Charge conversional ternary polyplex

Abstract

Disclosed is a polymer composite (polyplex) that contains nucleic acid, a cationic polymer, and an anionic polymer. The anionic polymer covers the surface of the composite comprising the cationic polymer and nucleic acid, has a negative charge at neutral pH, and can change so as to have a positive charge at mildly acidic pH.

Description

TECHNICAL FIELD[0001]The present invention relates to a polymer complex (polyplex) containing a nucleic acid for use as a nonviral synthetic vector capable of delivering the nucleic acid to a target cell. More particularly, the present invention relates to a charge conversional ternary polyplex comprising a nucleic acid, a cationic polymer and an anionic polymer, wherein the anionic polymer covers the surface of a complex comprising the cationic polymer and the nucleic acid.BACKGROUND ART[0002]DNA or RNA delivery to a target cell mediated by a nonviral synthetic vector (lipoplex and polyplex) has been widely recognized as a promising alternative method for delivery that uses a viral vector which has been confronting safety issues specific to biological properties[1]. Nonetheless, even in the case of a nonviral vector, the main concern is the conflict between the delivery efficiency and the safety issues (in particular, chemical toxicity). While most vectors having high transfection ...

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Benefits of technology

[0004]Accordingly, the present inventors have examined a new approach for a polyplex design that exerts both properties of high transfection activity and low toxicity by integrating a charge conversional moiety into a polyplex structure. Although maleic acid amide derivatives, i.e., cis-aconitic acid amide and citraconic acid amide, bear negative charges at neutral pH, they are rapidly degraded at weak acidity of pH 5.5 and expose the positively-charged amine[8]. Therefore, a ternary polyplcx (a polyplex having pDNA / polycation / polyanion having degrading side-chain) formed by the present inventors by covering the surface of a positively-charged polyplex with a polymer derived from degrading amide, by which the ternary polymer maintains neutrally- to negatively-charged properties outside the cell. On the other hand, in the acidic environment of endosomes, the charge conversional moieties are expected to charge positively and promote endosomal escape of the polyplex owing to membrane disruption. This scheme is illustrated in FIG. 1.

[0005]The present invention has an objective of providing a polymer that is negatively charged outside the cell but that undergoes charge conversion and positively charged once entering the endosome. The present invention also has an objective of providing a complex (polyplex) comprising the polymer, namely a ternary polyplex which overcomes the problem (dilemma) existing between gene transfection efficiency and safety and which can fulfill low toxicity and high gene transfection efficiency.

[0006]In order to solve the above-described problems, the present inventors have gone through keen examination. As a result, they found that a polymer obtained by causing a polycation to react with citraconic anhydride or cis-aconitic anhydride is negatively charged under a neutral condition but its charge is converted to give a cation under an acidic condition, and therefore the polymer shows low toxicity and high gene transfection efficiency, thereby accomplishing the present invention.

[0021]In the field of gene vector studies, transfection into a toxicity-sensitive primary cell such as HUVEC is extremely difficult. A polyplex of the present invention is capable of delivering a nucleic acid such as DNA or siRNA to such types of cells in a highly efficient manner without evoking toxicity. Medically speaking, major drawbacks of nucleic acid delivery include low efficiency, high toxicity and instability in the blood. The charge conversional ternary polyplex of the present invention has very low toxicity since it is negatively charged outside the cell and thus is extremely useful as a gene vector in vivo.

Problems solved by technology

Nonetheless, even in the case of a nonviral vector, the main concern is the conflict between the delivery efficiency and the safety issues (in particular, chemical toxicity).

While most vectors having high transfection efficiency show high toxicity, vectors with low toxicity are often associated with low transfection efficiency.

However, due to the toxicity of these polycations, polyplexes are used only for limited applications[2].

On the contrary, the positively-charged property of a polyplex could cause non-specific interaction with a negatively-charged serum component, thereby producing a thrombus in the blood capillary.

This has a risk of disturbing construction of a plasma membrane and a risk of inducing high cytotoxicity and excess immune response[4].

In this case, however, severe loss in transfection efficiency cannot be avoided primarily due to decrease in the cellular uptake and deterioration of the endosomal escape ability.

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