Mechanism of cellular uptake of highly fluorescent conjugated polymer nanoparticles.

EXPERIMENTAL Reagents The J774A.1 mouse macrophage-like cell line was obtained from American Type Culture Collections. Texas red dextran (TR-dex; M r = 10,000 MW), TRIZOL reagent, and SuperScript First Strand synthesis system for RT-PCR were obtained from Invitrogen. The conjugated polymer PFBT (M r = 10,000; Polydispersity = 1.7) was purchased from American Dye Source (Quebec, Canada). PCR primers were synthesized by Integrated DNA Technologies. GoTaq Master Mix and Cell Titer Blue were purchased from Promega. Interferon gamma and LPS were purchased from Fisher Scientific and Sigma-Aldrich respectively. Monoclonal antibodies against Clathrin Heavy Chain (D36C) and Caveolin-1 (D46G3) were obtained from Cell Signaling Technology as well as goat anti-rabbit IgG F(ab′) 2 fragment conjugated with Alexa Fluor 647 as a secondary antibody. The rat anti-mouse LAMP-1 antibody conjugated with allophycocyanin (APC) was purchased from Southern Biotechnology. All other chemical reagents used in this study were obtained from Fisher Scientific.

Nanoparticle preparation and characterization

Conjugated polymer nanoparticle preparation was carried out as described previously 27 . Briefly, 20 mg of the conjugated polymer PFBT was dissolved in 10 g of HPLC-grade tetrahydrofuran (THF) by stirring overnight under inert atmosphere. The solution was then filtered through a 0.7 micron glass fiber filter in order to remove any insoluble material. Then 400 μL of the solution above (2000 ppm) was injected by pipette into 8 mL water under mild sonication using a commercial ultrasonic bath (Bransonic) for 1 minute. This solution was then heated in a water bath (~70°C) under constant N 2 -bubbling until THF and half of the water was removed. Next, a second batch (400 μL into 8 mL) of a prepared nanoparticle solution was added, mixed with the previously concentrated solution and heated and under constant N 2 bubbling for another time period to remove THF. 5 mL of the resulting nanoparticle solution remained after THF removal and this solution was dialyzed against 0.01 M borate buffer (pH = 8.5) twice for 12 h. Finally, 5.5 mL PFBT nanoparticle solution in 0.01 M borate was obtained. Nanoparticles were characterized by atomic force microscopy (AFM); PFBT nanoparticle solutions were dried on mica or prepared glass cover slips and height images were recorded as described previously 21 . AFM size distributions were determined from these images to be 18 ± 5 nm. Nanoparticle concentrations were estimated from the mass of conjugated polymer starting material diluted into aqueous solution and the volume of individual particles, assuming complete polymer to nanoparticle conversion. Specifically, particle heights measured by AFM were used to calculate nanoparticle volume, assuming a spherical shape. Nanoparticle volumes were converted to single nanoparticle mass, assuming a nanoparticle density of 1 g/cm 3 (actual density is between 0.95 and 1.05 g/cm 3 ). The total mass of conjugated polymer diluted in the reprecipitation, divided by the mass of a single nanoparticle, yields the number of nanoparticles formed, and is easily converted to moles of nanoparticles. Moles of nanoparticles divided by the final volume of the preparation solution yields the final molar concentration of nanoparticles. Concentration calculations do not take into account small (≤3%) reductions in yield that result from filtration (UV-vis measurements are made before and after filtration) and may therefore be a slight overestimate. Based on these calculations the nanoparticle concentration was 250 ppm/140 nM in 0.01 M borate buffer. From the calculated concentration and the measured absorbance, an absorbance cross section was calculated for the PFBT nanoparticles in water (1.8×10 −13 cm 2 ), consistent with previously reported values 21 . The maximum volume of PFBT nanoparticles added to cells in these studies was 10% of the total culture volume to minimize any effects of the vehicle. Cell Culture The J774A.1 mouse macrophage-like cell line was obtained from American Type Culture Collections and grown in high glucose Dulbecco’s Modified Eagle Medium (DMEM; Mediatech) supplemented with 10% heat inactivated fetal bovine serum (FBS; Hyclone), penicillin/streptomycin, sodium pyruvate, and L–glutamine at 37°C in a humidified incubator with 5% CO 2 until reaching 80–90% confluence. Cells were propagated by incubating for ≥ 5 minutes in ice cold phosphate buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.2) followed by gentle pipetting then split in a 1:4 to 1:8 ratio.

Flow cytometry

Cells were analyzed using flow cytometry by first resuspending them in ice cold Ringer’s buffer (RB; 155 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 2 mM NaH 2 PO 4 , 10 mM glucose, 10 mM HEPES, pH 7.2–7.4) with gentle pipetting and were analyzed on a Becton-Dickenson FACScan flow cytometer. For all experiments, excitation utilized the 488 nm line from a 15 mW argon ion laser and the green fluorescence channel (FL1). For each sample, 10,000 cells were measured. Data was analyzed using FlowJo software (Treestar).

