Fernando A De Oliveira

Lindomar J C Albuquerque

Gwendoline Delecourt

Véronique Bennevault

Philippe Guégan
email: philippe.guegan@sorbonne-universite.fr

Dr Fernando Carlos Giacomelli
email: fernando.giacomelli@ufabc.edu.br

Current Designs of Polymeric Platforms towards the Delivery of Nucleic Acids inside the Cells with Focus on Polyethylenimine

Keywords: nanomedicine, gene therapy, gene delivery, non-viral vectors, polymers, polyethylenimine, polyplexes

we review the gene delivery pathway using non-viral vectors, focusing on the progresses with regard to the use of polyethylenimine and derivatives.

INTRODUCTION

Molecular therapy is an encouraging strategy for the treatment of human diseases. Particularly, gene therapy aiming to modify the gene expression in target cells is attractive for treating a variety of malignancies which are linked to the absence, or presence of abnormal genes. [START_REF] Seymour | Gene Therapy Matures in the Clinic[END_REF] The approach can be employed, for instance, in the deactivation of oncogenes, replacement of nonfunctioning tumor suppressor genes, and to transfer genetic materials directly into target cells for permanent changes in their phenotypes. Accordingly, these therapies consider nucleic acids as the active agent and therefore, the active site is inside the cell. The transfer of genetic material into cells can be accomplished essentially by using two approaches: i) in in vivo gene editing, the nucleic acids are directly administrated into the body whereas ii) in ex vivo gene editing, the cells are previously isolated, edited, and further transplanted into the patient in a more complex procedure. [START_REF] Shim | Therapeutic gene editing: delivery and regulatory perspectives[END_REF] In such an approach, the reverse transfection procedure can also be employed where instead of adding nucleic acids to cells, cells are added to preplated nucleic acids. [START_REF] Erfle | Reverse transfection on cell arrays for high content screening microscopy[END_REF] The cellular uptake of naked nucleic acids is nevertheless inefficient due to electrostatic repulsion between their anionic phosphate backbones and the negatively charged plasma membranes. [START_REF] Lomas | Efficient Encapsulation of Plasmid DNA in pH-Sensitive PMPC-PDPA Polymersomes: Study of the Effect of PDPA Block Length on Copolymer-DNA Binding Affinity[END_REF] Additionally, whenever nucleic acids are directly administered in vivo, they can be rapidly cleared by specific enzymes. [START_REF] Houk | Pharmacokinetics of Plasmid DNA in the Rat[END_REF][START_REF] Yin | Non-Viral Vectors for Gene-Based Therapy[END_REF] Accordingly, cargo-delivery platforms are required to provide extracellular protection and stability to the biomacromolecules, allowing further cell internalization and the release of the therapeutic agent in the right intracellular compartment.

In this framework, viral vectors have been formerly demonstrated to be highly effective. Nevertheless, viral delivery systems suffer from significant drawbacks, including the unfavorable interaction with the immune system, [START_REF] Hollon | Researchers and Regulators Reflect on First Gene Therapy Death[END_REF] besides the production price incompatible with the society financial means, with average costs sometimes reaching almost $1 billion per patient. [START_REF] Orkin | Paying for Future Success in Gene Therapy[END_REF][START_REF] Qtaish | Niosome-Based Approach for in Situ Gene Delivery to Retina and Brain Cortex as Immune-Privileged Tissues[END_REF] These entities can also be oncolytic and mutagenic. [START_REF] Rolland | Gene Medicines: The End of the Beginning?[END_REF][START_REF] Waehler | Engineering Targeted Viral Vectors for Gene Therapy[END_REF][START_REF] Berns | Good News for Gene Therapy[END_REF] Hence, strategies to design non-viral gene carriers have been called. The synthetic vectors usually contain cationic units able to bind to negatively charged nucleic acids and drive them towards the intracellular milieu. The polycation polyethylenimine (PEI) and derivatives are recognized as highly efficient gene delivery systems, although they still cannot compete with the cell entry mechanism of viral vectors. [START_REF] Deng | Revisit the Complexation of PEI and DNA -How to Make Low Cytotoxic and Highly Efficient PEI Gene Transfection Non-Viral Vectors with a Controllable Chain Length and Structure?[END_REF][START_REF] Han | Polyplex Formation of Calf Thymus DNA with Branched and Linear Polyethyleneimine[END_REF][START_REF] Zhang | History of Polymeric Gene Delivery Systems[END_REF] The supramolecular assemblies based on PEI are however typically linked to significant cytotoxicity due to the membrane disruptive property. [START_REF] Monnery | Cytotoxicity of Polycations: Relationship of Molecular Weight and the Hydrolytic Theory of the Mechanism of Toxicity[END_REF][START_REF] Fischer | In Vitro Cytotoxicity Testing of Polycations: Influence of Polymer Structure on Cell Viability and Hemolysis[END_REF][START_REF] Grandinetti | Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?[END_REF] Truly, cytotoxicity is a common characteristic of many polycations in this regard.

Taking into account the abovementioned considerations, it is currently accepted that more fundamental understanding and optimizations are required. We herein review mainly the recent designs of polyethylenimine-based platforms to deliver nucleic acids inside the cells. Firstly, we explore the delivery pathway of non-viral gene delivery vectors, and afterwards, we briefly underline the main classes of non-viral vectors as well as the main subclasses of polymeric vectors with potential application in gene therapies. We further contextualize the evolution of PEI-based synthetic vectors, and subsequently, we exploit essentially the most recent achievements as well as challenges and opportunities towards enhanced efficacy.

