Clonogenic survival of F98 glioma cells following treatment with either free cisplatin or liposomal cisplatin for 4 or 24 h.

<p>Surviving fractions (S.Fs) were determined for the F98 glioma cells treated with (A) CHEMS lipsomes, (B) free cisplatin following a 4 h (•) or 24 h (○) exposure. Each data point represents the mean of 3 replicates ± the standard deviation.</p

Neuropathologic changes associated with i.c. CED of Lipoplatin™ or its “hollow” counterparts or free cisplatin (A).

<p>(A) Lipoplatin™ (1.8 µg). Rat was euthanized 2 hr after CED. There is cerebral edema and a single focus of hemorrhage in the R cerebral hemisphere, adjacent to the medial boundary of the R lateral ventricle and another in the lateral hypothalamic area. (B) Lipoplatin™ (0.9 µg cisplatin). The rat was euthanized 4 days after CED. There is extensive necrosis with a dense infiltrate of foamy macrophages. (C) Lipoplatin™ (0.9 µg cisplatin) euthanized on d 4. There is a 6×4 mm zone of advanced necrosis with a peripheral zone of microglial reaction. There is edema of the neurophil and vacuolization, but no inflammatory response. (D) and (E) Lipoplatin™ “hollow” liposomes (100×). There is a 7×4 mm zone of hemorrhage associated with necrosis and edema of the neuropil. (E) There is an infiltrate of macrophages and hyperplasia of endothelial cells of adjacent small vessels. (F) Free cisplatin (3 µg), rats euthanized on d 7. Higher power (400×) view of (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048752#pone-0048752-g004" target="_blank">Fig. 4</a>H). There is hemorrhage, necrosis and infiltration with lipid laden macrophages. Scattered larger blood vessels show early fibrin deposition in the walls.</p

Neuropathologic changes associated with i.c. CED of cisplatin containing CHEMS liposomes (A-E), their “hollow” counterparts (F), or free cisplatin (H).

<p>(H&E) stained coronal sections at 400× magnification unless otherwise noted. (A) and (B) CHEMS – cisplatin, (4.8 µg), euthanized at 2 wks. (A) Although no necrosis is seen there are clear, possibly lipid containing vacuoles scattered reactive astrocytes and a mild infiltrate of macrophages. (B) There is focus of incipient necrosis and a moderate infiltrate of macrophages. Neurons consistent with late neuronal injury, proliferation of astrocytes and ependymal cells along the wall of the ventricle are also seen, but not in this photomicrograph. (C) and (D) CHEMS – cisplatin (9 µg), euthanized on d 10. There is focal intense inflammation with large numbers of macrophages, scattered lymphocytes, hemorrhage and necrosis and (E) prominent neovascularization. (F) and (G) “Hollow” CHEMS liposomes, euthanized on d 10. There is prominent necrosis with an intense inflammatory response consisting of lymphocytes and macrophages. Small vessels are engorged with blood and show reactive endothelial cells. (G) GFAP immunostaining revealed paraventricular foci of reactive astrogliosis. (H) Free cisplatin (3 µg), euthanized on d 7. Low power view (100×) shows a prominent focus (7×7 mm) of confluent hemorrhage with disruption of adjacent necrotic white matter.</p

Biodistribution of liposomes following CED to F98 glioma bearing rats.

a<p>F98 glioma cells were implanted into the right caudate nucleus of 8 Fischer rats. Twelve to 14 d later, they received CED of cisplatin containing CHEMS liposomes (9.6 µg in 10 µl over 30 min) and were euthanized either immediately following (t = 0 h.) CED or 24 h later. The tumors, BAT and normal brain and blood samples were collected and Pt concentrations were determined by ICP-OES.</p>b<p>Brain around tumor arbitrarily included an area of 1 mm beyond the dissected margins of the tumor.</p>c<p>Means and standard deviations (SD) were calculated on values obtained from 4 rats for each time point.</p>d<p>The P-value<0.05 compared to that determined at t = 0. Other than this, there were no significant differences in the 0 and 24 h Pt values.</p

Formulations of liposomal cisplatin preparations.

a<p>Hollow liposomes (before cisplatin loading).</p>b<p>Liposome/cisplatin measured immediately after preparation.</p>c<p>After lyophilization, 10 d storage at ambient temperature, and resuspension.</p>d<p>In water.</p>e<p>In 5% dextrose.</p

Comparison of the toxicity against F98 glioma cells of Lipoplatin™ and CHEMS cisplatin liposomes and their “hollow” counterparts.

a<p>F98 glioma cells were exposed to the test agent for 4 hours, following which clonogenic assays were carried.</p>b<p>Surviving fractions were determined following 7 days incubation at 37°C in a CO₂ incubator.</p>*<p>P-value is computed using two-sample t-test.</p