Transfer of molecules into the cytosol of cells

Details Transfer of molecules into the cytosol of cells Transfer of molecules into the cytosol of cells

1997-03-28

1999-03-02

Campell, Bruce R.

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Cell membrane or cell surface is target

435375, 514 44, 514410, 540145, 935 52, C12N 1300

Patent

active

058769897

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a method for introducing molecules in cells by disrupting endosomal and lysosomal membranes using photodynamic treatment, without killing the majority of the cells by the photodynamic treatment.
The majority of molecules do not readily penetrate cell membranes. Methods for introducing molecules into the cytosol of living cells are useful tools for manipulating and studying biological processes. Among the most commonly used methods today are microinjection, red blood cell ghost mediated fusion and liposome fusion, osmotic lysis of pinosomes, scrape loading, electroporation, calcium phosphate and virus mediated transfection. These techniques are useful for investigations of cells in culture, although in many cases impractical, time consuming, inefficient or they induce significant cell death. They are thus not optimal for use in biological and medical research or therapeutics in which the cells shall remain functional.
It is well known that porphyrins and many other photosensitizing compounds induce cytotoxic effects on cells and tissues. These effects are based upon the fact that the photosensitizing compound upon light exposure releases singlet .sup.1 O.sub.2 which decomposes the membranes of the cells and cell structures and eventually kill the cells if the destruction is extensive. These effects have been utilized to treat several types of neoplastic diseases. The treatment is named photodynamic therapy (PDT) and is based on injection of a photosensitizing and tumorlocalizing dye followed by exposure of the tumor region to light. The cytotoxic effect is mediated mainly through the formation of singlet oxygen. This reactive intermediate has a very short lifetime in cells (<0.04 .mu.s). Thus, the primary cytotoxic effect of PDT is executed during light exposure and very close to the sites of formation of .sup.1 O.sub.2. .sup.1 O.sub.2 reacts with and oxidize proteins (histidine, tryptophan, methionine, cysteine, tyrosine), DNA (guanine), unsaturated fatty acids and cholesterol. One of the advantages of PDT is that tissues unexposed to light will not be affected. There is extensive documentation regarding use of PDT to destroy unwanted cell population, for example neoplastic cells. Several patents relate to photodynamic compounds alone or conjugated with immunoglobulins directed to neoplastic cell receptor determinants making the complex more cell specific. Certain photochemical compounds, such as hematoporphyrin derivates have furthermore an inherent ability to concentrate in malign cells. These methods and compounds, which are directed to destroy the unwanted cells are described in the Norwegian patent NO 173319, in Norwegian patent applications Nos 90 0731, 90 2634, 90 1018, 94 1548, 85 1897, 93 4837, 92 4151 and 89 1491.
In PCT/US93/00683 a drug delivery system is described which is comprised of an anticancer drug and a photoactivatable drug attached to copolymeric carriers. Upon administration this complex enters the cell interior by pinocytosis or phagocytosis and will be located inside the endosomes and lysosomes. In the lysosomes the bond between the antineoplastic compound and the polymer is hydrolyzed and the former can diffuse passively through the lysosome membrane into cytosol. Thus this method limits the method to small molecular compounds which are able to diffuse across the lysosome membranes. After allowing a time lag for diffusion a light source of appropriate wavelength and energy is applied to activate the photoactivatable compound. The combined effect of the anticancer drug and photoactivatable drug destroy the cell. Thus all use of photoactivatable compounds known is directed to extensively destroy cell structures leading to cell death It is not known of a method to release membrane unpermeable molecules into the cytosol after localized rupturing of endosomal/lysosomal membranes.
The object of the present invention is thus to provide a method to transport molecules into cytosol of living cells, in culture or in tissues, by exposing the cells to a pho

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Kopecek, J., B. Rihova and N.L. Krinick, "Targetable photoactivatable polymeric drugs", Journal of Controlled Release v. 16, pp. 137-144.

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