Nucleic Acid Therapeutics Using Polyplexes: A Journey of 50 Years (and Beyond).

Review pubs.acs.org/CR

Nucleic Acid Therapeutics Using Polyplexes: A Journey of 50 Years (and Beyond) Ulrich Lac̈ helt*,†,‡ and Ernst Wagner*,†,‡ †

Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig Maximilians Universität, 81377 Munich, Germany Nanosystems Initiative Munich (NIM), 80799 Munich, Germany 1. INTRODUCTION

‡

Since the elucidation of the molecular structure of nucleic acids and understanding of their role in encoding and translating genetic information into biological function,1 the knowledge about nucleic acid biology has continuously increased. The sequencing of human genomes revealed a surprising interindividual heterogeneity due to single-nucleotide polymorphisms and genomic copy number variations.2 Identification of genetic profiles which are associated with the risk of developing certain diseases will have an enormous impact on individualized medicine and modern disease-preventive healthcare.3 Beyond the central dogma of molecular biology which describes that genes are being transcribed in RNA in a highly regulated fashion and subsequently translated into proteins, additional functions of RNA have been identified.4 Noncoding RNAs such as microRNA or short interfering RNA (siRNA) were found to act as additional endogenous regulators of gene expression.5 Altogether, the increasing knowledge about these natural nucleic acid functions also opened new opportunities for therapeutic interventions at the nucleic acid level. As individual nucleic acid-encoded variations can directly cause illnesses or be associated with disease development, artificial therapeutic nucleic acids should be able to attenuate the severity or even cure diseases on a causal basis. Indeed, within about 25 years of more than 2000 clinical gene therapy trials, the first nucleic acid nanomedicines reached medicinal market authorization.6 Developments were much slower than initially expected. As outlined below, delivery of the various therapeutic nucleic acids to their required cellular site of action appeared as a dominating bottleneck for successful medical development. Complexation of nucleic acid with polymeric carriers into polyplex nanoparticles is one possible approach to facilitate the delivery.

CONTENTS 1. Introduction 2. Therapeutic Nucleic Acids 2.1. Gene Therapy with Gene Expression Constructs 2.2. Modulation of Gene Expression by Antisense or RNAi Nucleic Acids 2.3. Protein-Interacting Therapeutic Nucleic Acids 2.4. Challenge of Nucleic Acid Delivery 3. Delivery Pathway of Polyplexes 3.1. Extracellular Delivery Requirements 3.2. Intracellular Delivery Requirements 4. Cationic Core Polymers 4.1. First-Generation Polycations 4.2. Nature-Derived Polymers 4.3. Biodegradable Polymers 4.4. Well-Tolerated Copolymers and Dendrimers 4.5. Precision Polymers and Sequence-Defined Oligomers 5. Functional Delivery Domains 5.1. Shielding Domains 5.2. Targeting Ligands 5.3. Endosomolytic Functions 5.4. Nuclear Import and Retention 6. Multifunctional Dynamic Polyplexes 7. Conclusion and Prospects Author Information Corresponding Authors Notes Biographies Acknowledgments Dedication References

A A A B D D E E F G G I I J J L L O P Q S V V V V V V V V

2. THERAPEUTIC NUCLEIC ACIDS Therapeutic nucleic acids can act at the different stages of the gene expression process, as outlined in Figure 1 and the following subsections. 2.1. Gene Therapy with Gene Expression Constructs

Gene therapy in its classical meaning, in terms of the substitution of defective genes by introduction of functional genetic versions, was the first concept for the therapeutic usage of nucleic acids.7 After two decades with worldwide more than 2000 approved, ongoing, or completed clinical gene therapy trials,8 the first gene Special Issue: Nanoparticles in Medicine Received: November 30, 2014

© XXXX American Chemical Society

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DOI: 10.1021/cr5006793 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Different stages of therapeutic intervention by nucleic acids. Exogenous DNA and mRNA can be introduced into cells for the expression of transgenes and new proteins. Splice-switching oligonucleotides (SSO) redirect the splicing process by specific antisense binding to exon splice sites of the pre-mRNA. Antisense oligonucleotides and RNAi nucleic acids (exogenous miRNA or siRNA) suppress the target mRNA translation by sterical blockade and/or RNA degradation.

therapeutic effects in experimental genetic disease animal models.12 mRNA delivery has also been applied as a therapeutic concept in experimental tumor models.13

therapy product alipogene tiparvovec (Glybera), based on an adeno-associated viral (AAV) vector encoding a human lipoproteinlipase (LPL) gene variant, got marketing authorization by the European Commission in 2012.6d Additional successful clinical results have been obtained with AAV vectors in patients with inherited serious genetic defects for the cure of Leber’s congenitial amaurosis (LCA), an inherited form of blindness,9 and hemophilia B.10 Retroviral vectors were used for the cure of the inherited severe combined immunodeficiencies ADA SCID and X1 SCID.8b Moreover, multiple gene therapy developments are ongoing for treatment of acquired noninherited serious diseases such as life-threatening viral infections or cancer.11 Among the possible gene vectors, viruses which have been optimized by nature for the purpose of nucleic acid transfer into host cells represent the most efficient delivery vehicles. Over two-thirds of the clinical trials worldwide are based on viral delivery vectors. Moreover, only virus-based vectors have been successfully applied for stable introduction of therapeutic genes required for the persistent therapy of inherited diseases. Despite their high transfer efficiency, viral vectors also exhibit significant limitations, such as immunogenicity, limited cargo capacity, restricted cell tropism, or sophisticated analytics and production. Efficient nuclear delivery of genes into nondividing target cells and long-term incorporation as genetic DNA information which does not disturb the natural host genome are serious technical and biosafety issues for all gene transfer systems. In this respect, the direct transfer of mRNA, which does not require location and transcription in the nucleus, provides an interesting alternative approach. Although single application of such a “gene therapy without genes” is not expected to provide long-lasting genetic cures, recent technical developments such as SNIM (stabilized nonimmunogenic mRNA) based on incorporation of modified nucleosides into the mRNA have demonstrated improved cytosolic persistence and protein expression, resulting in

2.2. Modulation of Gene Expression by Antisense or RNAi Nucleic Acids

Gene expression can also be modulated at various levels of the natural mRNA biosynthesis and function. An early stage of posttranscriptional regulation is represented by splicing. For larger gene products like the muscle dystrophin protein, multiple introns are deleted from the transcribed RNA by the natural splicing process, generating the mature mRNA out of the remaining exons. Malfunctioning genetic defective exons can be removed by “exon skipping”, resulting in slightly shorter but functional gene products. This process is mediated by spliceswitching oligonucleotides (SSOs), which can specifically block an exon splice site by complementary (“antisense”) Watson− Crick base pairing and thus trigger alternative splicing to the next exon. On the basis of this great therapeutic potential, SSOs have been clinically evaluated for the treatment of Duchenne muscular dystrophy.14 Antisense oligonucleotides can also mediate shutdown of gene expression on the mRNA level by steric blockade or RNase Hdependent degradation of the complementary target mRNA which suppresses translation of the target protein. Fomivirsen (Vitravene) represents the first marketed antisense therapeutic, which received FDA approval for the local treatment of CMV retinitis in 1998. 15 It presents an oligodeoxynucleotide phosphorothioate 21-mer with complementary sequence to the mRNA transcript of a cytomegalovirus (CMV) gene. A more recent example of a successful antisense therapeutic development is mipomersen, a phosporothioate oligonucleotide targeting the apolipoprotein B mRNA, approved in 2013 as adjunct medication in patients with homozygous familial hyperB


C

a

nucleic acid vectors associated with magnetic nanoparticles exposed to a magnetic field

magnetical enhancement (“magnetofection”)

silica, gold, calcium phosphate, carbon nanotubes, metal−organic frameworks

direct interaction of genetic material with inorganic nanoparticles or with functionalization units of inorganic/ organic hybrid systems

complexation of genetic material by cationic polymers or direct covalent conjugation

transient permeabilization of cell membranes by electric pulses

electroporation

polymeric transfection agents

rapid injection of genetic material in a large volume

hydrostatic pressure

encapsulation of genetic material into the inner volume of liposomes or complexation by cationic lipids and lipidoids

transport of genetic material into cells with microprojectiles accelerated to high velocity

particle bombardement (“gene gun”)

lipid-based transfection agents

direct injection of genetic material into cellular compartments

microinjection

principle

This class is the subjectof the review. Details and references can be found in the text.

inorganic nanoparticles

organic delivery systems

physical methods

class

Table 1. Outline of Nonviral Nucleic Acid Delivery Approaches

+ individual characteristics and advantages depending on the type of material (e.g., porosity, crystallinity, controllable size and shape, magnetism, biochemical inertness, cellular uptake, biodegradability) + versatile functionalization in combination with organic compounds + suitable for systemic delivery − biocompatibility and toxicity have to be considered

+ self-assembly of polyplexes + cytosolic delivery facilitated by ionic membrane interactions and endosomal buffering + high flexibility for variation and functionalization + suitable for systemic delivery − biocompatibility and toxicity have to be considered

+ self-assembly of lipid-particles + cytosolic delivery facilitated by hydrophobic membrane interactions + high flexibility for variation and functionalization + suitable for systemic delivery − biocompatibility and toxicity have to be considered

+ fast transfection kinetics and high efficiencies + magnetically guided vector deposition in target tissues + high flexibility in combination with diverse nonviral (or viral) vectors − in vivo use restricted to localized target tissues − low penetration depth of magnetic force in the body

+ use of naked genetic material + suitability for a wide range of different cells and tissues − in vivo use restricted to localized target tissues

+ use of naked genetic material + simple and cost effective − low efficiency − limitation of potential target tissues

+ suitable for a wide range of different cells and tissues + application in genetic vaccination and gene transfer over the skin − in vivo use restricted to local treatments

+ control over localized intracellular deposition + high efficiency on the single cell level − elaborative treatment of single cells − no in vivo applicability

characteristics

37

a

13,24,36

35m−q

35i−l

35f−h

35b−e

35a

ref

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which bind target molecules with affinities comparable to antibodies and can be engineered in vitro using evolutionary selection processes.27 Because of their small to medium size they are readily accessible by chemical synthesis. As the first FDAapproved therapeutic aptamer, the polyethylene glycol (PEG) conjugated antivascular endothelial growth factor (VEGF) aptamer pegaptanib (Macugen) entered the market for the treatment of age-related macular degeneration in 2004.6c A series of clinical developments against target proteins in the bloodstream involved in coagulation, such as factor IXa, thrombin, or von Willebrand factor, is ongoing. In drug and nucleic acid delivery research, aptamers also have been evaluated as cell receptor targeting ligands.28 Human beings have developed strong innate and acquired immune responses against invading foreign nucleic acids. Specialized proteins such as Toll-like receptors (TLRs)29 and cytosolic sensors like cyclic GMP-AMP synthase (cGAS)30 recognize subtle differences of almost any type of nonmammalian nucleic acids, both in structure as well as in cellular location, and trigger immune and cellular suicide responses. Knowledge about these processes, on the one hand, enables synthetic modification of therapeutic nucleic acids to bypass undesired cellular responses. On the other hand, immunostimulatory nucleic acids, such as unmethylated cytosine-phosphateguanosine (CpG) oligodeoxynucleotides, can be used as adjuvants for vaccination.31 Nucleic acids which trigger also tumor-specific cell killing by activation of membrane-bound and cytosolic pattern recognition receptors have great potential for cancer immunotherapy approaches. For example, the intracellular delivery of double-stranded artificial RNA polyinosinicpolycytidylic acid poly(I:C)32 simulates a viral infection and mediates immune response via endosomal Toll-like receptor 3 (TLR3), cytosolic dsRNA-dependent protein kinase (PKR), or the melanoma differentiation-associated gene 5 (MDA5).29b,c,33 The cytosolic retinoic acid-inducible gene 1 (RIG-I) recognizes 5′-triphosphate-modified RNA (differing from the cap structure of natural mRNA). Attachment of this motif has boosted antitumoral action of siRNA.34

