polyethyleneimine Chemical Properties

Density 

1.08 g/mL at 25 °C

vapor pressure 

9 mm Hg ( 20 °C)

refractive index 

n20/D 1.5240

Flash point:

>230 °F

solubility 

Chloroform (Sparingly), DMSO (Sparingly), Methanol (Slightly)

form 

Oil

color 

Colourless

InChI

InChI=1S/C2H8N2.C2H5N/c3-1-2-4;1-2-3-1/h1-4H2;3H,1-2H2

InChIKey

SFLOAOINZSFFAE-UHFFFAOYSA-N

SMILES

C(N)CN.C1NC1

EPA Substance Registry System

1,2-Ethanediamine, polymer with aziridine (25987-06-8)

Safety Information

Hazard Codes 

Xn

Risk Statements 

22-36-52/53-43-41

Safety Statements 

26-36-61-36/37/39

RIDADR 

UN 3082 9 / PGIII

WGK Germany 

3

polyethyleneimine Usage And Synthesis

Overview

Polyethyleneimines (PEIs) are highly basic and positively charged aliphatic polymers, containing primary, secondary and tertiary amino groups in a 1:2:1 ratio. Every third atom of the polymeric backbone is therefore an amino nitrogen that may undergo protonation. As the polymer contains repeating units of ethylamine, PEIs are also highly watersoluble. PEIs are available in both linear and branched forms with molecular weights ranging from 700 Da to 1000 kDa.
For a long time, PEI has been also used in non-pharmaceutical processes, including water purification, paper and shampoo manufacturing. It has been also reported that PEI is relatively safe for internal use in animals and humans ^[1]. PEI is widely used to flocculate cellular contaminants, nucleic acids, lipids and debris from cellular homogenates to facilitate purification of soluble proteins^[2-4]. Enzymatic reactions in bioprocesses constitute another field in which PEI was used: as an immobilizing agent for biocatalysts^[5], as a soluble carrier of enzymes^[6] or in the formation of macrocyclic metal complexes mimicking metalloenzymes^[7]. PEI is also a common ingredient in a variety of formulations ranging from washing agents to packaging materials.
PEIs have been extensively studied as a vehicle for nonviral gene delivery and therapy. Since its introduction in 1995^[8], PEI (Fig. 1) has been considered the gold standard for polymer-based gene carriers because of the excellent transfection efficiencies of its polyplexes (complex of nucleic acid and polymer) in both in vitro and in vivo models [9]. Polycation-mediated gene delivery is based on electrostatic interactions between the positively charged polymer and the negatively charged phosphate groups of DNA. In aqueous solution, PEI condenses DNA and the resulting PEI/DNA complexes, carrying a net positive surface charge, can interact with the negatively charged cell membrane and readily internalized into cells^[10]. PEI retains a substantial buffer capacity at virtually any pH and it has been hypothesized that this simple molecular property is related to the efficiency of the complex multistage process of transfection. As a matter of fact, the ‘proton sponge’ nature of PEI is thought to lead to buffering inside endosomes. The proton influx into the endosome, along with that of counter-anions (generally chloride anions), maintains the overall charge neutrality even if an increase of ionic strength inside the endosome is expected. This effect generates an osmotic swelling and the consequent physical rupture of the endosome, resulting in the escape of the vector from the degradative lysosomal compartment. The proton sponge hypothesis has been a subject of debate, speculation and research without reaching a general consensus about the real mechanism involved^[11].

