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2826 Current Medicinal Chemistry 2008 15 2826-2839

0929-867308 $5500+00 copy 2008 Bentham Science Publishers Ltd

Polyethylenimine In Medicinal Chemistry

Paola Vicennati Antonella Giuliano Giancarlo Ortaggi and Andrea Masotti

Chemistry Department SAPIENZA University of Rome Ple Aldo Moro 5 00185 Rome - Italy

Abstract Polyethylenimine (PEI) an organic branched or linear polyamine polymer has been successfully used in the past for DNA complexation and transfection in vitro and in vivo into several cell lines and tissues PEI was also applied in different fields from gene therapy and several studies have emphasized the importance of this polymer in medicinal chem-istry In this brief critical review the uses and applications of this versatile polymeric molecule will be discussed

Keywords Polyethylenimine polymer DNA transfection gene therapy drug delivery medicinal chemistry biomedical ap-plications

1 INTRODUCTION

Polyethyleneimines (PEIs) are highly basic and posi-tively charged aliphatic polymers containing primary sec-ondary and tertiary amino groups in a 121 ratio Every third atom of the polymeric backbone is therefore an amino nitro-gen that may undergo protonation As the polymer contains repeating units of ethylamine PEIs are also highly water-soluble PEIs are available in both linear and branched forms with molecular weights ranging from 700 Da to 1000 kDa

PEIs have been extensively studied as a vehicle for non-viral gene delivery and therapy Since its introduction in 1995 [1] 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 nu-cleic acid and polymer) in both in vitro and in vivo models [2] Polycation-mediated gene delivery is based on electro-static interactions between the positively charged polymer and the negatively charged phosphate groups of DNA In aqueous solution PEI condenses DNA and the resulting PEIDNA complexes carrying a net positive surface charge can interact with the negatively charged cell membrane and readily internalized into cells [3] PEI retains a substantial buffer capacity at virtually any pH and it has been hypothe-sized that this simple molecular property is related to the efficiency of the complex multistage process of transfection As a matter of fact the lsquoproton spongersquo 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 en-dosome resulting in the escape of the vector from the degra-dative lysosomal compartment The proton sponge hypothe-sis has been a subject of debate speculation and research without reaching a general consensus about the real mecha-nism involved [4-7]

However it has been shown that both the efficacy and toxicity of PEI are strongly correlated with its molecular weight (MW) as well as its structure (branched or linear b-PEI or l-PEI respectively) Efficacy and adverse reactions seem thereby to be strongly associated A good compromise

Address correspondence to this author at the Chemistry Department SAPIENZA University of Rome Ple Aldo Moro 5 00185 Rome ndash Italy E-mail andreamasottiuniromalit

between efficiency and toxicity was found for branched PEI with a molecular weight of 25 kDa It is for this reason that PEI 25 kDa was the most widely used in several applica-tions

Fig (1) Chemical structure of branched polyethylenimine (PEI)

For a long time PEI has been also used in non pharma-ceutical 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 [8]PEI is widely used to flocculate cellular contaminants nu-cleic acids lipids and debris from cellular homogenates to facilitate purification of soluble proteins [9-11] Enzymatic reactions in bioprocesses constitute another field in which PEI was used as an immobilizing agent for biocatalysts [12] as a soluble carrier of enzymes [13] or in the formation of macrocyclic metal complexes mimicking metalloenzymes [14] PEI is also a common ingredient in a variety of formu-lations ranging from washing agents to packaging materials

Several papers reported the use of PEI in the field of me-dicinal chemistry and in this brief critical review examples appeared in the last decade were considered and discussed (Table 1)

PEIrsquos application fields may be divided according to its use in

11 Use of PEI as a drug

12 Use of PEI for delivery of small drugs and for the photo dynamic therapy (PDT)

13 Use of PEI for antimicrobial coating

Polyethylenimine in Medicinal Chemistry Current Medicinal Chemistry 2008 Vol 15 No 27 2827

14 Use of PEI for the preparation of nanosized delivery vectors

15 Use of PEI for non-invasive optical imaging devices

11 Use of PEI as a Drug

Only few papers reported the pharmacologic potential of PEI used as a drug It is worth citing a couple of papers by Tansy [15 16] appeared in the literature in the late 70rsquos concerning the effects of the ingestion of these polymers on gastrointestinal motor activity in view of the therapeutic effects and hygienic usefulness of the commercial polyethyl-eneimines The original work focused on polyethylenimine and gastric emptying in rodents and dogs Experiments were

conducted on fasted rats to determine the effect of ingested polyethyleneimines upon gastric emptying A dose related long lasting (4 hours) and reversible delay in gastric empty-ing was detected within 15 minutes from the administration of branched polyethyleneimine while the linear polymer has no observable effect The branched PEI polymer with all the primary amino groups selectively acetylated has a low activ-ity as well Thus the study of Tansy demonstrated that poly-amines have a profound impact on the motility of the gastro-intestinal tract suggesting that polyamine pharmacophores are excellent candidates for the manufacturing of antitransit and antidiarrheal drugs However PEI caused severe retch responses in dogs and due to the structural similarity with other natural polyamines Tansy continued to study the effect of spermidine spermine and other polyamine analogues

Table 1 Applications of Polyethylenimine and Literature References

Paragraph Specific applications Cited reference


Ingestion of polyethylenimines and study of their effect on gastrointestinal motor activity [15-16]

Use of polyethylenimines to block fibrin formation (anticoagulant activity) [17]

Effect of polyethylenimines on the permeability properties of the Gram-negative bacterial outer membrane (OM) [18-19]


Coating of alginate beads for controlled drug delivery of low molecular weight therapeutic agents [20]

Use of polyethylenimines to enhance nasal absorption of negatively charged drug [21]

Use of polyethylenimines and photo dynamic therapy (PDT) as a possible treatment for localized infections [22-23]

Use of polyethylenimines in cancer antiangiogenic photodynamic therapy [24-25]


Polyethylenimines as bactericidal coating materials [26-27]

Use of polyethylenimines as bactericidal molecule able to kill gram positive and gram negative bacteria [2528-32]

Use of polyethylenimines in dental clinic for replacement of hard tissues [33]

Polyethylenimines as amphiphilic antimicrobial polymers [34-35]

Polyethylenimines able to protect against infection of implanted materials by bacteria (complication following pros-thetic surgery)

[36]

Polyethylenimines as suitable coating for biomedical 316L stainless steel [37]

14 Use of PEI for the preparation of Nanosized delivery vectors

Polyethylenimines in nanosized dendritic core-shell architectures encapsulating drugs [38]

Polyethylenimines in dendritic core-shell architectures derivatized with poly(ethylene glycol) chains [39]

Polyethylenimines derivatized with different fatty acids in dendritic core-shell architectures [40-41]

nanosized cationic hydrogels for drug delivery [42]

Polyethylenimine and poly(DL-lactide-co-glycolide) in micelle-like polymer aggregates preparation [43]

Polyethylenimines for nanostructured delivery systems for proteins [44-4648]

Polyethylenimines in nanoparticle-mediated nuclear drug delivery [47]


Polyethylenimines for quantum dots coating [49]

Polyethylenimines for non-invasive optical imaging (Near Infrared NIR) assessment of caspasesrsquo activity in vitro [50]

Protein-phosphorylation-responsive cell-permeable and biocompatible polyethylenimines [51]

Conjugation of polyethylenimine with a NIR-dye to obtain multifunctional delivery vectors for DNA delivery in vivo [52]

2828 Current Medicinal Chemistry 2008 Vol 15 No 27 Vicennati et al

An important biological function of PEI was reported by Chu et al showing that PEI readily blocks fibrin formation thus exhibiting anticoagulant activity [17] This study dem-onstrated that even at a nanomolar concentration PEI sig-nificantly blocks thrombin-catalyzed fibrin formation in vitro accounting for its anticoagulant property This uncom-petitive inhibition was independent on the concentration of fibrinogen (FBG) thrombin or NaCl PEI showed no effect on thrombin amidolytic activity suggesting that the blockade of thrombin interaction with FBG could explain the inhibi-tion on fibrin formation PEI also drastically depressed rabbit brain thromboplastin procoagulation as assessed by a single-stage clotting assay using human plasma In a THP-1 mono-cytic hypercoagulation cell line a 4-hours exposure to bacte-rial endotoxin or Ca2+ ionophore A23187 resulted in a 5- or 10-fold enhancement in monocytic tissue factor (mTF) pro-coagulation respectively Monocytic TF hypercoagulation was offset by PEI included in the assay mixture that is able to arrest mTF hypercoagulation with an IC50 = 12 nM

