13 research outputs found
Schematic illustration of the conjugation of the cell-penetrating peptide (CPP) TAT to lyophilisomes.
<p>(1) Primary amine groups of lyophilisomes react with Sulfo-GMBS introducing reactive maleimide groups. (2) CPPs (cysteine-functionalized TAT-peptides; C-Ahx-YGRKKRRQRRR) are conjugated to maleimide-conjugated lyophilisomes, resulting in stable CPP-conjugated lyophilisomes. Sulfo-GMBS â=â sulfo-<i>N</i>-[Îł-maleimidobutyryloxy]sulfo succinimide ester; Ahx â=â aminohexanoic acid; TAT â=â trans-activating transcriptional activator.</p
Sorting of lyophilisomes by fluorescence-activated cell sorting.
<p>a/b) A representative size distribution of the initial lyophilisome population (a) and sorted lyophilisomes (b) is depicted, showing smaller lyophilisomes after sorting. Note the difference in x and y axes. c) Initial lyophilisome population depicted in a FACS dot plot with forward (size)/FITC-positive lyophilisome (FL1 channel) scatter where gated FITC-positive lyophilisomes were sorted. d) After sorting, the scatter showed merely small lyophilisomes, as large lyophilisomes were removed.</p
Improved Intraoperative Detection of Ovarian Cancer by Folate Receptor Alpha Targeted Dual-Modality Imaging
Complete resection of tumor lesions
in advanced stage ovarian cancer patients is of utmost importance,
since the extent of residual disease after surgery strongly affects
survival. Intraoperative imaging may be useful to improve surgery
in these patients. Farletuzumab is a humanized IgG1 antibody that
specifically recognizes the folate receptor alpha (FRα). Labeled
with a radiolabel and a fluorescent dye, farletuzumab may be used
for the intraoperative detection of ovarian cancer lesions. The current
aim is to demonstrate the feasibility of FRα-targeted dual-modality
imaging using <sup>111</sup>In-farletuzumab-IRDye800CW in an intraperitoneal
ovarian cancer model. Biodistribution studies were performed 3 days
after injection of 3, 10, 30, or 100 ÎŒg of <sup>111</sup>In-farletuzumab-IRDye800CW
in mice with subcutaneous IGROV-1 tumors (5 mice per group). In mice
with intraperitoneal IGROV-1 tumors the nonspecific uptake of <sup>111</sup>In-farletuzumab-IRDye800CW was determined by coinjecting
an excess of unlabeled farletuzumab. MicroSPECT/CT and fluorescence
imaging were performed 3 days after injection of 10 Όg of <sup>111</sup>In-farletuzumab-IRDye800CW. FRα expression in tumors
was determined immunohistochemically. Optimal tumor-to-blood-ratios
(3.4â3.7) were obtained at protein doses up to 30 ÎŒg.
Multiple intra-abdominal tumor lesions were clearly visualized by
microSPECT/CT, while uptake in normal tissues was limited. Fluorescence
imaging was used to visualize and guide resection of superficial tumors.
Coinjection of an excess of unlabeled farletuzumab significantly decreased
tumor uptake of <sup>111</sup>In-farletuzumab-IRDye800CW (69.4 ±
27.6 versus 18.3 ± 2.2% ID/g, <i>p</i> < 0.05).
Immunohistochemical analyses demonstrated that the radioactive and
fluorescent signal corresponded with FRα-expressing tumor lesions.
FRα-targeted SPECT/fluorescence imaging using <sup>111</sup>In-farletuzumab-IRDye800CW can be used to detect ovarian cancer <i>in vivo</i> and could be a valuable tool for enhanced intraoperative
tumor visualization in patients with intraperitoneal metastases of
ovarian cancer
Internalization of lyophilisomes with and without TAT peptide into HeLa cells.
<p>FACS showed a large number of lyophilisome-positive cells for TAT-conjugated lyophilisomes after 1 h (67±3%) without trypan blue and a cellular uptake of 25±1% with trypan blue. Values for lyophilisomes without TAT peptide were low. When lyophilisomes were incubated for 4 h, TAT-conjugated lyophilisomes conserved the large number of lyophilisome-positive cells (79±8%) with an increased internalization of 59±14%, while unmodified lyophilisomes still showed few lyophilisome-positive cells and little cellular uptake. *p<0.01 **p<0.001. CPP â=â cell penetrating peptide; TAT â=â trans-activating transcriptional activator.</p
Cellular binding and internalization of unmodified lyophilisomes and TAT-conjugated lyophilisomes.
<p>HeLa, OVCAR-3, Caco-2 and SKOV-3 cells incubated with TAT-conjugated and unmodified lyophilisomes resulted in 86±3% and 12±4%, 87±3% and 16±8%, 97±3% and 19±3%, and 95±10% and 67±20% lyophilisome-positive cells, respectively. TAT â=â trans-activating transcriptional activator. *p<0.01 ***p<0.0001.</p
Cellular uptake of TAT-conjugated lyophilisomes as analyzed by transmission electron microscopy.
<p>HeLa cells were incubated with unmodified (a) and TAT-conjugated lyophilisomes (b-d) for 4 h. a) No attachment or uptake was observed using unmodified lyophilisomes. b-d) TAT-conjugated lyophilisomes (white arrows) showed various processes required for effective drug delivery systems, such as attachment (b) and uptake (c). Additionally, signs of degradation of the capsule inside the cell were visualized (black arrows, d). Scale bar represents 1.0 ”m. TAT â=â trans-activating transcriptional activator.</p