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Branched and linear poly(ethylene imine)-based conjugates: synthetic modification, characterization, and application Michael Ja¨ger,ac Stephanie Schubert,bc Sofia Ochrimenko,bc Dagmar Fischer*bc and Ulrich S. Schubert*acd Received 10th January 2012 DOI: 10.1039/c2cs35146c Poly(ethylene imine)s (PEIs) are widely used in different applications, but most extensively investigated as non-viral vector systems. The high ability of cationic PEIs to complex and condense negatively charged DNA and RNA combined with their inherent proton sponge behavior accounts for the excellent efficiency in gene delivery. Further chemical modifications of the polymer expand the application potential, primarily aiming at increased transfection efficiency, cell selectivity and reduced cytotoxicity. Improvements in the synthesis of tailor-made PEIs in combination with new in-depth analytical techniques offer the possibility to produce highly purified polymers with defined structures. The contemporary strategies towards linear and branched poly(ethylene imine)s with modified surface characteristics, PEI-based copolymers as well as conjugates with bioactive molecules will be discussed. In this regard, the versatile branched PEIs have been successfully modified in a statistical manner, whereas the linear counterparts open avenues to design and synthesize well-defined architectures, in order to exploit their high potential in gene delivery. a

Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich-Schiller-University Jena, Humboldtstraße 10, 07743 Jena, Germany. E-mail: [email protected]; Web: http://www.schubert-group.com; Fax: +49 3641-948-202 b Institute of Pharmacy, Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, Otto-Schott-Straße 41, 07745 Jena, Germany. E-mail: dagmar.fi[email protected] c Jena Center for Soft Matter (JCSM), Humboldtstraße 10, 07743 Jena, Germany d Dutch Polymer Institute (DPI), John F. Kennedylaan 2, 5612 AB, Eindhoven, The Netherlands

Michael Ja¨ger was born in 1979 in Jena (Germany). He studied chemistry at the Friedrich-Schiller-University of Jena (Germany) and McGill University (Canada). For his PhD studies, he moved to Uppsala University (Sweden) to gain further insight into fundamental photosynthetic principles. After a short research stay with J.-P. Sauvage in Strasbourg (France), he finished his PhD thesis in 2009. His research interest Michael Ja¨ger covers photoactive compounds and the controlled modular assembly of functional molecules in (polymeric) architectures. Currently, he is working at the Department of Organic and Macromolecular Chemistry at the University in Jena on projects to utilize end-functionalized poly(ethylene imine)s. This journal is

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1. Introduction The high content of amino groups in poly(ethylene imine)s (PEIs) enables the usage of PEIs in different fields of application, e.g. as a chelating agent for metal ions,1–3 in waste water treatment,4 or as flocculation aid in the pulp and paper industry.4 In addition, PEIs have gained enormous attention in novel drug delivery systems, driven by the great advancements in chemistry to modify polymers and in biochemistry to

Stephanie Schubert (ne´e Hornig) was born in 1981 in Zwickau (Germany). She obtained her MS in chemistry at the Friedrich-SchillerUniversity of Jena (Germany) in 2005. After research activities at Virginia Tech (Blacksburg, USA), she finished her PhD studies in 2008 in the field of polysaccharide chemistry at the University in Jena in the group of T. Heinze. During a postdoctoral training with J. M. J. Freche´t at UC Berkeley Stephanie Schubert (USA), she gained further experiences in polymers as gene delivery devices. She is currently working on projects related to nanoparticles for drug delivery and sensor applications at the Pharmaceutical Department at the University in Jena.

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study their physiological action in depth.5,6 Among the many opportunities, therapies relying on DNA or RNA as drugs are considered to be highly promising treatment strategies in the future.7–13 For a safe and efficient gene delivery, cationic polymers offer great potential due to their inherent complexation of negatively charged nucleic acids and their protective effect against degradation.14,15 PEIs display a severe cytotoxicity and reduced blood compatibility depending on the structure (molar mass, branching) due to strong electrostatic interactions with cell membranes and the extracellular matrix. Hence, the investigation of the underlying mechanisms of cytotoxicity is of high importance to identify the critical parameters and design promising materials in the future materials.16–18 Several structural parameters such as molar mass, type, number, and density of charges as well as the three-dimensional structure and flexibility of the PEI molecules were identified to influence their safety and efficiency.14,19

Sofia Ochrimenko was born in 1983 in Jena (Germany). She studied pharmacy at the Friedrich-Schiller-University Jena (Germany) from 2001 to 2005. She passed her practical year in a pharmacy in Ilmenau and in the Spitalpharmacy of the University Hospital Basel (Switzerland). From 2007–2009 she worked as a pharmacist at the hospital pharmacy of the SRH WaldKlinikum Gera (Germany). In 2010, she joined the research Sofia Ochrimenko groups of Dagmar Fischer and Ulrich S. Schubert as a PhD student at the Friedrich-SchillerUniversity Jena (Germany) in the NanoConSens project.

Dagmar Fischer was born in Coburg (Germany) in 1967. She studied pharmacy at the University of Wu¨rzburg and received her PhD at the University of Marburg in 1997. After a scientific visit at the Texas Tech University Health Sciences Center in 2002 and 2003, she obtained her Habilitation at the University of Marburg in 2004. From 2004–2008, she was employed as Head of Preclinical Research and Development at Antisense Dagmar Fischer Pharma GmbH. Since 2008, she is Professor for Pharmaceutical Technology at the FriedrichSchiller-University Jena. Her research is focused on the field of polymeric gene delivery and bacterial nanocellulose as drug delivery systems, all combined with intense toxicological investigations.