The antibacterial properties of PEIs have been investi-gated in details and was applied in the development of coated materials (see also further) Helander [18] studied the effect of PEI on the permeability properties of the Gram-negative bacterial outer membrane (OM) using Escherichia coli Pseudomonas aeruginosa and Salmonella typhimurium as target organisms The OM is present in all Gram-negative bacteria and due to the presence of lipopolysaccharide (LPS) in the outer leaflet of the membrane it forms a permeability barrier against hydrophobic substances and macromolecules For this reason Gram-negative bacteria exhibit higher resis-tance to detergents and hydrophobic antibiotics than do Gram-positive bacteria Sensitization of Gram-negative bac-teria to hydrophobic antibiotics is a property shared by all polycationic permeabilizing agent which either release con-siderable amounts of LPS from the OM or bind to LPS with-out releasing it into the medium 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 gml PEI increased the bacterial uptake of 1-N-phenylnaphthylamine a hydrophobic fluorescent probe indicating an increased hydrophobic per-meation of the outer membrane PEI also increased the sus-ceptibility 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 ani-onic detergent SDS when bacteria are opportunely pre-treated with the polymer No sensitization to lysozyme was observed with PEI in agreement with similar findings on the inability of other polycationic permeabilizers All these re-sults show that PEI is able not only to disorganize the OM in a transient way but also in an irreversible manner most likely by intercalating in the LPS layer similarly to po-lymyxin B nonapeptide

Recently Gao [19] prepared and characterized quater-narized polyethyleneimine (QPEI) and investigated their antibacterial properties mainly by using Escherichia coli as model bacterium and the colony count method The experi-ment shown that QPEI has outstanding activity due to the antibacterial groups on the macromolecular chains The antibacterial ratio reached 100 for bacterium suspensions

of 109 CFUml with a polymer concentration of 15 mgl for a contact time of 4 min The cationic degree influences the antibacterial ability of QPEI greatly and the higher the cati-onic degree the stronger the antibacterial activity The ex-perimental data indicated that the pI of the E coli protein is probably 45 When pH gt 45 the antibacterial activity of QPEI increased with increasing pH and when pH gt 6 the antibacterial ratio reached a maximum and remained nearly constant The enzyme activity measurements revealed that the antibacterial action of QPEI was essentially due to a sterilization process Similarly to the small quaternary am-monium salt QPEI causes cell death by disrupting cell membranes and releasing the intracellular contents

As a permeabilizing agent the mechanism of polyeth-ylenimine antibacterial activity most likely consists in the osmotic swelling after its internalization and membrane disruption PEI is an efficient proton-sponge and after inter-nalization into endosome compartments an influx of counte-rions takes place to balance that of H+ Protons and counteri-ons definitely determine a swelling of the polymer and the collapse of the endosome structure After this event drugs and polymer are released into the cytosol where they can exert their specific effect

12 Use of PEI for Delivery of Small Drugs and for the Photo Dynamic Therapy (PDT)

Setty [20] studied the effect of coating alginate beads with polycations for controlled drug delivery of low molecu-lar weight therapeutic agents As polycation PEI was se-lected for its several advantageous properties (hydrophylic-ity biocompatibility and thermal stability) and furosemide was chosen as a model water-insoluble drug The fu-rosemide-loaded calcium alginate (ALG) calcium alginate-polyethyleneimine (ALG-PEI) and alginate-coated ALG-PEI (ALG-PEI-ALG) beads by ionotropicpolyelectrolyte com-plexation method to achieve controlled release of the drug were prepared Drug-loading efficiency (DLE) of ALG beads varied from 9716 to 10049 and variation in for-mulation factors (incubation CaCl2 concentration initial drug load) was found not to influence the DLE of ALG beads Anyway release of furosemide from ALG beads in simulated intestinal fluid (SIF phosphate buffer solution of pH 68) was rapid and complete in 25 hours irrespective of the variation in formulation factors The drug release was accompanied with a rapid swelling and erosiondisintegra-tion of ALG beads and this phenomenon has been considered as a major disadvantage of ALG beads in sustaining drug release in SIF PEI treatment of ALG beads however pro-longed the drug release considerably Increase in both PEI concentration and exposure time decreased DLE Release of furosemide from ALG-PEI beads was prolonged considera-bly compared with that from ALG beads Ionic interaction between alginate and PEI led to the formation of polyelectro-lyte complex membrane the thickness of which was depend-ent on the conditions of PEI treatment (PEI concentration and exposure time) The membrane acted as a physical bar-rier 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 Furthermore it


was shown that the encapsulated drug was not degraded by PEI treatment

The hypothesis that PEIs could enhance nasal absorption of the negatively charged drug was tested by Ashan and coworkers [21] They reasoned that since DNA and low molecular weight heparins (LMWHs) have similar charge distribution properties (LMWHs are negatively charged oli-gosaccharides used in the treatment of deep vein thrombosis and pulmonary embolism) PEI should also be able to form a complex with LMWH via electrostatic interactions If so this should neutralize the drugs surface charge and facilitate its absorption via mucosal routes Therefore they designed a study to test this hypothesis They found that PEI can en-hance nasal absorption of enoxaparin a LMWH and that such enhancement occurs through neutralization of the nega-tively charged glycosaminoglycan unit of the drug In this regard enoxaparin was formulated with PEIs of different molecular weights and the efficacy of PEI in enhancing nasal absorption of LMWH was tested in a rodent model It was shown that PEIs neutralize the negative surface charge of LMWHs This neutralization may weaken or diminish the coulombic repulsion effect between the negatively charged cell membrane and the drug and consequently increase drug absorption across the epithelium Otherwise PEI could competitively bind with the negatively charged molecules of the cell surface and subsequently release the drug after endo-cytosis as it is widely believed to happen in PEI-enhanced DNA transfection The efficacy of PEIs in enhancing the bioavailability of nasally administered LMWH was studied by administering LMWH formulated with PEI into the nose of anesthetized rats and monitoring the drug absorption They found that the efficiency may be ranked as PEI-1000 kDa PEI-750 kDagtPEI-25 kDa When PEI-1000 kDa was

used at a concentration of 025 there was a 4-fold increase in both the absolute and relative bioavailabilities of LMWH compared to the control formulation Thus the authors con-cluded that polyethylenimines can be used as potential carri-ers for nasally administered LMWHs after nasal administra-tion

Hamblinrsquos research group has been involved in the use of photo dynamic therapy (PDT) as a possible treatment for localized infections [22] They shown that covalent conju-gates (Fig 2) between PEI and chlorin(e6) (ce6) can be used as a potent broad-spectrum antimicrobial photo sensitizers (PS) resistant to protease degradation and therefore constitut-ing an alternative to the previously described poly-L-lysine chlorin(e6) (pL-ce6) conjugates [23] They prepared a novel set of second-generation polycationic conjugates between chlorin(e6) and three molecular forms of polyethyleneimine (PEI) a small linear a small cross-linked and a large cross-linked molecule The conjugates were synthesized charac-terized and tested for their ability to kill a panel of patho-genic microorganisms the gram-positive Staphylococcus aureus and Streptococcus pyogenes the gram negative Es-cherichia coli and Pseudomonas aeruginosa and the yeast Candida albicans after exposure to low levels of red light The large cross-linked molecule efficiently killed all organ-isms while the linear conjugate killed gram-positive bacteria and C albicans The small cross-linked conjugate was the least efficient antimicrobial PS and its remarkably low activ-ity could not be explained by reduced photochemical quan-tum yield or reduced cellular uptake In contrast to polylysine conjugates the PEI conjugates were resistant to degradation by proteases such as trypsin that hydrolyze ly-sine-lysine peptide bonds In fact this macromolecular vehi-cle does not contain peptide bonds and is therefore resistant