Figure 2. Common modification motifs for the chemical stabilization of synthetic nucleic acids.

cholesterolemia.16 Several more antisense therapeutics for the treatment of cancer or viral infections are currently in preclinical or clinical development stages. RNA interference (RNAi) therapeutics constitute the second class of therapeutic nucleic acids aiming at the shutdown of gene expression. It includes double-stranded (ds) short interfering RNA (siRNA)17 and micro RNA (miRNA).18 When delivered into the cytosol of target cells, one of the two 21−23 bases long RNA strands, the guide strand, is incorporated into RNAinduced silencing complexes (RISC) which can knock down disease-associated target mRNAs. In addition to the pure antisense recognition mechanism, which is based on the equimolar annealing of single-stranded oligonucleotides to target mRNA, siRNA RISC can mediate degradation of target RNA in a catalytic fashion. Subsequent to the discovery of RNA interference by dsRNA in nonmammalian eukaryotes,19 an analogous process was discovered in mammalian species.20 More than 1700 genetically encoded human miRNAs21 are naturally regulating about one-third of our genes by blocking translation, determining cellular fate and functions. For example, loss of tumor-suppressor miRNAs renders tumor cells to a more aggressive state. Therapeutic introduction of such miRNAs can compensate such deficiencies and reverse the malignant phenotypes. Conversely, endogenous tumor-promoting oncomiRNAs can be inactivated by antisense miR antagonists (antagomirs), which turn off the tumor-promoting activity, reactivating beneficial target gene expression.22 The list of siRNA and miRNA therapeutics in clinical development is steadily increasing.23 Tumor suppressor miR34 is downregulated in many human cancers leading to metastasis, antiapoptosis, chemoresistance, and tumor proliferation. A first synthetic miR-34 mimic delivered as lipid-based formulation has been clinically tested in patients suffering from primary liver cancer and liver metastasis from other cancers.24 In another oncological trial in patients with hepatic and extrahepatic metastases, the combination of two siRNAs, one targeting vascular endothelial growth factor A and the other one targeting kinesin spindle protein, was tested within a stable nucleic acid lipid particle (SNALP) formulation, providing evidence for gene silencing and encouraging clinical results.25 Transferrin-cyclodextrin-oligocation-based siRNA nanoparticles were tested for tumor-targeted delivery in humans, resulting in specific silencing of the target mRNA within tumor biopsies.26 Liver disease targets under clinical evaluation include gene silencing of apolipoprotein B (treatment of hypercholesteremia) or silencing of transthyretin (treatment of inherited TTR amyloidosis).23a Naked siRNAs were locally administered in clinical studies targeting viral infections or wet age-related macular degeneration (AMD).

2.4. Challenge of Nucleic Acid Delivery

The mentioned different kinds of therapeutic nucleic acids present a novel terrific arsenal for a specific and an effective fight against diseases if the delivery problem can be solved. All mentioned nucleic acids are far larger than conventional drugs. They cannot diffuse across lipid membranes into target cells and display very limited biological stability. Table 1 gives an overview over different classes of nonviral gene delivery systems including physical methods,35 polymeric or lipid-based transfecting agents,13,24,36 and inorganic nanoparticles.37 Elaborate reviews about the different approaches beyond polyplexes can be found elsewhere.38 Strongly depending on the size and nature of the therapeutic nucleic acid, three main delivery strategies can be considered:39 (i) chemical oligonucleotide backbone modifications, (ii) covalent conjugation with transport vehicles, and (iii) supramolecular assembly into nanosized formulations. Oligonucleotide-based drugs present far smaller molecules than DNA or RNA gene vectors. Like small drugs, they can be synthesized as precise molecules with chemical modification to improve transport across cellular membranes, stabilize them against degradation, improve their binding to complementary nucleic acid target sequences, and reduce the immunogenicity. These chemical modification strategies (Figure 2) are reviewed elsewhere.39a,40 Shortening of oligonucleotides may enhance

2.3. Protein-Interacting Therapeutic Nucleic Acids

Another class of therapeutic nucleic acids recognizes their molecular target by direct protein interaction and not via Watson−Crick base pairing. Aptamers are artificial nucleic acids D


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cellular delivery.41 Conformationally locked nucleic acids (LNAs), where chemical bridging locks ribonucleotides into the A form, display enhanced hybridization with target RNA. Thus, “tiny LNAs” of only eight nucleotides can effectively block complementary target miRNA.41b A recent exciting mode to reduce nucleic acid size presents the replacement of doublestranded siRNA by potent single-stranded siRNA (ss siRNA).42 This required the introduction of a 5′-vinylphosphonate as a metabolically stable mimic of 5′-phosphorylated RNA needed for RISC formation and optimal chemical stability of ss siRNA by introduction of alternating 2′-fluoro and 2′-methoxy nucleosides in the central strand, 2′-methoxyethyl at the ends, and phosphorothioates in most positions. Effective gene silencing by stabilized ss siRNA was reported in vivo after intravenous administration in the liver42a or upon intracerebral spinal fluid administration in the CNS of a Huntington disease mouse model.42b Despite various chemical modifications, even for short nucleic acids it is difficult to imagine versions of base sequence analogs which diffuse into cells as easily as successful small drug compounds. Therefore, the covalent conjugation with delivery domains such as targeting or membrane translocating compounds presents a second encouraging delivery option for oligonucleotide and RNAi therapeutics. Noncovalent complexation and encapsulation into carrier nanoparticles are interesting alternatives for medium-sized and large nucleic acid such as gene expressing DNA or mRNA. Lipid-based nanoparticles include liposomal encapsulations or nucleic acid complexes with cationic liposomes, termed lipoplexes.43 Polymer-based nanosystems include polymerosomes and polymer micelles, hydrogels, and complexes of nucleic acids with cationic polymers, termed polyplexes.43 The latter will be the main subjects of the current review. In 1965 Vaheri and Pagano reported the improved transfection of phenol-extracted purified poliovirus RNA with cationic diethylaminoethyl (DEAE) dextran.44 Three years later, the laboratory applied the same polycation for transfection of SV40 viral DNA, demonstrating 100 000 times enhanced infectivity.45 Our account reports on a lively history of 50 years of polymer-based nucleic acid delivery;46 it outlines the discovery of biological delivery challenges, resulting in subsequent technical breakthroughs to resolve some of these hurdles. The continuous refinement of involved macromolecular and supramolecular chemistry, leading to the current strategies for further development of polyplexes into multifunctional precise nanostructures, is reviewed.

Figure 3. Barriers in the nucleic acid delivery pathway of polyplexes. (A) Formation of stable polyplexes, (B) avoidance of rapid clearance and unspecific interactions with blood components, and (C) cellular barriers.

been experimentally evaluated also for systemic delivery. For example, subcutaneous administration of ALN-TTRsc, an antiTTR siRNA conjugated with synthetic trimeric N-acetylgalactosamine ligand targeted to the hepatocyte asialoglycoprotein receptor, demonstrated very promising TTR gene silencing in rodents and humans.47 Even for direct intravenous administration, extracellular barriers are substantial. Free unmodified nucleic acids are degraded in blood by nucleases. Moreover, nucleic acids are rapidly cleared from the bloodstream and would not reach the target cells. The polyplex formation process,48 based on the entropy-driven ionic interaction between the polyanionic nucleic acid and the multivalent cationic polymers, is able to produce nanoparticles containing the nucleic acid payload in compacted and protected form. Polyplexes can be as small as 6 nm (average hydrodynamic diameter of single polymer-complexed siRNA molecules), 49 25 nm (single complexed plasmid DNA molecules),50 or larger sizes of one to several hundreds of nanometers (containing multiple copies of the nucleic acid equivalent to hundred kilo base pairs of packaged nucleic acid).51 The polyplex size is a critical parameter for systemic administration, with a big impact on the biodistribution and pharmacokinetics. Nanoparticles with a hydrodynamic diameter of below 6 nm are rapidly cleared by the kidneys.52 Particle sizes of up to 400 nm can facilitate accumulation in highly vascularized solid tumors. The mechanism is based on the enhanced permeability and retention (EPR) effect of tumor tissues containing leaky blood vessels,53 which enable particle passage and entrapment into tumors. However, the extent of passive accumulation and the size threshold of the porous tumor vasculature depend on the type of cancer.54 For example, testing polymeric micelles with hydrodynamic diameters ranging from 30 to 100 nm, only the smallest were able to accumulate in poorly

3. DELIVERY PATHWAY OF POLYPLEXES For efficient intracellular delivery, several barriers are faced and have to be overcome by polymer-based nucleic acid carriers (Figure 3). In the following the different extracellular and intracellular delivery requirements are described. 3.1. Extracellular Delivery Requirements

For nucleic acid therapeutics, both localized and systemic drug actions have been considered. For local action, direct injection (such as into the skin, retina, central nervous system, or tumors) or topical delivery (such as to the lung epithelium via airway inhalation) has been performed. Intravenous administration has been the main investigated route for systemic delivery. Like for other biomacromolecular drugs, oral or intestinal delivery would place an additional serious barrier which currently prevents practical use of this route for systemic nucleic acids delivery. Intraperitoneal, intramuscular, and subcutaneous injections have E