REQUIREMENTS AND CHALLENGES IN THE DELIVERY PATHWAY

The potential use of non-viral vectors in gene therapy has been kicked off as justified by the well-known weaknesses of viral counterparts, which are biological entities able to effectively deliver nucleic acids inside the cells at high yields. The most common viral vectors include adenovirus, adeno-associated viruses (AAV), lentiviruses and retroviruses. [START_REF] Lundstrom | Viral Vectors in Gene Therapy[END_REF] While being highly effective as transfecting agents, they are simultaneously associated with fundamental drawbacks, creating the need for synthetic vectors. In this regard, cationic entities that can bind electrostatically to the phosphodiester backbones of nucleic acids to form electrostatic complexes denote a promising class of delivery systems. Such vectors nevertheless must be capable to overcome multiple barriers to successfully delivery genetic material inside the cells, [START_REF] Shi | Challenges in DNA Delivery and Recent Advances in Multifunctional Polymeric DNA Delivery Systems[END_REF] and the nucleic acid binding and condensation is only the first pre-requisite of a nonviral gene delivery vector that can potentially be used in therapies. They must further resist the extracellular barriers, whatever the administration way, and this is particularly critical during intravenous administrations. The serum stability is already one main roadblock because cationic entities are susceptible to unspecific interaction whenever in contact with biological fluids. This may lead to the formation of large aggregates that are rapidly recognized, captured and cleared due to the action of the mononuclear phagocyte system (MPS). The binding of negatively charged proteins (abundant in the bloodstream) may also conduct to unwanted effects such as the activation of serum complement proteins, thus inducing immune responses. [START_REF] Merkel | In Vitro and In Vivo Complement Activation and Related Anaphylactic Effects Associated with Polyethylenimine and Polyethylenimine-Graft-Poly(ethylene glycol) Block Copolymers[END_REF] The required nucleic acid binding capacity and stability in biological fluids already exclude a non-negligible number of non-viral gene carrier candidates. This issue can be in principle overwhelmed by shielding positive surfaces, for instance, by using neutral elements, therefore reducing the interaction with the naturally negative environments. If these concerns are overcome, then the next step of the journey is the cell adhesion and efficient cellular uptake. The electrostatic complexes are mostly internalized by endocytosis pathways due to their relatively large sizes. [START_REF] Rejman | Size-Dependent Internalization of Particles via the Pathways of Clathrin-and Caveolae-mediated Endocytosis[END_REF] The eukaryotic cells commonly uptake nanoparticles in the range 10-300 nm via clathrin-mediated endocytosis, whereas caveolae-mediated cellular uptake has a smaller upper limit size around 80-100 nm. [START_REF] Wang | Size and Dynamics of Caveolae Studied Using Nanoparticles in Living Endothelial Cells[END_REF][START_REF] Canton | Endocytosis at the nanoscale[END_REF] The polymeric-based gene delivery carriers are most probably internalized via both mechanisms (clathrin and caveolae pathways), and the dominant is probably cell line-dependent since both clathrin [START_REF] Billiet | Gene Transfer by Chemical Vectors, and Endocytosis routes of Polyplexes, Lipoplexes and Lipopolyplexes in a Myoblast Cell Line[END_REF] and caveolae-dependent [START_REF] Rejman | Role of Clathrin-and Caveolae-Mediated Endocytosis in Gene Transfer Mediated by Lipo-and Polyplexes[END_REF] routes were already suggested to be correlated with higher transfection levels. Besides, other mechanisms such as flotillin-mediated endocytosis have been evidenced. [START_REF] Vercauteren | Flotillin-Dependent Endocytosis and a Phagocytosis-Like Mechanism for Cellular Internalization of Disulfide-Based Poly(amido amine)/DNA Polyplexes[END_REF] The uptake of nanoparticles via clathrin-mediated endocytosis is followed by the pH drop from 7.4 to roughly 6.0 in early endosomes, and 5.0 in late-endosomes and lysosomes.

Such an acidic process may lead to the degradation of the assemblies and their molecular bricks, consequently conducting to poor transfection efficiency. The caveolae-dependent route is frequently considered non-acidic and non-digestive, therefore less affected by lysosomal degradation, and possibly leading to higher transfection levels. [START_REF] Hossen | Switching the Intracellular Pathway and Enhancing the Therapeutic Efficacy of Small Interfering RNA by Auroliposome[END_REF] These considerations are debatable. [START_REF] Kiss | Endocytosis via Caveolae: Alternative Pathway with Distinct Cellular Compartments to Avoid Lysosomal Degradation?[END_REF] Regardless of the cellular uptake mechanism, the process can be restricted by the presence of shielding agents used to stabilize the supramolecular assemblies in the extracellular milieu. The attachment of specific ligands at their outer surface can then be used as an approach to provide cellular uptake at higher yields as well as targeting capability. This can be particularly useful in tumor treatment since the damaged cells typically overexpress growth factors and receptors that regulate the intracellular concentration of nutrients (transferrin and folate receptors are common examples).

Hence, ligands present at the outer surfaces of the assemblies, with high affinity to such receptors, are able to potentialize preferred cell capture. In the step after, the complexes flow towards the intracellular ambient in vesicles named endosomes that contain the engulfed material. The endosomal escape is further required to avoid lysosomal degradation of the therapeutic nucleic acids, which would lead obviously to reduced transfection efficiency. The mechanism used by electrostatic complexes to overcome the endosomal barrier is still debated, and we discuss the main hypothesis in the following section. Out of the endosomes, the supramolecular assemblies have to be unpacked and, at this stage, instead of extracellular stability, intracellular lability is desired since the dissociation of the complexes is essential to enable the therapeutic activity of the nucleic acids. Taking into account that the nucleic acid condensation is at least partially attributed to electrostatic interactions, the dissociation can be induced by high salt concentrations. The dissociation can be likewise potentialized by the presence of negatively charged polyelectrolytes competing for PEI binding such as observed for PEI/DNA polyplexes in the presence of the polysaccharide heparin. [START_REF] Clamme | Monitoring of the Formation and Dissociation of Polyethylenimine/DNA Complexes by Two Photon Fluorescence Correlation Spectroscopy[END_REF] The continuous expansion of linear PEI chains as pH reduces and protonation increases may also occur in endosomal conditions and impact the interaction of PEI with nucleic acids or membranes, thus accounting for the success of PEI as a gene delivery vector. [START_REF] Gallops | Effect of the Protonation Level and Ionic Strength on the Structure of Linear Polyethyleneimine[END_REF] The effectiveness of a gene delivery system also depends on the site of action. While plasmid DNA (pDNA) requires the nuclear import, various RNA molecules (siRNA and miRNA, for example) have the cytosol as the final destination. The CRISPR-Cas9 genome editing tool, which was the subject of the 2020 Nobel Prize in Chemistry, can similarly be used to treat a variety of genetic diseases, and it also requires the nuclei internalization. The search of efficient delivery vectors for the genome-editing agent represents the next challenge in vector design. [START_REF] Yin | Delivery Technologies for Genome Editing[END_REF] Truthfully, the cytosolic transport of the therapeutic agent to reach the nuclei membrane is still poorly understood, as it is the mechanism of nuclei internalization, although possibly only small-sized polyplexes (smaller than 50 nm) are allowed to easily enter. [START_REF] Andersen | AFM Visualization of Sub-50 nm Polyplex Disposition to the Nuclear Pore Complex Without Compromising the Integrity of the Nuclear Envelope[END_REF] This dimension nonetheless diverges from the optimum size (around 90 nm) suggested for efficient siRNA delivery (cytosolic delivery) which balances the circulation time and cellular uptake. Larger nanoparticles with over 100 nm were demonstrated to be quicker cleared, whereas smaller ones (about 40 nm) were not efficiently uptaken by tumor cells. [START_REF] Liang | Optimizing the Size of Micellar Nanoparticles for Efficient siRNA Delivery[END_REF] Taking into account the abovementioned considerations, it is clear that the gene delivery process by using non-viral vectors is very challenging, thus justifying the fairly limited success so far achieved. One has to face at least with i) nucleic acid packaging, ii) serum stability; iii) cell targeting and cellular uptake, iv) endosomal escape and intracellular unpacking and v) cytosolic transport and intranuclear delivery. The schematic representation of the main extracellular and intracellular barriers towards in vivo gene delivery using synthetic vectors is cartooned in Figure 1. In the next section, we explore in more detail the possible mechanisms enabling the supramolecular systems to escape the endosomes (step iv).

THE DEBATED MECHANISM OF ENDOSOMAL ESCAPE

Amongst the fairly long list of challenges for the successfulness of non-viral gene delivery vectors, the strategies to overcome the extracellular barriers are overall identified, nevertheless, the pathway used by the supramolecular systems to escape the endosomes is still debated. [START_REF] Bus | The Great Escape: How Cationic Polyplexes Overcome the Endosomal Barrier[END_REF] The step is a key factor determining the transfection efficiency provided by a polymeric material, [START_REF] Jiang | Quantitating Endosomal Escape of a Library of Polymers for mRNA Delivery[END_REF] and it notably attenuates the effectiveness of many cationic carriers. Therefore, more fundamental understanding of molecular biology is required to accurately comprehend the process. Hence, multidisciplinary teams are essential to move forward in the subject. The earliest investigations linked higher transfection efficiency of cationic non-viral gene delivery vectors to the so-called proton sponge effect. The pH-responsive vectors with pKa ~ 7 would enable nucleic acid binding and condensation at pH 7.4, and further protonation in the acidic intracellular environment. The increased ionic concentration inside the endosomes would conduct to an influx of ions (chloride) and water towards the inner cavity of the vesicles, and the osmotic swelling would inevitably lead to the rupture of endosomal membrane releasing the entrapped material into the cytosol. Such a hypothesis nevertheless was not able, for example, to justify the empirical evidences concerning the use of poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methylamino]-ethyl ester) -PDAMA (the molecular structure of PDAMA is provided in Figure 4). This homopolymer has two tertiary amine groups in each monomeric unit.