N

NH

N

NH

COOH

O

HN

COOH

N

Branched PEI

N

N

NH

N

NH2

NH2

NH2

NH2

HN

N

H2N

H2N

N

NH

N

NH

COOH

O

HN

COOH

NH

NH

Linear PEI

PEI-ce6

A B

Fig (2) Chemical structures of chlorin(e6) conjugated with A) linear or B) branched polyethylenimine (PEI)


to protease degradation The advantage of protease stability combined with the ready availability of PEI suggested that these molecules may be superior to polylysine-PS conjugates for photodynamic therapy of localized infections

PEI was also studied in cancer antiangiogenic photody-namic therapy mediated by polycation liposomes by Okursquos research group [24 25] These studies indicated that antian-giogenic photodynamic therapy (PDT) consisting in a laser irradiation at 15 min post-injection of photosensitizer in vivois effective for cancer treatment and a photosensitizer ben-zoporphyrin derivative monoacid ring A (BPD-MA) encap-sulated in polycation liposomes (PCLs) liposomes modified with cetylated polyethylenimine (cetyl-PEI) is more effec-tive than BPD-MA encapsulated in non-modified liposomes

13 Use of PEI for Antimicrobial Coating

In several papers PEIs are used as such or in the pres-ence of copolymers as bactericidal coating materials

Bourgeois [26] used PEI to build a specific delivery sys-tem for -lactamases The aim of that study was to provide a proof of concept of colon delivery of -lactamases by pectin beads aiming to degrade residual -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 -lactamases from degradation by proteases contained in the upper gastrointestinal tract The specific delivery system for -lactamases was composed of a core of calcium pectinate bead cross-linked at its surface with PEI [27] Pectin beads were prepared according to ionotropic gelation method using CaCl2 as a gelling agent Particles were then washed and soaked in PEI Beads thus obtained were solid with an ovoid shape and an internal

matrix-like structure Instantaneous gelation of pectin al-lowed an easy encapsulation of -lactamases in Ca-pectinate beads with an efficiency of 865 PEI improved the stabil-ity of Ca-pectinate beads protecting them from water pene-tration by cross-linking the free carboxylic functions of the Ca-pectinate network The cross-linking step do not influ-ence shape size and efficiency of encapsulation of -lactamases in beads Thus PEI made Ca-pectinate beads resistant to the denaturing effect of upper intestine condi-tions allowing to delay -lactamases release In vitro studies showed that -lactamases were released from pectin beads in a suitable colonic medium due to the action of pectinolytic enzymes When ampicillin was added to this medium the release of -lactamases induced as expected the antibiotic inactivation Finally after oral administration of loaded-beads to CD1 mice -lactamases were retrieved in high concentrations in faeces

Also Klibanov et al published several papers on this topic [28-32] They faced the problem to develop non-leaching permanent sterile surface materials to be used in hospitals and community settings by covalently functional-izing their surface with an antimicrobial compound They found that covalent attachment of long-chained moderately hydrophobic polycations (such as quaternarized N-alkyl-PEI) to surfaces of solid objects (Fig 3) renders the latter perma-nently bactericidal killing a broad range of pathogens gram positive and gram negative bacteria as well as fungi [25]

Concerning the mechanism flexible polymers apparently cross the microbial cell envelope delivering the active moi-ety into the membrane and killing the pathogen Only long-chained moderately hydrophobic immobilized polycations exhibited microbicidal activity The immobilized polycations were found to be unique and apparently with no analogues in nature They are not subject to existing mechanisms of resis-

Fig (3) The treatment by covalent attachment of long-chained hydrophobic quaternarized N-alkyl-PEI to surfaces renders the latter perma-nently bactericidal


tance such as multi-drug resistance pumps or multi-drug tolerant cells and no resistance develops upon repeated exposure to the polymer

In a follow-up work Klibanov replaced the surface-specific multistep immobilization techniques with a single-step general procedure similar to common painting [26] Glass or polyethylene slides were briefly dipped into organic solutions of certain optimally hydrophobic N-alkyl-PEI (where PEI stands for branched 750-kDa polyethylenimine) polycations followed by solvent evaporation The resultant polycation-coated slides were able to kill on contact all of the encountered bacterial cells This biocide effect was found not to be caused by N-alkyl-PEI molecules leached from the surface Further examination of the mechanism of this bacte-ricidal action suggested that the surface-deposited N-alkyl-PEI kills bacteria by rupturing their cellular membranes This conclusion was further supported by studies in which the molecular weight of PEI and the hydrophobicity of the alkyl moiety were varied

The work moved further reasoning that influenza virus belongs to a class of enveloped viruses and is than protected from the outside by a lipid membrane it was thought that the aforementioned hydrophobic polycations might damage it as well thereby inactivating the virus [27] In fact painting a glass slide with branched or linear NN-dodecyl methylpoly-ethylenimines (PEIs) and certain other hydrophobic PEI derivatives enables it to kill even influenza virus with essen-tially a 100 efficiency within minutes For most of the coating poly-ions this virucidal action is shown to be on contact ie solely by the polymeric chains anchored to the slide surface for others a contribution of the poly-ion leach-ing from the painted surface was supposed to increase the efficiency

Domb and coworkers applied PEIs to confer antibacterial properties to the composite resin materials widely used in the dental clinic for replacement of hard tissues [33] They wanted to test the hypothesis that the insoluble crosslinked quaternary ammonium polyethylenimine (PEI) nanoparticles in composite resin restorative materials have a stable and long-lasting antibacterial effect against oral bacteria Strep-tococcus mutans without affecting the flexural strength of the commercial materials Nanoparticles with N-octylNN-dimethyl ammonium groups were reproducibly prepared from PEI by crosslinking with 15-dibromopentane followed by alkylation with bromoctane and quaternarization with

methyl iodide Antimicrobial assays using S mutantsshowed that these PEI nanoparticles when incorporated in dental composite resins at low concentration exhibited a strong antibacterial effect against the tested bacteria regard-less of the commercial composite resin to which they were added They could exclude that the antibacterial activity was due merely to bioactive components released to the medium Furthermore results indicated that the addition of a small amount (1) of the PEI nanoparticles did not affect the me-chanical properties of the restoration composites Composite resin materials incorporated with PEI nanoparticles main-tained antibacterial properties over 1 month without leaching out and displayed no alteration of the original mechanical properties Results shown that for composite resin restora-tions incorporation of antibacterial nanoparticles may pre-vent biofilm formation and secondary caries

Moeller has reported a new approach for the preparation of amphiphilic antimicrobial polymers (Fig 4) based on a one-step multi-functionalization of PEI with derivatized cyclic carbonates [34 35] The aim of this work was to pre-pare and characterize water-soluble polymers with a strong affinity to lipid membranes The rationale was to substitute a water-soluble hyperbranched macromolecule (ie PEI) by alkyl chains and ammonium groups in such a way that the water solubility was preserved but with the polymers able to adsorb to lipid membranes at the same time These amphipa-thic molecules could be of interest for the preparation of new antimicrobial polymers and bactericidal or bacteria-repellent surfaces in order to provide solutions for one of the biggest problems of modern medicine Depending on their hydro-philichydrophobic balance the obtained polymers could be used as water-soluble disinfectants and for antimicrobial coating materials Primary amine groups of branched PEI were functionalized with quaternary ammonium groups alkyl chains of different length allylic and benzylic groups in a one-step reaction using a carbonate coupler The bacte-ricidal properties of some of the amphiphilic polymers against Gram-negative and Gram-positive bacteria were investigated in solution regarding the effect of (i) the length of the alkyl chains (ii) the hydrophilichydrophobic balance and (iii) the kind of spacer between the cationic moiety and the polymer Minimal inhibitory concentrations (a log 4 reduction of bacterial growth) against Escherichia coli and Bacillus subtilis were determined in the range of 03-04 mgmL and 003-004 mgmL for water-soluble polymers Glass slides coated with functionalized PEIs showed a reduc-

Fig (4) The preparation of amphiphilic antimicrobial polymers based on a one-step multi-functionalization of PEI with derivatized cycliccarbonates


tion of colony forming units of at least 95 up to 999 against E coli and B subtilis However it was observed that polymers leached out of the coating This suggested the authors to consider an improvement of the cross-linking method or the development of a covalent bond between the polymer and the surface