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Figure 4. First-generation polymers for nucleic acid delivery. DEAE-dextran, diethylaminoethyl-dextran;44,45 PLL, poly-L-lysine; PLO, poly-L-ornithine; PLR, poly-L-arginine;68 ASOR, asialoorosomucoid.71

permeable pancreatic tumors, whereas all micelles showed comparable penetration in another tumor model.54c The stability of polyplexes in blood and other biological fluids presents another critical issue. The interaction with electrolytes, proteins, or cellular surfaces can cause partial or complete polyplex dissociation.55 Loss of delivery efficacy can be one consequence; binding of positively charged polyplexes with serum complement protein and activation of innate immune system,56 self-aggregation into larger microstructures, or aggregation of erythrocytes and other blood cells57 could also result in life-threatening conditions. Therefore, more stable packaging of polyplexes (for example, by introduction of hydrophobic elements or covalent cross-links within the particle core) and shielding the positive surface charges against undesired specific interactions with the bioenvironment are important measures to overcome these serious problems. A stabilized shielded nanoparticle may reach well-vascularized target sites via systemic circulation and in the case of certain tumors may even reside and accumulate there (“passive targeting”). However, subsequent target cell binding and uptake might be rather limited. Polyplexes with positive surface potential, which for example in cell culture can efficiently induce internalization by electrostatic interaction with the negatively charged cell membranes,58 have only limited applicability in vivo due the reasons mentioned above. Even more challenging, some target tissues are far less accessible via the bloodstream; for example, the blood vessel structure in the brain is extremely tight, forming a blood−brain−barrier (BBB) which cannot be passed by passive processes. For the numerous endothelial and other tissue barriers, nature has developed very efficient and specific intracellular and transcellular delivery processes which can also be utilized for therapeutic nanoparticle transport. For this purpose, the attachment of targeting ligands to the surface of polyplexes is a convenient approach to enhance (i) target cell binding and (ii) cellular internalization in a receptor-specific fashion. The selection of target receptor and a corresponding ligand has to be based on a series of considerations. This includes the abundance of receptors on the target cells and other cells as well as their function (for example, endocytosis, transcytosis). Tumor cells with a high metabolic activity and excessive proliferation frequently overexpress various growth factor receptors and receptors for intracellular uptake of nutrients, including transferrin receptor (TfR) or folate receptor. Also, the BBB expresses multiple receptors and carriers for transcytosis of nutrients across the endothelial barrier. The affinity of ligands for

the receptor, the concentration of competing endogenous natural ligands, possibly a multivalency of multiple ligands presented on one nanoparticle, and the nanoparticle size may strongly influence the performance of targeted delivery. In addition, the incorporation of receptor ligands for different receptors involved at subsequent stages of the delivery process (for example, transcytosis across an endothelial barrier, followed by cellular uptake within the target tissue) can be considered. 3.2. Intracellular Delivery Requirements

The entry across the cell surface membrane presents the first delivery challenge for macromolecules which cannot passively cross lipid membranes. Direct transfer into the cytosol is possible for smaller molecules via protein carriers or channels. Protein transduction is a special natural case, where proteins like the Antennapedia homeobox protein59 with specific transduction domains can directly translocate cell membranes and internalize into neighboring cells. Also, some enveloped viruses can directly deliver their payload into the cytosol by docking and fusing their membrane with the target cell membrane. Physical transfection technologies such as electroporation trigger transient pore formation in the cell membrane, enabling drug or nucleic acid to cross into the cytosol. The window between transfer efficacy and cytotoxicity usually is narrow, as cell membrane integrity is a strict requirement for cell survival. Nonenveloped viruses and protein toxins use an alternative, two-step cell entry process: first, engulfment into intracellular vesicles; second, escape from such vesicles into the cytosol. Lipid-free nanoparticles are supposed to employ the same route, though endocytic vesicle escape often is inefficient. Following ligand-dependent or -independent uptake by one of the manifold different natural endocytosis processes,60 the payload arrives in primary endosomes or other intracellular vesicles with various process-dependent fates. Receptor and vesicle type, nanoparticle size and multivalency of presented ligands, and receptor cross-linkage influence the subsequent vesicle trafficking and sorting in various compartments, such as recycling to cell surface or transcytosis, trafficking into Golgi organelles and endoplasmic reticulum (“retrograde transport”), or maturation into lysosomes which enzymatically degrade their content. Obviously, the latter fate has to be avoided for productive delivery of nucleic acids and other biomacromolecules. Strategies which nature has followed for virus and protein toxin delivery61 include two alternatives: (i) early escape out of endosomes such as for diphtheria toxin62 and many viruses or (ii) avoiding endolysosomal sorting, retrograde delivery into ER, F


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followed by cytosolic release, such as for cholera toxin63 or SV40 virus.64 Beyond cytosolic delivery, intracellular hurdles include protection of the nucleic acid payload against enzymatic degradation and delivery to the final site of action on one hand and release in bioactive form on the other hand. Polyplex stability does not necessarily correlate with nucleic acid biofunction often a compromise in assembly/disassembly properties is required.65 Thus, the polycationic core of polyplexes should have a dynamic function; it should stably protect the payload during the whole extracellular and intracellular delivery process but should liberate the nucleic acid at its target site. This site of action differs depending on the type of therapeutic nucleic acid. Geneencoding plasmid DNAs (pDNA) or splice-switching oligonucleotides have their destination in the nucleus, whereas the targets of mRNA, antisense, siRNA, or miRNA therapeutics are located in the cytosol. Cytosolic migration and nuclear entry is size dependent. Oligonucleotides can diffuse in the cytosol and passively enter the nucleus, whereas nucleic acids of approximately 250 bp or larger cannot passively move.66 In an experimental setup, direct intranuclear microinjection of naked transgene DNA resulted in gene expression in the majority of cells whereas the efficiency of cytosolic injection was less than 0.1%.35a For efficient delivery, the DNA has to be actively guided by direct transport (like for adenoviruses) or intravesicular transport along microtubules toward the nuclear envelope to be imported into the nucleus by active mechanisms or alternative pathways and be retained there in active form. Improved delivery of DNA into the nucleus by active transport domains has been the aim of several interesting studies.67 Despite partial successes, to date no efficient solution exists for intranuclear delivery of polyplexes and related DNA formulations.

polyornithine, polylysine, and polyarginine for the transfection of exogenous mammalian DNA. They concluded from these uptake studies that polyornithine performed best.68 In the early 1980s, following the seminal work by Capecchi35a of intranuclear microinjection of cloned gene constructs into cells, the first polymer-based transfections of functional recombinant gene expression constructs were performed.69 Spectroscopic and electron microscopy studies in this early period provided substantial information on polyplex structures. Oligocations and polycations not only bind but also condense nucleic acids into surprisingly regular nanostructures, including rods and toroids of virus-like dimensions.48 Interesting reports have been continuously published which improve our knowledge of such interesting fundamental processes.70 When polymer-based transfection was redirected from delivery of infectious viral genomes to plasmid-based gene constructs, the moderate transfection efficiency of polymers became a critical issue. Several directions were taken for improvement. George and Catharine Wu conjugated polylysine with asialoorosomucoid (Figure 4B), which they used in pDNA polyplexes for asialoglycoprotein receptor-mediated delivery to hepatocytes in vitro and in vivo.71 The investigators could demonstrate receptor specificity and improved cellular uptake by endocytosis. The success of the concept boosted many further ligand-polylysine-based strategies as reviewed by Zauner and colleagues,72 including also the first human clinical gene therapy studies with pDNA polyplexes.73 Another direction for polyplex improvement focused on the mechanism of the polycation-facilitated transfection process. A better understanding of the biological fate of transfected DNA would be helpful for further optimization. Investigators initially focused on cellular uptake and polyplex stability. However, neither DNA/polycation polyplex stability nor cellular uptake showed a perfect correlation with transfection activity. In 1983, Luthman and Magnusson74 demonstrated that chloroquine treatment during the first hours of polyoma DNA/DEAEdextran transfections considerably enhanced transfection. The antimalaria agent chloroquine accumulates in lysosomes, where it may reduce the activity of lysosomal enzymes. Blockade of DNA degradation by endolysosomal nucleases was assumed as the reason for the improved transfection, but the mechanisms were not completely clear. In 1984, Lopata and colleagues69c reported that a dimethyl sulfoxide or glycerol shock treatment of cells can strongly improve DEAE−dextran-mediated DNA transfection, resulting in a 50-fold improved high-level chloramphenicol acetyl transferase reporter gene expression. Such shock treatments were known to transiently destabilize or disrupt extra- and intracellular membranes. Polycations were known to be able to interact with lipid membranes and destabilize them. Collectively, the chloroquine and shock treatments however indicated that polyplexes had difficulties in crossing cellular membranes. It took researchers quite a long time to understand this key limitation. The problem became especially evident in transfections with polylysine-transferrin polyplexes,75 where the conjugated large serum protein transferrin prevents exposure of the polycationic charges at the polyplex surface. Despite very efficient transferrin-receptor-mediated intracellular uptake, polyplexes resided largely in endolysosomal vesicles, resulting in moderate gene transfer.75a,b Cotten and Birnstiel demonstrated that application of chloroquine (but not other endolysosomal inhibitors) dramatically enhanced gene transfer in erythroleukemic K562 cells.75c The chloroquine effect was particularly effective in erythroid cells due to an earlier low pH of

4. CATIONIC CORE POLYMERS Cationic polymers can be regarded as perfect noncovalent interaction partners for nucleic acids, providing similar size dimensions but opposite ionic charge. Our own life cycle, from oocyte and sperm cells, dividing cells to resting cells, is under impressive control of various polycations which compact the genome of every cell in a perfectly regulated manner. Polycations are also logical carriers for therapeutic nucleic acid delivery, because they inherently contain the majority of functions to fulfill the mentioned extracellular and intracellular delivery requirements. They can bind, condense and protect, and release nucleic acids; they can bind cell surfaces, trigger intracellular uptake, and mediate endosomal membrane destabilization required for cytosolic release and delivery to the intended site of action. Polymers however cannot fulfill these crucial actions in the temporal sequence required in the sophisticated delivery process. Moreover, within the bioenvironment they are subjected to many undesired interactions, resulting in loss of activity and toxicity. Five decades of polyplexes provided empirical and rational advances on the central question “How to tailor cationic polymers for delivery?” As outlined below, better knowledge of the biological challenges and improved macromolecular chemistry contributed to design of polycations with strongly improved delivery characteristics. 4.1. First-Generation Polycations

First-generation polymers are depicted in Figure 4. Since 1965 DEAE-dextran-based transfection44,45 was found to be very useful for the delivery of various infectious viral genomes. In 1975 Farber and colleagues evaluated DEAE-dextran, spermine, G