One was supposed to provide nucleic acid binding and condensation ability at physiological pH, while the other would offer endosomal buffering capacity. The titration of acidified solutions of PDAMA, PDMAEMA and PEI was performed and a polyelectrolyte behavior was observed for the three polymers, suggesting comparable binding and buffering abilities. Nevertheless, almost negligible in vitro transfection activity has been demonstrated for PDAMA/DNA polyplexes without the addition of membrane disruptive peptides. The authors conclude that the proton sponge mechanism cannot be generalized to all the polymers. [START_REF] Funhoff | Endosomal Escape of Polymeric Gene Delivery Complexes Is Not Always Enhanced by Polymers Buffering at Low pH[END_REF] Additionally, it has been demonstrated that the presence of PEI presumably does not induce changes in lysosomal pH. [START_REF] Benjaminsen | The Possible "Proton Sponge " Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH[END_REF] Besides, realistic calculations suggested that the osmotic pressure generated by the proton sponge effect is not sufficient to solely conduct to the endosome lysis process, [START_REF] Won | Missing Pieces in Understanding the Intracellular Trafficking of Polycation/DNA Complexes[END_REF] and other contributions have to be simultaneously involved. Recently, the direct polyplex-endosomal membrane interaction to explain the endosomal escape has been proposed, [START_REF] Rehman | Mechanism of Polyplex-and Lipoplex-Mediated Delivery of Nucleic Acids: Real-Time Visualization of Transient Membrane Destabilization without Endosomal Lysis[END_REF] and soon after, this has been updated as caused mainly by the interaction of free polycations (not complexed) with the lipid bilayers. The free chains are supposed to be able to interact with anionic phospholipids thereby disrupting the endosomal membrane and assisting the release. [START_REF] Deng | Revisit the Complexation of PEI and DNA -How to Make Low Cytotoxic and Highly Efficient PEI Gene Transfection Non-Viral Vectors with a Controllable Chain Length and Structure?[END_REF][START_REF] Albuquerque | Gene Transfection Mediated by Catiomers Requires Free Highly Charged Polymer Chains To Overcome Intracellular Barriers[END_REF][START_REF] Vaidyanathan | Cationic Polymer Intercalation into the Lipid Membrane Enables Intact Polyplex DNA Escape from Endosomes for Gene Delivery[END_REF][START_REF] Bonner | Crosslinked Linear Polyethylenimine Enhances Delivery of DNA to the Cytoplasm[END_REF] Furthermore, larger molar mass polycations were suggested to interact with membrane proteins, blocking the intervesicular fusion, thus avoiding the trapping in the lysosomes which would result in the degradation of the nucleic acids. [START_REF] Yue | Progress and Perspectives in Developing Polymeric Vectors for In Vitro Gene Delivery[END_REF] Overall, the current knowledge points out that the endosomal escape is possibly mediated by a combination of osmotic-pressure induced membrane rupture as aided by the presence of free polycations, and the last ones also play an important role by interacting with membrane proteins. Nevertheless, there seems not to be a consensus in the scientific community with regard to the main driving force. [START_REF] Vermeulen | The Proton Sponge Hypothesis: Fable or Fact?[END_REF]

MAIN CATEGORIES OF NON-VIRAL VECTORS FOR

GENE THERAPY

The class of non-viral vectors for gene therapy is comprehensive and it includes mainly cationic molecules that can interact electrostatically with the negatively charged phosphate groups of the nucleic acids, therefore leading to the formation of electrostatic complexes. The use of lipids, peptides, lipopeptides and polymers will be briefly reviewed and afterwards, we mainly focus on PEI and its derivatives.

Lipids

The supramolecular structures produced by the complexation of lipids and nucleic acids are named lipoplexes, and the transfection procedure is defined as lipofection. The lipids used as transfecting agents are composed by a hydrophilic, and commonly cationic head, linked to a hydrophobic domain. The amphiphilic feature of such molecules enables their self-assembly into different morphologies depending on the packing parameter (vesicles and micelles, for instance). Typically, vesicles (named liposomes) are used in the field of gene delivery. They interact via electrostatic forces with nucleic acids producing different hierarchical structures. The lamellar and hexagonal arrangements are composed by an alternation of lipid and nucleic acid domains. The liposomes composed by phosphatidylserine were demonstrated to efficiently deliver DNA into cells [START_REF] Fraley | Introduction of Liposome-Encapsulated SV40 DNA into Cells[END_REF] and later, high transfection rates were also reached by using the monovalent cationic lipids 1,2-dioleoyl-3-trimethylammoniumpropane (DOTMA), [START_REF] Felgner | Lipofection: a Highly Efficient, Lipid-Mediated DNA-Transfection Procedure[END_REF] 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP) [START_REF] Leventis | Interactions of Mammalian Cells with Lipid Dispersions Containing Novel Metabolizable Cationic Amphiphiles[END_REF] and dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE). [START_REF] San | Safety and Short-Term Toxicity of a Novel Cationic Lipid Formulation for Human Gene Therapy[END_REF] Additionally, lipids having a neutral head, for instance, dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), have been investigated as "helpers" in combination with cationic counterparts. [START_REF] Farhood | The Role of dioleoyl Phosphatidylethanolamine in Cationic Liposome Mediated Gene Transfer[END_REF][START_REF] Du | The Role of the Helper Lipid on the DNA Transfection Efficiency of Lipopolyplex Formulations[END_REF] Besides permanently positively charged cationic lipids, examples of ionizable monovalent and multivalent lipids used towards the same goals include 1,2-dioleoyl-3dimethylammonium propane (DODAP), [START_REF] Semple | Lipid-Based Formulations of Antisense Oligonucleotides for Systemic Delivery Applications[END_REF] dioctadecylamido-glycylspermine (DOGS), [START_REF] Behr | Efficient Gene Transfer Into Mammalian Primary Endocrine Cells with Lipopolyamine-Coated DNA[END_REF] 2,3-dioleyloxy-N-[2(spermi-necarboxamido)ethyl]-N,N-dimethyl-lpropanaminium tri-fluoroacetate (DOSPA) [START_REF] Balazs | Liposomes for Use in Gene Delivery[END_REF] and cholesterol derivatives. [START_REF] Gao | A Novel Cationic Liposome Reagent for Efficient Transfection of Mammalian Cells[END_REF][START_REF] Ciani | DOTAP/DOPE and DC-Chol/DOPE Lipoplexes for Gene Delivery: Zeta Potential Measurements and Electron Spin Resonance Spectra[END_REF] Figure 2 provides the molecular structure of the main lipids used in the formulation of non-viral gene delivery vectors. The class of lipid-based DNA therapeutics has been recently reviewed, [START_REF] Buck | Lipid-Based DNA Therapeutics: Hallmarks of Non-Viral Gene Delivery[END_REF] also with particular focus on the treatment of gliomas. [START_REF] Luiz | Gene Therapy Based on Lipid Nanoparticles as Non-Viral Vectors for Gliomas Treatment[END_REF] Figure 2. Molecular structure of main lipids used in non-viral gene delivery formulations (ionizable lipids are displayed as protonated species).