PEI has been used in the construction of polyelectrolyte multilayer films that are able to coat any type of surface (eg metals and plastics) without shape limitation (eg planar spherical or curved) This layer-by-layer technique was recently developed with the aim of using it for different biological applications in the fields of biosensors cell signal-ing control and anti-adhesive surfaces Egles [36] carried out studies concerning biofunctionalized interfaces able to protect against infection of implanted materials by bacteria one of the most serious complications following prosthetic surgery They developed a new strategy based on the inser-tion of an antimicrobial peptide (defensin from Anopheles gambiae mosquitoes) into films built by the alternate deposi-tion of polyanions and polycations with the aim to isolate the peptides while retaining their bioactivities Polyethyle-neimine (PEI) poly(sodium 4-styrenesulfonate) (PSS) poly(allylamine hydrochloride) (PAH) poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) were used to build the films Multilayer films were prepared either on 12-mm glass coverslips or in 96-well plastic plates PEI-(PSS-PAH) 2-(PGA-PLL)2-PGA films had a thickness of about 40 nm Defensin peptides were embedded in the multilayer structure by adsorption during film construction Noticeably the bio-functionalization could be achieved only when positively charged poly(L-lysine) was the outermost layer of the film likely because the close interaction of the bacteria with the positively charged ends of the films allows defensin to inter-act with the bacterial membrane structure Antimicrobial assays were performed with two strains Micrococcus luteus (a gram-positive bacterium) and Escherichia coli D22 (a gram-negative bacterium) The inhibition of E coli D22 growth at the surface of defensin-functionalized films was found to be 98 when 10 antimicrobial peptide layers were inserted in the film architecture Thus the combination of good biocompatibility found for polyelectrolyte multilayer films and their high degree of stability together with the possibility of varying the number of adsorbed active proteins or peptides and their amounts could lead to biomedical ap-plications ranging from the protection of several tools used for medical applications such as catheter needles surgical

tools and tubes to all types of materials that come into con-tact with wounds for a restricted period of time

The research of Ji and coworkers advanced in the same field [37] They explored the possibility to form albumin multilayers as a coating for biomedical 316L stainless steel with the goal of developing a fast easy processing and shape-independent method for non-thrombogenic coating The 316L stainless steel is widely used in coronary stents but the exposure to flowing blood of the metallic stents can result in thrombus formation A confluent layer of albumin is believed to be both anti-thrombogenic and anti-infective The ldquoelectrostatic self assemblyrdquo (ESA) method which is based on the alternating physisorption of oppositely charged polyelectrolytes represents a new alternative solution for biomaterial The buildup is easy and the procedure can be adapted to almost any type of surface Moreover the method is still valid whatever is the shape of the solid Multilayer films consisting of polyethylenimine (PEI) and albumin were successfully prepared on biomedical 316L stainless steel surface they were found stable in TrisndashHCl (pH 735) buffer solution for 21 days whereas less than 10 albumin was eluted by citrate phosphate buffered saline in 45 days Static platelet adhesion experiments indicated that the PEIalbumin deposited on stainless steel could resist platelet adhesion effectively Such an easy processing and shape-independent method may have good potential for surface modification of cardiovascular devices

14 Use of PEI for the Preparation of Nanosized Delivery Vectors

In several papers 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 capil-laries up to cytoplasm allowing also an efficient drug accu-mulation 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

So Haag and coworkers used functionalized hyper-branched PEI to build nanosized dendritic core-shell archi-tectures (Fig 5) able to encapsulate guest molecules poten-tially useful as drug delivery systems [38] To this scope the release of the encapsulated species occured as a result of the pH drop in tumor and infected tissues (pH 5-6) It is well

Fig (5) Functionalization of hyperbranched PEI to build nanosized dendritic core-shell architectures able to encapsulate guest moleculesand to act as drug delivery systems


known that imine bonds are sensitive to pH changes in a pH range of 5ndash7 thus they attached two different carbonyl com-pounds to the terminal amino groups of hyperbranched PEI leading to the formation of imine compounds in high yields (70-90) and in multigram quantities The transport capaci-ties of these molecular nanocarriers were first determined using the congo red dye as an easily detectable polar model compound used as pH indicator and as a neuroprotective drug Results showed that a minimum core size (ca 3000 g mol-1) and a highly branched architecture are required for successful encapsulation of the guest molecules similarly to what found for other dendritic core-shell architectures For efficient transport the degree of alkylation should be about 45-50 and the alkyl chains should have a minimum length (gtC10) The transport properties of these nanocarriers were tested with different guests Many other organic dyes such as bromophenol blue methyl orange methyl red and fluo-rescein all of which contain polar anionic sulfonate or car-boxylate groups and sodium counterions were readily en-capsulated and transported In contrast cationic dyes such as the triphenylmethane-based malachite green with an oxalate counterion were not transported at all The complexation of an antitumor drug (mercaptopurine) several oligonucleo-tides as well as bacteriostatic silver compounds have been studied for the potential use of these nanocarriers in drug and gene delivery Successful encapsulation and transport were observed in all cases by the PEI-based nanocarriers

The higher selectivity for large anionic guest molecules can be explained by the strong interaction of such species with the polar groups in the core of these dendritic macro-molecules Using several buffer solutions it was found that the imine-based nanocarriers are very sensitive to an external drop in the pH value the hydrolysis of the shell and the release of the encapsulated guest congo red occurs spontane-ously at pHlt7 whereas it is stable over several weeks at neutral pH opening the possibility of selectively release the encapsulated guest molecules in a physiologically relevant pH range In a further work [39] they described the synthesis of readily accessible dendritic core-shell architectures with biocompatible poly(ethylene glycol) chains Although the toxicity of PEI is relatively high it has recently been demon-strated that polyethylenglycol (PEG) modification of these structures can reduce their toxicity dramatically even for invivo applications PEIs can be modified with different shells such as polyamidoamine (PAMAM) or polyethylene glycol to build water-soluble core-shell architectures with PEI as the core Surprisingly these shell-coated nanocarriers exhib-ited much higher transport capacities than the unfunctional-ized PEI which suggests that the core-shell architectures can further stabilize the host-guest system The stability of the nanocarriers was evaluated with three different buffer solu-tions (pH 5 7 and 8) showing that pH labile shell can be cleaved by lowering the pH to a value of 5ndash6 The pH-sensitivity is in the same range as observed in malignant tissues (tumor infection) or endosomes and hence could be used as a trigger to release the encapsulated drugs from these nanocarriers

In another approach dendritic core-shell architectures were synthesized by simple melt reactions of polyethylen-imine (PEI) with different fatty acids [40] The degree of functionalization of the PEI amides could be simply con-

trolled by the stoichiometrical amounts of acid and PEI ap-plied in bulk These values were used to calculate the respec-tive molecular weight The structure-activity relationship between the guest and the host molecules was examined with various dyes and drug molecules It was found that the trans-port capacity rises with decreasing concentration of the polymer in solution resulting in a maximum transport of around 3g dyeg polymer or up to 150 guest molecules per dendritic core-shell architecture Larger aggregates were formed at higher concentration resulting in a decreased num-ber of encapsulated guest molecules due to more polymer-polymer interaction The maximum encapsulation of the guest molecule is achieved at a pH between 5 and 8 The absence of water lead to a decreased number of encapsulated guest molecules due to the formation of small aggregates

Thuumlnemann and coworkers [41] developed nanoparticle systems based on PEI as carriers for hydrophobic drugs In these systems the polymer caused the complexation of a fatty acid as well as the stabilization of the particles Thus PEI was used for the complexation of dodecanoic acid (C12) resulting in a poly(ethylene imine) dodecanoate complex (PEIndashC12) with a stoichiometry of amino functions to carboxylic acid functions 21 a lamellar nanostructure and a repeating unit of 29 nm at room temperature PEIndashC12 was doped with coenzyme Q10 and the hormone triiodothyronine as typical hydrophobic and pharmacological active com-pounds respectively The PEI-C12 was shown to act as a guest matrix that dissolves the above mentioned molecules up to 20 (ww) and 15 (ww) respectively forming homogeneous structures stable at room temperatures for at least 3 months

