Recombinant modular transporters for cell‐specific nuclear delivery of locally acting drugs enhance photosensitizer activity

Abstract

The search for new pharmaceuticals that are specific for diseased rather than normal cells in the case of cancer and viral disease has raised interest in locally acting drugs that act over short distances within the cell and for which different cell compartments have distinct sensitivities. Thus, photosensitizers (PSs) used in anti-cancer therapy should ideally be transported to the most sensitive subcellular compartments in order for their action to be most pronounced. Here we describe the design, production, and characterization of the effects of bacterially expressed modular recombinant transporters for PSs comprising 1) alpha-melanocyte-stimulating hormone as an internalizable, cell-specific ligand; 2) an optimized nuclear localization sequence of the SV40 large T-antigen; 3) an Escherichia coli hemoglobin-like protein as a carrier; and 4) an endosomolytic amphipathic polypeptide, the translocation domain of diphtheria toxin. These modular transporters delivered PSs into the nuclei, the most vulnerable sites for the action of PSs, of murine melanoma cells, but not non-MSH receptor-overexpressing cells, to result in cytotoxic effects several orders of magnitude greater than those of nonmodified PSs. The modular fusion proteins described here for the first time, capable of cell-specific targeting to particular subcellular compartments to increase drug efficacy, represent new pharmaceuticals with general application.

Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import

“…NLS sequences from SV40 large T-antigen (T-ag 111-135 [T-agNLS], T-ag 111-135 containing the mutation K 128 T, and T-ag 114-135 ), human cytomegalovirus UL44 protein (UL44 422-433 ), and retinoblastoma protein (Rb 860 -876 ) were produced by PCR from previously described vectors (Efthymiadis et al, 1997;Alvisi et al, 2005;Wagstaff and Jans, 2006). Multimodular (DLC-AS-T-agNLS) cassettes were produced using BamHI/BglII cloning as previously described (Rosenkranz et al, 2003) and inserted into mammalian expression plasmids pEPI-GFP or pEGFPC1 fused in frame C-terminal to the coding sequence of green fluorescent protein (GFP; Figure 1). pExpress-dsRed-DLC-1 (dsRed-LC8; Finke et al, 2004) was supplied by K. Conzelmann (Ludwig Maximilians University, Munich, Germany).…”

“…Treatment with taxol did not affect the appearance of cells expressing GFP-RPP 139-297 alone, but in cells coexpressing GFP-RPP 139-297 and dsRed-LC8, the filamentous structures on which the transfected proteins were colocalized became more defined. RPP, PTHR, and p53BP1 DLC-ASs were cloned in frame upstream of SV40 large T-antigen (T-ag) residues 111-135, which constitute the modular T-agNLS and which has previously been used out of context to facilitate the delivery of molecules of interest to the nucleus (Rosenkranz et al, 2003). When fused to the T-agNLS, all of the DLC-AS-containing fusion proteins were predominantly nuclear ( Figure 4B).…”

“…AVs are intended to overcome these problems by incorporating modules that mimic efficient nuclear targeting by viruses (e.g., cell-specific binding/uptake, endosomal escape, and nuclear import; Cohen et al, 2005;Glover et al, 2005;Alvisi et al, 2006; Mastrobattista et al, 2006a,b), while avoiding the pathogenicity associated with viral approaches. We have successfully utilized modular approaches to achieve nuclear drug delivery relevant to chemotherapy (Rosenkranz et al, 2003). It has been suggested that DLC-ASs could optimize nonviral delivery of therapeutics by mimicking the MTdependent trafficking of viruses toward the nucleus (Cohen et al, 2005; Mastrobattista et al, 2006a,b), but this has been speculative given the prior lack of evidence that DLC association can enhance NLS activity.…”

Preparation, cytotoxicity, and in vivo antitumor efficacy of ¹¹¹In-labeled modular nanotransporters

“…Moreover, MNTs possess interchangeable component domains, permitting relatively facile exchange of receptor recognition domains, which ultimately might be adapted for different diseases or might help improve homogeneity of drug delivery by targeting several tumor-specific molecules for personalized medicine. MNTs reactive with a variety of tumor-associated receptors have shown promise for delivering short range of action therapeutics including photosensitizers, 13 Ga,17 as they were demonstrated to colocalize in the nuclei of target cancer cells in vitro (up to 60% of internalized found in nuclei) 13,14,16,17 and in vivo, 14 as well as enhance significantly cytotoxicity of all the above-mentioned cargoes in vitro 13,[15][16][17][18] and the therapeutic potential of different photosensitizers in vivo.…”

“…18 Recent efforts in Auger electron-targeted radiotherapy have been primarily focused on applications involving 111 In 8,9,11,12 based, in part, on its ready availability, commercial and clinically compatible 2.8-day half-life, and the wealth of experience in patients with 111 In-labeled compounds as imaging agents. With regard to its low-energy electron spectrum, 111 In emits 14.7 Auger and Coster-Kronig electrons per decay; 19 moving its site of decay from the cell surface to the nucleus would increase the radiation dose delivered to the cell nucleus by more than tenfold for typical cell geometries.…”