Concerning the cellular uptake and intracellular pathway, lipoplexes are internalized presumably by endocytosis, and the endosomal escape occurs via the socalled flip-flop mechanism. The lipid-nucleic acid complexes are able to destabilize the endosomal membranes inducing a flip-flop of anionic lipids from the cytoplasmic-facing monolayer which diffuse into the complex and form a charge neutral ion pair with the positively charged lipids. The nucleic acid-cationic lipid interaction is weakened and ultimately, the therapeutic biomacromolecule is released into the cytoplasm. [START_REF] Xu | Mechanism of DNA Release from Cationic Liposome/DNA Complexes Used in Cell Transfection[END_REF] Apart from the sufficient transfection levels, the ability of lipids to disrupt the cellular membrane may lead to off-target effects and cytotoxicity depending on the chemical nature and concentration.

[60]

Amino Acids and Peptides

The main amino acids used in the intracellular delivery of nucleic acids include lysine (Lys, K), arginine (Arg, R) and histidine (His, H). Typically, poly(amino acid)s are used for such purpose as hereafter discussed in more details. The use of peptidebased nanocarriers has been encouraged due to the possibility of precisely control the syntheses steps, therefore building desired sequences, as well as incorporating targeting ligands. The introduction of solid-phase peptide synthesis (SPPS) by Merrifield was a milestone in this regard. [START_REF] Merrifield | Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide[END_REF] The molecules are bound to a solid support (resin), and the synthesis takes place step-by-step using protecting group chemistry. The intermediate peptides can be eliminated by washing and filtration. In this category of non-viral vectors for gene delivery, the cell penetrating peptides (CPPs) are also evoked. [START_REF] Taylor | Cell Penetrating Peptides, Novel Vectors for Gene Therapy[END_REF][START_REF] Khan | Cell Penetrating Peptides: A Versatile Vector For Co-Delivery of Drug and Genes in Cancer[END_REF] These are chains consisting of 4 to 40 highly charged amino acids (Arg and Lys, for example) or sequences with hydrophilic and hydrophobic domains (amphipathic molecules). [START_REF] Langel | Cell-Penetrating Peptides[END_REF] They have the ability to translocate the plasma membrane and can be used in a variety of biomedical applications. [START_REF] Derakhshankhah | Cell Penetrating Peptides: A Concise Review with Emphasis on Biomedical Applications[END_REF] The classical example of CPP is the TAT sequence which is rich in positively charged Arg and Lys residues. [START_REF] Sabouri-Rad | The Effect of Cell Penetrating Peptides on Transfection Activity and Cytotoxicity of Polyallylamine[END_REF] The protein penetratin [START_REF] Derossi | The Third Helix of the Antennapedia Homeodomain Translocates Through Biological Membranes[END_REF] is another example of CPP that has been used to optimize the intracellular delivery of nucleic acids. Penetratin was demonstrated to notably augment the transfection yields of polymethacrylate-based non-viral gene delivery systems. [START_REF] Christiaens | Enhancement of Polymethacrylate-Mediated Gene Delivery by Penetratin[END_REF] 4.

Lipopeptides

The fatty acids can be coupled to sequence-defined-peptide chains by SPPS to produce lipopeptides. The use of lipopeptides in nucleic acid delivery has been explored

by Wagner et al. for instance using artificial units such as succinoyl tetraethylene pentamine (Stp) and succinoyl pentaethylene hexamine (Sph). [START_REF] Schaffert | Novel Fmoc-Polyamino Acids for Solid-Phase Synthesis of Defined Polyamidoamines[END_REF][START_REF] Zhang | Lipo-Oligomer Nanoformulations for Targeted Intracellular Protein Delivery[END_REF] The building blocks were combined with cationic amino acids (such as lysine) and fatty acids. The entities were demonstrated to be useful in the delivery of pDNA, siRNA, mRNA and antisense oligonucleotides. [START_REF] Zhang | Lipo-Oligomer Nanoformulations for Targeted Intracellular Protein Delivery[END_REF][START_REF] Truebenbach | Co-delivery of Pretubulysin and siEG5 to EGFR Overexpressing Carcinoma Cells[END_REF][START_REF] Troiber | Stabilizing Effect of Tyrosine Trimers on pDNA and siRNA Polyplexes[END_REF][START_REF] Liu | Lächelt, pH-Reversible Cationic RNase A Conjugates for Enhanced Cellular Delivery and Tumor Cell Killing[END_REF][START_REF] Luo | Artificial Peptides for Antitumoral siRNA Delivery[END_REF][START_REF] Wang | Double Click-Functionalized siRNA Polyplexes for Gene Silencing in Epidermal Growth Factor Receptor-Positive Tumor Cells[END_REF] The importance of tyrosine trimers to improve the serum stability and buffer capacity of manufactured sequences, as well as enhanced effectiveness by using cysteine residues, have been also highlighted. [START_REF] Troiber | Stabilizing Effect of Tyrosine Trimers on pDNA and siRNA Polyplexes[END_REF][START_REF] Schaffert | Solid-Phase Synthesis of Sequence-Defined T-, i-, and U-Shape Polymers for pDNA and siRNA Delivery[END_REF] Sequencedefined pH-sensitive lipopeptides containing a cationic head, amino acid residues and lipophilic tails were equally investigated. [START_REF] Wang | Novel Polymerizable Surfactants with pH-Sensitive Amphiphilicity and Cell Membrane Disruption for Efficient siRNA Delivery[END_REF][START_REF] Malamas | Design and Evaluation of New pH-Sensitive Amphiphilic Cationic Lipids for siRNA Delivery[END_REF] These arrangements were able to condense siRNA into compact nanoparticles with low cytotoxicity, and provided high transfection yields.

Dendrimers

Dendrimers are hyperbranched structures with globular morphology and, whenever they have a cationic character, they can bind to nucleic acids thereby forming the so-called dendriplexes. The dendrimer poly(amido amine) (PAMAM) [START_REF] Haensler | Polyamidoamine Cascade Polymers Mediate Efficient Transfection of Cells in Culture[END_REF] has been widely investigated in this framework. PAMAM is only moderated protonated at physiological pH while highly protonated at acidic environments. Other non-viral gene delivery vectors belonging to the same class include dendritic polylysine (dPLL) [START_REF] Ohsaki | In Vitro Gene Transfection Using Dendritic Poly(L-lysine)[END_REF] and polypropylenimine (PPI). [START_REF] Studzian | Mechanisms of Internalization of Maltose-Modified Poly(propyleneimine) Glycodendrimers into Leukemic Cell Lines[END_REF][START_REF] Dufès | Dendrimers in Gene Delivery[END_REF] The molecular structures of these dendrimers are provided in Figure 3. The polypropylenimine was demonstrated to hold a relevant degree of cytotoxicity, which can nevertheless be circumvented by reducing its molar mass with a balanced effect in the transfection rate. [START_REF] Zinselmeyer | The Lower-Generation Polypropylenimine Dendrimers Are Effective Gene-Transfer Agents[END_REF] The dendritic PLL is relevant in this class of non-viral gene delivery vectors. Indeed, the architecture was reported to have a notable influence on the transfection rate of poly(amino acid)s, and higher yields were demonstrated for dPLL compared to the linear configuration. [START_REF] Yamagata | Structural Advantage of Dendritic Poly(l-lysine) for Gene Delivery into Cells[END_REF] The evidences were linked to the lower pKa of the globular architecture, resulting in weakened dendrimergene interaction, thus allowing an easier intracellular nucleic acid unpacking. Besides, one can still find macromolecules based on dendritic carbosilane [START_REF] Weber | Characterization of Carbosilane Dendrimers as Effective Carriers of siRNA to HIV-Infected Lymphocytes[END_REF] and glycodendrimers [START_REF] Cloninger | Biological Applications of Dendrimers[END_REF] evaluated as potential gene delivery vectors. Nevertheless, they are normally effective only when conjugated with positively charged segments. [START_REF] Arima | Sugar-Appended Polyamidoamine Dendrimer Conjugates with Cyclodextrins as Cell-Specific Non-Viral Vectors[END_REF]