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5 in endosomes.76 Later, a glycerol shock which stimulates endosomal burst also was found to boost polylysine-based transfections in fibroblasts.77 Use of other endosome-destabilizing domains and agents is reviewed in a subsequent section of the review. The effect of chloroquine78 is largely based on the natural acidification process of endosomes by the vacuolar ATPdependent vATPase. vATPase generates a proton gradient with a lumenal pH between approximately 5.9 and 6.5 in early endosomes (sorting endosomes and endocytic recycling compartment) and down to pH 5.0 in late endosomes and lysosomes.79 The weakly basic drug chloroquine in its unprotonated form can diffuse across lipid membranes into the cell, where in acidifying endosomes and lysosomes it is entrapped as protonated form. With continued vATPase activity, the protonated drug increasingly accumulates in the vesicles and influx of chloride counteranions via specific carriers and water result in osmotic pressure and swelling of endolysosomes, triggering membrane destabilization in the presence of polyplexes. Optimum dosage of chloroquine is difficult due to a narrow window between efficacy and cytotoxicity. In their search for better transfection agents, Jean-Paul Behr and collaborators wanted to utilize the endolysosomal protonation in a polymer-based way and screened cationic polymers with proton-sponge characteristics.80 Polyamines which are only partly protonated at neutral extracellular pH would still bind nucleic acid by interelectrolyte interactions, but they would increase their protonation and charge density within acidifying endosomes. This process should destabilize the polyplex containing endosomal vesicle. The criteria would not be fulfilled by the classical transfection polymers polylysine, polyornithine, or polyarginine due to their already complete cationization at physiological pH which enables nucleic acid binding but not endosomal buffering. The criteria also would not be fulfilled by polyhistidine, which can act as buffer but is not sufficiently protonated at neutral pH for nucleic acid binding. This hypothesis-driven approach resulted in the discovery of polyethylenimine (PEI) (Figure 5) as a very potent and commonly used transfection polymer.80 The transfection efficiency is relatively high both in vitro and in vivo at dosages where the polymer displays no or only moderate cytotoxicity. Optimized versions of PEI and derivatives have already been applied in human clinical studies in localized applications for cancer treatment and anti-HIV vaccination.81 PEI can be generated in various branched and linear forms of different molecular weight ranges, which allows tuning of polyplex stability, polymer efficacy, and toxicity.65a,82 Since the nucleic acid has to be liberated at the intracellular site of action, high polyplex stability does not necessarily correlate with transfection. Intracellular trafficking studies of pDNA polyplexes with poly-Llysine (PLL), linear PEI (LPEI), or branched PEI (BPEI) revealed that, in contrast to PLL, both LPEI and BPEI were capable of mediating endosomal escape, but the subsequent disintegration of LPEI/pDNA polyplexes correlated with a higher and faster transgene expression, as compared with BPEI polyplexes with restricted cargo release.65a The apparently more flexible LPEI polyplexes were also far better in intranuclear delivery in nondividing cells, whereas BPEI polyplexes required dividing cells in or before mitosis (associated with the breakdown of nuclear envelope) for efficient transfection.67f,g,i Significant evidence83 supports the original proton sponge hypothesis by PEI and related transfection agents (Figure 5B). The process was found to be strongly dependent on the V-

ATPase-driven proton transport; specific inhibitors such as bafilomycin blocked the transfection, whereas chloroquine which itself becomes protonated has a rather beneficial effect on the transfection. Free PEI molecules, which are not attached to polyplexes, nevertheless support the endosomal escape by codelivery with the polyplexes. Intracellular vesicle studies have confirmed delayed kinetics of acidification, chloride accumulation, and occasional disappearance of vesicles (presumably by rupture). Some debate about mechanistic details of the proton sponge hypothesis has been ongoing, resulting in refined mechanistic models.39g,46v,84 For example, Benjaminsen and colleagues84 reported that the PEI proton sponges do not buffer lysosomes, i.e., do not significantly change the lysosomal pH. This however does not contradict but rather supports the model that cationization of PEI amines takes place. Important critical calculations indicate that the osmotic pressure built up by the proton sponges would not suffice to blow up an endosomal vesicle. Moreover, some cationic DNA binding polymers with proton sponge properties do not mediate transfection;85 even PEI polyplexes when surface shielded by PEG molecules lose their transfection activity, and the activity is recovered using PEG molecules which are removable in endosomes.86 These findings are consistent with hypothetical models where, in addition to the osmotic consequence of proton sponge effect, a protonationdependent higher cationization of the PEI polymer triggers direct endosomal phospholipid membrane interaction and permeabilization. Such lytic effects have been observed with high-density polycations and plausibly could synergize with an effect based on increase of osmotic pressure and membrane tension. A series of other synthetic transfection polymers was evaluated in the 1990s, including poly(N-alkyl-4-vinylpyridinium) salts,87 poly[(2-dimethylamino)ethyl methacrylate] and poly[(2trimethylamino)ethyl methacrylate],88 and dendritic polyamidoamines (PAMAM dendrimers).89 Because of their high charge density PAMAM dendrimers possess proton sponge activity and however also significant generation-dependent cytotoxicity. Linear 10 kDa amphoteric PAMAM were found to possess good biocompatibility and favorable transfection activity.90 Building on the proton sponge hypothesis, histidines or other imidazole derivatives with a pKa of around 6 have been incorporated into polymers such as polylysine, PEI, or dendrimers to increase their endosomal buffer capacity. Various reports demonstrate the beneficial effects of these buffering units on transfection efficiency.91 Polymers like PEI and PAMAM are valuable transfection reagents but display drawbacks: they are nondegradable and significantly toxic in a molecular weight-dependent manner. As analyzed in detail for PEI,92 cytotoxicity includes generation of defects in cell surface and mitochondria or nuclear membranes, triggering necrosis and apoptosis, and inhibition of mitochondrial ATP synthesis. In addition to direct cellular toxicity, many polycations such as PEI, PAMAM, or polylysine react with the innate immune system and trigger complement activation as demonstrated in vitro.56a Recently, intravenous administration of PEI in pigs demonstrated analogous complement effects in vivo, triggering strong anaphylactic reactions.56b Realizing such possible problems, both papers introduced PEGylation as a solution to block the complement activation.56 This type of masking cationic charges has been an intensively studied subject for shielding polyplexes, as reviewed in a subsequent section. In summary, the lessons learned from first-generation transfection polymers were applied in later developments. The observed cytotoxicity should be reduced by generation of (i) H


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repeated administrations. It had been found that the cytotoxicity of polycations such as polylysine, PEI, or PAMAM dendrimer is highly molecular weight dependent. For example, PEI low molecular weight (LMW) 800 (less than 20 monomer units) or PAMAM dendrimer generation 4 polymers display low cytotoxicity but also low transfection activity, while PEI 22K or PAMAM generation 6 dendrimers are far more potent but significantly toxic. Thus, novel strategies aimed at ligation of welltolerated LMW polymers by bioreversible linkers into larger polymer conjugates with improved capacity for stabilizing nucleic acid polyplexes.46l,107 For bioreversible cross-linkage of LMW PEI (Figure 8), ester bonds,108 disulfides,109 ketals,110 imines,111 polyglutamic acid amide, and other amide linkages112 were applied. Modification of the polymer backbone with either ester bonds hydrolyzable in many extra- and intracellular locations, endosomally cleavable acetal or imine bonds, or disulfide bonds bioreducible in the cytosol greatly reduced cytotoxicity but retained or even enhanced transfection efficacy. Instead of introducing biocleavable links into known effective transfection polymers such as PEI, also novel polymers with biodegradable backbones were designed (Figure 9). In early pioneering work of the laboratory of Sung Wan Kim113 biodegradable and nontoxic poly(alpha-(4-aminobutyl)-L-glycolic acid) (Figure 9A) was designed. Formally it represents the polyester analog of polylysine (with the amide bonds replaced by ester bonds). A 2-fold-enhanced transfection activity over polylysine was obtained with reduced cytotoxicity. Like for polylysine the efficacy was still moderate, consistent with the lack of efficient endosomal escape functionality. More effective betaamino ester-based polymers were synthesized by Michael addition of amines to acrylate esters. On the basis of TRIS (tris(hydroxymethyl)methanamine), hyperbranched networktype poly(amino esters) termed n-PAE (Figure 9B) were generated, which showed efficacy and endosomolytic properties equivalent to PEI but far better biocompatibility.114 Also, linear beta-amino ester polymers were optimized as effective transfection polymers by reacting 4-aminobutanol with 1,4-butanediol diacrylate or 1−6-hexanediol diacrylate.115 This development in the laboratory of Robert Langer was followed by another conceptual milestone; the Michael addition approach was extended into a library design, where a series of primary amines or secondary diamines were reacted with a variety of bisacrylates. Parallel synthesis generated more than 100 polymers and, including semiautomated processes, more than 2000 novel beta-amino polyesters (Figure 9C).116 The strategies were extended to other library chemistries such as addition of aliphatic hydrocarbon-linked epoxides, which in combination with highthroughput screening resulted in the generation of a series of hydrophobic oligoamines termed lipidoids used for siRNA delivery.36e,117 The Michael addition strategy was applied by the groups of Kim, Feijen, and Engbersen for the generation of disulfide-based bioreducible polymers.46r,118 Cystamine bis-acrylamide was reacted with small oligoethylenimine building blocks as amine reactants, including triethylene tetramine (Figure 10A). Another interesting synthetic strategy to such bioreducible oligoamines was followed by Lu and colleagues who first used solid-phaseassisted assembly for generating defined oligomers (Figure 10B) containing triethylene tetramine, histidines, and two terminal disulfide-forming cysteines which were subsequently oxidized into polymers.119

nature-derived biopolymers, (ii) analogs of existing effective polymers which can biodegrade into lower molecular weight fragments, (iii) novel well-tolerated polymer backbones and detoxifying modifications of effective polymers, and (iv) precision polymers and sequence-defined oligomers. 4.2. Nature-Derived Polymers

Transfection agents based on natural polymers have the inherent advantage of biodegradability and increased biocompatibility.46h Protein-based carriers include chromosomal proteins such as unmodified or modified histones93 or HMG1.93c,94 Also, collagen-derived gelatin95 and atelocollagen96 have been the proteinaceous basis of nucleic acid carriers. Atelocollagen/siRNA nanoparticles were found effective in systemic delivery into bonemetastatic tumors. Other nature-derived polymers like DEAE-dextran (see previous section) are based on carbohydrates (Figure 6). Chitosan (poly-D-glucosamin, generated by deacetylation of chitin) and derivatives have been successfully applied for pDNA and siRNA delivery by numerous investigators.97 Quarternization of chitosan amines by methylation and PEGylation resulted in improved formation and colloidal stabilization of pDNA polyplexes.97f,j Additional modifications with beneficial effects include thiolation and targeting ligands for enhanced cellular uptake97d,i or histidinylation for improved endosomal escape.97k Cyclodextrin (CD) conjugates were developed based on two important characteristics of these molecules: the biocompatibility of these degradable molecules and the option to act in host−guest complexes. The efficacy of PAMAM dendrimers was enhanced by conjugation with alpha-, beta-, and gammacyclodextrins.98 Conjugation of linear and branched PEI with beta-CD reduced the cytotoxicity of pDNA polyplexes without reducing the transfection efficiency.99 Treatment with adamantyl-PEG,100 which inserts by CD-adamantane host−guest interaction, resulted in PEGylated PEI polyplexes used for systemic gene transfer to the liver. Recently, analogous low molecular weight PEI-CD conjugates were used for formation of pDNA polyplexes which were loaded with an adamantylmodified targeting peptide for the FGF receptor and adamantyl-disulfide-linked PEG for shielding.101 Hwang et al.102 synthesized linear cationic polymers of β-CDs cross-linked by amidine groups. The CD moieties mediated a far higher biocompatibility than the analogous CD-free polycations. Further on, the CD moieties of these polycations were used for docking the targeting protein transferrin via adamantane guest− host interaction.103 This polyplex design has been further developed into therapeutic transferrin-receptor-targeted, PEGshielded siRNA polyplexes104 which were also evaluated in human clinical trials, demonstrating gene silencing in distant tumors (Figure 7).26 Polycationic CDs were also generated by direct introduction of heptakis-pyridylamino substitutions.105 The degree of cationic modification is critical for nucleic acid polyplex formation. In this respect, the unique characteristics of α-CDs can be utilized to form polyrotaxanes, with monocationic CDs assembled on biodegradable linear polymer chains.106 Thus, degradable polycationic polyrotaxane polymers for DNA transfection were generated. 4.3. Biodegradable Polymers

