Materials and Instrumentation
All chemicals unless indicated were obtained from Sigma Aldrich and used as received. Air sensitive materials were handled in an Omni-Lab VAC glove box under dry nitrogen atmosphere with oxygen levels <0.2 ppm. All solvents were spectrophotometric grade and purchased from EMD Biosciences. Amine-bearing compounds were visualized on thin layer chromatography (TLC) plates using a ninhydrin solution. Acrylate compounds bearing terminal vinyl groups were visualized on TLC using KMnO4. All other TLC plates were visualized by iodine staining. Flash column chromatography was performed on a Teledyne Isco CombiFlash Companion. 1H NMR spectra were recorded on a Bruker DRX 401 NMR Spectrometer. UV-Vis absorbance spectra were taken using an HP 8453 diode array spectrophotometer. Photoluminescence and absorbance spectra were recorded with a BioTek Synergy 4 Microplate Reader. Dynamic light scattering analysis was performed on a Malvern Instruments ZetaSizer ZS90 in a low volume 12 μL quartz cuvette, with QD concentrations between 1-3 μM. Polymer molecular weights were determined in DMF solutions on an Agilent 1100 series HPLC/GPC system with three PLgel columns (103, 104, 105 Å) in series against narrow polystyrene standards.
To a stirred solution of acrylic acid (1.00 g, 13.88 mmol) and N-hydroxysuccinimide (NHS) (1.91 g, 16.65 mmol) in 40 mL of dry THF was added dropwise a solution of dicyclohexylcarbodiimide (DCC) (3.43g, 16.65 mmol) in 10 mL dry THF with stirring at 4 °C. The solution was warmed to room temperature and stirred for 2 h. Precipitates were removed by filtration, and the solvent was evaporated in vacuo. Ethylacetate (50 mL) was added to facilitate further precipitation of reaction byproducts, and the solution was filtered once more. The solvent was evaporated and the product dissolved in either 10 mL of anhydrous DMF or dry THF to create a stock solution, which was used in later reaction steps without further purification.
To an aqueous solution of sodium bicarbonate (50 mL, 0.3 M) was added DMF (50 mL) and histamine dihydrochloride (2.50 g, 13.59 mmol). To this solution was added compound 2 (2.75 g, 16.3mmol) in a solution of DMF, with stirring at 4 °C. The reaction was monitored via TLC by ninhydrin stain for primary amines, and confirmed to be complete after 30 min to give the crude compound 3. The solvent was removed in vacuo, and the product redissolved in DMF (50 mL). The solution was filtered, and triethylamine was introduced (2.27 mL, 16.30 mmol). Di-tert-butyl dicarbonate was added dropwise at 4 °C, and the solution was stirred overnight at RT. Water was added and the solution extracted with CHCl3 (3 × 25 mL). The organics were combined and dried over sodium sulfate, and the solvent removed in vacuo. The crude product was purified by silica column (ethyl acetate/hexanes gradient 50:50 to 100:0, v/v) to give the pure product as a clear oil (2.59 g, 72% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) 7.95 (s, 1H), 7.10 (s, 1H), 6.19 (dd, J1 = 1.8 Hz, J2 = 17.0 Hz, 1H), 6.07 (dd, J1 = 9.8 Hz, J2 = 17.0 Hz, 1H), 5.53 (dd, J1 = 1.8 Hz, J2 = 10.0 Hz, 1H), 3.53 (dt, 2H), 2.72 (t, 2H), 1.54 (s, 9H).
Neat methoxy poly(ethylene glycol) (10 g, 18.18 mmol, average MW 550 g/mol) was degassed at 80 °C for 1 h with stirring to remove traces of water. The flask was back-filled with N2 and cooled on an ice bath before thionyl chloride (1.98 mL, 27.27 mmol) was slowly added. The solution was warmed to 25 °C and stirred for 2 h. The conversion was monitored by the disappearance of the broad O–H stretch at 3,500 cm−1 and the appearance of a C–Cl stretch at 730 cm−1 in the IR spectrum. The product was diluted with DMF (20 mL) and the solvent removed under reduced pressure. This was repeated three times to remove all residual traces of thionyl chloride. The sample was dissolved in a solution of sodium azide (1.77 g, 27.27 mmol) in 100 mL DMF and stirred overnight at 85 °C. The solvent was removed under reduced pressure and 200 mL of dichloromethane was added. The precipitate was removed by vacuum filtration and the solvent evaporated in vacuo to yield the intermediate mono-azide. The sample was dissolved in 150 mL of tetrahydrofuran (THF), and triphenylphosphine (7.15 g, 27.27 mmol) was added. The solution was stirred at 25 °C for 4 h before adding 1 mL of water and stirring overnight. The THF was removed in vacuo and 100 mL of water was added. The precipitate was removed by vacuum filtration and the filtrate washed with toluene (3 × 50 mL). The water was removed in vacuo to yield the pure product as light yellow oil (9.67 g, 95%). 1H NMR (400 MHz, CDCl3): δ (ppm) 3.69 – 3.46 (m, 46H), 3.37 (s, 3H), 2.85 (t, 2H).
