Cytoplasmic Delivery and Nuclear Targeting of Synthetic Macromolecules.
Aparna Nori, Monica Tijerina, Keith D. Jensen, Pavla Kopeckova, Jindrich Kopecek.
Seventh European Symposium on Controlled Drug Delivery, Noordwijk ann Zee, Netherlands, April 3-5, 2002, Proceedings, p. 31.


Introduction. The rationale for the use of water-soluble polymers as drug carriers is based on the mechanism of cell entry. Whereas low-molecular weight drugs enter the cell interior by diffusion, macromolecular therapeutics are internalized by endocytosis and are lysosomotropic [1]. However, the endocytic route is not optimal for the delivery of therapeutic molecules such as genes, antisense oligonucleotides, peptides and proteins, which are susceptible to degradation by lysosomal enzymes. In addition, the activity of many drugs relates to their subcellular location. To develop second-generation macromolecular therapeutics, which would localize at a predetermined subcellular location, a detailed knowledge of the relationship between their structure and their internalization and subcellular fate is needed.

To examine the possibility to manipulate the internalization and subcellular fate of macromolecular therapeutics, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing a protein transduction domain (PTD) [2] or a nuclear localization signal (NLS) [3] were designed, synthesized, and their internalization and subcellular fate in human ovarian carcinoma cells was evaluated.

Tat-Mediated Cytoplasmic Delivery of HPMA Copolymer Conjugates. Self-translocating peptide sequences such as the Tat peptide derived from the HIV-1 Tat protein that can mediate cytoplasmic transport of other compounds to which they are tethered [4], have opened new channels for macromolecular drug delivery. Attachment of the Tat peptide mediated the cytoplasmic transport of proteins [5], microspheres [6], and liposomes [7]. It was reasonable to hypothesize that attachment of the Tat PDT to synthestic macromolecules may result in their cytoplasmic delivery. To this end, the subcellular distribution of HPMA copolymer-Tat (GRKKRRQRRRGYK(FITC)C) conjugates in A2780 human ovarian carcinoma cells was monitored by confocal fluorescence microscopy and subcellular fractionation methods. The results revealed the transport of the conjugate by a Tat-mediated energy independent process, to both the cytoplasm and the nucleus. For the first time, Tat-mediated cytoplasmic accumulation of a polymer bound anticancer drug doxorubicin was also demonstrated. These findings hold great promise for the development of polymer-based systems for the cytoplasmic delivery of therapeutic molecules such as genes and antisense oligonucleotides.

Nuclear Delivery of HPMA Copolymer Conjugates Utilizing a Nuclear Localization Signal. Photodynamic therapy (PDT) is a mode of anticancer treatment involving the selective activation of photosensitive compounds by light in tumor tissues generating oxidative damage and ultimately cell death. Directing photosensitizers to subcellular sites within tumor cells has been shown to enhance efficacy by generating damage within sites sensitive to PDT [8]. The nuclear delivery of HPMA copolymer-mesochlorin e6 monoethylenediamine (Mce6) conjugates (P(NLS)-Mce6) was investigated utilizing a nuclear localization signal (NLS; PKKKRKVK(FITC)C) resembling simian virus SV40 large tumor antigen. HPMA copolymer-Mce6 conjugates (P-Mce6) without NLS were used as control. Subcellular localization of (P(NLS)-Mce6) in human ovarian carcinoma A2780 cells was analyzed by confocal microscopy and subcellular fractionation. Anticancer activity was assessed utilizing an MTT bioassay. Diffuse cytoplasmic staining was visible in cells treated with (P(NLS)-Mce6) with some observable punctate staining. In contrast, non-targeted HPMA copolymers (P-Mce6) showed only punctate fluorescence consistent with lysosomal accumulation. Conjugates containing the NLS sequence showed enhanced localization in the plasma membrane, cytosol, and nuclei. Cytotoxic evaluation described approximately an 8-fold decrease in the IC50 for P(NLS)-Mce6 compared to P-Mce6. Conjugation of NLS to HPMA copolymer-Mce6 significantly altered subcellular distribution and considerably lowered the IC50 compared to endocytically accumulated P-Mce6.

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Acknowledgements. This research was supported in part by NIH grant CA51578 from the National Cancer Institute and an Advanced Predoctoral Fellowship in Pharmaceutics from the PhRMA Foundation.

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