The design of biodegradable polymers may serve as a solution for two problems: elimination of acute toxicities of larger polycations occurring within a few hours to days after administration and reducing negative long-term effects which might potentiate in I


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4.4. Well-Tolerated Copolymers and Dendrimers

comparison of linear, hyberbranched, and perfectly dendritic polylysines were made.130e Branching favorably improves transfection properties by altering amine pK values and improving endosomal buffering. However, amidation of lysines at both the α and the ε amino groups results in loss of enzymatic biodegradability and increase of cytotoxicity of polymers. Hyperbranched polylysines still contain enzymatically cleavable lysine chains and thus display best overall transfection/ biocompatibility properties. Dendritic carriers have also been designed by Haag and colleagues, who used well-tolerated dendritic oligoglycerol cores which they modified with oligoamine shells. These carriers displayed encouraging activity for siRNA transfection.132 Similarly, Russ et al. started with a LMW branched PEI or a PPI dendritic core which was modified by degradable hexanediol diacrylate ester linkages with a cationic oligoamine shell, including spermine or oligoethylenimine.133 These pseudodendritic structures displayed lower polydispersity than randomly cross-linked polymer preparations, low cytotoxicity, and good biodegradability and in mouse models in vivo gene transfer into tumors.

In parallel to the development of novel biodegradable polymers, the collected information about existing polymers resulted in approaches to tune their biocompatibility and also potency by chemical derivatization. In this respect, the synthesis of block copolymers by incorporation of noncationic hydrophilic segements such as PEG or poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA) strongly reduced the cytotoxicity of the generated polycations.120 Cationized polymethacrylates88a−c (Figure 11), such as poly[(2-dimethylamino)ethyl methacrylate], pDMAEMA, or poly[(2-trimethylamino)ethyl methacrylate], pTMAEMA, can be prepared with convenient yield and size distributions. Coblocks of PEG or pHPMA improved the biocompatibility.88b−e Apart from stable polymethacrylate polycations, also acid-degradable analogs were designed for pDNA polyplex formation, which under endosomal acidic conditions release the cationic groups from the polymer backbone, resulting in release of the cargo pDNA and transfection.121 The cationic block copolymer p(HPMADMAE)-b-PEG, with pHPMA modified at the hydroxyl group with dimethylaminoethyl carbonate, presents another interesting biodegradable version generated by the group of Wim Hennink.122 Introduction of N-(pyridyldithioethyl) methacrylamide (PDTMA) as (approximately 15%) copolymer units resulted in p(HPMA-DMAE-co-PDTEMA)-b-PEG cationic materials which after pDNA polyplex formation can be crosslinked with dithiols into disulfide-stabilized polyplexes (Figure 11D). Due to the lability of the carbonate esters, it was possible to remove the cationic DMAE groups by treatment at pH 8.5 and 37 °C for 6 h, retaining decationized polyplexes stabilized merely by the remaining disulfide cross-links.122 Decationized pDNA polyplexes showed excellent biocompatibility as well as enhanced blood circulation times and tumor accumulation when compared with their cationic precursors. Kataoka and colleagues optimized PEG-poly(aspartic acid) for nucleic acid delivery by amidation with various oligoamines (Figure 12) including diethylene triamine (DET).46v,65b,123 In this development they made a series of very notable observations: the oligoamide modification triggered enhanced biodegradation of the pAsp backbone by transamidation; the PEG-containing PEG-pAsp(DET) showed high biocompatibility and better tissue diffusion properties as demonstrated in three-dimensional cell spheroid cultures;123a polymers containing even units of protonatable aminoethane units (such as DET) showed best efficiency in pDNA transfections,123e whereas odd repeats display best efficacy for mRNA delivery.65b Also, statistical modifications of existing polymers have demonstrated advantageous potential. For example, the transfection efficacy of 25 kDa branched PEI for siRNA is limited. Simple derivatization of 10% of PEI nitrogens by succinylation,124 20% amidation with tyrosine residues,125 or pyridylthiourea grafting126 converted PEI 25 kDa into a potent siRNA transfection agent. Because of their smaller and globular size, also modified dendrimers and dendrimer-like carriers provide an opportunity for better tolerated transfection agents.127 Different dendrimers based on polyamidoamine (PAMAM),89a,c,128 polypropylenimine (PPI),129 or dendritic polylysine130 were generated. Transfection efficacy but also cytotoxicity was largely dependent on the dendrimer generations. Conjugation of dendrimers with a large protein ligand such as transferrin reduced cytotoxicity but maintained the transfection activity, enabling also systemic in vivo gene delivery.131 For polylysine, analyses and direct

4.5. Precision Polymers and Sequence-Defined Oligomers

As described in the previous sections, several barriers have to be overcome for effective nucleic acid delivery. Just like natural viruses, the artificial polymer-based nucleic acid carriers have to be responsive to a changing bioenvironment, and several different functional elements may have to be conjugated in a reversible manner. The properties of a resulting macromolecular conjugate are not determined only by the presence or absence of certain functional subunits. The exact size, topology (linear, branched, comb, hyperbranched, dendritic), number, and attachment sites of subunits can play a decisive role for the biological activity.82a,c,130e,134 Therefore, the production of multifunctional materials requires a precise synthetic strategy, both for pharmaceutical reasons and for the necessity to identify clear-cut structure−activity relationships. Several of the abovedescribed multifunctional polymer preparations suffer from the problem of insufficient precision: polymer conjugates often represent inhomogeneous mixtures in macromolecule sizes, topologies, and chemical conjugation sites. Improved polymerization chemistries such as controlled radical polymerization and specific ligation strategies result in a narrow polymer size distribution, low polydispersity, controlled architectures, and subunit composition.135 Such processes already made their way to the generation of multifunctional, “well-defined” polymers for nucleic acid delivery.136 Nevertheless, at the molecular nanolevel, parameters such as the exact number and order of monomers and attached subunits are still hard to control in polymerization reactions. Nature has developed a compelling evolutionary mechanism: the natural nucleic acids and proteins present macromolecules with unique sequence-defined precision. Also, for artificial transfection polymers with multiple different subunits, a maximal degree of precision might be achieved by “sequence definition”, where a defined monomer sequence can be used for the unique macromolecule identification and definite discrimination between isomers. Such a sequence provides all compound information and descriptive parameters, including the monodisperse molecular weight, exact monomer order, orientation, and topology. Especially in the context of synthetic materials for clinical application and their regulatory compliance, J


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Figure 5. “Proton sponge” polymers. (A) First proton-sponge transfecting agents include polyethylenimine in linear (LPEI) or branched (BPEI) form and poly(amidoamine) (PAMAM) dendrimers (generation 1 is shown for simplicity reasons). (B) Schematic illustration of the hypothesized protonsponge effect mediating endosomal escape due to buffering and osmotic vesicle swelling and cation-triggered membrane disruption.83

ornithine within defined linear or branched structures was obtained.141a,b Incorporated cysteines provided polyplex stabilization by covalent disulfide cross-links;141c,g endosomalbuffering histidines or amphipathic membrane-destabilizing peptide motifs141d−j triggered improved pDNA and siRNA delivery by endosomal escape. Analogous to classical peptide synthesis, together with natural amino acids also artificial building blocks such as triethylene tetramine or fatty acids were incorporated.119,142 Laura Hartmann, Hans Börner, and collaborators adopted solid-phase peptide synthesis for the assembly of completely unnatural building blocks.143 Instead of using protected amino acids, alternating coupling of diamines (3,3′-diamino-N-methyl-dipropylamine or a bis tBoc-protected spermine) and a diacid (succinic acid anhydride) yielded sequence-defined oligo(amidoamines). Optionally, at a precise position a disulfide linkage or a terminal PEG chain was introduced, and the precise oligomers were used for pDNA polyplex formation. To obtain the best compatibility of sequence-defined oligomer synthesis (Figure 13) with standard automated Fmoc peptide synthesis, Schaffert et al. designed artificial oligoamino acids with appropriate internal tBoc and amino-terminal Fmoc protecting groups.144 These amino acids (Figure 13A) contain 3−5 repeats of the aminoethane motif which is also the proton sponge basis of the efficient transfection polymer PEI. These novel building blocks can be assembled with classical peptide synthesis methodology (simplified illustration in Figure 13B) in combination with commercially available Fmoc α-amino acids into a multitude of defined oligo(ethanamino)amide sequences and topologies.49,134c,144b,145 Reported oligomer architectures (Figure 13C) which were realized with the solid-phase synthesis (SPS) strategy include oligomers with linear,134c,145a twoarm,145a three-arm,145a,c four-arm,144b,145a,d and comb architectures134d as well as PEGylated two-arm compounds with targeting ligands.49,145d,e Branching points were introduced by lysines, which provide two amines (α, ε) after deprotection during synthesis. Terminal cysteines served for the bioreversible lateral polyplex stabilization based on the formation of

this high degree of precision, reproducibility, and compound identification is desirable. Like in the process of translation of a RNA sequence into a protein, the sequence-defined mechanism requests a sequential assembly of the macromolecule on a template. An intriguing artificial concept of template-assisted synthesis has been recently reported, where monomers are converted into sequence-defined oligomers in a single step due to a sequence-specific prearrangement at a DNA template.137 More practically, the classical solidphase peptide synthesis, as developed by Robert Bruce Merrifield,138 presents a highly efficient example of a sequencedefined oligomer assembly controlled by timely feeding the different building blocks to the solid-phase-attached growing macromolecule. Over the last five decades solid-phase-assisted synthesis has made tremendous progress. Nowadays synthesis of peptides and oligonucleotides is routine, and the synthesis of whole proteins such as the glycoprotein erythropoietin139 and even a whole bacterial DNA genome140 has been achieved. Nature has demonstrated how to solve the challenge of “multifunctionality with high precision”; the intracellular delivery domains included in viruses or protein toxins have been optimized by biological evolutions of such sequences. An analogous chemical sequence-based evolution process could be applied to the carrier polymer design. First, building blocks have to be identified which serve as microdomains useful in a particular delivery process; these building units can be artificial compounds as already used in polymer synthesis or can be natural protected amino acids or lipids. These units have to be assembled into various defined macromolecular sequences and topologies and be evaluated in relevant delivery assays. The structure−activity relations obtained with these precise oligomers can be utilized for the next round of carrier design and selection. Various researchers have already implemented elements of such a “chemical evolution” process. First, sequence-defined structure−activity relations were observed for peptide-based transfection agents.141 Important information about required numbers and sequence effects of nucleic acid binding amino acids such as lysine, arginine, or K