Polymers

Cationic polymers (polycations) can bind via electrostatic forces to nucleic acids producing supramolecular aggregates with overall dimensions comparable to those of viruses. The phosphate groups of the nucleic acids normally interact with positively charged synthetic vectors leading to the formation of electrostatic complexes (named polyplexes). Amongst the subclasses of polymers that can be used in nucleic acid delivery, the most important are nitrogen-based polycations including polypeptides, polymethacrylates, polysaccharides, poly(β-amino esters), polyvinylamines and polyamines (mostly, polyethylenimine and derivatives). The last one is indeed the main focus of this review nevertheless, we briefly describe other subclasses of nitrogen-based polycations investigated towards the same goal in the following section.

MAIN CLASSES OF POLYMER-BASED NON-VIRAL VECTORS FOR GENE THERAPY

Poly(amino acid)s

Regarding the class of poly(amino acid)s, poly-L-lysine (PLL) is certainly the most commonly found in the literature. The polymer can be synthesized by ringopening polymerization of -benzyloxycarbonyl-L-lysine N-carboxyanhydride (Lys(Z)-NCA). [START_REF] Chen | Peptide-Based and Polypeptide-Based Gene Delivery Systems[END_REF] The polyplexes produced by using PLL are efficiently uptaken by cells however, they suffer of a serious disadvantage which is the poor ability to escape the endosomes due to the low buffering capacity of PLL below physiological pH (pKa ~ 10.5), [89] thereby resulting in reduced transfection efficiency. This could be to some extent circumvented by grafting histidine or poly-L-histidine (PLH) into the main chain, then providing buffering capacity due to the histidine protonation at pH < 6. [START_REF] Midoux | Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes[END_REF] Poly-L-histidine holds buffering capacity, but poor nucleic acid binding capacity at physiological pH due to insufficient cationization. The poly(amino acid)s poly-Lornithine (PLO) [START_REF] Conejos-Sánchez | Polyornithine-Based Polyplexes to Boost Effective Gene Silencing in CNS Disorders[END_REF] and poly-L-arginine (PLR) [START_REF] Zhou | Progress in Arginine-Based Gene Delivery Systems[END_REF] are also considered as gene carriers.

Indeed, polyplexes produced using PLO were evidenced to provide overall better results compared to those manufactured using PLL as attributed to the greater affinity of PLO to pDNA, therefore condensing the biomacromolecule at lower N/P ratios. [START_REF] Ramsay | Polylysine and Polyornithine Gene Transfer Complexes: A Study of Complex Stability and Cellular Uptake as a Basis for their Differential in-vitro Transfection Efficiency[END_REF] The use of PLR is useful particularly towards enhanced cellular uptake as inspired by cellpenetrating peptides. Although a couple of poly(amino acid)s demonstrated reasonable transfection rates, the polyplexes suffer of a common drawback which is the high level of cytotoxicity, particularly when the molar mass is elevated (this is frequently evidenced for polycations as previously stated). The coating by using poly(ethylene glycol) (PEG), such as in the construction of the gene carrier CK30PEG10K, attenuates cytotoxicity. This macromolecule consists of a 30 mer PLL conjugated to 10 kDa PEG.

Electrostatic complexes produced using CK30PEG10K were demonstrated to be nonimmunogenic, able to condense large DNA chains and efficiently deliver them into RPE cells, therefore with potential application in ocular gene therapies. [START_REF] Kelley | DNA Nanoparticles Are Safe and Nontoxic in Non-Human Primate Eyes[END_REF][START_REF] Han | Comparative Analysis of DNA Nanoparticles and AAVs for Ocular Gene Delivery[END_REF][START_REF] Boylan | Highly Compacted DNA Nanoparticles with Low MW PEG Coatings: In Vitro, Ex Vivo and In Vivo Evaluation[END_REF] Figure 4 provides the molecular structure of the main polymers investigated as potential gene delivery vectors, including the class of poly(amino acid)s.

Polymethacrylates

Polymethacrylates containing amino groups in their structure have been evaluated as gene delivery vectors. These polymer-based carriers are commonly synthesized via controlled polymerization techniques, thus enabling the synthesis of well-defined chains. The investigations are essentially centered on poly [2-(dimethylamino) ethyl methacrylate] (PDMAEMA) which contains a tertiary amino group and can be found in different architectures (linear and star-shaped, for instance).

Although polyplexes produced from PDMAEMA can be internalized efficiently by cells, the endosomal escape has been demonstrated to be the main bottleneck for its effectiveness. [START_REF] Layman | Influence of Polycation Molecular Weight on Poly(2-dimethylaminoethyl methacrylate)-Mediated DNA Delivery In Vitro[END_REF][START_REF] Funhoff | Cationic Polymethacrylates with Covalently Linked Membrane Destabilizing Peptides as Gene Delivery Vectors[END_REF] The transfection efficiency is notably influenced by the molar mass [START_REF] Layman | Influence of Polycation Molecular Weight on Poly(2-dimethylaminoethyl methacrylate)-Mediated DNA Delivery In Vitro[END_REF] and the architecture. [START_REF] Synatschke | Influence of Polymer Architecture and Molecular Weight of Poly(2-(dimethylamino)ethyl methacrylate) Polycations on Transfection Efficiency and Cell Viability in Gene Delivery[END_REF] Beneficial synergic effect in this regard was reached by using a mixture of PDMAEMA and poly(-amino ester) (this class of potential nonviral gene delivery vectors is discussed hereafter). [START_REF] Santo | Combination of Poly[(2-dimethylamino)ethyl methacrylate] and Poly(β-amino ester) Results in a Strong and Synergistic Transfection Activity[END_REF] Furthermore, the transfection efficiency of polymethacrylate-based polyplexes has been recently demonstrated to be enhanced by using higher contents of primary amino groups in the chains, whereas higher amounts of tertiary amino groups hamper the process. [START_REF] Trützschler | Beyond Gene Transfection with Methacrylate-Based Polyplexes-The Influence of the Amino Substitution Pattern[END_REF] The authors additionally highlighted that the endosomal escape is likely to be related to a strong polymer-membrane interaction rather than the popular proton sponge effect. In this category, we recall the attempts concerning PDAMA, [START_REF] Funhoff | Endosomal Escape of Polymeric Gene Delivery Complexes Is Not Always Enhanced by Polymers Buffering at Low pH[END_REF] as the reported results were important to deepen the discussions with respect to the actual mechanism of endosomal escape.

5.3.