Figure 1 the chemical structure of polyethyleneimines

Features

Polyethylenimine(PEI) is one of the most widely used synthetic polycations in various applications because of its chemical functionality arising from the presence of cationic primary (25%), secondary (50%), and tertiary amines (25%)^[12,13]. PEI is formed by the linking of iminoethylene units and can have linear, branched, comb, network, and dendrimer architectures depending upon its synthesis and modification methods, which greatly influences its properties, both physical and chemical^[14]. Furthermore, these synthetic approaches enable PEI to be available in a wide range of molecular weights. At room temperature, branched PEI (BPEI) is a highly viscous liquid while linear PEI (LPEI) is a solid. PEI has several attractive features for its use in widespread applications, such as low toxicity, ease of separation and recycling, and (last but not least) it being odorless. In addition to these attractive features, there is a distinct feature of PEI which places it ahead of other polyions (e.g. polyallylamine or chitosan) when it comes to loading, and which justifies its widespread use in fields as varied as detergents, adhesives, water treatment, cosmetics, carbon dioxide capture,^[15-18] as a DNA transfection agent, and in drug delivery^[19,20] despite being a weak polymeric base with pKa values between 7.9 and 9.6, it possesses a high ionic charge density, which in practical terms translates into being a more cost-effective material. This derives from the possibility of either reaching the same loadings with reduced amounts of the polymer (which would colloquially mean "getting a bigger bang for the buck") or reaching loadings that are beyond the reach of the aforementioned examples while avoiding enzyme agglomeration thanks to its multi-branched network.

Applications

PEI’s application fields may be divided according to its use in:
Use of PEI as a drug
An important biological function of PEI was reported by Chu et al. showing that PEI readily blocks fibrin formation, thus exhibiting anticoagulant activity^[23]. This study demonstrated that even at a nanomolar concentration, PEI significantly blocks thrombin-catalyzed fibrin formation in vitro, accounting for its anticoagulant property. The antibacterial properties of PEIs have been investigated in details and were applied in the development of coated materials. Helander^[24] studied the effect of PEI on the permeability properties of the Gramnegative bacterial outer membrane (OM) using Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium as target organisms. Due to the polycationic nature of PEI, it could be expected that this polymer may act as an efficient OM-permeabilizing agent. As expected, even at a concentration lower than 20 μg/ml PEI increased the bacterial uptake of 1-N-phenylnaphthylamine, a hydrophobic fluorescent probe, indicating an increased hydrophobic permeation of the outer membrane. PEI also increased the susceptibility of bacteria toward other hydrophobic antibiotics like clindamycin, erythromycin, fucidin, novobiocin and rifampicin, without being bactericidal itself. Moreover, PEI is able to sensitize the bacteria to the lytic action of the anionic detergent SDS when bacteria are opportunely pretreated with the polymer.
Use of PEI for delivery of small drugs, and for the photodynamic therapy (PDT)
