Site-specific cell perforation technique

Abstract

A technique for controlling membrane denaturation reactions other than physical shear force was developed. For example, the present invention provides, a method for causing membrane disruption at a specific site by reacting a stimulus such as light with a compound that is activated by the stimulus, where the reaction occurs on a membrane such as a biomembrane. It also provides a membrane structure such as cells in which a specific site has been disrupted, which are obtained by the present method. Introduction of substances such as genes also became possible by using this membrane structure. Further provided is a membrane-destroying member for disrupting a membrane at a specific site. Thus, the present invention enabled, for example, easy membrane penetration using components constituting microelectrodes, micromanipulators, and microinjectors, which were conventionally hardly usable in penetrating cell membranes.

1 . A method of denaturing or perforating a specific site of a membrane, the method comprising contacting the whole or part of the membrane with a reagent containing a specific compound that induces a membrane-denaturing reaction by a specific stimulation, and giving said stimulation.  
     
     
         2 . The method of  claim 1 , wherein the membrane is a cell membrane, a cell wall, a biomembrane, or an artificial membrane.  
     
     
         3 . The method of  claim 1  or  2 , wherein the region stimulated is, included in the region contacted with the reagent.  
     
     
         4 . The method of  claim 1  or  2 , wherein the region contacted with the reagent is included in the region stimulated.  
     
     
         5 . The method of  claim 4 , wherein the reagent is contacted using a support.  
     
     
         6 . A method of any one of  claim 1  to  5 , wherein the specific stimulation is light, and the compound is a photosensitizing compound.  
     
     
         7 . A membrane obtained by the method of any one of  claims 1  to  6 , wherein the specific site has been perforated or denatured, or a membrane structure containing said membrane.  
     
     
         8 . The membrane or membrane structure containing said membrane of  claim 7 , wherein the membrane is a cell membrane, a biomembrane, or an artificial membrane.  
     
     
         9 . The membrane structure of  claim 7  or  8 , which is a cell, a micelle, or a liposome.  
     
     
         10 . A method of injecting a compound into a structure, the method comprising mixing the structure of any one of  claims 7  to  9  with a complex comprising a compound to be injected and a carrier.  
     
     
         11 . The method of  10 , wherein the carrier is a liquid or solid.  
     
     
         12 . A method of  claim 10  or  11 , wherein the substance is a nucleic acid or protein.  
     
     
         13 . A membrane-destroying member for denaturing or perforating a specific site of a membrane, which comprises a support, and a membrane denaturation promoting portion comprising a membrane denaturation force other than physical shear force, which was formed on at least one site on the surface of the support.  
     
     
         14 . The membrane-destroying member of  claim 13 , wherein the support is rod-shaped, tube-shaped, needle-shaped, or spherical.  
     
     
         15 . The membrane-destroying member of  claim 13  or  14 , wherein the membrane denaturation promoting portion is coated or fixed with a compound that generates the membrane denaturation reaction at the membrane denaturation promoting portion.  
     
     
         16 . The membrane-destroying member of  claim 15 , which utilizes a sequential peroxidation reaction of the membrane components, in which the membrane denaturation reaction is started by a direct/indirect production reaction of reactive oxygen species.  
     
     
         17 . The membrane-destroying member of  claim 15  or  16 , wherein the membrane denaturation reaction is induced by a specific stimulation and reaction precursor, and includes the reaction that denatures or perforates the membrane.  
     
     
         18 . The membrane-destroying member of  claim 17 , wherein the specific stimulation is light, and the reaction precursor is a photosensitizing compound.  
     
     
         19 . The membrane-destroying member of any one of  claims 13  to  18 , wherein the support penetrates the membrane following membrane denaturation or disruption, and said penetrated membrane closely contacts with said support.  
     
     
         20 . The membrane-destroying member of any one of  claims 13  to  19 , wherein the membrane is a cell membrane, a biomembrane or an artificial membrane.