Kabanov has recently reviewed the progress in the field of nanosized cationic hydrogels for drug delivery summariz-ing preparation methods properties and interactions with cells [42] These hydrogels belong to the family of nanoscale materials based on dispersed networks of cross-linked ionic and nonionic hydrophilic polymers This work was focused on the nanosized cationic network of cross-linked poly(ethylene oxide) (PEO) and polyethyleneimine (PEI) PEO-cl-PEI nanogels These appeared to be promising and versatile systems for drug delivery The synthesis of micro- and nano-gels was carried out following different approaches (emulsion polymerization or copolymerization at elevated temperature using rapidly stirred solutions addition or step-growth polymerization of the polyfunctional monomers in solution using a wide range of cross-linking systems) By varying the reaction conditions including the type and dis-persity of the media addition of surfactants temperature and ratio of reagents the size of the particles could be con-trolled In particular PEO-cl-PEI nanogels were synthesized by cross-linking of branched PEI (25 kDa) with bis-activated PEO (8 kDa) molecules Fine dispersed systems were ob-tained when the cross-linking reaction was performed by a modified solvent emulsificationevaporation method leading to the formation of particles of 30 to 300 nm size Formation of polyion-complexes leads to the collapse of the dispersed gel particles (Fig 6) However the complexes form stable aqueous dispersions due to the stabilizing effect of the PEO chain These systems allow for immobilization of negatively charged biologically active compounds such as retinoic acid indomethacin and oligonucleotides (bound to polycation


chains) or hydrophobic molecules (incorporated into nonpo-lar regions of polyionndash surfactant complexes) leading to the formation of nanocomposite materials in which the hydro-phobic regions from polyion-complexes are joined by the hydrophilic PEO chains The average hydrodynamic diame-ters observed for the loaded PEO-cl-PEI nanogel (size within 100 nm) fitted well within the preferred size range for drug delivery systems In fact particles with diameters of less than 5ndash10 nm are rapidly removed through extravasation and renal clearance while larger particles (ranging from ca 10 to 70 nm) are still small enough to penetrate even the very small capillaries within the body tissues and therefore may offer the most effective distribution in certain tissues To allow for the targeted delivery of nanogels in the body the surface of the nanogel particles have been modified with various biospecific ligands polypeptide ligands to enhance receptor-mediated delivery The advantages of these systems include simplicity of formulation with the drugs high load-ing capacity and stability of the resulting formulation in dispersion These systems allow for the immobilization of biologically active compounds of diverse structure including charged drugs low molecular mass hydrophobic molecules and biopolymers Furthermore nanogels can be chemically modified to incorporate various ligands for targeted drug delivery The in vitro studies suggest that nanogels can be used for efficient delivery of biopharmaceuticals in cells as well as for increasing drug delivery across cellular barriers

Nam [43] proposed new micelle-like polymer aggregates prepared from oligomeric polyethylenimine and poly(DL-lactide-co-glycolide) (PEIndashPLGA) di-block copolymers and investigated their micellar characteristics in aqueous media PEIndashPLGA were synthesized by directly coupling PLGA with a carboxy-terminal group of PEI The block copolymers were prepared by varying the length of the hydrophobic PLGA block (Mn = 6 10 and 21 K) while that of the hy-drophilic PEI block (Mn =423) was fixed PEIndash PLGA block copolymers were found to be self-assembled in water by using a PLGA segment as a hydrophobic aggregate block and a PEI segment as a hydrophilic corona-forming block The block copolymers formed micelle-like aggregates with critical association concentration (CAC) in the range of 154ndash25710-3 gl in water It was found that the size and CAC of the aggregates depended on the hydrophobic block length as the hydrophobic PLGA block length became higher lower CAC values were obtained The dependency of

the aggregate size and morphology on interactive small molecules (eg ions) could provide a key parameter to con-trol aggregate structures by simply changing the medium composition during the manufacturing process The cellular uptake behavior of PEIndashPLGA aggregates was compared with that of plain PLGA showing that PEIndashPLGA aggregates were readily adsorbed onto the cell surfaces and translocated into the cytoplasm implying their versatile applicability as a drug carrier

A couple of papers by Middaugh [44] and Yamada [45] reported the use of PEIs for protein transduction Middaugh et al developed a nanoparticle delivery systems (Fig 7)which have the potential to deliver proteins by improving their stability increasing the duration of their therapeutic effect as well as permitting administration through non-parenteral routes [40] In fact a major problem in proteins administration is that they are often marginally stable and consequently easily damaged during their formulation as drugs The authors described nanoparticles made by polyeth-ylenimine (PEI) and dextran sulfate (DS) under aqueous conditions and stabilized by crosslinking with Zn2+ ions The oppositely charged polymers self-assemble through phase separation and form nanoparticles at room temperature Thus this technique is in principle applicable to a broad range of labile drugs and bioactive macromolecules includ-ing proteins and has been applied to low molecular weight small molecule drugs [46] In this case the author considered the possibility of incorporating a protein into this nanoparti-cle system while maintaining the proteinrsquos conformation and stability using insulin as a model protein drug since its structure stability and physicochemical characteristics have been extensively studied and it has a wide therapeutic utility It was found that the pH of PEI solutions the weight ratio of the two polymers and zinc sulfate concentration play sig-nificant roles in controlling particle size Spherical particles of 250 nm mean diameter were produced under optimal conditions with a zeta potential of approximately +30mV The association of insulin with the particles appears to be an efficient process Most of the formulations studied demon-strated an entrapment efficiency of 80ndash90 Furthermore results suggest that there was no significant insulin degrada-tion upon incorporating insulin into PEIndashDS particles and no significant conformational changes compared to free insulin under optimized formulation conditions Rapid release char-acteristics were observed in in vitro dissolution studies in

Fig (6) The formation of polyion-complexes leads to the collapse of the dispersed gel particles These systems allow for immobilization of negatively charged biologically active compounds (ie retinoic acid indomethacin oligonucleotides or hydrophobic molecules)


most formulations insulin was completely released from nanoparticles within 5 min independently of the PEI solu-tionrsquos pH and the polymer ratio Biological activity in strep-tozotocin-induced diabetic rats however exhibited a pro-longed hypoglycemic effect

Shen and coworkers [47] were interested in nanoparticle-mediated nuclear drug delivery and PEI nanoparticles are well suited to this scope considering their wide use in gene delivery However PEI is a highly positive charged mole-cule at physiological pH and may elicit some adverse reac-tion in vivo Thus they reported PEI nanoparticles with a negative-to-positive charge-reversal triggered by the solid tumor acidity (pHlt7) or lysosomal (pH 4ndash5) for nuclear drug delivery To this scope polycaprolactone (Mn=3800)-block-PEI(Mn=1800) (PCL-PEI) was synthesized and its PEI blocks reacted with 12-cyclohexanedicarboxylic anhydride to convert the primary and secondary amines into correspon-dent amides (PCL-PEIamide) The PEI block with 20 of its primary and secondary amines converted into their am-ides was found optimal in terms of the charge-reversal kinet-ics of the resulting nanoparticles Folic acid (FA) moieties were also conjugated to the PEI block to form PCL-b-PEIamide-FA for folate-receptor targeting (Fig 7) The PCL-PEIamide-FA formed nanoparticles of about 210 nm in

diameter in water The nanoparticles were about 120 nm in diameter if loaded with 146 wt doxorubicin (DOX) The hydrolysis kinetics of the amides in the PCL-PEIamide was tested at several pH values (the amides were hydrolyzed to more than 75 and 50 at pH values of 50 and 60 respec-tively after 24 hours) and the charge reversal of the PCL-PEIamide micelles was confirmed by measuring their potentials at different acidities corroborating the hypothesis that amides with -carboxylic acids can hydrolyze in acidic conditions to regenerate the amines giving rise to a nega-tive-to-positive charge reversal In vitro experiment showed that targeted charge-reversal nanoparticles TCRNsDOX are more effective in killing SKOV-3 cancer cells than the free doxorubicin

