Enhanced antiproliferative efficacy by hydrolysable BSA-MTX conjugate is CNS-1 glioma cell line.

<p>Dose dependent toxicity experiments were conducted on CNS-1 cells as described in the Methods section. MTX (A), BSA---NH-CO-MTX (B) and BSA-S-MAL-(CH₂)₂-CONH-(CH₂)₆- NHCOMTX (C) were added to the cell culture for 72 hrs before MTT toxicity assay was applied to determine their antiproliferative efficacies. Data are presented as mean±SEM of 3–4 experiments in quadruplicates.</p

Rate of hydrolysis of MTX-CONH-(CH₂)₆-NH₂ from PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX at acidic and neutral pH values.

<p>Rate of hydrolysis was determined by the decrease in the absorbance of the PEG₄₀-MTX conjugate at 305nm as a function of time during dialysis against 1mM HCl (pH 3.0) or 50 mM Hepes buffer (pH 7.4). The experimental protocol described in the experimental part was applied. Data are presented as mean±STDEV (n = 4).</p

MTX and Fmoc-L-glutamic acid structures.

<p>Illustrations of MTX (A) and Fmoc-L-glutamic acid (B).</p

Schematic presentation of the proposed mechanisms involved in the chemical hydrolysis of methotrexate-amino derivatives covalently linked to carboxylate moieties of macromolecules (A) at acidic (B) and neutral (C) pH values.

<p>Schematic presentation of the proposed mechanisms involved in the chemical hydrolysis of methotrexate-amino derivatives covalently linked to carboxylate moieties of macromolecules (A) at acidic (B) and neutral (C) pH values.</p

Principle macromolecules conjugates, prepared and investigated in this study.

<p>Principle macromolecules conjugates, prepared and investigated in this study.</p

An antiproliferative efficacy of BSA-MTX conjugates against MTX-sensitive CNS-1 cells.

<p>An antiproliferative efficacy of BSA-MTX conjugates against MTX-sensitive CNS-1 cells.</p

Rate of hydrolysis of F-moc-L-glutamic-hexamethylenamine linked to PEG₃₀-----COOH at acidic and neutral pH values.

<p>The rates of hydrolysis were determined by the decrease in absorbance of the conjugates at 280nm as a function of time during dialysis against 1mM HCL (pH 3.0, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158352#pone.0158352.g005" target="_blank">Fig 5A and 5C</a>) or PBS (pH 7.4, Fig 6B). Data are presented as mean±STDEV (n = 4).</p

Dose response inhibition of dihydrofolate-reductase by MTX-hexamethylenamine, released from PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX at acidic conditions.

<p>The indicated concentrations of MTX and its derivatives were analyzed for their potencies to inhibit the reduction of dihydrofolate to tetrahydrofolate by DHFR. In this process NADPH is oxidized to NADP, and the extent of this oxidation is monitored by the decrease in absorbance at 340 nm (experimental section).</p

Engineering a cysteine-specific MTX-containing reagent- Rates of hydrolysis following its conjugation to PEG₂₀-SH or to HSA at acidic pH.

<p>PEG₂₀-S-MAL-(CH₂)₂-CONH(CH₂)₆-NHCO-MTX and HSA-S-MAL-(CH₂)₂-CONH(CH₂)₆-NHCO-MTX were dialyzed against 1L of 1mM HCl and the decrease in the absorbance at 305nm in the dialysis tube was recorded applying the protocol as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158352#pone.0158352.g001" target="_blank">Fig 1</a>. Data are presented as mean±STDEV (n = 4).</p

Decrease in the absorbance of PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX with time during dialysis against 1mM HCl.

<p>PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX and PEG₄₀-CONH-(CH₂)₆-NHCO-MTX (0.2 μmole/ml of each) were dialyzed against one liter of 1mM HCl (pH 3.0). At the indicated time points, 30 μl aliquots were withdrawn from the dialysis tube, diluted 20 folds and their absorbance at 305 nm was monitored. Data are presented as mean±STDEV (n = 3).</p