As polycation, PEI was selected for its several advantageous properties (hydrophylicity, biocompatibility and thermal stability) and furosemide was chosen as a model water-insoluble drug[20]. The furosemide-loaded calcium alginate (ALG), calcium alginatepolyethyleneimine (ALG-PEI) and alginate-coated ALG-PEI (ALG-PEI-ALG) beads by ionotropic/polyelectrolyte complexation method to achieve controlled release of the drug were prepared^[25]. Release of furosemide from ALG-PEI beads was prolonged considerably compared with that from ALG beads. Ionic interaction between alginate and PEI led to the formation of polyelectrolyte complex membrane, the thickness of which was dependent on the conditions of PEI treatment (PEI concentration and exposure time)^[25]. The membrane acted as a physical barrier to drug release from ALG-PEI beads. The coating of ALG-PEI beads further prolonged the release of the drug by increasing membrane thickness and reducing swelling of the beads possibly by blocking the surface pores. Hamblin’s research group has been involved in the use of photodynamic therapy (PDT) as a possible treatment for localized infections^[26]. They shown that covalent conjugates between PEI and chlorin (e6) (ce6) can be used as a potent broad-spectrum antimicrobial photo sensitizers (PS) resistant to protease degradation and therefore constituting an alternative to the previously described poly-L-lysine chlorin (e6) (pL-ce6) conjugates^[27].
Use of PEI for antimicrobial coating
Bourgeois^[28] used PEI to build a specific delivery system for beta-lactamases. The aim of that study was to provide a "proof of concept" of colon delivery of beta-lactamases by pectin beads aiming to degrade residual beta-lactam antibiotics, in order to prevent the emergence of resistant bacterial strains. Pectin is almost totally degraded by pectinolytic enzymes produced by colon microflora, but it is not digested by gastric or intestinal enzymes. In addition, pectin beads could efficiently protect beta-lactamases from degradation by proteases contained in the upper gastrointestinal tract. The specific delivery system for beta-lactamases was composed of a core of calcium pectinate bead, cross-linked at its surface with PEI^[29]. PEI improved the stability of Ca-pectinate beads, protecting them from water penetration by cross-linking the free carboxylic functions of the Ca-pectinate network. The cross-linking step does not influence shape, size and efficiency of encapsulation of betalactamases in beads. Thus, PEI made Ca-pectinate beads resistant to the denaturing effect of upper intestine conditions, allowing to delay beta-lactamases release.
Use of PEI for the preparation of nano-sized delivery vectors
In several papers^[30-33], PEI takes part to the composition of nanoparticles used for drug delivery. The advantages of using nanoparticles for drug delivery result from their small size, that allow for the penetration through even small capillaries up to cytoplasm, allowing also an efficient drug accumulation at the target sites in the body. Furthermore, the use of biodegradable materials for nanoparticles preparation allows for sustained drug release within the target site over a period of days or even weeks after injection.
Use of PEI for non-invasive optical imaging devices
PEI is also an important polymer for non-invasive optical imaging devices (Near Infrared, NIR) enabling the assessment of several cellular functions like caspases' activity in vitro^[34]. The cell-permeable branched polyethylenimine (25 kDa), was modified with deoxycholic acid (DOCA) hydroxysuccinimide ester, resulting in PEI-DOCA nanoparticles. After attaching the effector caspase-specific near-infrared (NIR) fluorescence probe (Cy5.5-DEVD) to amphiphilic bile acid-modified polymer backbone, this polymeric nanoparticle system can be easily controlled with the optical imaging technique. The imaging-probe entry into cells is an important area in apoptosis imaging because the caspases’ reaction occurs in the cytoplasm. Thus, the tracking of the fluorescein isothiocyanate (FITC)-labeled Cy5.5DEVD26-PEI-DOCA20 nanoparticles in HeLa cells allowed for the monitoring of both caspase-3 and caspase-7 activity. Therefore, this polymeric nanoparticles can be used to measure apoptosis in cell-based high-throughput screens for inhibitors or inducers of apoptosis.

