q&more
My watch list
my.chemie.de  
Login  
Fischer_Heinze_980x442

The targeted transport of DNA and RNA using vectors (mostly made from synthetic polymers) in cell cultures has become part of routine practice in biological R&D – a fact highlighted by the multitude of commercial kits now available. To date, however, obstacles relating to use in patients have beset many laboratory studies and – in particular – the transfer to clinical practice. In many cases, such issues are related to safety concerns and the limited biodegradability of such carriers. At FSU Jena, new bio-based and natural systems using polysaccharides are being developed with the aim of solving these specific problems.

Vectors used to transfer DNA and RNA must be “all-rounders”, capable of performing a wide range of discrete tasks. These include maintaining the stability of their nucleic acid “cargo”, achieving an efficient and selective uptake by the target cells, and with high efficiency (transfection) while simultaneously ensuring excellent tolerability. Particularly in terms of use in patients, both their safety and their biocompatibility must be assured at all times [1].

Dextrans: familiar sugars, rediscovered

Dextrans have been successfully deployed in pharmacy and medicine for many decades now as plasma expanders, tableting excipients and stabilizers for colloidal formulations: both their toxicology and biodistribution are therefore adequately well-studied. The polysaccharides are formed biosynthetically as a branched or unbranched molecule in sucrose-rich media by the activity of the dextransucrase enzyme, which is produced by various Leuconostoc strains. Dextrans are degraded in the organism and metabolized in the body [2]. Since they are not themselves capable of interaction with DNA and RNA, the logical next step was the covalent binding of cationic pendant groups to accomplish an electrostatic interaction with the negatively charged DNA and RNA [3]. To date, however, such side chains have often consisted once again of synthetic polymers rather than biogenic functionalities. Such polymers are neither biodegradable nor excretable and occasionally exhibit high toxicity.

Sugars and amino acids: two natural partners

In contrast to the above, introducing naturally-occurring functionalities with amine and ammonium groups appeared to be a highly promising approach. Work at Jena on polysaccharide chemistry has resulted in the development of efficient reactions under moderate conditions that permit the introduction of almost any carboxylic acid into polysaccharides [4, 5].

Fig. 1 Development of a dextran amino acid ester library with controllable, high-quality characteristics

One pioneering achievement was the reproducible production of dextran amino acid esters with controllable molar mass and configurable content (average degree of substitution, DS) for the ester substituents – a synthesis variant that can easily be upscaled. In this approach, amino acids are activated with commercially available N,N-carbonyldiimidazole and undergo a homogeneous reaction to produce the corresponding dextran derivatives with a high level of purity and without appreciable polymer degradation (the average degree of polymerization (DP) of the initial dextran is preserved) or toxic byproducts. Based on this synthesis model, we gain access to a library of monofunctional and multifunctional dextran amino acid esters, which can also act as carriers of other functional groups: a construction kit for a practically unimaginable number of dextran derivatives offering considerable structural diversity. Products selected to date have exhibited outstanding characteristics as DNA transporters and interest is currently focused on β-alanine-substituted dextrans.

Alanine-substituted dextrans as DNA transporters

Alanine-substituted dextrans are excellently water-soluble, can be stored permanently at room temperature following freeze-drying, and can be reconstituted quickly and completely in buffers and cell culture media. In standardized toxicity tests (according to DIN ISO 109935) using L929 mouse fibroblasts, they exhibited outstanding levels of tolerability that were higher by a factor of two than those for known synthetic polymers such as the commonly used polyethyleenimines [6]. If one investigates interactions with blood cells such as erythrocytes, which are the first contact partners within the organism following injection, no hemolytic effects or aggregations of blood cells – i.e. no thrombotic events – are observed in any therapeutically relevant quantities.

Fig. 2 Amino-acid substituted dextrans as biocompatible and highly efficient transfection systems

The simple pipetting of DNA or RNA into the solutions of alanine-substituted dextrans causes the spontaneous formation of complexes that are deployable within a few minutes. With an overall cationic charge and a size of approximately 100 nm under optimized conditions and batch ratios, the complexes meet all of the requirements for good uptake via the cell membrane. They can be deployed in serum-rich media and protect the nucleic acids against enzymatic attack from nucleases in blood just as well as they stabilize against the uncontrolled and undesirable displacement of the nucleic acids by serum proteins. They are not only capable of transfecting mammal cells but have the unusual property of causing no effects of toxicological concern even at high doses.

What does the future hold?

Modification of the complexes based on dextran-alanine for the intended application cannot be achieved merely by varying the production conditions and varying the formulation of the complex. The model can be re-used for a range of cationic amino acids and their combination.

Properties such as the binding strength of the complexes with DNA and RNA, stability, toxicity and transfection efficiency can be controlled by the selection and combination of various types of amino acids. While lysine is more advantageous for binding DNA, for example, an excess of alanine has an especially positive effect on the transfection of cells. To sum up, the new synthesis strategy offers numerous approaches for structural variations. Amino acid-modified dextrans offer a simple way of bypassing numerous obstacles such as poor biodegradability, unacceptable toxicity and time-consuming instructions for synthesis experiments.

___________________________________________________________________________________________

Infobox

Center of Excellence for Polysaccharide Research
Friedrich Schiller University Jena

The Center of Excellence for Polysaccharide Research is a leading research institution originally formed by six multinationals at the Friedrich Schiller University Jena and the Thuringian Institute of Textile and Plastics Research (TITK) at Rudolstadt in 2002, and which received financial support from this consortium of companies until 2007. The Center’s activities focus on joint R&D work on polysaccharides as functional raw materials for the future.