To a solution of compound 5 (2.20 g, 3.94 mmol) in dry THF was added compound 2 (1.00 g, 5.92 mmol) in a solution of dry THF, with stirring at 4 °C. The reaction was monitored via TLC by ninhydrin stain for primary amines, and confirmed to be complete after 30 min. The solution was filtered and the solvent evaporated in vacuo. The crude product was purified by silica column (methanol/ethyl acetate gradient 0:100 to 5:95, v/v) to give the pure product as a pale yellow oil (1.88 g, 78% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) 6.68, 6.19 (dd, J1 = 2.0 Hz, J2 = 17.0 Hz, 1H), 6.08 (dd, J1 = 9.8 Hz, J2 = 17.0 Hz, 1H), 5.52 (dd, J1 = 2.0 Hz, J2 = 9.8 Hz, 1H), 3.56 – 3.37 (m, 48H), 3.27 (s, 3H).
To a solution of 4,7,10-trioxa-1,13-tridecanediamine (10.00 g, 45.45 mmol) in DCM (25 mL) was added dropwise di-tert-butyl dicarbonate (1.98 g, 9.09 mmol) at 4 °C. The solution was allowed to warm to RT and stirred overnight. The solution was washed with water (3 × 20 mL) to remove unreacted starting material. TLC analysis with ninhydrin staining shows mostly mono-substituted product in the organic phase. The organics were dried over sodium sulfate and solvent removed in vacuo. The crude product (3.80 g, 17.27 mmol) was dissolved in a mixture of aqueous sodium bicarbonate buffer (20 mL, 0.3 M), and DMF (20 mL), to which compound 2 (3.38 g, 20.00 mmol) was added dropwise in a solution of DMF with stirring at 4 °C. The reaction was monitored via TLC by ninhydrin stain for primary amines, and confirmed to be complete after 30 min. Water was added and the solution extracted with CHCl3 (3 × 25 mL). The organics were combined and dried over sodium sulfate, and the solvent removed in vacuo. The crude product was purified by silica column (ethyl acetate/methanol gradient 100:0 to 95:5, v/v) to give the pure product as a clear oil (4.52 g, 27% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) 6.23 (dd, J1 = 2.0 Hz, J2 = 17.0 Hz, 1H), 6.08 (dd, J1 = 9.8 Hz, J2 = 17.0 Hz, 1H), 5.56 (dd, J1 = 2.0 Hz, J2 = 9.8 Hz, 1H), 3.65 – 3.44 (m, 14H), 3.17 (t, 2H), 1.83 – 1.65 (m, 4H), 1.39 (s, 12H).
To a solution of O-(2-Aminoethyl)-O′-[2-(Boc-amino)ethyl]decaethylene glycol (0.50 g, 0.78 mmol) in dry THF (25 mL) was added triethylamine (0.086 g, 0.85 mmol) and compound 2 (0.20 g, 1.16 mmol) dropwise in a solution of THF with stirring at 4 °C. The reaction was monitored via TLC by ninhydrin stain for primary amines, and confirmed to be complete after 30 min. The solution was filtered and the solvent removed in vacuo. The crude product was purified by silica column (DCM/MeOH gradient 100:0 to 95:5, v/v) to give the pure product as a clear oil (0.38 g, 70% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) 6.28 (dd, J1 = 2.0 Hz, J2 = 17.0 Hz, 1H), 6.16 (dd, J1 = 9.8 Hz, J2 = 17.0 Hz, 1H), 5.59 (dd, J1 = 2.0 Hz, J2 = 9.8 Hz, 1H), 3.70 – 3.50 (m, 46H), 3.30 (q, 2H), 1.42 (s, 9H).