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In addition, the topology seems to play a crucial role in in vivo applications.20 A 25 kDa branched PEI and a 22 kDa linear PEI are both the commonly used in vitro transfection agents. Actually, 22 kDa linear PEI is used in clinical trials due to its high transfection ability and acceptable biocompatibility.14,21,22 For clinical applications an extensive characterization in particular with regard to identity and purity of the polymers is required. Additionally, polymer modifications are necessary to optimize the performance of PEIs especially for systemic administration regarding biocompatibility, long blood circulation times, specific cell targeting, and a high stability during storage and in vivo distribution. In combination with other macromolecular species, an improvement in biocompatibility and transfection could already be achieved as demonstrated, e.g., for PEI-poly(ethylene glycol) (PEG) copolymers.23 Furthermore, PEIs conjugated with targeting functions open the possibility to selectively address the sites of interest.24–29 Most of these promising systems, however, lack a sufficient purity of the polymers and/or a detailed characterization from a polymer scientist’s point of view.30 Moreover, modern polymer chemistry offers a much broader range of polymerization techniques and polymer analogous reactions for the creation and modification of PEIs of interest. In this contribution, the improvements in the synthesis of well-defined PEIs are reviewed with an emphasis on recent strategies to design and access tailor-made derivatives. Such materials are envisioned to minimize the negative side effects while maintaining the high potential in gene delivery.

2. General biological aspects The rational development and design of non-viral vector systems for the transport of nucleic acids are based on the characteristics of the biological barriers, which inhibit the safe and efficient transport of DNA and RNA.14,31,32 After application

Ulrich S. Schubert was born in Tu¨bingen (Germany) in 1969. He studied chemistry in Frankfurt and Bayreuth (both Germany) and the Virginia Commonwealth University, Richmond (USA). His PhD studies were performed at the Universities of Bayreuth and South Florida/Tampa (USA). After postdoctoral training with J.-M. Lehn at the University in Strasbourg (France), he moved to the TU Mu¨nchen (Germany) and obtained his Habilitation Ulrich S. Schubert in 1999. From 1999–2000 he was Professor at the Center for NanoScience, University of Munich (Germany), and from 2000–2007 Full-Professor at TU Eindhoven (The Netherlands). Currently he holds a chair at the Friedrich-Schiller-University Jena with research interest in nanoparticle systems as sensor and drug delivery devices, supramolecular chemistry, inkjet printing of polymers, polymers for energy, and self-healing materials. This journal is

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into the body, an ideal polymeric non-viral vector system should be (i) resistant to degradation while circulating in the blood and, therefore, able to protect the nucleic acid drug against nucleases, (ii) able to selectively find and penetrate the target cell membrane and gain access to the intracellular target structure, and (iii) efficient in endosomal release and, when necessary, nuclear translocation. PEIs can complex nucleic acid drugs by electrostatic interactions forming small sized particles and masking the anionic charges of DNA. These effects are prerequisites for an efficient uptake of polynucleotides into cells, cytoplasmic movement, and protection against degradation in blood and cytoplasm. The strength of the complexation depends on the polymer characteristics, e.g. molar mass, branching, and number of cationic charges. Hence, the distribution of cationic charges within the polymer determines their access to nucleic acids and also to the surface of the target cells, in addition to the composition of the formed complex, e.g. the ratio of polymer to DNA.14,15,33 PEIs with a high degree of branching, high molar mass, high cationic charge densities, and a highly flexible structure are characterized by strong electrostatic interactions with negatively charged nucleic acids, resulting in the formation of small and enzymatically stable polyplexes with high cellular uptake.6,34–36 After cellular uptake, PEIs can accomplish the release of polyplexes from the endosomal/lysosomal compartment into the cytoplasm by the so-called proton-sponge effect based on their intrinsic pH dependent buffer capacity.5,37 The basicity and protonation were also reported to be influenced by the molar mass and the degree of branching of PEIs. Although for in vitro use many studies were performed and structure–activity relationships were established with PEIbased polyplexes, the in vivo situation is considerably more complex, in particular in the case of systemic administration. Only a few clinical studies have been performed using PEIs, all of them focussing on local application. A diphtheria toxin A expressing plasmid was polyplexed with a 22 kDa linear PEI (jetPEI) and locally successfully used in the bladder of two patients with recurrent superficial transitional cell carcinoma.38 A PEG-PEI-cholesterol derivative complexed with an interleukin-12 expressing plasmid showed only limited success in patients with recurrent ovarian cancer.39 After a successful clinical phase 1 with the DermaVir patch and a topical vaccine for HIV treatment in clinical phase 2,40 a linear 22 kDa PEI modified with mannose and dextrose was used to transfect a plasmid encoding for several HIV proteins into target antigen-expressing cells.21 Currently, a pilot study for the treatment of locally advanced pancreatic adenocarcinoma with intratumoral injection of jetPEI/DNA complexes with antitumoral effects and chemosensitizing activity for Gemcitabine is recruiting patients.41 A clinical trial using a combination of plasmid DNA, siRNA and jetPEI is currently announced in relapsed or refractory patients for the treatment of multiple myeloma.42 These first clinical results give reason for optimism, although the overall properties of the polymers are not ideal yet. Hence, future research in the PEI field will be focussed on the design of biodegradable and more biocompatible derivatives, enhancement of targeting specificity after systemic administration, This journal is

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transfection efficiencies on par with viruses, and long term action by sustained release mechanisms. For the development of a PEI system showing a breakthrough in biocompatibility, targeted gene delivery, and suitable handling for human administration, the whole structural diversity of PEIs should be taken into account. Synthetic concepts are presented in the following paragraphs with emphasis on the synthesis, chemical modification, and analytics of the PEI based systems.