Poly(β-amino ester)s

Poly(-amino ester)s are polymers with properties of tertiary amines and esters, therefore presumably able to bind to nucleic acids at physiological pH, and holding degradable and pH-responsive properties. They are typically synthesized by Michael addition from a diacrylate and an amine. Accordingly, the large portfolio of amine and diacrylate monomers as well as post-polymerization approaches enable the manufacturing of large polymer libraries with different properties concerning pH responsiveness and degradability. The linear form of poly(-amino ester)s (PAEs) was introduced by Langer et al., [START_REF] Lynn | Degradable Poly(β-amino esters): Synthesis, Characterization, and Self-Assembly with Plasmid DNA[END_REF] and it has been demonstrated that the chemical nature of the terminal groups significantly impacts cytotoxicity and transfection rates. [START_REF] Anderson | Structure/Property Studies of Polymeric Gene Delivery Using a Library of Poly(β-amino esters)[END_REF][START_REF] Akinc | Synthesis of Poly(β-amino ester)s Optimized for Highly Effective Gene Delivery[END_REF] This feature encouraged the synthesis of branched PAEs, therefore with a higher number of available terminal groups permitting further optimizations. [START_REF] Huang | Tailoring Highly Branched Poly(β-amino ester)s: a Synthetic Platform for Epidermal Gene Therapy[END_REF][START_REF] Liu | Highly Branched Poly(β-amino ester) Delivery of Minicircle DNA for Transfection of Neurodegenerative Disease Related Cells[END_REF] The polymer structure, especially the degree of hydrophobicity, influences the degradation and DNA condensation ability in this class of potential nonviral gene delivery vectors. [START_REF] Zhong | A Versatile Family of Degradable Non-Viral Gene Carriers Based on Hyperbranched Poly(ester amine)s[END_REF] Truly, the history of PβAEs in gene delivery is still fairly recent, and there is no a clear scenario concerning clinical trials at the moment.

The synthesis, formulation and biomedical applications of PβAEs have been recently

reviewed, [START_REF] Liu | Poly(β-Amino Esters): Synthesis, Formulations, and Their Biomedical Applications[END_REF] as well as the use in gene delivery formulations. [START_REF] Cordeiro | Poly(β-amino ester)-Based Gene Delivery Systems: From Discovery to Therapeutic Applications[END_REF]

Polysaccharides

The polysaccharides overall do not hold cationic charges nevertheless, one exception is chitosan. This justifies the fairly high number of gene delivery investigations based on such natural polymer. [START_REF] Cao | Recent Advances in Chitosan-Based Carriers for Gene Delivery[END_REF] This polysaccharide is composed by β-(1→4)-linked D-glucosamine and N-acetyl-D-glucosamine as depicted in Figure 4.

Chitosan is the deacetylated form of chitin which is obtained by enzymatic deacetylation or chemical hydrolysis. [START_REF] Younes | Chitin and Chitosan Preparation from Marine Sources[END_REF] The DNA condensation is promoted, as usual, by the interaction of the phosphate groups of nucleic acids and the amino groups of the natural polymer. The ability of chitosan to provide gene transfection is nevertheless critically affected by the environmental pH. [START_REF] Lichtenberg | Efficacy of Chitosan/Double-Stranded RNA Polyplex Nanoparticles for Gene Silencing Under Variable Environmental Conditions[END_REF] These authors moreover highlighted that the particle size does not notably influence gene knockdown, nevertheless, the behavior is markedly influenced by the particle charge and interaction with organic matter. Indeed, at least to some extent due to its pKa ~ 6.5, [START_REF] Mohammed | An Overview of Chitosan Nanoparticles and Its Application in Non-Parenteral Drug Delivery[END_REF] chitosan itself has a fairly limited solubility as well as limited nuclei acid binding capacity at physiological pH (7.4). Nevertheless, the functional amine groups can be used as active sites, and chemical modifications are relatively easy to be accomplished. Hence, a variety of attempts have been conducted towards improving its performance. The transfection rates of chitosan-based polyplexes depend notably on the degree of deacetylation. Such a parameter must be precisely selected in order to compromise transfection levels and the formation of stable complexes. [START_REF] Köping-Höggård | Chitosan as a Nonviral Gene Delivery System. Structure-Property Relationships and Characteristics Compared with Polyethylenimine In Vitro and After Lung Administration In Vivo[END_REF][START_REF] Kiang | The Effect of the Degree of Chitosan Deacetylation on the Efficiency of Gene Transfection[END_REF] Besides the fairly common investigations concerning chitosan, and particularly its derivatives (such as in combination with PEI, for instance), [START_REF] Nam | Target Gene Delivery From Targeting Ligand Conjugated Chitosan-PEI Copolymer for Cancer Therapy[END_REF] a number of other investigations dealing with different polysaccharides including dextran and cyclodextrin (CD) can be found in the literature. Nevertheless, these natural polymers poorly interact with nucleic acids and they are typically found as segments of hybrid gene delivery vectors, generally grafted to nitrogen-based cations or polycations. Cyclodextrin-based nucleic acid delivery systems has been recently reviewed, [START_REF] Xu | Versatile Types of Cyclodextrin-Based Nucleic Acid Delivery Systems[END_REF] and dextran-spermine, [START_REF] Azzam | Polysaccharide-Oligoamine Based Conjugates for Gene Delivery[END_REF] dextran-PEI, [START_REF] Sun | Synthesis of (Dex-HMDI)-g-PEIs as Effective and Low Cytotoxic Nonviral Gene Vectors[END_REF] dextran-histidine [START_REF] Thomas | Unraveling the Intracellular Efficacy of Dextran-Histidine Polycation as an Efficient Nonviral Gene Delivery System[END_REF] and dextran-chitosan [START_REF] Park | Galactosylated Chitosan-graft-Dextran as Hepatocyte-Targeting DNA Carrier[END_REF] were also investigated.

Polyvinylamines

The polyvinylamines (PVAm) are obtained generally by controlled hydrolysis of polyvinylamides previously prepared by radical polymerization [START_REF] Dréan | Far Beyond Primary Poly(vinylamine)s Through Free Radical Copolymerization and Amide Hydrolysis[END_REF] and recently, progresses in the polymerization protocols allowed for well-defined polyvinylaminebased polymers with controlled molar masses. [START_REF] Dréan | Controlled Synthesis of Poly(vinylamine)-Based Copolymers by Organometallic-Mediated Radical Polymerization[END_REF] Polyvinylamine and poly(Nmethylvinylamine), respectively bearing primary and secondary amines, demonstrated high transfection yields and low levels of cytotoxicity. [START_REF] Dréan | Use of Primary and Secondary Polyvinylamines for Efficient Gene Transfection[END_REF] The effectiveness of polyvinylamines with secondary amines is molar mass-independent, whereas remarkable influence of the molar mass was observed for the case of primary amines.

Furthermore, it has been demonstrated beneficial effects potentialized by the incorporation of imidazole and guanidine groups in the polymer chains. [START_REF] Dréan | Poly( N-methylvinylamine)-Based Copolymers for Improved Gene Transfection[END_REF] The efficacy is nonetheless dependent on the chemical modifications performed, particularly the degree of substitution, and transfection levels compared to lPEI in a variety of cell lines can be reached by playing with such parameter. Although an old family of cationic entities, this class of polymers has been seldom explored in the framework of gene delivery, and further advances are expected to be shown shortly.

Polyethylenimine

Concerning polymeric gene vectors, certainly the most investigated polymer is polyethylenimine (PEI) in different architectures and derivatives. This is justified by the high levels of transfection activity evidenced, although frequently linked to high degrees of cytotoxicity. Accordingly, strategies for optimizing its performance and overcome such drawback are still a field of active research. The PEI chains are easily substituted by using a variety of different chemical groups, and this feature encouraged the manufacturing of different derivatives. We review the main approaches towards overcoming different extracellular and intracellular barriers in the following section.