To a solution of O-(2-Aminoethyl)-O′-[2-(Boc-amino)ethyl] decaethylene glycol (0.50 g, 0.78 mmol) in DMF (150 mL) was added biotin (0.21 g, 0.86 mmol) and EDC (0.13 g, 0.86 mmol). The solution was stirred overnight, and the solvent removed in-vacuo. The crude product was purified by silica column (DCM/MeOH 98:2, v/v) to give the pure product as a colorless oil (0.63 g, 85% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) 4.23 (m, 1H), 4.43 (m, 1H), 3.48 (m, 4H), 3.52-3.61 (m, 40H), 3.24 (m, 2H), 3.35 (m, 2H), 2.82 (dd, J1 = 12.8 Hz, J2 = 4.9 Hz, 1H), 3.06 (m, 1H), 1.37 (s, 9H), 2.68 (d, J = 12.8 Hz, 1H), 2.16 (t, J = 7.5 Hz, 2H), 1.60 (m, 4H), 1.40 – 1.32 (m, 2H).
To compound 10 (0.50 g, 0.57 mmol) was added 4 M HCl in dioxane, and stirred for 1 h at room temperature. The solvent was removed in vacuo, and the crude product dissolved into a solution of 0.25 M aqueous sodium bicarbonate with DMF. To this solution was added dropwise a solution of compound 2. The reaction was monitored via TLC by ninhydrin stain for primary amines and confirmed to be complete after 30 min. The solvent was removed in vacuo, and the crude product was purified by silica column chromatography (DCM:MeOH 98:2, v/v) to give the product as a colorless oil (0.31 g, 65% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) 6.28 (dd, J1 = 2.0 Hz, J2 = 17.0 Hz, 1H), 6.17 (dd, J1 = 9.8 Hz, J2 = 17.0 Hz, 1H), 5.61 (dd, J1 = 2.0 Hz, J2 = 9.8 Hz, 1H), 4.49 (m, 1H), 4.30 (m, 1H), 3.48-3.72 (m, 44H), 3.42 (m, 2H), 3.13 (m, 1H), 2.89 (dd, J1 = 12.8 Hz, J2 = 4.9 Hz, 1H), 2.74 (d, J = 12.8 Hz, 1H), 2.22 (t, J = 7.4 Hz, 2H), 1.66 (m, 4H), 1.43 (m, 2H).
Typical PIL Polymerization
All monomers were kept as dilute stock solutions between 30-100 mg/mL in either ethylacetate or methanol. Stock solutions of RAFT agent 12 were prepared at 220 mg/mL in DMF, and AIBN was prepared at 50 mg/mL in DMF. All reagents were weighed out volumetrically. In a typical polymerization, monomers 4 (33 mg, 0.13 mmol) and 6 (77 mg, 0.13 mmol) were added to an 8 mL vial. The solvent was removed in vacuo and 50 μL of dry DMF along with RAFT agent 12 (2.53 mg, 0.0088 mmol), and AIBN (1.43 mg, 0.0088 mmol) were added. The contents of the vial were mixed, centrifuged at 5000 g for 2 min, and then transferred to a 1 mL ampoule. The ampoule was subjected to 4 cycles of freeze-pump-thaw, and sealed under vacuum using a butane torch. The vial was heated to 70 °C on an oil bath for 1.5-3 h, after which 0.5 mL of a 4 M HCl in dioxane solution was added to cleave the BOC protecting groups. After 1 h at RT, the HCl was removed in vacuo. The deprotected polymer was dissolved in MeOH, to which a solution of NaOH in MeOH (1M) was added dropwise to adjust the pH to be between 8-9. The solvent was removed in vacuo, and then CHCl3 was added to precipitate the salts. The solution was filtered through a 0.45 μm PTFE filter and the solvent removed in vacuo to yield the final polymer for QD ligand exchange.