Finally Yamada and coworkers [48] reported that cation-ized-proteins covalently modified with PEI (direct PEI-cationization) efficiently enter into cells They investigated if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a spe-cific affinity (indirect PEI-cationization) (Fig 8) PEI-cationized avidin (PEI-avidin) streptavidin (PEI-streptavidin) and protein G (PEI-protein G) were prepared by a carbodiimide reaction and the delivery of biotinylated proteins and antibodies into living cells was investigated

Fig (7) The structure of the targeted charge-reversal nanoparticle (TCRN) and its pH-triggered charge reversal

Fig (8) Protein cationized directly with PEI by carbodiimide reaction (A) Biotinylated protein complexed with PEI-avidin or PEI-streptavidin (B) Antibody bound to PEI-protein G (C)


Results showed that PEI-avidin (andor PEI-streptavidin) is able to convey very efficiently a biotinylated GFP (green fluorescent protein) into various mammalian cells A GFP variant containing a nuclear localization signal was found to enter in the nucleus These results indicate that indirect PEI-cationization using non-covalent interaction could be as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their func-tions in the cytosol

15 Use of PEI for Non-Invasive Optical Imaging Devices

Nie [49] used PEI to coat the surface of quantum dots (QD) The main difficulty of using QDs for cell-labeling resides in their delivery into the cytoplasm However PEI is not only able to move across cell membranes through rapid endocytosis but is also able to disrupt intracellular organelles through a ldquoproton spongerdquo effect (see Introduction) Hyper-branched copolymer ligands such as polyethylene glycol (PEG) grafted polyethylenimine (PEI-g-PEG) were found to encapsulate and solubilize luminescent quantum dots through direct ligand-exchange reactions (Fig 9) The grafted PEG segment was found essential for reducing the cytotoxicity of PEI as well as for improving the overall nanoparticle stability and biocompatibility In comparison with previous QDs encapsulated with amphiphilic polymers the cell-penetrating QDs were smaller in size and considera-bly more stable in acidic environments Cellular uptake and imaging studies revealed that QDs coated with PEI-g-PEG2 are rapidly endocytosed and are initially stored in vesicles then a slow endosomal escape and the release into the cyto-plasm was observed

PEI is also an important polymer for non-invasive optical imaging devices (Near Infrared NIR) enabling the assess-ment of several cellular functions like caspasesrsquo activity invitro [50] The cell-permeable branched polyethylenimine (25 kDa) was modified with deoxycholic acid (DOCA) hydroxysuccinimide ester resulting in PEI-DOCA nanopar-ticles (Fig 10) After attaching the effector caspase-specific near-infrared (NIR) fluorescence probe (Cy55-DEVD) to amphiphilic bile acid-modified polymer backbone this po-lymeric 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 caspasesrsquo reaction occurs in the cytoplasm Thus the track-

ing of the fluorescein isothiocyanate (FITC)-labeled Cy55-DEVD26-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 meas-ure apoptosis in cell-based high-throughput screens for in-hibitors or inducers of apoptosis

Fig (10) Polyethylenimine (25 kDa) was modified with deoxy-cholic acid (DOCA) hydroxysuccinimide ester resulting in PEI-DOCA nanoparticles The effector caspase-specific near-infrared (NIR) fluorescence probe Cy55-DEVD was conjugated to am-phiphilic bile acid-modified polymer backbone

The use of PEI was also described in a recent paper where protein-phosphorylation-responsive cell-permeable and biocompatible polymeric nanoparticles for visualizing protein phosphorylation by protein kinase A (PKA) were reported (Fig 11) [51] Protein kinase A is one of the best studied and most important kinases in single cells The po-lymeric nanoparticles possess a PKA specific peptide motif (Leu-Arg-Arg-Ala-Ser-Leu-Gly LRRASLG termed kemp-tide) and are easily prepared by the self-assembly of a poly-ion-induced complex (PIC) composed of both positively and

Fig (9) Schematic diagram showing the encapsulation and the solubilization of CdSeCdSZnS quantum dots and the direct exchange reac-tion between the octadecylamine capping ligands and the multivalent copolymer ligands (PEI-g-PEG)


negatively charged polyelectrolytes as previously reported To produce a phosphorylation-responsive polyelectrolyte a positively charged polymer conjugate by chemical coupling of kemptide and Cy55 to poly(ethyleneimine) (25 kDa) was synthesized Upon protein phosphorylation PIC nanoparti-cles dissolve because negatively charged phosphate groups are incorporated into the serine residue of kemptide result-ing in polyelectrolyte solubilization This new cellular imag-ing system allows to explore protein kinase activities in various single living cells that express protein kinases The technique may also be applied to high-throughput cell-based drug-screening systems targeting protein kinases

In our recent work [52] the conjugation of PEI with a near infrared (NIR) dye is aimed to obtain a multifunctional delivery vector whose localization can be monitored in vivowith non invasive techniques and that may serve to identify potential transfection sites and assess its efficiency to deliver DNA To obtain the NIR-PEI conjugate the indocyanine dye IR-820 was employed The heterocyclic nitrogen atoms of IR-820 bear two alkyl-sulfonate groups that improve photo-stability and provide a sphere of solvation in water prevent-ing this dye from aggregation The NIR dye-polymer conju-gate (IR820-PEI) is highly soluble in water absorbs at 665 nm and emits at 780 nm displaying a large Stokesrsquo shift (115 nm) These characteristics make this system more suitable for use as fluorescence probes than do common cyanine dyes This multifunctional polymeric molecule enabled to monitor the DNA delivery in vivo with optical imaging tech-niques

Even if other devices are actually available in several cel-lular and molecular research fields the versatility of poly-ethylenimine and its derivatives not only resides in the abil-ity to complex DNA [61] siRNA miRNA PNA [53] and

other molecules and deliver them in vitro and in vivo but also in the possibility to obtain multifunctional delivery systems with improved properties (ie superparamagnetic) following straightforward functionalization reactions An example of PEI-based vectors bound to iron oxide nanoparti-cles having superparamagnetic properties was recently re-ported [54 55] Finally several efforts have been made to render this polymeric molecule ldquotissue-specificrdquo by coupling it with suitable monoclonal antibodies obtaining targeted DNA delivery systems Therefore polyethylenimine and its numerous derivatives will pave the way to great promises in a near future both in basic sciences and biomedical applica-tions

2 CONCLUSIONS

This critical review was aimed to discuss the versatility of polyethylenimine as a powerful molecule in several me-dicinal chemistry applications ranging from drug delivery (other than gene therapy) use of PEI per se or as a versatile moiety for imaging devices or other multifunctional systems

The reviewed works indicate that PEI is a versatile mole-cule PEI may be successfully employed in a large variety of biomedical applications However some intrinsic character-istics limit the use of this polymer as is In particular in vitrostudies have demonstrated that PEI (branched 25 kDa) may be cytotoxic most likely due to lack of biodegradability [56 57] This could be attributed to non-degradable methylene backbone Moreover recently PEI and poly(L-lysine) were reported to behave as apoptotic agents [58 59]

Despite these limitations suitable chemical derivatization and functionalization may diminish the cytotoxic effect of these polyamine polymers [60] Other PEI hydrophobic

Fig (11) Protein-phosphorylation-responsive cell-permeable and biocompatible polymeric nanoparticles for visualizing protein phosphory-lation by protein kinase A (PKA)


functionalizations have been demonstrated to diminish the transfection efficiency of slightly derivatized polymers but their ability to deliver other molecules like siRNA miRNAs PNAs and other drugs remained unaltered [61]

All of these results clearly indicate that more studies are needed to completely understand the mechanisms of actions the suitable dosage for eliminating side effects and study all the potential biomedical and clinical application of such a versatile polymer

REFERENCES

[1] Boussif O Lezoualcrsquoh F Zanta M A Mergny M D Scher-man D Demeneix B Behr J P A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo polyeth-ylenimine Proc Nat Acad Sci USA 1995 92 7297

[2] Godbey WT Mikos AG Recent progress in gene delivery using non-viral transfer complexes J Control Release 2001 72 115

[3] Godbey WT Wu KK Mikos AG Poly(ethylenimine) and its role in gene delivery J Control Release 1999 60 149