3. Synthesis and characterization of PEIs 3.1.

Ring-opening of aziridine

A branched poly(ethylene imine) is readily prepared by polymerization of aziridine as first described in the patent literature in the 1940s (Scheme 1, top). The polymerization was investigated by Zomlefer et al. and proceeds via the cationic ringopening of aziridine,43 leading inevitably to branched chains with primary, secondary, and tertiary amino groups. The general mechanism is depicted in Scheme 1, and is initiated by an electrophilic attack of a Lewis acid (or protons) on an aziridine monomer (1). The generated active aziridinium ion is easily attacked by a nucleophilic species to yield the ringopened product. In the case of aziridine as the nucleophile, the resulting product contains a secondary amino group and a new aziridinium species at the terminus (2). Multiple consecutive attack of aziridine results in the linear propagation of the chain (3). However, the secondary (and tertiary) amino groups generated during polymerization may also react and consequently lead to branched structures (4a and 4b). In addition,

Scheme 1 (top) Polymerization of aziridine to branched poly(ethylene imine) (brPEI), (bottom) mechanism of the cationic ring-opening polymerization of aziridine: initiation step (1), linear propagation via initial (2) and consecutive attack (3) of aziridine, branching reactions with secondary (4a) and tertiary (4b) amino groups, deprotonation of aziridinium (5a) and tertiary ammonium (5b), and terminal attack of a nucleophile (6).

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the deprotonation steps (5a and 5b) may initiate new chains by protonation of the aziridine monomer. Finally, the polymerization is terminated by the attack of a nucleophile, e.g. upon addition of water or methanol (6). Hence, the interplay of the involved steps determines the exact ratio of primary, secondary, and tertiary amines. It was calculated that a branched PEI contains a theoretical ratio of primary/secondary/tertiary amines of 1 : 2 : 1. However, it was shown later that commercially available compounds contain a ratio closer to 1 : 1 : 1 indicating a more highly branched structure.44,45 Different reaction conditions are likely to cause such deviations from the theoretical values. Comparison of low molar mass (LMM) and high molar mass (HMM) PEI samples also revealed a higher degree of branching for HMM-PEI.36 This is expected because the depletion of monomer vs. formed secondary (4a) and tertiary (4b) amino groups statistically favors branching at high conversions.44 In addition, intramolecular branching (=cross-linking) increases the molar mass distribution further. Molar masses up to 1000 kDa with a polydispersity index (PDI) B4 are reported, but subsequent chemical cross-linking allows the preparation of materials with molar masses of virtually unity. In practice, the different solubility behavior enables the fractionation of large batches of branched PEI (brPEI) and a subsequent determination of the molar masses and polydispersity indices. However, the lack of structural control in brPEIs during synthesis limits the use to study structure– property relationships of PEIs, because no defined systematic variation of the molar mass, degree of branching etc. is possible. In addition, the handling of aziridine requires safety precautions, which are often not available in a standard chemistry laboratory. 5–25 kDa brPEIs were reported to be the most suitable for gene transfer. Larger brPEIs (>25 kDa) revealed a strong electrostatic interaction with DNA and RNA, but the biocompatibility decreased with increasing molar masses. Although less toxic, PEIs with lower molar masses were not able to form compact complexes and protect the nucleic acids. As an exception, a low molar mass 2.7 kDa PEI was reported to be highly effective which was related to a lower degree of branching compared to commercial products.46 Kra¨mer et al. synthesized pseudo-dendrimers based on branched PEIs and reported the lowest cytotoxicity for a degree of branching of about 60%.47 More details can be found in a review by Neu et al. and the references cited therein.15 3.2.

N-substituted aziridine

Later work has focused on the controlled polymerization of N-substituted aziridines (Scheme 2). Although the undesired re-initiation step upon proton transfer is suppressed (the related alkyl transfer does not occur), branching and termination of the tertiary amine can still occur. In this way, cyclic structures can be formed as reported during the (co-)polymerization of N-alkyl-substituted aziridine.48 Hence, more bulky substituents favor the propagation vs. the termination step to yield linear but N-substituted poly(ethylene imine). Various functionalized monomers bearing alkyl-,49,50 benzyl-,51 and even perfluoroacyl-52 or sulfonyl-53 groups were successfully polymerized. 4758

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Scheme 2 Polymerization of N-functionalized aziridines including alkyl-, benzyl-, pyranyl-, perfluoroacyl- and sulfonyl-substituents.

Upon subsequent cleavage of the former ‘‘protecting’’ moieties, it is possible to generate the corresponding lPEI. Besides the chemically inert alkyl-substituent, reductive de-benzylation or hydrolytic cleavage of acyl- and sulfonyl-substituents is reported.54 However, unwanted side reactions due to the harsh reaction conditions or incomplete cleavage may occur. Demember and Taylor reported the clean removal of the perfluoroacyl-groups to yield exclusively the lPEI similar to that obtained via the oxazoline-route (vide infra).52 Goethals and co-workers reported an elegant route to lPEIs taking advantage of the acid-labile tetrahydropyranyl-group (Scheme 2).49 Full hydrolysis was achieved using 0.1 M hydrochloric acid at room temperature, and the molar masses were determined by low-angle laser light-scattering (LALLS). Although pure lPEI samples between 8 to 20 kDa were obtained, the practical value of the approach suffers from the preparation and handling of the aziridines and was not tested in gene delivery studies. 3.3.

lPEIs via substituted oxazolines

A viable alternative approach to lPEIs was reported independently by several groups in the 1960s.55–57 The cationic ring-opening polymerization of 2-oxazoline yielded the corresponding acylated lPEI (Scheme 3), which is subject to acidic58–60 or alkaline hydrolysis54,61 to the respective lPEI.62,63 Particularly 2-substituted oxazolines are attractive, as they offer the possibility to adjust the stability of the formed amide group. Unsubstituted 2-H-poly(oxazoline) was shown to hydrolize under basic conditions,54 while incomplete hydrolysis is observed for alkyl-substituted poly(oxazoline)s.60 Noteworthily, an increased stability against hydrolysis for longer aliphatic or aromatic side chains is observed. In addition, examples with partial reductive cleavage of benzylprotected PEIs are reported.64,65 Wang et al. also reported on the partial enzymatic hydrolysis of a poly(ethyl-oxazoline) block.66 In general, a strong acidic medium (conc. HCl) and elevated temperatures (100 1C) are required to prepare lPEIs from parent methyl- and ethyl-substituted poly(oxazoline).60,67 However, the resulting PEI hydrochloride precipitates and retards full cleavage even under forcing conditions. Tauhardt et al. recently reported a short and reproducible microwave-assisted

Scheme 3

Linear PEI via substituted 2-oxazolines.