6.

POLYETHYLENIMINE-BASED NON-VIRAL

VECTORS FOR GENE THERAPY

The Discovery of PEI as a Potential Non-Viral Gene Delivery Vector

The PEI-based polycations are considered as a second generation of polymers investigated as potential gene delivery vectors. [START_REF] Boussif | A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and In Vivo: Polyethylenimine[END_REF] The second-generation embraces PEI and the already mentioned dendrimer PAMAM. The amine groups in the PEI chains are only partially protonated, and there are many of them not protonated at physiological pH, which are capable of picking up more protons when pH reduces. [START_REF] Gallops | A Polymer Physics Perspective on Why PEI Is an Effective Nonviral Gene Delivery Vector[END_REF] This is a notable difference compared to polycations of the first generation (PLL, for instance) which is fully protonated at physiological pH, therefore with limited endolysosomal buffering capacity, and then requiring the addition of lysosomotropic additives or membrane disruptive agents to promote the endosomal escape. Indeed, PEI has started to be vastly investigated towards the intracellular delivery of nucleic acids precisely due to the sufficient residual positive charges at physiological pH enabling its binding to nucleic acids, besides the buffering capacity at lower pH. The PEI chains are fairly flexible in the chemical point of view and then, their features can be tuned to some extent to optimize polyplex stability, cytotoxicity, cellular uptake and transfection efficiency. Nevertheless, although PEI is able to mediate endosomal escape of polyplexes and further gene transfection, the efficiency is only moderate compared to viruses. Truly, the PEI discovery allowed a deeper understanding of the gene delivery pathway along with a list of requirements for the successfulness of non-viral gene delivery vectors based on polymeric materials. We underline in the following sections essentially PEI derivatizations hypothesized to optimize principally the cytotoxicitygene transfection balance.

Main Architectures of PEI Chains

The cationic polymer PEI is mostly found in two different architectures as depicted in Figure 5. This depends essentially on the synthetic strategy used to manufacture the material. The linear (lPEI) chains are synthesized typically via the cationic ring-opening polymerization of 2-ethyl-2-oxazoline (PEtOx) with further hydrolysis, whereas the acid-catalyzed polymerization of aziridine leads to the manufacturing of branched chains (bPEI). These are the configurations more commonly used to deliver nucleic acids inside the cells, although other topologies were investigated, such as comb-like [START_REF] Haladjova | Comblike Polyethylenimine-Based Polyplexes: Balancing Toxicity, Cell Internalization, and Transfection Efficiency via Polymer Chain Topology[END_REF] and cyclic chains. [START_REF] Cortez | The Synthesis of Cyclic Poly(ethylene imine) and Exact Linear Analogues: An Evaluation of Gene Delivery Comparing Polymer Architectures[END_REF] The PEI chains contain a very high density of nitrogen atoms which allows chain protonation, particularly in acidic ambients. The branched configuration (bPEI) contains primary, secondary and tertiary amines usually in the ratio 1:2:1 and with different pKa (4.5 for primary, 6.7 for secondary and 11.6 for tertiary amines). [START_REF] Willner | Photoinduced Electron-Transfer Processes Using Organized Redox-Functionalized Bipyridinium-Polyethylenimine-Titania Colloids and Particulate Assemblies[END_REF] The presence of tertiary amines presumably provides a high density of positive charge at physiological pH, thus allowing nucleic acid binding. We nevertheless highlight that these pKa values cannot be easily assigned due to the effect of local groups and polyelectrolyte behavior. Other numbers have been also suggested, such as the predominant protonation of primary [START_REF] Wang | Novel Branched Poly(Ethylenimine)-Cholesterol Water-Soluble Lipopolymers for Gene Delivery[END_REF] or secondary [START_REF] Jun | The Application of Polyethyleneimine Draw Solution in a Combined Forward Osmosis/Nanofiltration System[END_REF] amines at neutral pH. The degree of protonation anyway increases by reducing the pH. The linear configuration has only secondary amines and empirical data as well as theoretical calculations suggest that roughly 60% of the amino groups are protonated at nearly neutral pH, regardless of the chain architecture (linear or branched). [START_REF] Ziebarth | Understanding the Protonation Behavior of Linear Polyethylenimine in Solutions through Monte Carlo Simulations[END_REF][START_REF] Tang | The Influence of Polymer Structure on the Interactions of Cationic Polymers with DNA and Morphology of the Resulting Complexes[END_REF] This feature permits both architectures (lPEI and bPEI) to condense genetic material, and they mediate endosomal escape and gene transfection. The linear configuration has been reported to be more efficient for gene delivery purposes as attributed to the higher stability of bPEI polyplexes, leading then to less efficient unpacking of the biomacromolecules in the intracellular milieu.

[135]

Strategies to Enhance Effectiveness

The research concerning PEI-based polyplexes is highly active and the more we progress the more we understand the main challenges which still limit clinical outputs.

The transfection efficiency and cytotoxicity of the polymeric gene delivery vectors are usually linked to the chain´s molar mass. This is indeed a nasty correlation since usually the longer the chains, the higher the transfection rates, but higher levels of cytotoxicity are observed at the same time. Accordingly, endless efforts have been already dedicated to target more efficient polymeric vectors of lower cytotoxicity. The long way from the synthesis of a non-viral gene vector towards the delivery of the nucleic acid inside the cells is highly complex as underlined previously. Different steps of the delivery pathway require commonly conflicting properties on the chemical and structural points of view. Therefore, the requirements of non-viral vectors throughout the whole pathway (nucleic acid binding and condensation, serum stability, cell targeting and cellular uptake, efficient endosomal escape, intracellular unpacking, cytosolic migration and nuclei entry) have to be precisely understood to hypothesize chemical modifications able to optimize the delivery performance. The PEI chains can be manufactured by using different synthetic strategies depending on the desired configuration, and derivatives can be constructed during the polymerization procedures, or by using postpolymerization protocols.

Synthetic Approaches towards the Manufacturing of PEI Derivatives

The chemical modification of PEI chains is expected to optimize the properties of the non-viral gene delivery vectors. This can be theorized aiming a variety of goals within the delivery pathway. The functionalization with targeting ligands is a universal strategy in nanomedicine to target specific cells. The modifications are also useful to improve stealth properties and endosomal escape capacity, to enable the intracellular unpacking of the genetic material, as well as to reduce cytotoxicity. In this context, different strategies can be used to modify the chains. The synthesis of lPEI is carried out usually via the acidic or basic hydrolysis of poly(2-oxazoline)s. Functional groups can be introduced during the oxazoline polymerization using functional initiator in the initiation step and/or functional nucleophile during the termination step. [START_REF] Guillerm | How to Modulate the Chemical Structure of Polyoxazolines by Appropriate Functionalization[END_REF] This is not usually the preferred synthetic route, particularly to couple biological molecules, because these are frequently sensitive to environmental conditions and can be degraded easily during the hydrolysis step, therefore losing activity. Hence, the postfunctionalization of PEI chains is commonly employed despite being more tedious. The hydrolysis of POx creates secondary amines that can be used to graft residues able to improve biocompatibility, facilitate targeting and enhance buffering capacity, for instance. [START_REF] Jiang | Recent Advances in the Development of Polyethylenimine-based Gene Vectors for Safe and Efficient Gene Delivery[END_REF] The small molecules can be grafted at the polymer units and/or at the chain ends. [START_REF] Diehl | Thermo-Responsive Polyoxazolines with Widely Tuneable LCST[END_REF][START_REF] Xie | Targeted Delivery of siRNA to Activated T Cells via Transferrin-Polyethylenimine (Tf-PEI) as a Potential Therapy of Asthma[END_REF][START_REF] Pons | ω-Hydrazino Linear Polyethylenimine: A Monoconjugation Building Block for Nucleic Acid Delivery[END_REF] The amine groups allow a fairly large portfolio of postfunctionalization reactions, thus permitting the manufacturing of a variety of derivatives starting either form the linear or branched configuration.