[4] Kichler A Leborgne C Coeytaux E Danos O Polyethylen-imine-mediated gene delivery a mechanistic study J Gene Med 2001 3 135

[5] Kulkarni R P Mishra S Fraser S E Davis M E Single cell kinetics of intracellular nonviral nucleic acid delivery vehicle acidification and trafficking Bioconjug Chem 2005 16 986

[6] Akinc A Thomas M Klibanov A M Langer R Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis J Gene Med 2005 7 657

[7] Forrest M L Pack D W On the kinetics of polyplex endocytic trafficking implications for gene delivery vector design Mol Ther 2002 6 57

[8] Polyethylemine and ethylenimine Chemical Safety Information from Intergovernmental Organizations Document 12-13-05 (httpwwwinchemorgdocumentsjecfajecmonov20je08htm)

[9] Cordes RM Sims WB Glatz CE Precipitation of nucleic acids with poly(ethyleneimine) Biotechnol Prog 1990 6(4) 283

[10] Kirk N Cowan D Optimising the recovery of recombinant thermostable proteins expressed in mesophilic hosts J Biotechnol1995 42(2) 177

[11] Milburn P Bonnerjea J Hoare M Dunnill P Selective floccu-lation 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

[12] Bahulekar R Ayyangar NR Ponrathnam S Polyethyleneimine in immobilization of biocatalysts Enzyme Microb Technol 199113(11) 858

[13] 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

[14] Suh J Cho Y Lee K J Macrocyclic metal complexes built on polyethyleneimine J Am Chem Soc 1991 113 4198

[15] Melamed S Carlson G R Moss J N Belair E J Tansy M F GI pharmacology of polyethyleneimine I effects on gastric emp-tying in rats J Pharm Sci 1977 66(6) 899-901

[16] Tansy MF Martin JS Innes D L Kendall F M Melamed S Moss J N GI pharmacology of polyethyleneimine II motor activity in anesthetized dogs J Pharm Sci 1977 66(6) 902

[17] Chu A J Beydoun S Mathews S Hoang T Novel anticoagu-lant polyethylenimine inhibition of thrombin-catalyzed fibrin for-mation Arch Biochem Biophys 2003 415 101

[18] Helander I M Alakomi H-L Latva-Kala K Koski P Poly-ethyleneimine is an effective permeabilizer of gram-negative bacte-ria Microbiology 1997 143 3193

[19] Gao B Zhang X Zhu Y J Studies on the preparation and antibacterial properties of quaternized polyethyleneimine Biomat Sci Polym Ed 2007 18 531

[20] Setty C M Sahoo S S Sa B Alginate-coated alginate-polyethyleneimine beads for prolonged release of furosemide in simulated intestinal fluid Drug Dev Ind Pharm 2005 31 435

[21] Yang T Hussain A Bai S Khalil I A Harashima H Ahsan

F Positively charged polyethylenimines enhance nasal absorption of the negatively charged drug low molecular weight heparin J Control Release 2006 115 289

[22] Tegos G P Anbe M Yang C Demidova T N Satti M Mroz P Janjua S Gad F Hamblin M R Protease-stable poly-cationic photosensitizer conjugates between polyethyleneimine and chlorin(e6) for broad-spectrum antimicrobial photoinactivation An-timicrob Agents Chemother 2006 50(4) 1402

[23] Soukos N S Ximenez-Fyvie L A Hamblin M R Socransky S S Hasan T Targeted antimicrobial photochemotherapy Antim-icrob Agents Chemother 1998 42 2595

[24] Takeuchi Y Kurohane K Ichikawa K Yonezawa S Nango M Oku N Induction of intensive tumor suppression by antiangi-ogenic photodynamic therapy using polycation-modified liposomal photosensitizer Cancer 2003 97 2027

[25] Takeuchi Y Ichikawa K Yonezawa S Kurohane K Koishi T Nango M Namba Y Oku N Intracellular target for photo-sensitization in cancer antiangiogenic photodynamic therapy medi-ated by polycation liposome J Control Release 2004 97 231

[26] Bourgeois S Laham A Besnard M Andremont A Fattal E In vitro and in vivo evaluation of pectin beads for the colon deliv-ery of beta-lactamases J Drug Target 2005 13 277

[27] Bourgeois S Fattal E Andremont A Couvreur P 2003PCTFR0302474

[28] Lewis K Klibanov A M Surpassing nature rational design of sterile-surface materials Trends Biotechnol 2005 23 343

[29] Park D Wang J Klibanov A M One-step painting-like coating procedures to make surfaces highly and permanently bactericidal Biotechnol Prog 2006 22 584

[30] Haldar J An D Aacutelvarez de Cienfuegos L Chen J Klibanov A M Polymeric coatings that inactivate both influenza virus and pathogenic bacteria Proc Natl Acad Sci USA 2006 103 7667

[31] Milovi N M Wang J Lewis K Klibanov A M Immobilized N-alkylated polyethylenimine avidly kills bacteria by rupturing cell membranes with no resistance developed Biotechnol Bioeng2005 90 715

[32] Tiller J C Liao C-J Lewis K Klibanov A M Designing surfaces that kill bacteria on contact Proc Natl Acad Sci USA 2001 98 5981

[33] Beyth N Yudovin-Farber I Bahir R Domb A J Weiss E IAntibacterial activity of dental composites containing quaternary ammonium polyethylenimine nanoparticles against Streptococcus mutans Biomaterials 2006 27 3995

[34] Pasquier N Keul H Moeller M Polymers with specific adhe-sion properties for surface modification synthesis characterization and applications Des Monomers Polym 2005 8 679

[35] Pasquier N Keul H Heine E Moeller M From multifunction-alized poly(ethylene imine)s toward antimicrobial coatings Bio-macromolecules 2007 8 2874

[36] Etienne O Picart C Taddei C Haikel Y Dimarcq J L Schaaf P Voegel J C Ogier J A Egles C Multilayer polye-lectrolyte films functionalized by insertion of defensin a new ap-proach to protection of implants from bacterial colonization Antim-icrob Agents Chemother 2004 48(10) 3662

[37] Ji J Tan Q Fan D-Z Sun F--Y Barbosa MA Shen Fabri-cation of alternating polycation and albumin multilayer coating onto stainless steel by electrostatic layer-by-layer adsorption Col-loids Surf B Biointerfaces 2004 34 185

[38] Kraumlmer M Stumbyuml J-F Turk H Krause S Komp A De-lineau L Prokhorova S Kautz H Haag R pH-responsive mo-lecular nanocarriers based on dendritic core-shell architectures Angew Chem Int Ed 2002 41 4252

[39] Xu S Kraumlmer M Haag R pH-Responsive dendritic core-shell architectures as amphiphilic nanocarriers for polar drugs J Drug Target 2006 14 367

[40] Kraumlmer M Kopaczynska M Krause S Haag R Dendritic polyamine architectures with lipophilic shells as nanocompartments for polar guest molecules A comparative study of their transport behavior J Polym Sci [A1] 2007 45 2287

[41] Thunemann A F General S Nanoparticles of a polyelectrolyte-fatty acid complex carriers for Q10 and triiodothyronine J Con-trol Release 2001 75 237

[42] Vinogradov S V Bronich T K Kabanov A V Nanosized cationic hydrogels for drug delivery preparation properties and in-


teractions with cells Adv Drug Deliv Rev 2002 54 135 [43] Nam Y S Kang H S Park J Y Park T G Han S-H

Chang I-S New micelle-like polymer aggregates made from PEI-PLGA diblock copolymers micellar characteristics and cellular up-take Biomaterials 2003 24 2053

[44] Tiyaboonchai W Woiszwillo J Sims R C Middaugh R Insulin containing polyethylenimine-dextran sulfate nanoparticles Int J Pharm 2003 255(1-2) 139

[45] Kitazoe M Murata H Futami J Maeda T Sakaguchi M Miyazaki M Kosaka M Tada H Seno M Huh N Namba M Nishikawa M Maeda Y Yamada H Protein transduction assisted by polyethylenimine-cationized carrier proteins J Bio-chem 2005 137 693

[46] Tiyaboonchai W Woiszwillo J Middaugh CR Formulation and characterization of amphotericin B-polyethylenimine-dextran sulfate nanoparticles J Pharm Sci 2001 90 902ndash914