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Table 1 Selected analytical techniques used in the characterization of lPEI, including method of preparation, conditions of hydrolysis and molar masses Entry

Monomer

1

Pyranyl-aziridine

2

2-H-Oxazoline

3

2-Ethyl-oxazoline

a

Polymerization conditions In bulk, MeCN 65 to 25 1C DMF 80 1C In bulk or polar solvents 60 to 140 1C

Hydrolysis conditions 0.1 M HCl @ rt 98 1C, 3 h, 2.2 M NaOH54 (a) aq. NaOH (incomplete)69, or (b) conc. HCl, 100 1C60,67

Obtained molar masses 7–20 kDa (n = 170–460) 3 kDa (n = 65) 0.2–220 kDaa (n = 5–5000)

Analytical technique LALLS49 Vapor pressure osmometry68 SEC-RI60 SEC-MALLS with capillary viscosimetry70

Using commercial PEtOx (up to 500 kDa) yields lPEI (max. 220 kDa).

protocol to synthesize multi-gram batches of lPEIs with almost quantitative hydrolysis,71 including end group analysis by MALDI-ToF and ESI-ToF-MS. The advantage of the oxazoline-route originates from the commercial availability and convenient handling of the monomers (R = Me, Et), its living character to produce welldefined molar masses, and the robust polymerization protocols tolerating a variety of functional monomers, initiators, and quencher, including allyl and aryl groups.72 Unfortunately, most of the commonly incorporated functional groups are intolerant to the following harsh hydrolysis conditions; therefore their utilization (e.g. in PEGylation) is necessary prior to cleavage. Since virtually all biomedically attractive moieties are also unstable under the applied cleavage conditions, post-hydrolysis modification of PEIs has attracted great interest to create grafted materials for biomedical applications (see Section 4). Table 1 summarizes the key parameters of three synthetic routes to lPEIs: (a) the experimental conditions for the polymerization and (required) cleavage step, (b) typical molar masses and (c) exemplified analytical techniques of the obtained materials. For comparison, entry 1 lists the corresponding data for the cleavage of N-protected aziridine to lPEI (see Section 3.2). Up to now, lPEIs prepared via the oxazoline route are the materials that were already tested in clinical trials (see Section 2). lPEIs have several advantages compared to brPEIs both from the synthetic (more defined products) and also from the biological/clinical side (ongoing clinical trials). 3.4.

Characterization of PEIs

The classical characterization of PEIs and their conjugates is based on NMR data. The extent of branching can be determined by 13C-NMR spectroscopy, because the distinct chemical shifts of the carbon atoms in the linear and branched subunits are resolved.44,45 1H-NMR data are generally used to compare the area of the backbone signal vs. a known moiety within the polymer, e.g. the end groups. Hence, average molar masses can be obtained directly from 1H-NMR data. However, for higher molar masses, the intensity of the reference peak decreases and leads therefore to larger errors. In early studies, molar masses were also determined by LALLS49 or vapor pressure osmometry.68 With the advent of modern analytical techniques (Table 1, right column), particularly in combination with chromatography, PEI molecules were studied in greater depth. Unfortunately, simple analysis by size exclusion chromatography (SEC) is complicated by the insolubility This journal is

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in common eluents. In addition, protonation or complexation by eluent additives affects the hydrodynamic volume. Consequently, the calculated molar masses based on different standards are associated with a large systematic error. Furthermore, common commercial standards for cationic polymers, e.g. pullulan, are not applicable to the given SEC conditions. Lambermont-Thijs et al. reported SEC data using hexafluoroisopropanol as a solvent,60 but the question of a suitable standard for calibration remains to be answered. Kissel et al. combined SEC with a multi-angle laser light scattering detector (MALLS).70 Noteworthily, aqueous formic acid as an eluent was sufficient due to the better solubility of the investigated brPEI. Separate viscosity measurements allowed the determination of absolute molar masses, however, the high investment costs of the MALLS detector system hamper the use in daily routine analysis. A promising complementing technique is based on high resolution mass spectrometry, e.g. using MALDI-ToF- and ESI-ToF-MS.71,74 Although the direct quantification from the peak intensity is not strictly possible, valuable information of the relative occurrence of certain species can be obtained. Notably, the high resolution mass data of the parent and fragmented ions provide valuable information on the substructure of the polymer, e.g. the chain composition and the identification of the end-groups.74 In view of an efficient analysis of lPEIs and their conjugates, the development of automated SEC routine methods is expected to overcome the time-consuming analysis steps, and will reveal valuable information of PEI-conjugates in the future (see Section 4). 3.5.

Commercial sources of PEIs

Branched PEIs are available from a variety of suppliers, and the specified molar masses cover the complete range up to 750 kDa (Table 2, entry 1). As a consequence of the common preparation method of brPEIs via cationic ring-opening polymerization of aziridine, the reproducibility in the synthesis of identical batches is challenging (see Section 3.1). In contrast, lPEIs are predominantly prepared by acidic hydrolysis of poly(oxazoline)s. Table 2 lists a selection of common commercial sources, although an increasing number of new suppliers with custom-made PEIs exist. The reported molar mass range of available lPEIs covers 0.4 to 250 kDa (entries 4 and 5), but no PDI values are reported. Notably, the high PDI values of the precursor PEtOx (entry 3) are also reflected in the prepared lPEIs. In addition, reaching quantitative hydrolysis is challenging, and the degree of hydrolysis may vary among Chem. Soc. Rev., 2012, 41, 4755–4767

