Table 1. Characterization of Nontargeted HPMA Copolymer Conjugates, Polymeric Precursors, and Fluorescently Labeled HPMA Copolymer Conjugates.
| structures | mol % of side chainsa |
mmol of ligand/ g of polymer conjugate |
no. of ligands/ polymer chain (conjugate) |
apparent Mw (kDa)b |
|---|---|---|---|---|
| P-GFLG-Mce6 | 2.04 | 0.125 | 2.9 | 23 |
| P-GFLG-SOS | 1.62 | 0.106 | 3.4 | 32 |
| P-(GFLG-Mce6)-NH2 | 2.62 | 0.153 (Mce6) | 1.7 (Mce6) | 11 |
| 8.11c | 0.472 (NH2) | |||
| P-(GFLG-SOS)-NH2 | 4.60 | 0.270 (SOS) | 2.7 (SOS) | 10 |
| 4.70c | 0.272 (NH2) | |||
| P-(GFLG-Mce6)-MAL | 2.60 | 0.142 (Mce6) | 2.0 (Mce6) | 14 |
| 6.02d | 0.329 (MAL) | |||
| P-(GFLG-SOS)-MAL | 4.14 | 0.236 (SOS) | 5.2 (SOS) | 22 |
| 3.36d | 0.192 (MAL) | |||
| P-(GFLG-Mce6)-FITC | f | 0.060e | 1.0 (FITC) | f |
| P-(GFLG-SOS)-FITC | f | 0.038e | 0.6 (FITC) | f |
| P-(GFLG-Mce6)-(Fab′-FITC)g | f | 0.063e | 3.1 (FITC per conjugate) | f |
| P-(GFLG-SOS)-(Fab′-FITC)g | f | 0.050e | 2.5 (FITC per conjugate) | f |
Determined by UV spectrophotometry in methanol: ε395 = 158000 M−1 cm−1 for Mce6, and ε358 = 33000 M−1 cm−1 for SOS.
Apparent molecular weight (Mw) of polymers was estimated by size exclusion chromatography using AKTA/FPLC (Pharmacia) system equipped with a Superose 6 column, calibrated with polyHPMA fractions. PBS buffer pH 7.3 + 30% (v) acetonitrile and 0.1 M acetate buffer pH 5.5 + 30% (v) acetonitrile were used for polymer conjugates containing Mce6 and polymer conjugates containing SOS, respectively.
Determined by ninhydrin assay.
Determined by 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein assay (SAMSA assay, Molecular Probes).
Determined by spectrophotometric determination of FITC (ε497 = 73 000 M−1 cm−1 in 0.1 M sodium borate buffer).
Not determined.
Fluorescently labeled targeted conjugates were prepared by reacting P-(GFLG-Mce6)-Fab′ and P- (GFLG-SOS)-Fab′ (Table 2) with 5-SFX.