Products and methods are investigated and developed both within basic research and application-oriented research. Here, the Center pursues various strategies of biopolymer derivatization under homogeneous and heterogeneous reaction conditions, and of regioselective functionalization. The systems available also permit methods to be upscaled to the level of the process laboratory. The work of the Center also guarantees the provision of initial and advanced training to students in the field of bioorganic chemistry.

Leiter des Kompetenzzentrums:
Prof. Dr. Thomas Heinze

___________________________________________________________________________________________

(First publication of the article: Fischer, D., Heinze, T. (2015) labor&more 8, 26–29)

Literature:
[1] Schlenk, F. et al. (2013) Recent developments and perspectives on gene therapy using synthetic vectors, Ther Deliv. 4(1), 95 –113, DOI: 10.4155/tde.12.128
[2] Heinze, T. et al. (2006) Functional Polymers Based on Dextran, in: Klemm D. (eds) Polysaccharides II, Adv Polym Sci 205, Berlin, Heidelberg: Springer, 199 –291, DOI: 10.1007/12_100
[3] Ochrimenko, S. et al. (2014) Dextran-graft-linear poly(ethylene imine)s for gene delivery: Importance of the linking strategy, Carbohydr Polym 113, 597 –606, DOI: 10.1016/j.carbpol.2014.07.048
[4] Varshosaz, J. (2012) Dextran conjugates in drug delivery, Expert Opin Drug Deliv., 9(5), 509 –523, DOI: 10.1517/17425247.2012.673580
[5] Heinze, T. et al. (2006) Esterification of Polysaccharides, Springer-Verlag Berlin Heidelberg, 2006 ISBN 3-540-32103-9, DOI: 10.1007/3-540-32112-8
[6] Jaeger M. et al. (2012) Branched and linear poly(ethylene imine)-based conjugates: synthetic modification, characterization, and application, Chem Soc Rev 41(13), 4755 –4767, DOI: 10.1039/c2cs35146c

Date of publication: 02-Oct-2018

Facts, background information, dossiers

  • polysaccharides
  • transfection
  • sugars
  • dextrans
  • DNA transporters

More about Uni Jena

  • News

    When Proteins Shake Hands

    Be it in spider silk, wood, the spaces between body cells, in tendons, or as a natural sealant for small wounds: protein fibres are found virtually everywhere in nature. These small protein fibres, also referred to as protein nanofibres by experts, often have outstanding properties such as ... more

    High resolution without particle accelerator

    A visit to the optometrist often involves optical coherence tomography. This imaging process uses infrared radiation to penetrate the layers of the retina and examine it more closely in three dimensions, without having to touch the eye at all. This allows eye specialists to diagnose disease ... more

    Tunable materials clear the way for advanced optics

    Now you see it, now you don’t: In books and movies, wizards use magic spells to easily change things from a solid to see-through state. However, in reality, materials with properties called phase transition can pull off a similar trick, changing from clear to cloudy depending on the tempera ... more

  • q&more articles

    Highly-prized components

    The isolation of bioactive plant ingredients, essential oils or dyes and flavourings of plant origin requires costly and sophisticated procedures. Several applications do not actually require isolation of the individual components, however – their concentration is sufficient. Moreover, for ... more

    Molecules in the mirror

    In 1871 the children‘s book “Through the Looking-Glass – And What Alice Found There” by the English author Lewis Carroll, in which Alice enters a world behind the mirror, was published. She explains to her cat: “First there’s the room you can see through the glass – that’s just the same as ... more

  • Authors

    Prof. Dr. Thomas Heinze

    Thomas Heinze, born in 1958, studied chemistry at FSU Jena. After receiving his doctorate there in 1989 and subsequent postdoc work at KU Leuven (Belgium), he completed his habilitation in 1997. In 2001, he accepted a professorship in Macromolecular Chemistry at the University of Wuppertal ... more

    Prof. Dr. Dagmar Fischer

    Dagmar Fischer is a licensed pharmacist before obtaining her doctorate in pharmaceutical technology and biopharmacy from the Philipps University of Marburg in 1997. After a period spent at Texas Tech University Health Sciences Center (USA), she gained several years' experience as Head of Pr ... more

    Prof. Dr. Gerhard K. E. Scriba

    born 1956, studied pharmacy in Bonn and in 1980 received the licence to practise pharmacy. He received his doctorate in 1984 at the Westphalian Wilhelms-University in Munster, where, in 1995, he qualified as a professor in the subject of pharmaceutical chemistry. In 1999 he was appointed to ... more

Topics A-Z

All topics

q&more – the networking platform for quality excellence in lab and process

The q&more concept is to increase the visibility of recent research and innovative solutions, and support the exchange of knowledge. In the broad spectrum of subjects covered, the focus is on achieving maximum quality in highly innovative sectors. As a modern knowledge platform, q&more offers market participants one-of-a-kind networking opportunities. Cutting-edge research is presented by authors of international repute. Attractively presented in a high-quality context, and published in German and English, the original articles introduce new concepts and highlight unconventional solution strategies.

> more about q&more

q&more is supported by:



Your browser is not current. Microsoft Internet Explorer 6.0 does not support some functions on Chemie.DE