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Table 2 Selected commercial sources73 of linear forms of PEI and the parent PEtOx used in pharmaceutical studies with available analytical data (as provided by the supplier) Entry Commercial name (supplier)

Mn in kDa

PDI

1

Up to 750

1.1a

5 50, 200, 500 0.4 2.5, 25, 250 2.5 (4)c 25 (40)c 22

— 3–4 — — — —

22



2 3 4 5 6 7 8

brPEI (Sigma Aldrich, PolySciences, BASF) PEtOx (PolySciences) PEtOx (Sigma-Aldrich, PolySciences) lPEIb lPEI (PolySciences) Polyethyleneimine MAX (Polysciences) ExGen500 (Fermentas, Thermo Scientific) jetPEI (Polyplus Transfection)

a

PolySciences, for Mn = 1.2 and 1.8 kDa. b Sigma-Aldrich, not available anymore. c Free base form, number in parentheses refers to the respective hydrochloric salts.

different batches of commercial lPEIs (>90%). Hence, an additional hydrolysis step is performed to promote full amide cleavage (entry 6). Recently, a series of improved lPEIbased transfection agents became commercially available (entries 7 and 8). Besides the varying degree of hydrolysis among different batches, the high PDI values and the available molar masses are relevant parameters to control. In this regard, a sustainable synthetic procedure for the preparation of lPEI is highly desired to assure identity and purity for in vivo and clinical applications.

4. Modification of PEIs A current topic of high interest is the modification of PEIs with bioactive substances, besides the broad application of PEI copolymers. The efficient formation of conjugates requires selective chemical reactions for attachment of the functions while retaining their activity. 4.1.

General aspects of PEI modification

The modification of PEIs with various functionalities greatly expands the potential application possibilities of PEIs, e.g. in targeted gene delivery to specific cells in vivo, long-term blood circulation or controlled drug release. The general strategy relies on the modification of the amino groups. Commonly, a 25 kDa brPEI is used in the studies and the chemical modification is expected to occur preferentially at the terminal primary amino groups. However, with an increasing degree of functionalization, reaction of the secondary and/or tertiary amino groups is also possible. In addition, the degree of functionalization is rarely quantitative due to steric reasons, which is a common observation in polymer-analogue reactions. Hence, most biological studies focused on partially modified PEI conjugates. The reported methods are categorized in Scheme 4: alkylation of PEIs has been achieved via (a) reductive N-methylation using formaldehyde/formic acid,58,75 (b) nucleophilic substitution with alkylhalogenids,76 (c) ring-opening of epoxides,77 and Michael-type addition of (d) acrylates47,78 or (e) acrylnitriles.47 Acylation has been achieved via (f) isocyanateaddition to form carbamates34 and (g) carboxylic esters activated by N-hydroxysuccinimide79–82 or N,N 0 -carbonyldiimidazole.83 By using these principal reaction pathways, 4760

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Scheme 4

Chemical transformation of the amino groups of PEI.

a large variety of PEI derivatives becomes accessible. As a consequence, numerous publications deal with the defined functionalization of PEI in order to obtain insights into structure–property relationships for the development of a more efficient, more specific, and less toxic transfection agent. For example, acetylation of the primary and secondary amines results in PEI structures with different buffer capacities, which play an essential role in the endosomal release of genes.84 Further studies revealed similar results showing that a higher content of secondary to tertiary amines leads to better complexation efficiencies and, hence, higher transfection rates.15 The pKa as well as the buffer capacity increased proportionally to the number of primary and secondary amines. A systematic investigation of various PEI derivatives by Thomas and Klibanov revealed significantly enhanced transfection efficiency with low cytotoxicity for alanine acylated PEIs and dodecyl modified PEIs at the primary amine, which leads to the conclusion that not only the amine content and functionality are crucial for polyplex formation, but also the hydro-/lipophilicity and the resulting polymer conformation and constitution.85 The use of bifunctional reagents allowed cross-linking and/or introduction of pendant functional groups, which may be used for further reactions. Noteworthily, biodegradable bridges (e.g. disulfide,86 imines,87 amides,82 or esters78,81) were introduced to accomplish elimination from the body and to decrease accumulation and toxic effects of PEI-conjugates.88 With these methods in hand, a multitude of structures can be designed and synthesized, although poly(ethylene glycol)modified poly(ethylene imine) (PEG-PEI) is the most prominent approach. A recent review by Mintzer and Simanek describes the application of PEI-based conjugates as non-viral vectors for gene delivery.14 A further modification beyond the covalent attachment can be accomplished using the electrostatic interactions, e.g. with genes to introduce targeting functions.89 Another strategy is the exploitation of specific hydrophobic interactions of attached ligands that are known to enhance physical attraction between the components, e.g. PEG-avidin/biotin-PEI90 or PEI-cyclodextrin/palmitatemodified insulin.91 The PEGylation via the avidin–biotin bridge results in reduced cytotoxicity but also lower transfection efficiencies. The cleavability of the avidin/biotin complex at increased biotin concentrations after systemic administration This journal is

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might be advantageous for in vivo studies.90 The concept of avidin/biotin interactions was also used for the production of cystamine-bis-acrylamide crosslinked PEIs (bearing a redox sensitive S–S bond), which were functionalized with avidin/ 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and complexed with biotinylated transferrin leading to tumor targeted transfection.92 A further example is the modification of PEIs with biotinylated PEG in order to complex a conjugate of the rat anti-mouse antibody 8D3 and streptavidin for targeting the transferrin receptor.93 Cyclodextrin/palmitate complexation for modification of PEIs with insulin was shown to enhance the gene expression by an order of magnitude without any cytotoxic effects.91 This concept can be used for the conjugation of additional relevant targeting functions (e.g. TAT peptide for targeting of placenta mesenchymal stem cells),94 while the cyclodextrin function can also serve as a drug carrier.95 A chitosan grafted PEI functionalized with cyclodextrin was shown to efficiently transfect plasmid DNA and siRNA depending on the supramolecular PEGylation with adamantane modified PEG.96 However, the stability of such systems based on hydrophobic or electrostatic interactions represents an important parameter.

