Flow Cytometry of HEK 293T Cells Interacting with Polyelectrolyte Multilayer Capsules Containing Fluorescein-Labeled Poly(acrylic acid) as a pH Sensor

Abstract

Polyelectrolyte multilayer sensor capsules, 5 microm in diameter, which contained fluorescein-labeled poly(acrylic acid) (PAAAF) as pH-sensitive reporter molecules, were fabricated and employed to explore their endocytotic uptake into HEK 293T cells by flow cytometry. The percentage of capsules residing in the endolysosomal compartment was estimated from the fluorescence intensity decrease caused by acidification. Capsules attached to the extracellular surface of the plasma membrane were identified by trypan blue quenching. The number of capsules in the cytoplasm was rather small, being below the detection limit of the method. The advantages of polyelectrolyte multilayer capsules are that the fluorophore is protected from interaction with cellular compartments and that the multilayer can be equipped with additional functions.

Uptake of Colloidal Polyelectrolyte‐Coated Particles and Polyelectrolyte Multilayer Capsules by Living Cells

“…Comparative uptake and deformation study by TEM 2 (PDADMAC/PSS) 6 Au (20 nm) in the shell SiO 2 , 3.0 mm dissolved -3.0 mm 5 5 n m [9] Comparative uptake and deformation study by TEM 3 (PDADMAC/PSS) 8 Au (20 nm) in the shell SiO 2 , 3.0 mm dissolved -3.0 mm 105 nm [9] Comparative uptake and deformation study by TEM 4 (PDADMAC/PSS) 2 Au (20 nm) in the shell SiO 2 , 3.0 mm undissolved -3.0 mm Solid particle Comparative uptake and deformation study by TEM 5 (PDADMAC/PSS) 4 Alexa Fluor 488 dextran in the cavity SiO 2 , 4.5 mm dissolved Heat treatment 3.0 mm 4 5 n m [9] Comparative uptake and deformation study by TEM 6 (PSS/PAH) 4 Alexa Fluor 488 in the shell MF, 5.0 mm dissolved -5.0 mm 8 -1 0n m [47] Uptake study by CLSM 7 (PSS/PAH) 4 Alexa Fluor 488 in the shell MF, 5.0 mm undissolved -5.0 mm 8 -1 0n m [47] Uptake study by CLSM 8 (PSS/PAH) 5 SNARF-1-dextran in the cavity CaCO 3 , 4-6 mm dissolved -4-6 mm À100 nm [48] Uptake study by pH sensitive capsules 9 (PSS/PAH) 2 TRITC in the shell CaCO 3 , 4-6 mm dissolved -4-6 mm À60 nm [48] Intracellular localization study (imunostaining) 10 (PDADMAC/PSS) 4 TRITC label in the shell, Alexa Fluor 488 dextran in the cavity SiO 2 , 4.5 mm dissolved Heat treatment 3.0 mm 4 5 n m [9] Cargo-release study their big size. This has been demonstrated by confocal microscopy for the human breast carcinoma cell line MDA-MB-435S, [20] the human breast adenocarcinoma cell line MCF-7, [29,30] the human embryonic kidney cell line HEK 293T, [31,32] the human colon carcinoma cell line LIM1215, [11] the African green monkey kidney cell line VERO-1 [21] and VERO, [33] and bone-marrow-derived primary dendritic cells. [34] It has also been claimed before that capsules get deformed upon incorporation by cells.…”

“…3). [17,32] Upon incubation of the capsules with cells their fluorescence indicates a change from slightly alkaline (the cell medium; red fluorescence of SNARF) to acidic (endosomal/lysosomal/phagosomal compartments inside cells, green fluorescence of SNARF). All capsules' outside cells always displayed red fluorescence, i.e., were in alkaline environment.…”

Interaction, uptake, and processing of LbL-coated microcarriers by PMNs

“…(300 ll) solutions were rapidly mixed for 1 min (26). After a sedimentation step of about 10 min, the resulting particles were centrifuged for 1 min at 1,300 3 g. Afterward the particles were washed several times and stored in 0.1 M NaCl for subsequent coating.…”

Surface Functionalized Colloidal Microparticles for Fast Endocytotic Cell Uptake

“…As recently described, the intracellular pH can be used as indicator for particle localization in different cell compartments due to variation of the pH value from about pH 4.5-5 within (endo)lysosomes to pH 7.5 in the cytoplasm. [17] Thus, the pH-sensitive FITC label allows particle tracking within the cell by FCM. To allow a pH calculation from the detected particle fluorescence within the cell, a calibration curve was detected for each particle design using citrate/phosphate (CIP) buffer as model environment.…”

“…As shown recently, pH sensor molecules can be additionally integrated in a colloidal carrier system to localize particles within different cell compartments. [16][17][18] With this particle design, fast and more pronounced endosomal particle uptake and an increased particle number in endolysosomes could be obtained. In addition, TAMRA labeled CPP as surface layer were used to investigate the layer stability of ingested particles and particles in cell supernatant.…”