For gene delivery

The potential of PEI as a gene delivery vector was first discussed in 1995^[35] following which there have been numerous studies reporting its application in gene delivery both in vitro and in vivo. PEIs of molecular weights ranging from 800 to 25 kDa have been investigated in gene delivery^[36-38]. The results showed that PEIs having molecular weight 25 kDa were the most suitable for transfection. Higher molecular weight increases cytotoxicity due to cell surface aggregation of the polymer^[39]. Though low molecular weight PEIs is less toxic, they do not display effective transfection property. Due to the low positive charge, low molecular weights PEIs are incapable of condensing DNA effectively. Also, the low surface charge of the PEI/DNA complexes does not induce effective cellular uptake through charge-mediated interactions^[38].
PEI polymers can be broadly classified into branched and linear PEI. Compared to linear PEI, highly branched PEI forms stronger and smaller complexes with DNA^[40,41]. The complexation of branched PEI with DNA is less dependent on the buffer conditions than the high molecular weight linear PEI, which is dependent on the buffer condition. Linear PEI (22 kDa) on complexation with DNA in a high ionic strength solution has been observed to form larger-sized complexes (1 μm), whereas in 5% glucose, the complex size was found to be 30 60 nm. The in vivo studies showed that linear PEI/DNA complexes prepared in high salt conditions were less efficient in transfection than those formed in low salt condition^[42].
The transfection efficiency/cytotoxicity profile of PEIs is largely influenced by their molecular weight, degree of branching, zeta potential and particle size. With increase in molecular weight, branched PEIs exhibit high transfection efficiency; however, cytotoxicity has also been found to increase concurrently^[38]. To overcome the cytotoxicity associated with PEIs, different strategies have been studied. These include using linear high molecular weight PEI, substituting or linking high molecular weight branched PEIs with polysaccharides, hydrophilic polymers such as PEG, disulfide linkers, lipid moieties, etc. PEI and its derivatives have been used to deliver nucleic acids in vivo and the results are promising and have been used in cancer and RNA interference (RNAi) therapy. There are several reports suggesting delivery of nucleic acids by PEI derivatives in vivo that showcase the potential of PEI in delivery of therapeutics. These derivatives are either polysaccharide-decked PEIs or cross-linked PEI nanoparticles. The use of polysaccharides such as chondroitin sulphate, hyaluronic acid, gellan gum or dextran to modify branched PEI (Mw 25 kDa) has made the resulting polymers less toxic thereby improving their transfection efficiency in vivo^{[43, 44]}. Also, the linkers such as polyglutamic acid, polyethyleneglycol-bis (aminoethylphosphate), piperazine-N, N¢-dibutyric acid, butane-1, 4-diol bis glycidyl ether (BDG) when used to crosslink PEI (25 kDa) resulted in the formation of vectors with significantly enhanced transfection efficacy in vivo^[45,46]. Subsequent sections will elaborate on low and high molecular weight branched and linear PEI-mediated delivery of therapeutic genes to various tissues in a specific manner.