Therefore, covalent attachment appears as the preferred route for functionalization. The modification of PEI with active ligands (e.g. targeting functions, markers) holds the main part in PEI modification and will be discussed in the following chapters. 4.2.

PEI-conjugates

The following section details the direct modification of the PEI backbone with bioactive ligands as a common tool towards target-specific PEI-conjugates (Scheme 5). In this manner, efficient and specific coupling agents and optimized reaction conditions are required to tolerate any remaining functional groups in the conjugate. For example, activation of PEI with N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) represents a common strategy to bind proteins or antibodies via their thiol or amine moieties. The pyridyl dithiol group of SPDP can either react directly with thiol-containing ligands97 or can be reduced using dithiothreitol (DTT) to generate a free thiol group, which subsequently reacts with SPDP bound to amino groups (Scheme 5a)98 or other thiol-reactive groups such as maleimides or iodoacetals.99 In this way, a variety of

Scheme 5 Strategies for the preparation of PEI-conjugates modified with bioactive ligands.

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PEI-conjugates have been prepared, e.g. with epidermal growth factors,97 monoclonal antibodies,98 RGD peptides for integrin mediated endocytosis,100,101 peptides for binding growth factor receptors,102 sugars, and many more. The use of SPDP is highly efficient and enables the tuning of the functionalization degrees by simply varying the molar ratios. However, this method suffers from the instability of the disulfide bridge. It was shown that thiols such as mercaptoethanol and cysteine at physiological concentrations cause a rapid cleavage of the ligand from the PEIs.101 Alternatively, maleimides are versatile linkers for the binding of thiol containing ligands by formation of thioether bonds, as demonstrated for succinimidyl trans-4-(maleimidylmethyl) cyclohexane-1-carboxylate (SMCC, Scheme 5b),102 N-(maleimidobutyryloxy)succinimide (GMBS)103 and N-(maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS, Scheme 5c)104 with very high yields for functionalization and a good control over the reaction. Sulfo-KMUS can also be used for the coupling of primary amines via thiolation of PEI using 2-iminothiolane and subsequent reaction of sulfo-KMUS activated amines with the thiolated PEI.104 Dithiobis(succinimidylpropionate) (DSP) is another coupling agent applied for the immobilization of PEIs with amine functionalities (Scheme 5d), however, the conjugate still contains sensitive dithiols.105 A further strategy for the conjugation of PEIs with amines is described for clenbuterol (Scheme 5e).106 The secondary amine of clenbuterol was activated with the product of ethylchloroformate and maleimidopropionic acid, and was subsequently allowed to react with PEIs. The immobilization of ligands containing carboxylic acids can efficiently be achieved by applying carbodiimide chemistry (Scheme 5f). In this approach, the carboxylic groups are activated to reactive intermediates that form stable amide bonds after reaction with the amine nucleophiles. The use of dicyclohexylcarbodiimide (DCC) or the water-soluble EDC in combination with N-hydroxysuccinimide (NHS) provides an efficient coupling of targeting functions with carboxy groups such as folate,107 hyaluronic acid,108 uronic acids,109 or the prostaglandin analogue iloprost.110 NHS is useful to prevent undesired side reactions by forming more stable NHS esters as the reactive acylating agents.99 A further convenient synthesis strategy relies on the reductive amination, leading to a direct linkage of the ligand to the polymer without the requirement of any spacer unit. For this purpose, aldehyde functions are generated by mild oxidation with sodium periodate, e.g. in lactoferrin,25 whereas sugar based targeting functions (e.g. glucose, galactose, mannose, maltose, lactose) can be employed via their open-chain form without any pretreatment (Scheme 5g).87,111 The intermediate imine products (Schiff bases) then have to be reduced, which is usually achieved using sodium cyanoborohydride. A less toxic alternative is claimed to be titanium(IV) isopropoxide in combination with sodium borohydride.112 The functionalization with sugar molecules to specifically interact with the cell membrane can also be realized using phenylisothiocyanate functionalized sugar moieties.113 Another approach is mainly applied for cyclodextrins using the tosylated derivative for coupling to PEI, which resulted in a significantly promoted route of caveolae-mediated endocytosis.114 4762

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In general, the degree of substitution of PEI-conjugates usually depends on the amount of accessible primary amines of PEIs. However, the conjugation reactions are not completely selective for primary amines, and a non-negligible amount of secondary amines may also be modified. Conjugation ratios from less than one ligand per PEI chain up to several tens can be reached for a 25 kDa brPEI (ca. 580 repeating units). The ratios are generally determined by 1H-NMR spectroscopy, UV/Vis- and fluorescence spectroscopy in a photospectrometric assay, or chromatographic methods for ligand detection. However, there is a severe lack of more detailed molecular characterization approaches. A closer look at the structure (block- or random distribution, molar mass after reaction, molecular dynamics, molecular assemblies, etc.) would probably lead to a better understanding of the transfection ability, and, hence, to a more efficient design of PEIconjugates. It is important to realize that, despite the efficient coupling steps, all methods discussed above enable only the stoichiometric control of the composition and preclude the exact spatial control of the conjugate’s architecture. As a consequence, the optimization of amount and location of bioactive ligands on the PEI is usually an empirical endeavour and has not allowed a defined functionalization yet. 4.3.

