Intracellular targeting of polymer-bound drugs for cancer chemotherapy.

Macromolecules have been traditionally employed as drug carriers due to their ability to selectively accumulate in malignant tissues compared to healthy tissues by either passive or active targeting, thus precluding undesirable side effects generated by free drug. The therapeutic activity proffered by such conjugates requires that the drug concentrate at its specific subcellular target such as the nucleus. Thus, the suitability of macromolecules as carriers also extends to their propensity to deliver the drug to a predetermined intracellular location. As binding a macromolecule to a drug facilitates cellular uptake by endocytosis, various approaches have been employed to either guide the drug to targets different from endosomal/lysosomal compartments by mediating vesicular escape, or to directly accomplish intracellular (cytoplasmic and nuclear) localization. This review discusses the utility of macromolecules in drug delivery and describes the numerous modalities (with a focus on cell-penetrating peptides) currently available for achieving effective intracellular drug delivery.

Free and N-(2-hydroxypropyl)methacrylamide copolymer-bound geldanamycin derivative induce different stress responses in A2780 human ovarian carcinoma cells.

The effects of geldanamycin (GA), 17-(3-aminopropylamino)-17-demethoxygeldanamycin (AP-GA), and N-(2-hydroxypropyl)methacrylamide copolymer-AP-GA conjugate [P(AP-GA)] on A2780 human ovarian carcinoma cells at an equitoxic dose (2x IC(50)) were compared by the gene expression array analysis. All treatments resulted in similar gene expression profiles up to 12 h (e.g., down-regulation of CDK4 and up-regulation of APAF-1), although P(AP-GA)-treated cells showed delayed gene expression because of time-dependent internalization of the conjugate and intracellular drug release from P(AP-GA). However, AP-GA-treated cells showed elevated expression of HSP70 and HSP27 after 6 h compared with that observed by GA and P(AP-GA) treatments. Depletion of C-Raf, an HSP90 client protein, was observed in all treatments up to 12 h. Confocal microscopy using mesochlorin e(6) as a model drug revealed that drug release caused by the lysosomal cleavage of glycylphenylalanylleucylglycine oligopeptide spacer, used as GA derivative copolymer attachment/release point, was moderately fast. These results suggested that AP-GA treatment may activate stress-response pathways, whereas P(AP-GA) treatment may suppress them and trigger signaling pathways essential to cell growth arrest and death by inducing an HSP90-active factor. Although GA and P(AP-GA) treatments induced a time-dependent increase in HSP70 and HSP27 protein expression (detected by Western blotting analysis), AP-GA treatment resulted in more rapid and more intense expression of both proteins. Our results suggest that conjugation of AP-GA to N-(2-hydroxypropyl)methacrylamide copolymer may be able to modulate the cell stress responses induced by AP-GA because of differences in its internalization mechanism, subcellular localization, and intracellular concentration gradients.

Subcellular trafficking of HPMA copolymer-Tat conjugates in human ovarian carcinoma cells.

One of the main obstacles to efficient intracellular delivery of therapeutic macromolecules is the barrier posed by the plasma membrane. In this study, the cell penetrating peptide Tat was conjugated to a synthetic macromolecule based on N-(2-hydroxypropyl)methacrylamide (HPMA) and its subcellular distribution in human ovarian carcinoma cell lines was studied. The Tat peptide mediated uptake resulted in cytoplasmic and nuclear localization and was found to be energy independent. Time and concentration studies verified the rapidity and dependence of the transport process on these parameters. Enhanced uptake of a polymer bound anticancer drug doxorubicin was also demonstrated. These results were corroborated independently by subcellular fractionation.

Cytoplasmic delivery and nuclear targeting of synthetic macromolecules.

Delivery of macromolecular drugs (e.g. antisense oligonucleotides, polymer-drug conjugates, etc.) designed to work in specific sites inside cells is complicated as macromolecules typically have access to fewer biological compartments than small molecules. To better understand the fate of macromolecules in cells and begin to alter that fate, we investigated the internalization and subcellular fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers and HPMA copolymer-drug conjugates in Hep G2 and A2780 cells. The subcellular fate of fluorescently labeled polymers was monitored by confocal microscopy and subcellular fractionation. Initially, the HPMA copolymers and HPMA copolymer-drug conjugates were internalized by endocytosis and remained in endosomes/lysosomes. At longer incubation times (>8 h), small amounts of the HPMA copolymers were observed to enter the cytoplasm and accumulate in the nucleus of the cells. Nuclear accumulation was confirmed after cytoplasmic microinjection. Oligonucleotides conjugated via lysosomally degradable spacers entered into the cytoplasm and nucleus of the cells faster than the polymers. The effect of the subcellular location was correlated to the toxicity of the photosensitizer, mesochlorin e(6) (Mce(6))-HPMA copolymer conjugates. The plasma membrane and late endosomes were more sensitive to damage by Mce(6). Targeting the polymer conjugates to the nucleus with the nuclear localization sequence (NLS) as well as conjugating the Mce(6) via a degradable spacer increased cell adhesion and uptake, promoted their entry into the cytoplasm and nucleus of the cells, and increased their toxicity. To further promote entry of the polymers into the cytoplasm and nucleus of the cells, the protein transduction domain, Tat peptide, was conjugated to the HPMA copolymers. This resulted in high binding to the cell membrane, but also facilitated rapid (<5 min) entry of the macromolecules into the cytoplasm and nucleus of cells. These results will prove valuable in the future design of macromolecular therapeutics.

Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells.

We have synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cell penetrating peptide Tat conjugates and evaluated their subcellular distribution in A2780 human ovarian carcinoma cells by confocal fluorescence microscopy and subcellular fractionation. Our data indicate the transport of these conjugates by a single Tat molecule to both the cytoplasm and nucleus via a nonendocytotic and concentration independent process. The uptake was observed to occur within 3 min, as confirmed by live cell microscopy. In contrast, HPMA copolymers lacking the Tat peptide were internalized solely by endocytosis. For the first time, Tat-mediated cytoplasmic delivery of a polymer bound anticancer drug, doxorubicin, was also demonstrated. These findings establish the feasibility of overcoming major cellular and subcellular obstacles to intracellular macromolecular delivery and hold great promise for the development of polymer-based systems for the cytoplasmic delivery of therapeutic molecules.