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Figure 6. Nature-derived cationic polymers. (A) Chitosan,97 (B) conjugate of α-, β-, or γ-cyclodextrin with a G2 PAMAM dendrimer (schematic illustration),98 (C) conjugates of β-cyclodextrin with branched and linear PEI.99

5.1. Shielding Domains

bioreducible disulfides. Optionally, hydrophobic domains consisting of bis (acyl)-modified lysines145a−c or tyrosine trimers145f were included for hydrophobic polyplex stabilization. Because of the precision of the chemical design, which was not available for classical polymers such as PEI, simple questions on structure−activity relationships could be addressed. Linear sequences of the building block Stp (succinyl tetraethylene pentamine, containing three protonatable nitrogens per unit) were generated to investigate the effect of increasing molecular weight of linear PEI-like oligomers.134c Beyond a critical length of more than 10 Stp units (representing 31 protonatable nitrogens and 50 nitrogens in total), efficient pDNA polyplex formation and gene transfer was found. At an optimum length of 30 Stp units (representing 91 protonable nitrogens and 150 nitrogens in total), a 6-fold higher transfection efficiency was observed compared with standard linear PEI 22 kDa, which contains approximately 500 (±200) protonable nitrogens. Cytotoxicity was very low but molecular weight dependent and 10-fold lower for the larger oligomers with 20−40 Stp units as compared with linear PEI.134c

Efficient complexation of nucleic acids with a surplus of polycations usually results in formation of nanoparticles with positive zeta potential. In cell culture transfections this positive charge can be advantageous for binding negatively charged cell surfaces58 and also may help in endosomal escape.39g,46v In the extracellular space, this positive surface charge of polyplexes however mediates many undesired reactions, especially in intravenous administration.55−57 Acute toxicity triggered by partial dissociation and aggregation of nanoparticles and blood cells, unspecific interactions with complement and other blood components, or nontarget cells can be reduced by providing polyplexes with a coat of hydrophilic macromolecules, which shield the surface potential from the exterior environment (Figure 14).146 The most common shielding agent has been PEG.54b,86,88e,97d,100,103,120b,d,123a,b,145d,147 PEG-polylysine/ pDNA polyplexes were the first polymer-based formulations which were evaluated in human clinical in vivo gene therapy studies in epithelia of cystic fibrosis patients.147j,l Alternative hydrophilic polymers used in the context of nucleic acid delivery are pHPMA,88b,e,136b,146,148 hydroxyethyl starch (HES),149 hyaluronic acid,150 or polysarcosine (Figure 14A).151 Stable or reversible hydrophilic coatings have been incorporated into polyplexes in several ways; strategies for both pre- or postintegration of hydrophilic polymer blocks have been pursued. For preintegration, block-co-polymers such as PEGpLys, pHPMA-pTMAEM, or PEG-pAsp(DET) were used in

5. FUNCTIONAL DELIVERY DOMAINS Polymer backbones may (or may not) inherently fulfill several nucleic acid delivery functions. In the following, the current state of the art in improving individual specific delivery tasks is reviewed. L


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Figure 7. Schematic illustration of a self-assembling multicomponent siRNA delivery system based on electrostatic nucleic acid complexation and cyclodextrin/adamantane host−guest interaction.104

Figure 8. Biodegradably cross-linked oligoethylenimines. (A) Ester bonds,108 (B) imines,111 (C) disulfides,109 (D) ketals,110 and (E) polyglutamic acid amides.112a Biodegradable cross-linking elements are indicated in red.

polyplex formation. This type of block-co-polymers was the first one evaluated in a human clinical in vivo polyplex study, treating

airway epithelial tissue with PEG-pLys/pDNA polyplexes.147j−l The block copolymers have the inherent capacity to form “core/ M


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Figure 9. Biodegradable cationic polyesters. (A) Poly(α-(4-aminobutyl)-L-glycolic acid) (PAGA),113 (B) network poly(amino ester) (n-PAE),114 and (C) combinatorial synthesis of β-amino polyesters by Michael addition.115,116

Figure 10. Disulfide-based bioreducible cationic polymers. (A) Cross-linked oligoamines obtained by Michael addition to cystamine bis-acrylamide118 and (B) defined oligoamine-peptide conjugates assembled on solid phase and subsequently cross-linked by oxidation.119

shell polymer micelles”152 containing an inner cationic core neutralized by the nucleic acid and an outer hydrophilic polymer shell (Figure 14B). The length and ratio of hydrophilic to cationic polymer within the block copolymer controls polyplex characteristics (Figure 14C).49,153 For example, larger compacted polyplexes are formed with PEG−PEI at a lower PEG: PEI ratio, whereas at high PEG:polycation ratios and lower chain length of the polycation segment the compaction of nucleic acids, proceeding by close alignment of several polyanionic nucleic acid helices using the polycation as “electrostatic glue”,70 is prevented. Monomolecular copolymer-decorated rod- or spaghetti-like structures (depending on the number of nucleic acid base pairs) are obtained. For small nucleic acids such as siRNA a rod

length of only 8 nm might be favorable,49 for pDNA spaghettilike structures of several hundred nanometers in length suffer from suboptimum compaction.145e In postintegration strategies, first nonshielded polycation/ nucleic acid polyplexes are formed, and in a second step the hydrophilic shell is subsequently attached. Thus, the compaction of nucleic acid is not compromised by the shielding polymers. Subsequent incorporation of the hydrophilic coat may take place via ionic electrostatic noncovalent association150,154 or monocovalent (for PEG) and multicovalent (in the case of pHPMA) attachments. The downsides of postintegration strategies may appear in scale-up, for example, less chemical control on the coat attachment as compared to the use of block copolymers, possibly N


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Figure 11. Cationized poly(methacrylates) used for nucleic acid delivery. (A) pDMAEMA homopolymer,88a (B) random copolymers of cationic DMAEMA or TMAEM and neutral hydrophilic comonomers,88c−e (C) block copolymers,88b,e,122 and (D) three-step assembly of decationized polyplexes with p(HPMA-DMAE-co-PDTEMA)-b-PEG.122 DMAEMA, N,N-dimethylaminoethyl methacrylate; TMAEM, N,N,N-trimethylaminoethyl methacrylate; NVP, N-vinylpyrrolidone; HPMA, N-(2-hydroxypropyl) methacrylamide; PEG, polyethylene glycol.

requesting improved polyplex purification methods for removal of nonincorporated reagents. Hydrophilic polyplex shielding reduces nanoparticle aggregation, strongly improves biocompatibility, and significantly extends blood circulation time,147d,g however not to the extent as has been observed for correspondingly modified polymeric and liposomal drugs. Lateral polyplex stabilization by additional covalent cross-linkage155 or, as recently demonstrated, decationization122c improves polyplex circulation times. Stable polyplex shielding from unspecific extracellular interactions however may also reduce intracellular efficacy in transfecting the target cells (“PEG dilemma”). For example, PEGylation of PEI polyplexes strongly reduced their transfection activity, which was recovered by introducing endosomal pHsensitive pyridylhydrazone bonds for PEG attachment.86 Other pH-sensitive links which generated favorable polyplex characteristics include acetals110b,156 and dialkylmaleic acid monoamides.157 5.2. Targeting Ligands

Figure 12. Polyaspartimides with different oligoamine substitutions in the side chain.65b,123 Neighboring protonatable amines are indicated in blue. The classification according to an even or odd number of protonatable amines per oligoamine segment has an impact on the endosomal buffering characteristics and transfection efficiency.

The introduction of biochemical asialoorosomucoid-poly-Llysine conjugates for pDNA polyplex formation and receptormediated gene transfer into hepatocytes presented a conceptual breakthrough.71 Early on several other investigators started to extend the strategy to other receptor−ligand systems.75,158 Transferrin, an iron transport protein which targets the O


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various target tissues; details can be found in reviews.159 Ligands (Table 2) range from small chemical compounds such as vitamins or drugs49,122b,158c,160 and carbohydrates157b,161 to synthetic peptides,145e,162 proteins,163 antibodies,164 and aptamers.165 Ligand effects go beyond specific cellular receptor binding; the extent and kinetics of nanoparticle uptake by endocytosis (Figure 15) as well as the intracellular fate can be modulated by the selected ligand(s) and multivalency of their presentation at the polyplex surface.166 5.3. Endosomolytic Functions

The endosomal escape is considered to be a major limitation for the intracellular delivery of polyplexes.167 After cell entry by endocytosis, polyplexes are entrapped in vesicles and have to be released to reach their cytosolic or nuclear site of action in intact form and to evade degradation in late endo- and lysosomes or recycling to the cell surface. The strongly beneficial effect of the hydrophobic weak base chloroquine, which accumulates in acidic endolysosomal vesicles, on DEAE-dextran or polylysine polyplexes (see section 4.1) indicated the great endosomal bottleneck. Hypothesized mechanisms for the enhanced endosomal escape by chloroquine are vesicle swelling and the generation of osmotic pressure, the inhibition of endolysosomal maturation due to pH buffering, and a direct effect on nucleic acids by intercalation.78 Chloroquine-related quinolone derivatives have been directly incorporated into peptide-based nucleic acid carriers.142b The improved endosomal escape of proton sponge polymers such as PEI is based on similar effects. Incorporation of histidine residues (which provide excellent buffer capacity around pH 6) further improve endosomolytic proton sponge characteristics (Figure 16) and were found to greatly improve the transfection efficacy of various transfection polymers, including polylysine91a,c,134e,168 or PEI91e,f and several other carriers.134d,141g,145d,169 As commented in an early article by Jean-Paul Behr,80b the “proton sponge is a trick to enter cells the viruses did not exploit”. The differing but very potent alternative viral strategies also have been exploited to improve endosomal escape of polyplexes. Many viruses possess specific endosomal membrane-destabilizing domains. The presence of noninfectious viral particles such as inactived adenoviruses170 or rhinovirus particles171 during transfection of receptor-targeted polylysine polyplexes enhanced efficiency up to more than 1000-fold. Entering via their own endocytosis pathway, viruses met internalized polyplexes and facilitated their cytosolic release. To ensure the colocalization, polyplexes were directly coupled with viral particles via various biochemical linkages.172 In logical continuation, instead of whole virus particles, synthetic virus-derived or artificial membraneactive peptides were incorporated into polyplexes.167,173 These included pH-responsive rhinovirus VP1 peptide171,174 or influenza virus hemagglutinin HA2 derived INF peptides,49,161a,b,174,175 apitoxin-derived lytic melittin peptidic derivatives,141i,176 or amphipathic artificial sequences such as KALA or EALA repeats.89a,141h Alternatively, endosomal pH-triggered membrane destabilization by hydrophobic polymer or lipid domains142a,145a,c,156b,157b,177 has resulted in pH-specific lytic activity and enhanced cytosolic delivery of the cargo. A further, physically triggered strategy termed “photochemical internalization (PCI)” is the photoinduced endosomal escape, where photosensitizers located together with polyplexes in endosomal vesicles upon irradiation very fast and effectively release endocytic materials into the cytosol.178 This process was found to strongly improve polyplex transfections.

