Vladimir Omelyanenko

Targetable HPMA copolymer-adriamycin conjugates. Recognition, internalization, and subcellular fate

Recognition, internalization, and subcellular trafficking of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing N-acylated galactosamine (GalN) or monoclonal OV-TL16 antibodies (Ab) have been investigated in human hepatocarcinoma HepG2 and ovarian carcinoma OVCAR-3 cells, respectively. The intrinsic fluorescence of fluorescein or adriamycin (ADR) attached to HPMA copolymers permitted us to follow the subcellular fate of HPMA copolymer conjugates by confocal fluorescence microscopy and fluorescence spectroscopy. The pattern of fluorescence during incubation of HPMA copolymer-ADR-GalN conjugate containing lysosomally degradable tetrapeptide (GFLG) side-chains with HepG2 cells was consistent with conjugate recognition, internalization, localization in lysosomes, followed by the release of ADR from the polymer chains and ultimately diffusion via the cytoplasm into the cell nuclei. A similar pattern was observed in OVCAR-3 cells for Ab targeted HPMA copolymer conjugates. To test our hypothesis that HPMA-copolymer-bound anticancer drugs will be inaccessible to the energy-driven P-glycoprotein efflux pump in multidrug resistant (MDR) cells, we have compared the internalization of the HPMA copolymer-ADR conjugates by sensitive (A2780) and ADR-resistant (A2780/AD) ovarian carcinoma cell lines. Preliminary data on relative retention of ADR in MDR (A2780/AD) cells indicate a higher intracellular ADR concentration after incubation with HPMA copolymer-ADR conjugate when compared to incubation with free (unbound) ADR.

HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. II. Processing in epithelial ovarian carcinoma cells in vitro

The binding, internalization, subcellular trafficking and in vitro cytotoxicity of N‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymer–anti‐cancer drug–OV‐TL16 antibody (Ab) conjugates in the ovarian carcinoma OVCAR‐3 cell line have been investigated. Adriamycin (ADR) and meso chlorin e6 mono(N‐2‐aminoethylamide) (Mce6) photosensitizer were used as anti‐cancer drugs. Targeted (Ab‐containing) conjugates were compared with non‐targeted HPMA copolymer–drug conjugates and with free drugs. Targeted conjugates were taken up rapidly by cells and detected within lysosomes by confocal fluorescence microscopy. The ADR attached to polymer chains via a degradable GFLG spacer was released from the conjugate, diffused via the lysosomal membrane into the cytoplasm and ultimately accumulated in the cell nuclei. In contrast, conjugates containing ADR bound via the GG spacer accumulated in the lysosomes, but no fluorescence could be detected in the cell nuclei. Binding the drugs to a non‐targeted HPMA copolymer decreased their cytotoxicity in vitro. The IC50 dose increased from 2 μM for free ADR to 150 μM for P(GFLG)–ADR (P is the HPMA copolymer backbone) and from 0.34 μM for free Mce6 (with light) to 290 μM for P–(GG)–Mce6. However, attachment of OV‐TL16 Abs rendered HPMA copolymer–drug conjugates biorecognizable by OVCAR‐3 cells and markedly increased their cytotoxicity. The IC50 doses were 4.4 and 0.38 μM for the targeted conjugates P(GFLG)–ADR–Ab and P(GG)–Mce6–Ab (with light), respectively. Biorecognition was shown to be specific by inhibition experiments with free Ab. The findings indicate the potential of these conjugates as effective agents in the treatment of ovarian cancer. Int. J. Cancer 75:600–608, 1998.

