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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 8, 2003 as doi:10.1096/fj.02-0888fje. |
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,
,2
* Department of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, 119334, Moscow, Russia;
Department of Biophysics, Biological Faculty, Moscow State University, 119899, Moscow, Russia;
Laboratory of Molecular Diagnostics and Gene-Engineering Constructs, All-Russia Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, 127550, Moscow, Russia;
Laboratory of the Enzyme Systems, Bach Institute of Biochemistry, Russian Academy of Sciences, 119071, Moscow;
¶ Nuclear Signaling Laboratory, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia;
|| Department of Chemistry and Technology of Fine Organic Chemicals, Moscow Academy of Fine Chemical Technology, 117571, Moscow, Russia; and
Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3168, Australia
2Correspondence: Department of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334, Moscow, Russia. E-mail: asobol{at}1.biophys.bio.msu.ru and alexander_s_sobolev{at}hotbox.ru
SPECIFIC AIMS
The search for new pharmaceuticals that are specific to affected rather than normal cells has generated intense interest in locally acting drugs for which distinct cell compartments have different sensitivities. The present paper reports a novel strategy to deliver locally acting drugs such as photosensitizers (PSs), describing modular recombinant transporters (MRTs) that are capable of targeting drugs not only to specific cells, but also to the most sensitive subcellular compartment for the action of the drug, thus increasing both the drug efficacy and specificity of its effects.
PRINCIPAL FINDINGS
1. Design of modular plasmids encoding MRTs of PSs, synthesis, and purification of the MRTs
We designed gene modules encoding the corresponding polypeptide modules according to the scheme: BamHI site–module sequence–BglII site–stop codon–HindIII site. This structure allowed all gene modules to be placed at any position along the hybrid gene since the flanking BamHI and BglII restriction sites have identical sticky ends. 
-Melanocyte-stimulating hormone (MSH) was chosen as a ligand module conferring recognition and uptake by specific target cells (melanoma) through binding to and subsequent internalization mediated by overexpressed melanocortin receptors. The optimized SV40 T antigen (T-ag) nuclear targeting module had the sequence: Ser-Ser-Asp-Asp-Glu-Ala-Thr-Ala-Asp-Ala-Gln-His-Ala-Ala124-Pro-Pro-Lys-Lys128-Lys-Arg-Lys-Val-Glu-Asp-Pro135, where the numbers refer to the T-ag amino acid sequence, with the nuclear localization sequence (NLS; residues 126–132) underlined. It is recognized with high affinity by the importin /ß heterodimer that, in concert with other transport factors, mediates import into the nucleus. The endosomolytic modules used were either the 1) GALA peptide shown to make pores in membranes at acidic pH or 2) translocation domain of diphtheria toxin together with its natural spacer (DTox), capable of permeating endocytotic membranes at acidic pH when in supramolecular complexes. Thus, we generated plasmids encoding a number of MRTs, including pR522 (HMP-NLS-MSH), pR523 (GALA-HMP-NLS-MSH), and pR676 (DTox-HMP-NLS-MSH), where HMP is a hemoglobin-like protein from Escherichia coli that functions as a carrier for PSs. The extent of MRT expression ranged from 5–8% of total protein for construct pR523 to 20–30% for constructs pR522 and pR676, with 60–70% solubility. The MRTs were purified on blue Sepharose, the single-step purification providing 90–95% purity.
2. MRT modules are functional
Purified chimeric MRTs were tested to assess whether their individual modules retained functionality and were able to contribute to the overall goal of cell-specific nuclear delivery (Fig. 1
). Stimulation of melanogenesis in B16-F1 cells by the MRTs possessing the MSH module showed they were able to interact with the melanocortin receptors of these cells to evoke a biological response (melanogenesis), the concentrations producing a half-maximal effect (EC50) being 14 nM for HMP-NLS-MSH and 17 nM for DTox-HMP-NLS-MSH; MRTs lacking the MSH module did not induce melanogenesis in B16-F1 cells (Fig. 1A
).
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MRTs delivered to cells are internalized into membrane-enclosed endosome structures with weakly acidic internal pH, which they must exit in order to be targeted subsequently elsewhere in the cell. The propensity of a polypeptide to penetrate through membranes in an acidic medium can be assessed from its ability to effect leakage of dye-loaded liposomes at different pHs (Fig. 1B
). In experiments with calcein-loaded liposomes, maximal activity of GALA-HMP-NLS-MSH was observed at about pH 3.5–4, which is more acidic than in endosomes. Liposome leakage under the action of DTox-HMP-NLS-MSH was observed in two pH intervals, 3.5 to 4.5, which was attributable to the HMP since it alone showed a maximal activity at pH 3.5–4.5 and 4.5 to 6, which is close to the endosomal pH and was attributable to activity of the DTox moiety. This, together with the fact that GALA-HMP-NLS-MSH was more difficult to express in bacteria (see above), encouraged us to characterize DTox-HMP-NLS-MSH in more detail.