[47] Xu P Van Kirk E A Zhan Y Murdoch W J Radosz M Shen Y Targeted charge-reversal nanoparticles for nuclear drug delivery Angew Chem Int Ed 2007 46 4999

[48] Futami J Kitazoe M Maeda T Nukui E Sakaguchi M Kosaka J Miyazaki M Kosaka M Tada H Seno M Sasaki J Huh NH Namba M Yamada H Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization J Biosci Bioeng 2005 99 95

[49] Duan H Nie S Cell-penetrating quantum dots based on multiva-lent and endosome-disrupting surface coatings J Am Chem Soc2007 129 3333

[50] 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 bio-compatible polymeric nanoparticles for apoptosis imaging J Am Chem Soc 2006 128(11) 3490

[51] Kim J-H Lee S Park K Nam H Y Jang S Y Youn I Kim K Jeon H Park R-W Kim I-S Choi K Kwon I CProtein-phosphorylation-responsive polymeric nanoparticles for imaging protein kinase activities in single living cells Angew Chem Int Ed 2007 46 5779

[52] Masotti A Vicennati P Boschi F Calderan L Sbarbati L and Ortaggi G A Novel Near-Infrared Indocyanine Dye-Polyethylenimine Conjugate Allows DNA Delivery Imaging in vivo Bioconjug Chem 2008 19(5) 983

[53] Masotti A Ortaggi G Peptide Nucleic Acid-Polyethylenimine Conjugates Promising Multifunctional Therapeutic Tools for the Future Oligonucleotides 2008 18(3) 301

[54] Corti M Lascialfari A Marinone M Masotti A Micotti E Orsini F Ortaggi G Poletti G Innocenti C Sangregorio C Magnetic and relaxometric properties of polyethylenimine-coated superparamagnetic MRI contrast agents J Magn Magn Mater2008 320 e316

[55] Masotti A Pitta A Ortaggi G Corti M Innocenti C Lascial-fari A Marinone M Marzola P Daducci A Sbarbati A Mi-cotti E Orsini F Poletti G Sangregorio C Synthesis and Characterization of Polyethylenimine-based Iron Oxide Compos-ites as Novel Contrast Agents for MRI Magn Reson Mater Phy (MAGMA) 2008 (in press)

[56] Thomas M Ge Q Lu JJ Chen J Klibanov AM Cross-linked small polyethylenimines while still non-toxic deliver DNA efficiently to mammalian cells in vitro and in vivo Pharm Res2005 22 373

[57] Forrest ML Koerber JT Pack DW A Degradable Polyeth-ylenimine Derivative with Low Toxicity for Highly Efficient Gene Delivery Bioconjug Chem 2003 14 934

[58] Moghimi SM Symonds P Murray JC Hunter AC Debska G Szewczyk A A two-stage poly(ethylenimine)-mediated cyto-toxicity implications for gene transfertherapy Mol Ther 200511 990

[59] Symonds P Murray JC Hunter AC Debska G Szewczyk A Moghimi SM Low and high molecular weight poly(l-lysine)spoly(l-lysine)ndashDNA complexes initiate mitochondrial-mediated apoptosis differently FEBS Lett 2005 579 6191

[60] Xiong MP Forrest ML Ton G Zhao A Davies NM Kwon GS Poly(aspartate-g-PEI800) a polyethylenimine ana-logue of low toxicity and high transfection efficiency for gene de-livery Biomaterials 2007 28 4889

[61] Masotti A Moretti F Mancini F Russo G Di Lauro N Checchia P Marianecci C Carafa M Santucci E Ortaggi G Physicochemical and biological study of selected hydrophobic polyethylenimine-based polycationic liposomes and their com-plexes with DNA Bioorg Med Chem 2007 15(3) 1504

Received May 06 2008 Revised August 12 2008 Accepted August 20 2008


14 Use of PEI for the preparation of nanosized delivery vectors


11 Use of PEI as a Drug

Only few papers reported the pharmacologic potential of PEI used as a drug It is worth citing a couple of papers by Tansy [15 16] appeared in the literature in the late 70rsquos concerning the effects of the ingestion of these polymers on gastrointestinal motor activity in view of the therapeutic effects and hygienic usefulness of the commercial polyethyl-eneimines The original work focused on polyethylenimine and gastric emptying in rodents and dogs Experiments were

conducted on fasted rats to determine the effect of ingested polyethyleneimines upon gastric emptying A dose related long lasting (4 hours) and reversible delay in gastric empty-ing was detected within 15 minutes from the administration of branched polyethyleneimine while the linear polymer has no observable effect The branched PEI polymer with all the primary amino groups selectively acetylated has a low activ-ity as well Thus the study of Tansy demonstrated that poly-amines have a profound impact on the motility of the gastro-intestinal tract suggesting that polyamine pharmacophores are excellent candidates for the manufacturing of antitransit and antidiarrheal drugs However PEI caused severe retch responses in dogs and due to the structural similarity with other natural polyamines Tansy continued to study the effect of spermidine spermine and other polyamine analogues

Table 1 Applications of Polyethylenimine and Literature References

Paragraph Specific applications Cited reference


Ingestion of polyethylenimines and study of their effect on gastrointestinal motor activity [15-16]

Use of polyethylenimines to block fibrin formation (anticoagulant activity) [17]

Effect of polyethylenimines on the permeability properties of the Gram-negative bacterial outer membrane (OM) [18-19]


Coating of alginate beads for controlled drug delivery of low molecular weight therapeutic agents [20]

Use of polyethylenimines to enhance nasal absorption of negatively charged drug [21]

Use of polyethylenimines and photo dynamic therapy (PDT) as a possible treatment for localized infections [22-23]

Use of polyethylenimines in cancer antiangiogenic photodynamic therapy [24-25]


Polyethylenimines as bactericidal coating materials [26-27]

Use of polyethylenimines as bactericidal molecule able to kill gram positive and gram negative bacteria [2528-32]

Use of polyethylenimines in dental clinic for replacement of hard tissues [33]

Polyethylenimines as amphiphilic antimicrobial polymers [34-35]

Polyethylenimines able to protect against infection of implanted materials by bacteria (complication following pros-thetic surgery)

[36]

Polyethylenimines as suitable coating for biomedical 316L stainless steel [37]

14 Use of PEI for the preparation of Nanosized delivery vectors

Polyethylenimines in nanosized dendritic core-shell architectures encapsulating drugs [38]

Polyethylenimines in dendritic core-shell architectures derivatized with poly(ethylene glycol) chains [39]

Polyethylenimines derivatized with different fatty acids in dendritic core-shell architectures [40-41]

nanosized cationic hydrogels for drug delivery [42]

Polyethylenimine and poly(DL-lactide-co-glycolide) in micelle-like polymer aggregates preparation [43]

Polyethylenimines for nanostructured delivery systems for proteins [44-4648]

Polyethylenimines in nanoparticle-mediated nuclear drug delivery [47]


Polyethylenimines for quantum dots coating [49]

Polyethylenimines for non-invasive optical imaging (Near Infrared NIR) assessment of caspasesrsquo activity in vitro [50]

Protein-phosphorylation-responsive cell-permeable and biocompatible polyethylenimines [51]

Conjugation of polyethylenimine with a NIR-dye to obtain multifunctional delivery vectors for DNA delivery in vivo [52]














N

NH

N

NH

COOH

O

HN

COOH

N

Branched PEI

N

N

NH

N

NH2

NH2

NH2

NH2

HN

N

H2N

H2N

N

NH

N

NH

COOH

O

HN

COOH

NH

NH

Linear PEI

PEI-ce6

A B































































2 CONCLUSIONS








REFERENCES















































































N

NH

N

NH

COOH

O

HN

COOH

N

Branched PEI

N

N

NH

N

NH2

NH2

NH2

NH2

HN

N

H2N

H2N

N

NH

N

NH

COOH

O

HN

COOH

NH

NH

Linear PEI

PEI-ce6

A B































































2 CONCLUSIONS








REFERENCES































































































































2 CONCLUSIONS








REFERENCES





































































REFERENCES


































































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