Quantum Dot Synthesis
CdSe cores were synthesized according to previously reported procedures,20,48,49
and were overcoated with either Zn0.8
S alloy shells or pure CdS shells. The alloy shell overcoating procedure has been described previously,20,49
and was used here to obtain QDs emitting at 565 and 605 nm with QYs of ~80% when diluted in hexane. For pure CdS shells, we developed a successive ion layer adsorption and reaction (SILAR) procedure that is modified from those reported by Peng et al and Mews et al (Xie JACS).36,37
Briefly, CdSe cores with a first exciton feature at 491 nm were synthesized by heating a mixture of trioctylphosphine (TOP), trioctylphosphine oxide (TOPO), CdO (0.9 mmol), and tetradecylphosphonic acid (TDPA, 2.0 mmol) to 340 °C under nitrogen, removing evolved water in vacuo
at 160 °C, re-heating to 360 °C under nitrogen, and rapidly introducing trioctylphosphine selenide (TOPSe, 3.4 mmol) in trioctylphosphine (TOP), followed by cooling to room temperature. Cores isolated by repeated precipitations from hexane with acetone were brought to 180 °C in a solvent mixture of oleylamine (3 mL) and octadecene (6 mL). Aliquots of Cd and S precursor solutions were then introduced alternately starting with the metal (Cd), waiting 15 min between the start of each addition. The Cd precursor consisted of 0.6 mmol Cd-oleate and 1.2 mmol decylamine in a solvent mixture of octadecene (3 mL) and TOP (3 mL). The S precursor consisted of 0.6 mmol hexamethyldisilathiane [(TMS)2
S] in 6 mL TOP. The dose of each overcoating precursor aliquot was calculated to provide a single monolayer of ions to the QD surface. Addition of a total of 4 aliquots each of Cd and S yielded QDs with emission at 562 nm and a QY close to unity when diluted in hexane. A similar procedure was performed on larger CdSe cores to obtain CdSe(CdS) QDs emitting at 610 nm.
Ligand Exchange with poly(PEG)-PIL
QDs (2 nmol) were precipitated using MeOH and brought into 50 μL of CHCl3. The QD stock solution was mixed with solution of poly(PEG) (5 mg) in CHCl3 (30 μL), and stirred for 10 min at RT, after which 30 μL of MeOH was added followed by stirring for an additional 20 min. QD samples were precipitated by the addition of EtOH (30 μL), CHCl3 (30 μL), and excess hexanes. The sample was centrifuged at 4000 g for 2 min. The clear supernatant was discarded, and the pellet dried in vacuo, followed by the addition of PBS (500 μL, pH 7.4) was added. The aqueous sample was then filtered through a 0.2 μm filter syringe filter before use.
Fluorescamine assay of amines on surface of PIL-QDs
QDs emitting at 543 nm ligand-exchanged with various PILs were purified by dialysis 3x through a 50kDa MW cut-off spin concentrator. The QDs were adjusted to 1-2 μM concentration and placed into an eppendorf tube (240 uL). To the tube was added a solution of fluorescamine in acetone (10 uL, 28 mg/mL) followed by vigorous vortexing. The samples were incubated for 10 min at RT and the photoluminescence intensity of Fluorescamine was recorded at 480 nm with an excitation at 380 nm. The amine concentration versus fluorescence count was obtained via a calibration curve generated by performing the same assay on a serial dilution of a known concentration of compound 5.
Covalent Conjugation of Streptavidin to poly(aminoPEG11)25%-PIL QDs
Stretpavidin (50 μL, 10 mg/mL; Sigma Aldrich) was activated in MES buffer (pH 6.5) using Sulfo-NHS and EDC (20 eq) for 20 min at RT. The activated SA was mixed with poly(aminoPEG11)25%-PIL QDs in sodium bicarbonate buffer at pH 8.4 at a SA:QD ratio of 5:1 and allowed to react for 1 h. The samples were dialyzed 2x through a 50 kDa MW cut-off spin concentrator and then used for labeling experiments.
Quantum Yield Measurement
QY of 605 nm emitting QDs was measured relative to Rhodamine 640 (λex
= 535 nm). Solutions of QDs in PBS and dye in ethanol were optically matched at the excitation wavelength. Fluorescence spectra of QD and dye were taken under identical spectrometer conditions in triplicate and averaged. The optical density was kept below 0.1 between 300-800 nm, and the integrated intensities of the emission spectra, corrected for differences in index of refraction and concentration, were used to calculate the QYs using the expression QYQD
) × (Peak AreaQD
) × (nQD solvent
Gel Filtration Apparatus
GFC was performed using an ÄKTAprime Plus chromatography system from Amersham Biosciences equipped with a self-packed Superdex 200 10/100 glass column. PBS (pH 7.4) was used as the mobile phase with a flow rate of 1.0 mL/min. For amine functionalized polymers, the PBS buffer was supplemented with 50 mM of 2-(2-aminoethoxy)ethanol. Typical injection volumes were 100 μL. Detection was achieved by measuring the absorption at 280 nm.
