Figure 1.
(a) Chemical structure and solution behaviour of the PMPC-PDPA copolymer in water at different pHs. (b) Process of encapsulations for both hydrophilic, hydrophobic and amphiphilic molecules.
Figure 2.
Polymersomes intracellular delivery.
(a) Mechanism of polymersome-mediated cytosolic delivery. (b) Primary human dermal fibroblast (HDF) exposed to Rhodamine-loaded (red) polymersomes imaged at different focal levels (0 µm, 5 µm, 10 µm) by confocal laser scanning microscopy (40x lens, bar 0.02 mm) and compared to untreated cells. The cell lysosomes and DNA were also stained using yellow Lysotracker and green SYTO-9, respectively. Figure bar 20 µm.
Figure 3.
Tracking period and cytotoxicity induced by polymersome-mediated staining compared with commercial method.
(a) Cell viability determined by MTT assay at different times for HDF cells exposed to either Rhodamine-loaded polymersomes or CellTracker (n = 3, error bar = SEM; *p<0.05). (b) Fluorescence intensity from HDF cells plated at different initial densities and exposed to daily dose of Rhodamine-loaded polymersomes (0.005 mM) (n = 3, error bar = SEM). (c) Fluorescence intensity exhibited by HDF cells after a single dose of polymersomes loaded with varying amounts of Rhodamine or CellTracker (n = 3, error bar = SEM). (d) Fluorescence micrographs of HDF cells recorded for the same exposure at different days after a single dose of Rhodamine-loaded polymersomes. (e) Fluorescence micrographs of primary HDF cells after 24 h incubation with PMPC-PDPA polymersomes loaded with membrane-staining amphiphilic Rhodamine B octadecyl ester perchlorate, (f) BODIPY TR ceramide, (g) fluorescein 1,2-dihexadecylphosphatidylethanolamine (DHPE), (h) labelled NBD cholesterol, (i) DNA staining membrane-impermeable propidium iodide, (l) FITC-labelled antibody anti α-tubulin, (m) labelled nucleic acids, (n) or large quantum dots. Figure bar = 0.02 mm.
Figure 4.
Selected examples of cells treated with Rhodamine-loaded polymersomes.
(a) Live primary human dermal fibroblast (HDF), primary human epidermal keratinocytes (HEK), primary human endothelial cells (HE), primary human monocytes (HMC), primary human macrophages (HMP), primary human mesenchymal stem cells (HMSc), primary rabbit limbal epithelial (RLE) cells, primary rat cortical neurons (RCN), primary rat motor neurons (RMN), Chinese hamster ovary (CHO) cells, rat Shawnoma (nemap22) cell, preosteocytes (MLO-A5) cells, human melanoma (A375SM) cell, human head & neck cancer (KB) and (SCC4) cells have all been exposed to Rhodamine-loaded polymersomes and successfully stained. 3D reconstruction from confocal laser scanning optical stacks of HDF cells (b), HE cells (c), SCC4 cells (c), and RMN cells (d). Figure bar = 0.02 mm.
Figure 5.
HDF cells seeded in a pre-cast fibrin clot gel stained using Rhodamine B octadecyl ester-loaded polymersomes and subsequently monitored using fluorescent microscopy.
(a) Imaged after 7 days at the initial cell seeding area. (b) Imaged after 7 days at the interface between the initial cell seeding area and the pre-cast gel. (c) Imaged after 14 days at the initial cell seeding area. (d) Imaged after 14 days at the interface between the initial cell seeding area and the pre-cast gel. Figure bar = 0.1 mm. 3D volumetric reconstruction of HDF cells migrating within fibrin-clot gels imaged at the migration front areas (e) and at the initial cell seeding area (f). Details of single cells and their filipodia imaged at the seeding area (g) and the migration front (h). Figure bar = 0.01 mm.
Figure 6.
Tracking different cell types in 3D models.
(a) Tissue-engineered human oral mucosa imaged by confocal laser scanning microscopy after 3 days, (b) 7 days and (c) 10 days. The keratinocytes (HOKs) and the fibroblasts (HOFs) that comprise the mucosa were previously stained by incubating the cells with Rhodamine B octadecyl ester- (red) and (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate)- (white) loaded polymersomes. (d) The blue channel shows endogenous autofluorescence from the connective tissue component 3D cell imaging allows the extent of dye permeation to be determined over time (error bar = SEM, n = 3). Between 90 and 110 slices were acquired for each image. Slice thickness 0.003 mm.