Assessment of the recognition of the MRTs by the importin /ß heterodimer indicated that the NLS in the context of the MRTs is capable of mediating high-affinity interaction with importins (Fig. 1C
); the apparent dissociation constants Kdiss for importin binding were 1.9 and 2.5 nM, respectively, for HMP-NLS-MSH and DTox-HMP-NLS-MSH. By comparison, a control T-ag NLS peptide including the NLS and optimized phosphorylation site showed a Kdiss of 2 nM, whereas the non-NLS-containing MRT DTox-HMP (Fig. 1C
) displayed only very low importin binding.
Results for probing the pH of the intracellular environments of the MRTs by image ratio video-intensified microscopy showed that no acidic or mildly acidic regions were revealed in the vicinity of PS-DTox-HMP-NLS-MSH localization, whereas the PS-HMP-NLS-MSH conjugate lacking an endosomolytic module was found in the more acidic regions of the cell. Deconvolution fluorescence microscopic analysis indicated that the PS-HMP-NLS-MSH conjugate was found in the nuclei of only 12.2% of the cells, whereas PS-DTox-HMP-NLS-MSH was present in 87.5% of the nuclei (P<0.05).
3. Photosensitizer delivered by MRTs to the nuclei of mouse melanoma cells is significantly more effective than free PS
PSs used for anti-cancer photodynamic therapy are an example of locally acting drugs, with cytotoxic action not exceeding 40 nm from the site of subcellular localization and the cell nucleus being the most sensitive site for reactive oxygen species produced by PSs upon their photoactivation. PSs of medical interest localize in various cytoplasmic structures, but not in the cell nucleus, accordingly need to be transported to this most vulnerable site of the target cell. Evaluation of the photocytotoxic effect on mouse B16-F1 melanoma cells that overexpress MSH receptors, a property of many melanomas, showed that the efficacy of PS is greatly enhanced by its delivery to the cell nucleus (Fig. 2
A). Half-maximal effect (EC50) of (bacteriochlorin p6)-DTox-HMP-NLS-MSH was attained at a concentration of 22 nM, which is 230 times lower than that required for free PS bacteriochlorin p6 (EC50=4990 nM). (Bacteriochlorin p6)-DTox-HMP-NLS-MSH was not photocytotoxic on normal C3H/10T1/2 (Fig. 2A
) or NIH/3T3 (data not shown) mouse fibroblast lines demonstrating cell-specific activity of the MRT. (Bacteriochlorin p6)-HMP-NLS-MSH conjugate, lacking the endosomolytic module, was 5.3-fold less active than its DTox module-containing counterpart (bacteriochlorin p6)-DTox-HMP-NLS-MSH; PS-MRT conjugates lacking the NLS module showed less photocytotoxic activity than the above two conjugates (Fig. 2B
).
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CONCLUSIONS AND SIGNIFICANCE
Our results show it is possible to design modular chimeric genes encoding MRTs and to obtain these MRTs as 90–95% pure polypeptides after a simple purification procedure. The modules of the chimeric MRTs retain their functional activities so that they are able to interact with the corresponding internalizable receptors overexpressed on target cells (e.g., mouse melanoma), escape from acidic compartments such as endosomes after internalization, be transported into the cell nucleus, and carry PSs (Fig. 3
). It is not clear whether the PS-DTox-HMP-NLS-MSH conjugates detected in the nuclei of melanoma cells by the PS-generated fluorescence are fully intact, but since reactive oxygen species generation is evident within the nucleus, it can be concluded that PSs have been delivered into the nuclei by the MRT. These data clearly indicate that nuclear localization of PS enhances its activity, again confirming that the nucleus is a hypersensitive site for photodynamic damage. Cytotoxic activity of the MRT-linked PS exceeded free PS by several orders of magnitude. These results are indicative of the prospects of using recombinant chimeric multicomponent vehicles for these and possibly other locally acting drugs such as, for example, -particle emitting radionuclides. The different modules of the MRTs are interchangeable, meaning they can be tailored for different applications. Our modular/combinatorial approach to drug delivery based on MRTs, we believe, constitutes the first step toward developing a new generation of pharmaceuticals.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0888fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0888fje 
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), HMP-NLS-MSH (
), DTox-HMP-NLS-MSH (•), DTox-HMP (
); maximum (100%) melanin production corresponds to 26.1 ± 1.2 µg of melanin per well. B) Liposome leakage induced by MRTs: DTox-HMP-NLS-MSH (•), GALA-HMP-NLS-MSH (
), HMP-NLS-MSH (
). Egg yolk phosphatidylcholine liposomes were loaded with fluorescent calcein up to the concentration of fluorescence quenching; liposome leakage resulted in appearance of fluorescence. 100% liposome leakage (maximum) corresponds to the fluorescence intensity observed after the addition of Triton X-100 (0.5% final concentration) to the calcein-loaded liposomes. C) Binding, B, of 
); cytotoxicity in C3H/10T1/2 normal mouse fibroblasts of (bacteriochlorin p6)-DTox-HMP-NLS-MSH conjugate (
) and free bacteriochlorin p6 (
) is also shown. B) Influence of MRT-bacteriochlorin p6 conjugates lacking different modules on B16-F1 cell survival tested under the same experimental conditions as in panel A: (
), (bacteriochlorin p6)-HMP-MSH; (