Reference

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2. Cordes, R.M.; Sims, W.B.; Glatz, C.E. Precipitation of nucleic acids with poly(ethyleneimine). Biotechnol. Prog., 1990, 6(4), 283.
3. Kirk, N.; Cowan, D. Optimising the recovery of recombinant thermostable proteins expressed in mesophilic hosts. J. Biotechnol., 1995 , 42(2), 177.
4. Milburn, P.; Bonnerjea, J.; Hoare, M.; Dunnill, P. Selective flocculation of nucleic acids, lipids, and colloidal particles from a yeast cell homogenate by polyethyleneimine, and its scale-up. Enzyme Microb. Technol., 1990, 12(7), 527.
5. Bahulekar, R.; Ayyangar, N.R.; Ponrathnam, S. Polyethyleneimine in immobilization of biocatalysts. Enzyme Microb. Technol., 1991, 13(11), 858.
6. Cong, L.; Kaul, R.; Dissing, U.; Mattiasson, B. A model study on Eudragit and polyethyleneimine as soluble carriers of aamylase for repeated hydrolysis of starch. J. Biotechnol., 1995, 42, 75.
7. Suh, J.; Cho, Y.; Lee, K. J. Macrocyclic metal complexes built on polyethyleneimine. J. Am. Chem. Soc., 1991, 113, 4198.
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21. C. R. Dick and G. E. Ham, J. Macromol. Sci., Part A: Pure Appl. Chem., 1970, 4, 1301–1314.
22. S. Kobayashi, Prog. Polym. Sci., 1990, 15, 751–823.
23. Chu, A. J; Beydoun, S.; Mathews, S.; Hoang, T. Novel anticoagulant polyethylenimine: inhibition of thrombin-catalyzed fibrin formation. Arch. Biochem. Biophys., 2003, 415, 101.
24. Helander, I. M.; Alakomi, H.-L.; Latva-Kala, K.; Koski, P. Polyethyleneimine is an effective permeabilizer of gram-negative bacteria. Microbiology, 1997, 143, 3193.
25. Setty, C. M.; Sahoo, S. S.; Sa, B. Alginate-coated alginatepolyethyleneimine beads for prolonged release of furosemide in simulated intestinal fluid. Drug Dev. Ind. Pharm., 2005, 31, 435.
26. Tegos, G. P.; Anbe, M.; Yang, C.; Demidova, T. N.; Satti, M.; Mroz, P.; Janjua, S.; Gad, F.; Hamblin, M. R. Protease-stable polycationic photosensitizer conjugates between polyethyleneimine and chlorin(e6) for broad-spectrum antimicrobial photoinactivation. Antimicrob. Agents Chemother., 2006, 50(4), 1402.
27. Soukos, N. S.; Ximenez-Fyvie, L. A.; Hamblin, M. R.; Socransky, S. S.; Hasan, T. Targeted antimicrobial photochemotherapy. Antimicrob. Agents Chemother., 1998, 42, 2595.
28. Bourgeois, S.; Laham, A.; Besnard, M.; Andremont, A.; Fattal, E. In vitro and in vivo evaluation of pectin beads for the colon delivery of beta-lactamases. J. Drug Target, 2005, 13, 277.
29. Bourgeois, S.; Fattal, E.; Andremont, A.; Couvreur, P. 2003 PCT/FR03/02474.
30. Kra?mer, M.; Stumby?, J.-F.; Turk, H.; Krause, S.; Komp, A.; Delineau, L.; Prokhorova, S.; Kautz, H.; Haag, R. pH-responsive molecular nanocarriers based on dendritic core-shell architectures. Angew. Chem. Int. Ed., 2002, 41, 4252.
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34. Kim, K.; Lee, M.; Park, H.; Kim, J.-H.; Kim, S.; Chung, H.; Choi, K.; Kim, I.-S.; Seong, B. L.; Kwon, I. C. Cell-permeable and biocompatible polymeric nanoparticles for apoptosis imaging. J. Am. Chem. Soc., 2006, 128(11), 3490.
35. Boussif O, Lezoualc’h F, Zanta MA, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 1995;92:7297-301
36. Baker A, Saltik M, Lehrmann H, et al. Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery. Gene Ther 1997;4:773-82
37. Meunier-Durmort C, Grimal H, Sachs LM, et al. Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells. Gene Ther 1997;4:808-14
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39. Morimoto K, Nishikawa M, Kawakami S, et al. Molecular weight-dependent gene transfection activity of unmodified and galactosylated polyethyleneimine on hepatoma cells and mouse liver. Mol Ther 2003;7:254-61
40. Wightman L, Kircheis R, Rossler V, et al. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med 2001;3:362-72
41. Goula D, Remy JS, Erbacher P, et al. Size, diffusibility and transfection performance of linear PEI/ DNA complexes in the mouse central nervous system. Gene Ther 1998;5:712-17
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Chemical Properties

Polyethylenimines (PEI) are low to high molecular weight compounds with the general formula -[CH2-CH2-NH2]-, made by ring opening polymerization of aziridine. These polymers are available as linear, partly branched or repetitively branched polymers (dendrimers). The linear form contains only primary amines in the backbone whereas branched PEI also contains secondary and tertiary amines. Thus, these polymers have different properties and reactivities. Linear high MW PEI is usually solid at room temperature while branched PEIs are typically liquids at all molecular weights. All three forms are soluble in water, methanol, ethanol, and chloroform but insoluble in solvents of low polarity such as benzene, ethyl ether, and acetone.

Uses

Polyethyleneimine is a synthetic, water soluble, linear or branched polymer that has a high density of amino groups that can be protonated. At physiological pH, the polycation is very effective in binding to DNA and can mediate the transfection of eukaryotic cells. It is widely used in gene delivery system due to the ability to build a complex with DNA and support the release of endosomal through “proton sponge effect.” It is also facilitating the intracellular transport into nucleus. These properties succeed in polymer for MNPs coating and targeted therapy.

Application

Detergents, adhesives, water treatment, printing inks, dyes, cosmetics, and paper industry, adhesion promoter, lamination primer, fixative agent, flocculant, cationic dispersant, stability enhancer, surface activator, chelating agent, scavenger for aldehydes and oxides.

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• ETHYLENEDIAMINE MONOHYDRATE