PEI-PEG conjugates

A serious drawback of the ‘‘simple’’ modification of PEI with specific ligands is the superposition of the specific cellular interaction of ligands with the electrostatic attraction caused by the positively charged PEI at the cellular surface. Accordingly, the selectivity resulting from the attached ligands is negligible. In order to maintain the targeting function, PEG has been intensively investigated as a biocompatible and uncharged spacer unit. In this way, the shielding effect of PEG to prevent non-specific interactions can be coupled with the targeting function. The convenient synthesis benefits from readily available bifunctional PEG; e.g. PEG equipped with N-hydroxysuccinimide (NHS) and vinylsulfone that can react selectively with thiol groups of the peptide/protein/antibody on one side and the primary amines on the other side (Scheme 6a).79 Another example is the use of ortho-pyridyldisulfide PEG-succinimidyl propionic acid, which was linked to PEG and activated with 1,4-dithiothreitol (DTT) before coupling to a thiol-containing peptide (Scheme 6b).115 Alternatively, a pre-PEGylated PEI can be coupled directly with maleimide functionalized proteins, e.g. maleimide RGDmimetics (Scheme 6c).115 For immobilization of PEI with folate, which shows a high affinity to the overexpressed folate receptor in tumor cells, a diamino-endcapped PEG was reacted with NHS-activated folic acid on one side (Scheme 6d).107 Next, the thiol-functionalized PEI was created using Traut’s reagent (2-iminothiolane) and further coupled with the remaining amino group of PEG using the maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). A more convenient route is the conjugation of folic acid with NHS and N,N-dicyclohexylcarbodiimide (DCC) (Scheme 6e), which can easily be applied to various other functionalities possessing carboxyl functions.116 In fact, the usage of a spacer moiety between the PEI and the targeting ligand does not necessarily result in more specific This journal is

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Scheme 6 Strategies for the preparation of PEI-PEG-conjugates modified with bioactive ligands.

interactions and may even lead to lower transfection efficiencies compared to the ligand directly conjugated to the polymer backbone.79 By using different coupling strategies and PEG lengths, a broad variety of PEG-PEI-ligand conjugates with different ligand densities can be prepared. Consequently, structure–activity relationships can be derived and result in conjugates with optimized cell-specific interactions. 4.4.

Fluorescence- and radio-labelled PEIs

The modification of PEIs with labels is of general interest to trace the polymer during body transport and interaction with cells. The resulting information about biodistribution and cellular uptake is essential for the development of efficient and selective transfection agents. For this purpose, isocyanate chemistry is commonly applied to efficiently attach labels, e.g. fluorescein isothiocyanate (FITC) that easily reacts with amines.117 Due to the lack of pH stability of FITC and bleaching during fluorescence microscopy studies, several other fluorescent isocyanates-containing dyes were immobilized onto the PEI backbone, such as rhodamines117 or special chromophores for two-photon fluorescence correlation spectroscopy measurements (Scheme 7a).118 In addition, radio-labelling can be

performed via isocyanate chemistry using (2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA).115 The functionalization of PEI with DTPA for complexation, e.g. with 111indium, is possible by the reaction with DTPA anhydride (Scheme 7b).119 Alternatively, an 125iodine-label can be attached by reaction of PEI with iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester.120

5. Approaches to structurally well-defined PEI-conjugates The chemical transformations of the amino groups furnish the preparation of numerous PEI-conjugates. However, the mentioned methods allow only a stoichiometric control but lack the control over the site of reaction. In addition, the reproducibility of such protocols in view of more sophisticated architectures is highly questionable. This section deals with two conceptually different approaches to gain further control of the architectures: (a) employing a macro-initiator or -stopper and (b) strategies to utilize the available functional end groups of lPEIs. In conjunction with powerful analytical techniques, e.g. high resolution mass spectrometry, a detailed investigation of the materials becomes possible. The concurrent control and knowledge of structural as well as pharmaceutical properties enable a more detailed investigation of structure–activity relationships of novel sophisticated materials. 5.1.

Scheme 7 Fluorescence- and radio-labelling of PEI.

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PEG-block-PEI copolymers

In order to improve the physicochemical and biological stability of the polyplexes and to change the in vivo distribution patterns, a second generation of gene delivery systems was designed by attaching hydrophilic non-ionic water-soluble polymers, such as PEG,14,121 transferrin,24 pluronic,122 or poly(N-2-hydroxypropyl methacrylamide) (pHPMA).121,123 The highly mobile and Chem. Soc. Rev., 2012, 41, 4755–4767

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hydrated polymer strands seem to shield the surface charge of the polycationic complex, in order to sterically prevent nonspecific interactions with the biological environment and, thus, prolong the half life time of the carriers in the blood stream (‘‘stealth’’ or ‘‘brush’’-effect).5 It has been shown in various examples that PEGylation can enhance the storage stability due to steric stabilization, decrease the non-specific cellular uptake, reduce opsonization, uptake by the reticuloendothelial system and recognition by the immune system after systemic administration, and, consequently, leads to a prolonged systemic circulation.124 PEGylated PEIs revealed a dependency of their in vivo organ distribution and pharmacokinetic on the block length of PEG chains and the degree of substitution.125 For instance, a comparison between a 25 kDa brPEI and a PEGylated derivative (brPEI(25 kDa)-PEG(550 Da)50) demonstrated that immediately after intravenous injection the PEG-PEI reached only 50% of the values of the PEI in liver and spleen which correlated with prolonged circulation of PEGylated PEI in the blood.126 Whereas nonmodified DNA/ PEI-complexes were found to attach plasma proteins to their surfaces, PEGylated complexes were not able to interact with IgM, fibronectin, fibrinogen, complement factor C3, and did not aggregate even with erythrocytes.127 However, the PEG shielding also rendered the interaction with nucleic acids and therefore complexation as well as attachment of complexes to cells more difficult.128 These examples utilized stoichiometrically grafted PEIs, which preclude the precise control of PEGylation. Hence, the alternative attachment of PEG at the PEI terminus leads to PEG-block-PEI systems with a significantly better controlled architecture. The synthesis of PEG-block-PEI copolymers can be accomplished using a PEG-containing initiator (macro-initiator) or quencher (macro-stopper) (Scheme 8). Kissel et al. synthesized a brPEI-PEG copolymer using an amino-functionalized PEG to terminate the acidic aziridine polymerization.129 Although the gained control of architecture is overcompensated by the inherent branching during aziridine polymerization (see Section 3.1), this approach may be extended to the cationic ring-opening polymerization of oxazolines followed by acidic hydrolysis (see Section 3.3). Alternatively, Akiyama et al. used a PEG-initiator to prepare PEG-block-PMeOx, which was further hydrolyzed to the respective PEG-block-PEI.130 Later, this approach was extended to bifunctional initiators to yield PEI-block-PEGblock-PEI.131 As for any multifunctional initiator, the challenges arising from incomplete initiation or side reactions are inevitable and may result in less defined architectures.