Figure 13. Sequence-defined oligomers. (A) Artificial oligoamino acids used for Fmoc solid-phase synthesis (SPS) of sequence-defined oligo(ethanamino)amides.144 Structures presented in protected and deprotected form. (B) Illustration of the Fmoc SPS approach. (C) Examples of published oligomers realized by this strategy.49,134c,144b,145 K, lysine; A, alanine; C, cysteine; OAA, artificial oligoamino acid.

transferrin receptor commonly expressed on growing tumor cells, was used as targeting ligand in several human clinical studies. In 1994, the very first polymer-based human gene therapy study was performed for the purpose of anticancer vaccination using transferrin-polylysine for the delivery of interleukin-2 (IL-2) pDNA polyplexes into patient’s primary melanoma cell cultures. The IL-2-expressing cells were used in γirradiated live form as antimelanoma vaccine.73 More recently, transferrin was used for in vivo targeting PEGylated cationized cyclodextrin siRNA polyplexes to tumors of patients.26 The encouraging early achievements were the basis for enormous and still expanding explorations using a multitude of ligands for P


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Figure 14. Shielding of polyplexes. (A) Hydrophilic polymers used as shielding agents of polyplexes, (B) core/shell assembly of shielded polyplexes, and (C) influence of the polycation to PEG ratio on the nucleic acid condensation and polyplex formation process. PEG, polyethylene glycol; pHPMA, poly(N-(2-hydroxypropyl)methacrylamide); pSAR, poly(sarcosine); HES, hydroxyethyl starch; HA, hyaluronic acid. References may be found in the text.

nondividing cells. Passive diffusion of pDNA or polyplexes in the cytosol is restricted; therefore, this transport has to occur either within endosomes trafficking along microtubules to the perinuclear space166a,183 or actively within the cytosol. Midoux, Pichon, and colleagues67a selected an adenoviral peptide which mediates virus attachment to the microtubular motor protein dynein; conjugated with pDNA, this peptide triggered transport along microtubules in cells and enhanced transfection. At the nuclear envelope, the polyplex or the pDNA has to be transported across the nuclear pore by active import mechanisms or alternative pathways into the nucleus. Cell cycle studies indicate that this step is a real bottleneck in transfection.67f−i Depending on the selected polymer, a more or less strong cell cycle dependence of transfection was observed, which was up to >100-fold enhanced in the G2/M phase of cell cycle when the nuclear envelope disintegrates. Several investigators have addressed active nuclear import by incorporating nuclear localization signal peptide sequences, obtaining variable successes.67c−e For polyplexes with linear PEI which show lower cell cycle dependence,67g an active role of the polymer in nuclear uptake has been speculated.67i,92c Independent from whether the nuclear entry proceeds with or without mitosis, the cargo pDNA has to be actively retained there and expressed in active form. Incorporation of peptides such as histone H3 tails180c,d or NLS peptides184 or other cell devision-responsive peptides67b into polyplexes has been found beneficial for nuclear retention. For PEI polyplexes (but not lipofectamine lipoplexes), nucleic acid unpackaging after nuclear entry was the major determining factor for transgene expression.182b In a recent study, incorporation of histone 3 tail peptides with methylations known to signal transcriptional activation on chromosomal DNA (H3K4Me3) was shown to accelerate the onset and extent of protein expression as compared with the nonmethylated H3 pDNA polyplexes.185

At this point it should be reminded that alternative pathways for cytosolic entry exist which are conceptually different from endolysosomal escape. The direct entry across the cell surface membrane is an option for lipid-based systems179 which resemble enveloped viruses. For polyplexes and other systems without fusogenic lipid envelope, the transient perturbation of cell membrane integrity poses a big hazard for cell survival. Electroporation demonstrates that this window can be utilized but is rather narrow. The membrane leakage from various intracellular vesicles into the cytosol has been proven to be better tolerated. However, instead of optimizing endosomal escape, delivery strategies can be aimed at natural endocytosis processes which avoid lysosomal sorting and subsequent degradation of nucleic acid cargos, for example, retrograde transport into Golgi organelles and endoplasmic reticulum60,61,63,64 or nondegradative vesicles which might slowly release polyplexes. In this respect, interesting reports have been published demonstrating productive nucleic acid uptake pathways which are not based on clathrin-coated-pit-mediated endocytosis.147k,180 5.4. Nuclear Import and Retention

For delivery of pDNA-based gene expression constructs, nuclear delivery is an additional challenge beyond the other barriers. Although it has been clear for a long time from early transfection studies that this process successfully takes place, the involved mechanisms are far less understood. Apart from the different discussed intracellular uptake pathways, it even has been questioned whether a cytosolic transfer is involved at all.181 Also, the timing and location of the release of the nucleic acid cargo from the cationic polymers can be highly variable between different polyplexes; as determined by fluorescence energy transfer and related experiments, both free and complexed nucleic acids have been observed in all delivery compartments including the nucleus.65a,182 Provided that cytosolic delivery takes place via either endosomal or retrograde pathways, the pDNA has to be delivered to the pores of the nuclear envelope of Q


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Table 2. Examples of Receptors and Ligands Used in Targeted Polyplexes159 a target tissue

receptor

tumor

Transferrin R EGF R

brain

liver

lung

HER2/neu Folate R Integrin CD13 CXCR4 Bomb R LHRH R Neuroblastoma JL-1 HGF R/c-Met Transferrin R Lactoferrin R GABA(B) R NGFR TrkA LRP1 Leptin R Laminin R ASGP R Hepatocyte LDL R family Serpin Enzyme R Polymeric Ig R Airway cells PECAM β2-R IP1 Insulin R Lactoferrin R

targeting ligand

polymer

Transferrin, B6 peptide, T7 peptide EGF, EGF peptide, GE11 peptide, anti-EGF AB, TGF-α Anti-Her2 AB Folate, MTX RGD peptide NGR peptide Plerixafor (AMD) Bombesin peptide LHRH peptide ChCE7 AB Anti-JL1 AB c-Met binding peptides (cMBP) Transferrin Lactoferrin RVG29 peptide NGF peptide Angiopep peptide leptin peptide Streptococcus derived peptide ASOR, tri/tetrameric galactose, lactose-α-CD, tri-NAG Malaria protein CS RAP peptide Antisecretory component AB Surfactant A, B Anti-PECAM AB Clenbuterol Iloprost insulin lactoferrin

pLys, protamine, dend pLys, PEI, PEG−PAMAM, PEG−PEI, PEG-CD, PEG-STP pLys, PEI, PEG−PEI, PAMAM, PEG-STP

26,75a,145e,162f

ref

pLys, PEI PEG-pLys, PEG−PEI, PEG-STP pLys, PEG−PEI, PEI, PAE PEI/PEG PEG-polyAMD PEG-lipopeptide PAMAM, PEI, PPI pLys, PEG−PEI pLys PEG-STP PEG−PAMAM PEG−PAMAM PEG−PAMAM Lys10/PEI PEG−PAMAM PEG-dend pLys PEG-dend pLys pLys, PEI, PAMAM, PEG-masked DPC

164d,g,h 49,122b,158c,160a,c,f 145e,162a,e 162i 160e 162j 162k 164c 164f 162q 131d 131c 162l 162m 162n 162o 162p 47a,71a,157b,161a,e,f

pLys pLys pLys pLys pLys PEI PEI PEI PEI PEI

163a 163d 162b 158a 163g 164e 160b 160d 163f 163e

147c,162c,d,g,h,163b,c,164a

a

Abbreviations: AB, antibody; ASGP, asialoglycoprotein; ASOR, asialoorosomucoid; CD, cyclodextrin; dend pLys, dendritic polylysine; DPC, Dynamic PolyConjugate; HGF R, hepatocyte growth factor receptor; Ig, immunoglobulin; LDL, low-density lipoprotein; LHRH, luteinizing hormone releasing hormone; LRP, LDLR-related protein; NAG, N-acetyl galactosamine; NGF, nerve growth factor; PAMAM, poly(amido amine); PEG, polyethylene glycol; PEI, polyethylenimine; PPI, polypropylenimine; pLys, polylysine; R, receptor; RAP, receptor-associated protein; STP, Stpbased sequence-defined oligomer.

Figure 15. Cellular uptake of polyplexes visualized by live-cell fluorescence microscopy. (A) Cellular uptake kinetics of EGFR targeted and untargeted polyplexes. The presence and type of targeting ligand impact the internalization rate (EGF > GE11 > untargeted). (B) Cellular uptake route of EGF and GE11 conjugated polyplexes investigated by colocalization with endocytotic pathway markers. Transferrin-Alexa488 (TF488) served as marker for clathrin-mediated endocytosis; caveolin-GFP expressing cells were used to visualize caveolin mediated uptake. Colocalization is marked with yellow circles. Both EGFR targeting ligands are predominantly internalized by clathrin mediated endocytosis. Reprinted with permission from ref 166b. Copyright 2012 American Chemical Society.

R


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6. MULTIFUNCTIONAL DYNAMIC POLYPLEXES Different steps in the delivery process require different active functions as outlined above. The chemist is faced with the following challenges: to design one or several precise macromolecules covering all critical delivery functions, to direct supramolecular assembly of these macromolecules with the nucleic acid payload into well-defined multifunctional polyplexes, and to take care by chemical programming that the incorporated delivery functions become activated at the right locations in a programmed, timely fashion. Such a “programmed delivery”186 is required for obvious reasons. Polyplex chemistry has to be dynamic for delivery; nanoparticles have to be stable outside the cell but dissociate and release their payload at the proper location inside the cell. Polyplexes have to be generally inert against biological surfaces and molecules but sticky to their cell targets. Polyplexes may need to destabilize intracellular vesicle membranes for crossing barriers but must not destroy other cell surface, mitochondrial, or nuclear membranes.

Figure 16. Intracellular release of cointernalized calcein visualized by spinning disk confocal microscopy during transfections with (A) alanine- or (B) histidine-containing oligo(ethanamino)amides. Higher cytosolic calcein fluorescence in transfections with histidine-containing oligomers demonstrates the enhancement of endosomal escape. Reprinted with permission from ref 145d. Copyright 2014 Elsevier.

