDI and good molecular weight control was maintained (vide infra). The plot of [Monomer] : [RAFT] ratio versus measured polymer DP using GPC followed a linear relationship, showing good MW control (Figure 2A), and a narrow PDI <1.2 (Figure 2B) was observed. Figure 2A shows that the targeted DP of <30 for copolymer 13a was achieved using [Monomer]:[RAFT] ratios between 20 to 33:1, as measured by GPC. The same polymerization carried out in the absence of RAFT agent yielded a poorly controlled polymer (PDI > 3), confirming that the RAFT agent is responsible for mediating polymerization even under high relative initiator concentrations (Figure 2B). Because controlled polymerizations could be performed even under the relatively high [AIBN]/[RAFT] ratio of 1:1, new polymers could be rapidly prototyped due to the short reaction times required for near-complete polymer conversion.