Abstract Biodistribution, pharmacokinetics, and efficacy of prostate‐cancer‐targeted HPMA copolymer/DTX conjugates are evaluated in nude mice bearing prostate cancer C4‐2 xenografts. PSMA‐specific monoclonal antibodies 3F/11 are used as the targeting moiety. Control conjugates contain either non‐specific IgG or no IgG. The ratios of tumor accumulation to total background organs (heart, lung, kidney, liver, spleen and blood) accumulation increase substantially with time for the targeted conjugate, and the ratio at 48 h is 7‐fold higher than that at 6 h. Preliminary evaluation of the efficacy of the conjugates in vivo show tumor growth inhibition for all HPMA copolymer/DTX conjugates. magnified image
Semitelechelic (ST) polymers are low molecular weight linear macromolecules having a reactive functional group at one end of the polymer chain and one terminal end. To modify proteins or biomedical surfaces by one point attachment, ST polymers should be used. ST polymers with N-hydroxysuccinimide ester end groups ST-poly [N-(2-hydroxypropyl) methacrylamide] (ST-PHPMA)-COOSu were synthesized by esterification of ST-PHPMA-COOH with a large excess of N-HOSu with dicyclohexyl carbodiimide as coupling agent. The modification of charge transfer with narrow fractions of ST (2-hydroxypropyl) methacrylamide (HPMA) polymers produced conjugates possessing a uniform structure compared with those prepared from unfractionated ST macromolecules. ST HPMA polymers also effectively modify biomedical surfaces. The functional ST HPMA polymers can be readily prepared by free radical polymerization in the presence of functional mercaptans. The functional groups and chain length of the ST polymers can be controlled by the choice of a particular mercaptan and the reaction conditions.
The biological activities of sequential combinations of anticancer drugs, SOS thiophene (SOS) and mesochlorin e6 monoethylenediamine (Mce6), in the form of free drugs, nontargeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer−drug conjugates, P-GFLG-Mce6 and P-GFLG-SOS (P is the HPMA copolymer backbone and GFLG is the glycylphenylalanylleucylglycine spacer), and Fab′-targeted HPMA copolymer−drug conjugates, P-(GFLG-Mce6)-Fab′ and P-(GFLG-SOS)-Fab′ (Fab′ from OV-TL16 antibodies complementary to CD47), were evaluated against human ovarian carcinoma OVCAR-3 cells. Mce6, SOS, P-GFLG-Mce6, P-GFLG-SOS, P-(GFLG-Mce6)-Fab′, and P-(GFLG-SOS)-Fab′, when used as single agents or in binary combination, exhibited cytotoxic activities against OVCAR-3 cells, as determined using a modified MTT assay. The binding and internalization of P-(GFLG-Mce6)-Fab′ and P-(GFLG-SOS)-Fab′ by OVCAR-3 cells were visualized by confocal microscopy and flow cytometry. The results confirmed an enhanced biorecognition by OVCAR-3 cells of Fab′-targeted HPMA copolymer conjugates over nontargeted conjugates. The median-effect analysis and the determination of the combination index (CI) were used to describe the drug interaction and quantify the synergism, antagonism, or additivity in anticancer effects. The sequential combinations of SOS+Mce6 and P-GFLG-SOS+P-GFLG-Mce6 displayed very strong synergism to synergism in the entire range of cell inhibition levels (fa = 0.5 − 0.95). The P-(GFLG-SOS)-Fab′+P-(GFLG-Mce6)-Fab′ exhibited a strong synergism for fa values up to about 0.85, but showed synergistic effect and nearly additive effect at fa = 0.9 and 0.95, respectively. These observations support the continuation of in vivo investigations of these conjugates for the treatment of ovarian cancer.
Polyplexes are polyelectrolyte complexes of DNA and polycations, designed for potential gene delivery. We investigated the properties of new polyplexes formed from cholesterol-modified polycations and DNA. Three complexes were tested; their cholesterol contents were 1.4, 6.3, and 8.7 mol %. UV spectroscopy and fluorescence assay using ethidium bromide proved the formation of polyplexes. The kinetics of turbidity of polyplexes solutions in physiological solution showed that the colloid stability of polyplexes increases with increasing content of cholesterol in polycations. Dynamic, static, and electrophoretic light scattering, small-angle X-ray scattering, and atomic force microscopy were used for characterization of polyplexes. The observed hydrodynamic radii of polyplexes were in the range of 30−60 nm; they were related to the polycation/DNA ratio and hydrophobicity of the used polycations (the cholesterol content). The properties of polyplex particles depend, in addition to polycation structure, on the rate of polycation addition to DNA solutions.
The influence of different methods of binding the OV-TL16 antibody and its Fab′ fragment to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer—drug (adriamycin {ADR} or meso chlorin e6 mono(N-2-aminoethylamide) {Mce6} conjugates on the affinity of conjugates to an ovarian carcinoma (OVCAR-3) cell associated antigen was investigated. The binding of the antibody to HPMA copolymer—drug (ADR or Mce6) conjugates via amino groups resulted in conjugates which were heterogeneous in their antigen binding. Coupling the HPMA copolymer—Mce6 conjugate to the carbohydrate region of the antibody resulted in conjugates with a more homogeneous distribution of affinity constants than conjugates prepared by linking the antibody to the polymer via amino groups. However, both methods resulted in a decrease in the affinity constant compared to the native antibody. Conjugates prepared with the Fab′ fragment of the OV-TL16 antibody demonstrated a more homogeneous affinity than either conjugate prepared with the whole antibody. To verify the hypothesis that the changes in the binding affinity and homogeneity are a consequence of conformational changes in the antibody structure, a series of physicochemical methods where employed to characterize the conjugates. The excitation energy transfer between OV-TL16 antibody and drugs (ADR and Mce6) and the spectral properties of Mce6 were used to monitor the interactions between the antibody and drugs. The quenching of the intrinsic fluorescence of the antibody was also employed to study its conformational changes. An attempt has been made to correlate the biorecognition at the cellular surface with the interactions of drug with the antibody molecule and with changes in antibody conformation.
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