Fluorescence microscopy

All fluorescence images were acquired with an inverted fluorescence microscope (Olympus IX71) equipped with fluorescence excitation from a 300W Xe arc lamp coupled to the microscope via a liquid light guide (Sutter Instruments). Differential interference contrast (DIC) was used for transmitted light imaging. The microscope was equipped with both excitation and emission filter wheels (Sutter Instruments). A Sedat set consisting of a beam splitter, single band excitation filters [387 nm (11 nm band pass), 494 nm (20 nm band pass), 575 nm (25 nm band pass)], and single band emission filters [447 nm (60 nm band pass), 531 nm (22 nm band pass), 624 nm (40 nm band pass)] (Semrock) was used for all experiments. Finally, an Orca-ER CCD (Hamamatsu) was used for image acquisition. Control of all microscope components and all image processing was performed using Slidebook 5.0 (Intelligent Imaging Innovations). Nanoparticle uptake Cells were grown in 35 mm tissue culture dishes until ~ 70% confluent, then treated with PFBT nanoparticles. The maximum final concentration of nanoparticles was 10-fold less than the PFBT nanoparticle stock solution to avoid more than 10% dilution of media. Cells were washed 3X in ice cold RB and detached via gentle pipetting and analyzed by flow cytometry. For imaging of nanoparticle uptake, cells were plated at 100K/dish in glass bottom dishes and incubated with PFBT nanoparticles. Cells were washed 3X in RB and the optical bottom dishes were placed on the microscope in a microscope stage heater at 37°C (Warner Instruments) and images acquired. Colocalization with TR-dex J774A.1 cells were plated at 100K/dish in glass bottom dishes and incubated with 2 nM (4 ppm) PFBT nanoparticles and 250 nM TR-dex overnight. Cells were washed 3X in RB and the optical bottom dishes were placed on the microscope in a microscope stage heater at 37°C and images acquired.

Immunofluorescence and colocalization

The cells were plated at 100K/dish in glass bottom dishes and incubated with 2 nM (4 ppm) PFBT nanoparticles from 2–16 hours. Cells were washed in DMEM and chased from 0–4 hours. Following labeling with nanoparticles, the cells were fixed at 37°C with 4% paraformaldehyde in RB for 10 minutes then washed 3X in RB. The fixed cells were incubated in blocking buffer (BB; RB + 1.5% v/v bovine serum albumin (BSA) and 0.3% Triton-X) for 2 h at 4°C. Next, cells were incubated with the 1° antibody in RB + 1% BSA and 0.3% Triton-X for 16 h at 4°C. Dilutions of 1° antibodies were as follows: LAMP-1 (1:200); clathrin heavy chain (1:200); caveolin-1 (1:200). Following incubation with the 1° antibodies, cells were washed 3× 15 min in BB at room temperature and incubated with the Alexa fluor 647 2° conjugate (1:1000) for 2 h at room temperature in RB + 1% BSA and 0.3% Triton-X. Finally, the cells were washed 3 × 15 min in BB and images acquired. Blocking nanoparticle uptake using inhibitors of endocytosis Known inhibitors of various endocytic processes were used to help elucidate the mechanism of cellular uptake of CP nanoparticles. Cells were plated in 35 mm tissue culture plates and grown to ~ 70% confluence. The cells were preincubated with various inhibitors: methyl-β-cyclodextrin (2.5 mg/ml), wortmannin (100 ng/ml), LY294002 (20 μg/ml), cytochalasin D (10 μg/ml), nocodazole (10 μg/ml), genistein (10 μg/ml), and chlorpromazine (1 μg/ml) for 30 min followed by addition of 2 nM (4 ppm) PFBT nanoparticles for 1.5–2 h. Cells were then washed 3X in ice cold PBS and resuspended by gentle pipetting. Cells were analyzed by flow cytometry. Statistical analysis of the mean fluorescence for each treatment compared to the untreated and vehicle controls was done using ANOVA in Sigmaplot. An inhibitor cytotoxicity control was run using propidium iodide staining to insure that there was less than 10% cytotoxicity from the inhibitor alone and concentrations were adjusted as needed. Cytotoxicity of PFBT nanoparticles The Cell Titer Blue assay was used to assess cytotoxicity of PFBT nanoparticles. Cells were plated at 10K/well in a black 96-well plate and incubated with indicated concentrations of PFBT nanoparticles for 16 h. Cell Titer Blue reagent was added according to manufacturer instructions and incubated with the cells for an additional 2 h then the plate was read out using a fluorescence plate reader (Tecan). Statistical analysis of cell viability versus the untreated control was done using ANOVA in Sigmaplot RT-PCR of inflammatory markers J774A.1 cells were grown in 35mm tissue culture plates. When the cells were ~ 90% confluent they were treated with 2 nM (4 ppm) PFBT nanoparticles for 2 h. Control cells received vehicle alone treatment or treatment with 60 ng/ml interferon-γ and 100 g/ml LPS for 2 h. RNA was extracted and the Total RNA was analyzed by reverse transcriptase PCR for levels of gene expression of the proinflammatory markers TNFα and interleukin-1β (IL-1β). β-actin was used as a control. We used the following oligonucliotide primers for the RT-PCR: TNFα (forward: 5′-GAACTGGCAGAAGAGGCACT-3′/reverse: 5′-AGGGTCTGGGCCATAGAACT-3′); IL-1β (forward: 5′-AAATGCCTCGTGCTGTCTGACC-3′/reverse: 5′-CTGCTTGACAGGTGCTGATGTACC-3′), and β-actin (forward: 5′-TGTGATGGTGGGAATGGGTCAG-3′/reverse: 5′-TTTGATGTCACGCACGATTTCC-3′). Total RNA was extracted (1.0 mL of TRIZOL reagent per 10 6 cells) according to manufacturer instructions. The isolated RNA isolated was quantified by UV-Vis spectrophotometry (260 nm) and the RNA quality assessed by agarose gel electrophoresis and UV-Vis (260 nm/280 nm ratio). First strand synthesis was carried out with 3 μg of RNA in a 20 μL reaction using random hexamers as described by the manufacturer. Amplification of the targeted DNA was carried out in a 50 μl reaction as instructed by the manufacturer with 4 μl of the first strand synthesis reaction. The amplified DNA product was separated on a 2% agarose gel with 100 bp DNA ladder as a marker and visualized using SYBR green staining.

Supplementary Material 1_si_001