Targeted delivery of diagnostic agents by surface-modified liposomes

Surface-modified LS have been used for the specific delivery of heavy metal-based imaging agents. The liposome surface was modified with PEG, AMmAb, Dext-SA, chelating agent DTPA-PE, and with NGPE-modified chelating polymer: DTPA-NPLL-NGPE. The hypothesis is suggested attempting to explain the phenomenon of long circulation of PEG-coated LS from the point of view of statistical properties of flexible polymer molecule in solution. Direct experiments using fluorescent labels were performed to prove the hypothesis. The calculations performed on the basis of the hypothesis were designed to find the optimal concentration of PEG on the LS surface, and suggested that it not only provides a protective effect but also does not create steric hindrances for the surface-immobilized mAb. As a result, long circulating targeted ILS have been prepared. Intravenously administered 111In-labelled PEG-AMmAb-LS were targeted to the area of experimental myocardial infarction in rabbits under γ-scintigraphic control. Infarct-to-normal ratios of 111In radioactivity of about 20 were achieved. PEG-and Dext-modified liposomes with surface-incorporated Gd-labelled DTPA-PE or DTPA-NPLLNGPE were used for the subcutaneous administration and subsequent NMR-imaging of lymph nodes in rabbits. Two mechanisms of MR-signal enhancement were found for the surface-modified Gd-containing LS: the increase in signal intensity due to the increase in water quantity in the vicinity of Gd atoms because of PEG-associated water; and better lymph node accumulation of Dext-LS via receptormediated endocytosis. Surface modification of LS opens the possibilitiy for targeted delivery of heavy metal-based imaging agents.

Biorecognition of HPMA copolymer-adriamycin conjugates by lymphocytes mediated by synthetic receptor binding epitopes.

AbstractPurpose. The EDPGFFNVE nonapeptide (NP) was recognized as the CD21 (CR2) binding epitope of the Epstein-Barr virus (EBV) gp350/ 220 envelope glycoprotein which mediates the virus attachment to human B lymphocytes (Nemerow et al., Cell 56:369-377, 1989). Here we evaluated the targeting potential of a synthetic receptor binding epitope (NP) covalently attached to a water-soluble polymeric drug carrier. In particular, the biorecognition of N-(2-hydroxypropyl) metha-crylamide (HPMA) copolymer-NP conjugates by B- and T-cells and the cytotoxicity of HPMA copolymer-NP-adriamycin (ADR) conjugates toward B-cells, T-cells, and peripheral blood lymphocytes (PBL) were evaluated. Methods. HPMA copolymer-NP and optionally ADR conjugates varying in the NP density and mode of NP attachment were incubated with Raji B-cells (human Burkitts lymphoma), CCRF-CEM T-cells (acute human lymphoblastic leukemia), and CCRF-HSB-2 T-cells (human lymphoblastic leukemia). The kinetics of binding was studied, the Langmuir adsorption isotherms analyzed, binding constants calculated, and IC50 doses determined. Results. Flow cytometry studies revealed that binding was homogeneous to both cell types. The apparent binding constants to T-cells were about two times higher when compared to B-cells. The binding and cytotoxicity increased with increased amount of epitopes per polymer chain. Attachment of the NP via a GFLG spacer resulted in increased biorecognition when compared with conjugates containing NP bound via a GG spacer. HPMA copolymer-NP-ADR conjugates possessed specific cytotoxicity to T- and B-malignant cells. Concentrations, which were lethal to the latter, were not toxic for PBL. Conclusions. The data obtained seem to indicate the potential of the HPMA copolymer-NP conjugates as polymer anticancer drug carriers targetable to immunocompetent cells.

HPMA copolymer-modified avidin: immune response.

Protein-polymer conjugates to be used in the pretargeted delivery of a photosensitizer to cells were synthesized and characterized. Avidin was modified by N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing the photosensitizer, mesochlorin e6 mono(N-2-aminoethylamide) (Mce6). Synthesis of HPMA copolymer-avidin-Mce6 conjugates was carried out so that either predominantly single point attachment or multipoint attachment of copolymer chains to avidin would result. HPMA copolymer-avidin conjugates were used which retained specific binding activity to a lower affinity biotin analog. Antigen specific anti-avidin immune response was shown to be reduced six-fold in some HPMA copolymer-avidin conjugates when compared to immune response to unmodified avidin. HPMA copolymer itself was shown to elicit a very low (IgM) immune response.

Biorecognizable Biomedical Polymers

The design of biorecognizable biomedical polymers has to be based on a sound biological rationale. Using this rationale, the principles of the design, synthesis, and characterization of biorecognizable polymers are demonstrated on several examples including: targetable water-soluble carriers of bioactive compounds, the relationship between the supramolecular structure of polymer conjugates and their biorecognition, poly(ethylene glycol) conjugates, bioadhesive polymers for colon-specific drug delivery, and biodegradable hydrogels.