Figure 17. Polymers with bioresponsive elements for the assembly of dynamic polyplexes. (A) Copolymer of cationic DMAEMA, hydrophobic butyl methacrylate, and styrene with pH and redox cleavable PEG,156b (B) covalent siRNA conjugate with amphiphilic vinylether copolymer PBAVE, pH cleavable PEG, and N-acetylgalactosamine targeting ligands,157 and (C) covalent siRNA-PLL conjugate with pH-reversible masked lytic peptide melittin.176e Acid-labile groups are indicated in red and bioreducible groups in blue. S


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Figure 18. Multifunctional sequence-defined nucleic acid carrier systems. (A) Folic acid targeted oligomer with PEG segment, artificial oligoamino acid Stp, and cross-linking cysteines in combination with an endosomolytic siRNA-Inf7 conjugate,49 (B) oligomer with a dual-functional methotrexate ligand, PEG segment, artificial oligoamino acid Stp, cross-linking cysteines, and endosomal buffering histidines for the delivery of cytotoxic poly(I:C).160f The functions of the individual domains are highlighted in color: two colors indicate a dual function. (C) Intracellular distribution of methotrexate-conjugated poly(I:C) polyplexes acquired by confocal laser scanning microscopy. Poly(I:C) and oligomers were spiked with fluorescently labeled analogs. Colocalization of nucleic acids and oligomers demonstrates the intracellular codelivery of both therapeutic entities, poly(I:C) and MTX. Reprinted with permission from ref 160f. Copyright 2014 American Chemical Society.

Chemical programming of delivery function requires knowledge about unique properties of the biological microenvironment where the delivery function should be activated. For example, knowledge on biological acidification in endolysosomes or certain extracellular tumor areas has motivated researchers to include pH-sensing chemical elements such as acid-labile bonds which change the polyplex properties. Specific microenvironment triggers include the presence of unique enzymes such as proteases or kinases or specific redox environments. In the following, several examples illustrate programmed dynamic polyplex delivery (Figure 17). A pioneering case of utilizing the endolysosomal acidification as trigger for “smart drug delivery” was designed by Alan Hoffman, Patrick Stayton, and colleagues.156b,c,177 Among other structures, they generated polymethacrylate copolymers containing blocks of hydrophobic butyl methacrylate, cationic DMAE methacrylate, and PEG-modified polystyrene blocks (Figure 17A). The hydrophilic PEG molecules, which provide biological shield and masking of the other functions, were attached via a pH-labile acetal and bioreducible disulfide bridge. Upon endosomal acidification, removal of the shielding PEG

Figure 19. Dual (pH, reduction) responsive amphiphilic block copolymer of a hydrophobic poly(ε-caprolactone) (PCL) block with a hydrophilic statistical methacrylate copolymer segment.192 Endosomal buffering tetraethylenepentamine (TEPA) is conjugated to the glycidyl methacrylate (GMA) element, and the oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) units serve as shielding domain. The pH-responsive element is indicated in red and bioreducible group in blue.

T


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Table 3. Human Clinical Trialsa carrier

nucleic acid

route of administration

disease

Transferrin-polylysine (AVET)

Interleukin-2 (IL-2) pDNA

ex vivo gene transfer to patient cells

melanoma

PEG-polylysine

CFTR pDNA

local, nasal mucosa of patients

cystic fibrosis

PEI

Diphtheria toxin A pDNA, H19 promoter

intravesical (bladder)

bladder cancer

PEG-PEI-Chol

Interleukin-12 (IL-12) pDNA

intraperitonealoptionally with chemotherapy

ovarian cancer

Transferrin-cyclodextrinoligocation

siRNA against Ribonucleotide reductase M2

intravenous

solid tumors

PEI-mannose

pDNA coding for 15 HIV antigens

dermal

HIV

conjugate with tri-NAGoligoamide

siRNA against TTR

subcutaneous

inherited TTR amyloidosis

a

outcome first polymer-based human gene therapy study (1994) phase I cancer vaccine, safety first polymer-based in vivo gene therapy study phase I, safety, bioactivity phase I/IIa and IIb safety, bioactivity phase I safety phase I safety gene silencing in tumor phase I and II HIV vaccine DermaVir anti-HIV immune responses targeting to the liver asialoglycoprotein receptor efficient TTR gene silencing

Details may be found in the references listed in the text.

precise PEG unit modified with either folic acid (Figure 18A)49 or methotrexate (MTX) (Figure 18B)160f provided polyplex shielding and receptor-targeted endocytosis via the folate receptor. Two arms of four cationic units of tetraethylenepentamine (TEPA)-succinamide provided RNA binding and proton sponge activity for pH-responsive endosomal escape. Terminal cysteine residues, which stabilize polyplexes by formation of bioreversible disulfide cross-links, were required for polyplex stabilization. For siRNA delivery, the endosomolytic influenza peptide INF7 was attached to the siRNA via bioreducible linkage. This strongly improved the endosomal escape and gene silencing efficacy.49 For the delivery of cytotoxic dsRNA poly(I:C), instead of INF7 a series of histidines was incorporated into the cationic arms for boosting endosomal escape. Finally, MTX not only acted as receptor ligand but also acted as chemotherapeutic drug for killing MTX-resistant tumor cells in combination with the codelivered (Figure 18C) cytotoxic RNA cargo. Within the precise oligomer construct, an artificial oligoglutamate sequence was linked with MTX which intracellularly enhances the intoxication via the DHFR target enzyme.160f Pun and colleagues192 generated dual (pH, reduction) responsive stabilized pDNA polyplexes by designing precise amphiphilic ternary block copolymers (Figure 19). A hydrophobic poly(ε-caprolactone) (PCL) block was disulfide linked with a statistical coblock of TEPA-modified glycidyl-methacrylates and oligoethylene glycol (OEG) methacrylates. PCL was supposed to act as a hydrophobic polyplex-stabilizing polymer core unit, the TEPA-modified polymer units present cationic proton sponge units for nucleic acid binding and endosomal escape similar as described in related work,49,123e,145a,160f and the OEG units serve for polyplex shielding and improved biocompatibility. The attached TEPA and OEG monomers were polymerized as separate coblocks or as statistical copolymer, resulting in PCL-SS-p(GMA-TEPA)-b-pOEGMA, PCL-SS-pOEGMA-b-p(GMA-TEPA), or PCL-SS-p[(GMATEPA)-st-pOEGMA] (the latter displayed in Figure 19). Apparently, copolymers containing pOEGMA blocks suffered from the so-called “PEG dilemma”, which reduced their transfection activity. The statistical OEG/TEPA block proved to be a very interesting novel solution to overcome the PEG

unmasks the hydrophobic and cationic polymethacrylate blocks, which exert a lytic activity toward the endosomal lipid membrane. This lysis results in release of the cargo drug into the cytosol. The concept of breaking bonds to unmask an endosomolytic activity has been followed by several investigators, including pH-triggered de-PEGylation of lytic amphiphilic polymer poly(butyl/aminoethyl vinylether) (pBAVE)157 for improved receptor-targeted siRNA delivery (Figure 17B) or unmasking of lytic mellitin peptide (Figure 17C).176d,e Acidic cleavage includes also various other deshieldings of polyplexes,86,110b,156a removal of cationic head groups121 or stabilizing hydrophobic domains187 from polymers, or polymer backbone degradation.110a Successfully applied pH-sensing units without breaking covalent bonds include protonation-induced deshielding154 or conformational activation of endosomolytic fusion peptides.173,175b The bioreductive environment of the cytosol with approximately 100−1000-fold higher intracellular glutathione (GSH) levels can serve as a trigger for a dynamic change with nucleic acid carriers.188 Reducible disulfide cross-links can stabilize polyplexes extracellularly but be cleaved intracellularly, releasing the nucleic acid and smaller polymer fragments with increased biocompatibility. Some disulfide cleavage may also occur in the extracellular space,189 especially at the cell surface, mediated by protein disulfide isomerases (PDI). The extracellular stability of disulfides can be tuned by their chemical environment;190 bulky groups providing sterical hindrance or hiding the disulfide in internal cores of polymers may prevent cationic neighboring groups, and exposure in the nanosystem surface may accelerate bioreduction. Bioreductive cleavage at the cell surface would be a useful process for removing disulfide-bound PEG.101,191 In this respect, bioreducible (pDMAEMA-SS-PEG-SS-pDMAEMA) triblock copolymers were applied for reversible shielding of pDNA polyplexes. The bioreducible shield resulted in up to more than 20-fold higher gene transfer as compared with the stably shielded copolymer analogue.191c For biosensitive RNA polyplexes containing multiple functions, sequence-defined oligoaminoamides have been used (Figure 18).49,160f All incorporated five or six functions were shown to be required for full activity of these polyplexes. A U


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dilemma. Incorporation of the PCL block resulted in strongly improved polyplex stability in serum. The in vitro and in vivo transfection studies indicate a TEPA unit triggered endosomal escape into the cytosol, followed by a bioreductive cleavage of the PCL unit, which destabilizes polyplexes and enables pDNA release.

7. CONCLUSION AND PROSPECTS Within the first 50 years of polymer-mediated nucleic acid delivery, fascinating versions of multifunctional dynamic polyplexes have already been described, including technical options to prepare them in a precise fashion. Alternative assembly methods122c,162k,193 will provide an additional dimension in the design of supramolecular architectures. The learning curve of polyplex sciences is still in its rising phase, fueled by advances in macromolecular chemistry, high-resolution methods to determine nanoparticle and biological ultrastructures, as well as bioimaging in living organisms.194 Retrospectively, the field has benefited most from the recognition of specific biological barriers and improved understanding of how nature has found its own solution to overcome these hurdles. Medical translation of polyplexes (Table 3) is still in the beginning with a limited number of completed human clinical trials.23a,26,47b,73,81,147j The recent expansion of the repertoire of therapeutic nucleic acids from classical pDNA gene transfer constructs to many other gene expression and protein-modulating nucleic acids such as mRNA, miRNA, or siRNA is expected to generate an additional boost for further optimization of polymeric delivery systems.

Professor Ernst Wagner received his doctoral degree in Organic Chemistry from the Vienna Technical University in 1985. Since 2001 he is Full Professor of Pharmaceutical Biotechnology at Ludwig Maximilians University in Munich and since 2005 a member of the Munich Center for Nanoscience. He coordinates the Area “Biomedical Nanotechnologies” of the Excellence Cluster “Nanosystems Initiative Munich”. He is a current Editor of Pharmaceutical Research and Associate Editor of The Journal of Gene Medicine.

ACKNOWLEDGMENTS We acknowledge the financial support of our research from the DFG Cluster “Nanosystems Initiative Munich” and the EU IMI network COMPACT. We thank Wolfgang Rödl, Miriam Höhn for excellent technical support, Olga Brück for skillful assistance, and Christoph Hohmann (Nanosystems Initiative Munich, NIM) for graphic design of the cover art.

AUTHOR INFORMATION

DEDICATION This review is dedicated to the late Prof. Max L. Birnstiel (1933− 2014), a pioneer in molecular biology, who isolated the first eukaryotic genes in 1966 and, amongst many other contributions, developed receptor-mediated nonviral gene transfer in the early 1990s.

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

REFERENCES

Biographies

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Ulrich Lächelt studied pharmaceutics at the University of Heidelberg, where he graduated in 2009. He joined the group of Pharmaceutical Biotechnology at the Ludwig Maximilians University in Munich in 2010 and worked on his Ph.D. research about multifunctional sequencedefined nucleic acid carriers under the supervision of Prof. Ernst Wagner. He received his doctoral degree in pharmaceutical biology in 2014 and continues the research work in a postdoctoral position. V


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AJ


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