Scheme 8 Macro-stopper route to PEG-brPEI (top) and macroinitiator route to PEG-lPEI (bottom).

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5.2. End-functionalized lPEIs and orthogonal coupling strategies The method of preparation via acidic hydrolysis of substituted oxazolines strongly limits the available functional end-groups of lPEIs, e.g. primary amino- and hydroxyl-groups. A second constriction stems from the similar reactivity of hydroxyland amino groups. Although chemoselective protocols are reported for small organic molecules,132 such methods are only of limited applicability to the respective PEI-containing polymers. The practical problems originate from the removal of by-products (which is not possible by simple distillation, chromatography, or extraction), limited solubility in common solvents, and, in general, lower reaction rates of polymeranalogue reactions. Hence, reported examples of selective transformation of the end groups are scarce. First, Lee et al. treated hydroxyl-terminated lPEIs with an excess of tosylchloride to protect all secondary amino-groups as tosyl-amines, while the hydroxy-group is transformed into a good leaving group.133 Subsequent reaction with a proper nucleophile, e.g. amines or thiols, leads to exclusive functionalization at the chain end. The final hydrolysis regenerates the amino group within the backbone. However, this approach needs full protection of the amino groups, which requires a large excess of expensive chemicals and long reaction times to assure full conversion. Secondly, Pons et al. prepared hydrazine-terminated PEtOx as well as the respective lPEI, and demonstrated the selective conjugation via imine-formation with aldehydes.134 The utilization of the azide groups in the well-established copper-promoted azide-alkyne cycloaddition (CuAAC) reactions seems to represent a potential alternative. Liu et al. demonstrated the possibility of CuAAC,135 but the removal of toxic copper remaining in the material needs to be solved, as PEI itself is an excellent metal chelating agent. In this regard, metal-free versions of the ubiquitous ‘‘Click-reaction’’ are promising conjugation methods, in particular bioorthogonal reactions based on Diels–Alder reactions, dipolar addition reactions, or addition-extrusion reactions.136

6. Conclusions Poly(ethylene imine)s possess high application potential in gene delivery, as shown by the preclinical studies using brPEIs with a molar mass of 25 kDa or linear PEIs with 22 kDa. Further chemical modifications of the pristine polymer, e.g. conjugation with bioactive moieties or PEG, aim to enhance cell selectivity or decrease the inherent cytotoxic effects. In the past decade, novel PEI design concepts emerged, including dynamic and bioreversible linkages and efficient chemical protocols to modify the polymer. However, most of the conjugates only allow a stoichiometric control of functionalization of the PEI backbone, but lack a precise regiocontrol, e.g. via the polymer end-groups. In this perspective, the general synthetic routes and the analytical tools for molecular characterization of PEIs are described. Branched PEIs are readily prepared by acidic polymerization of aziridine, whereas linear PEIs are conveniently prepared via acidic hydrolysis of poly(oxazoline)s, e.g. by a reproducible microwave-assisted protocol for multi-gram This journal is

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batches of lPEIs. Noteworthily, an increasing number of commercial suppliers of (modified) PEIs exist, but detailed analytical data are often missing. The chemical routes to modify the amino groups are presented and complemented by an overview of modern conjugation methods with relevance to gene delivery. Although these powerful methods allow to efficiently modify the surface of brPEIs or the backbone of lPEIs by stoichiometric means, they usually lack a defined regioselectivity needed for precisely tailored architectures, in particular for lPEIs. This regiospecific manipulation can be achieved prior to hydrolysis via the macro-initiators or -stopper approach, respectively. However, the substrate scope is very limited due to the subsequent harsh acidic hydrolysis. Alternatively, the selective functionalization of the end-group leads to a broader range of well-defined conjugates. To date, lPEIs with molar masses up to 9 kDa (ca. 200 repeating units) with narrow PDI values have been reliably prepared, including defined end-groups, but their incorporation into polymeric architectures relies on efficient protocols. Future developments, e.g. orthogonal conjugation methods, are expected to enable facile access to tailored architectures in a modular fashion. In order to characterize any PEI conjugates in depth, powerful complementary analytical techniques are required: the general analytical characterization of PEIs and their conjugates is primarily based on NMR spectroscopy, but the advent of chromatographic methods coupled to light scattering (SEC-MALLS) or mass spectrometry (SEC-MS) offers many new possibilities for an efficient and in-depth analysis routine. In particular, the fragmentation data from high-resolution mass spectrometry are expected to fuel the future analysis of tailored PEI-conjugates. With the modern synthetic and analytical methods in hand, a multitude of well-defined assemblies based on lPEIs (e.g. block-, star-, and combcopolymers) can be designed with tailored properties. In combination with the exciting opportunities provided by already existing modification methods, a systematic investigation of structural features becomes feasible to address the subtle balance between minimal toxicity and high transfection efficiencies in gene delivery.

Acknowledgements The authors thank the Thu¨ringer Ministerium fu¨r Bildung, Wissenschaft und Kultur (TMBWK, ProExzellenz-Programm NanoConSens, B514-09049), the Carl-Zeiss-Stiftung (Strukturantrag JCSM), and the Dutch Polymer Institute (DPI, technology area HTE) for financial support.

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