Fluorescamine Assay of Amine Reactivity
Stock solutions of either amine-containing polymers were made at 20 mg/mL concentration. A serial dilution was made using 1, 2, and 4 uL of polymer stock into 240 uL of PBS buffer, followed by addition of 10 uL of a 30 mg/mL solution of fluorescamine. This mixture was vortexed and incubated at room temperature for 10 min before fluorescence analysis on a BioTek plate reader with excitation at 380 nm and detection at 480 nm. The recorded fluorescence intensity signals were calibrated against solutions of known concentrations of compound 5 (methoxyPEG-NH2).
HeLa cells were grown in DMEM (Mediatech) with 10% Fetal Bovine Serum (Invitrogen), 50 U/mL penicillin and 50 μg /mL streptomycin (Invitrogen). The cells were transfected using 1 μL Lipofectamine 2000 (Invitrogen), 0.2 μg of BirA-ER plasmid22,43
and 0.2 μg of AP-YFP-TM plasmid per well of an 8-well chamber slide (LabTek). 1 mM biotin was added to the media during plasmid expression. Cells were imaged under 4 °C PBS the day after transfection. 1% Bovine Serum Albumin (Sigma) was added to block non-specific binding during specific binding studies of ligand-coated quantum dots. Commercial BSA is known to contain biotin, and the stock BSA solution was dialyzed with a 3 kDa cutoff dialysis tube three times for 8 h in PBS pH 7.4, in 4 °C.
Non-specific binding of QDs to serum
565 nm emitting CdSe(CdZnS) QDs (5 μL) of various surface coatings were mixed with fetal bovine serum (95 μL) to a final concentration of ~0.5 μM. The mixture was incubated for 4 h at 37 °C with gentle mixing. The resultant QD size distribution was then measured using gel filtration chromatography. The mixture was injected into a Superose 6 GL10/300 column (GE Healthcare, Piscataway, NJ) on an Agilient 1100 series HPLC with an in-line degasser, autosampler, diode array detector, and fluorescence detector (Roseville, CA). PBS (pH 7.4) was used as the mobile phase with a flow rate of 0.5 mL/min and an injection volume of 50 μL. In order to selectively measure the signal from the QD rather than FBS, the fluorescence detection at 565 nm with 250 nm excitation was chosen.
Fluroescence and Phase Contrast Microscopy
Cells were imaged live using a Nikon TE2000-U inverted microscope with a 60x water-immersion lens and a Princeton Instruments MicroMAX Camera with an additional 1.5x magnification tube lens. Bright field images were collected using differential interference contrast and 10 ms exposure. Fluorescence images were collected with epifluorescent excitation provided by the 488 nm line of an Argon-Ion laser with the appropriate dichroic (Chroma, Z488RDC) and emission filters (QD605: D605/30M, YFP: D565/30m). Images were collected and analyzed using Image J version 1.41o. Typical exposure times were 0.1-0.5 s and fluorescence images were background-corrected.
Animal and tumor models
Orthotopic P008 mammary carcinoma models were prepared by implanting a small piece (1 mm3
) of viable tumor tissue from the source tumor animal into the mammary fat pad chamber44
of 10 – 12 weeks old female Tie2-GFP/FVB mice. The tumors were allowed to grow up to 5 mm in diameter. All animal procedures were carried out following the Public Health Service Policy on Humane Care of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital.
Intravital Multiphoton Imaging
To study tumor vasculature using QDs and their distribution dynamic in live animals, 150 μL poly(PEG)-PIL QD600 at a concentration of 5 μM were injected retro-orbitally into the tumor bearing mice and imaged with multiphoton laser scanning microscope.51
The images were recorded as 3D stacks (200 μm thickness, 1 μm step size) at 0 hour, 3 h and 6 h interval respectively and processed using the NIH ImageJ software. For the GFP channel, the emission filter used was 535±20 nm, and for QD600, the emission filter was 625±75 nm. All images were captured with a 20x water emersion lens (N.A. 0.95) and an excitation wavelength of 880 nm (500 mW).