Hydrophobization of PEI Chains

The manufacturing of PEI chains with attached hydrophobic units can improve the delivery performance of transfecting agents since polycations with optimized degree of hydrophobicity are able to favorably interact with cell membranes containing lipids and cholesterol moieties, thereby leading to enhanced cellular uptake. [START_REF] Liu | Hydrophobic Modifications of Cationic Polymers for Gene Delivery[END_REF] The hydrophobization of the chains are also capable to provide enhanced stability to the supramolecular assemblies. [START_REF] Liu | Hydrophobic Modifications of Cationic Polymers for Gene Delivery[END_REF][START_REF] Wang | Enhancing the In Vitro and In Vivo Stabilities of Polymeric Nucleic Acid Delivery Nanosystems[END_REF] The approach can also reduce the polymernucleic acid binding strength which is beneficial for intracellular nucleic acid unpacking and release of the genetic material. [START_REF] Gabrielson | Acetylation of Polyethylenimine Enhances Gene Delivery via Weakened Polymer/DNA Interactions[END_REF] The versatility of PEI chains enables the covalent link of various types of hydrophobic segments (alkyl chains, fatty acids and cholesteryl groups are some examples). The linking of cholesteryl chloroformate to primary amines was evidenced to increase PEI transfection efficiency, [START_REF] Han | Water-Soluble Lipopolymer for Gene Delivery[END_REF] although the substitution of the secondary amines was later suggested to be advantageous since primary amines would then remain available for DNA condensation. [START_REF] Wang | Novel Branched Poly(Ethylenimine)-Cholesterol Water-Soluble Lipopolymers for Gene Delivery[END_REF] The position of the substituent has indeed a notable impact in the transfection performance of hydrophobically-modified PEI chains. [START_REF] Thomas | Enhancing Polyethylenimine's Delivery of Plasmid DNA into Mammalian Cells[END_REF] Overall, polyplexes produced from cholesterol-modified PEI chains demonstrates augmented cellular uptake and transfection efficiency. The fluorination of PEI chains was demonstrated to be an efficient strategy to provide enhanced serum stability and cellular uptake, although the degree of substitution has to be precisely controlled to balance cytotoxicity and intracellular delivery issues. [START_REF] Lee | Fluorination Effect to Intermediate Molecular Weight Polyethylenimine for Gene Delivery Systems[END_REF] In the same framework, the modification of PEI with cholesterol (Chol) along with superhydrophobic perfluorinated (F) moieties (F-PEI-Chol) evidenced to provide enhanced stability, cellular uptake and siRNA silencing, as well as lower cytotoxicity compared to PEI, F-PEI and PEI-Chol. [START_REF] Wu | Combined Hydrophobization of Polyethylenimine with Cholesterol and Perfluorobutyrate Improves siRNA Delivery[END_REF] The hydrophobic modification of low molar mass PEI by using short propionic acid (PrA) has been also hypothesized to enhance transfection efficiency. Optimized cellular uptake and efficient siRNA-induced silencing has been achieved with moderate degrees of substitution, highlighting that the balance between benefits and drawback is tiny, and remarkable increase in surface hydrophobicity might cause deleterious effects. [START_REF] Meneksedag-Erol | A Delicate Balance When Substituting a Small Hydrophobe onto Low Molecular Weight Polyethylenimine to Improve Its Nucleic Acid Delivery Efficiency[END_REF] Likewise, higher transfection efficiency has been obtained by modifying PEI chains with shorter hydrophobic groups compared to longer ones, and relevant influence of the conjugation degree has been also underlined. [START_REF] Teo | Hydrophobic Modification of Low Molecular Weight Polyethylenimine for Improved Gene Transfection[END_REF] Beneficial effects have been also demonstrated via the substitution of low molar mass PEI with linoleic acid (LA) and αlinolenic acid (αLA) towards siRNA delivery to myeloid leukemia cells. [START_REF] Valencia-Serna | siRNA-Mediated BCR-ABL Silencing in Primary Chronic Myeloid Leukemia Cells Using Lipopolymers[END_REF] The grafting of oleic and stearic acids to branched PEI was evidenced to conduct to better siRNA binding and protection in serum-containing media, which accordingly assisted the siRNA delivery process. [START_REF] Alshamsan | Formulation and Delivery of siRNA by Oleic Acid and Stearic Acid Modified Polyethylenimine[END_REF] The modification of PEI chains by using different types of fatty acids led to efficient targeting of pulmonary microvascular endothelium as mainly driven by surface charge features. [START_REF] Dunn | Highly Efficient In Vivo Targeting of the Pulmonary Endothelium Using Novel Modifications of Polyethylenimine: An Importance of Charge[END_REF] Overall, the hydrophobization of PEI chains is generally reported to be a clever strategy to enhance the efficacy of PEI in nucleic acid delivery, as attributed at least to some extent to enhanced cellular uptake of the polyplexes. [START_REF] Shen | Screening of Efficient Polymers for siRNA Delivery in a Library of Hydrophobically Modified Polyethyleneimines[END_REF] The approach is also useful to assist intracellular steps of the gene delivery pathway, to reduce the cytotoxicity and to enhance the serum stability of resulting polyplexes. Nevertheless, it is almost unanimity that the degree of hydrophobicity is highly relevant and must be precisely tuned to balance benefits and drawbacks.

Conjugation of Amino Acids and Peptides to PEI Chains

Taking into account the conjugation of amino acids to PEI chains, the linear configuration of polyethylenimine (lPEI) was substituted with histidine via the Michael reaction, for instance. The histidinylated lPEI (His-lPEI) polyplexes allowed for efficient gene transfection as attributed to increased buffering capacity thanks to the presence of the imidazole function at the histidine moieties. The insertion of carboxylic functions also introduces negative charges to the polymeric gene delivery vector leading to lower cytotoxicity. [START_REF] Bertrand | Histidinylated Linear PEI: a New Efficient Non-Toxic Polymer for Gene Transfer[END_REF] Moreover, cryo-TEM analysis of His-lPEI polyplexes revealed a less organized system compared to lPEI counterparts. The amorphous structure along with enhanced buffering capacity provided by the histidine residues possibly favor the intracellular dissociation of the assemblies and better cytosolic availability of pDNA, thus conducting to higher transfection efficiency. [START_REF] Maury | Influence of pDNA Availability on Transfection Efficiency of Polyplexes in Non-Proliferative Cells[END_REF] Along these lines, it has been demonstrated recently that a mixture of lPEI and His-lPEI increases the transfecting activity in NIH3T3 fibroblasts and skeletal muscle cells, although at the current stage, the main driving force contributing to the phenomenon remains unclear. [START_REF] Gomez | Improved Histidinylated lPEI Polyplexes for Skeletal Muscle Cells Transfection[END_REF] The use of imidazole-containing units to improve transfection efficiency, particularly in serum environment, and reduce the cytotoxicity of PEI-based polyplexes was also demonstrated with other agents (apart of amino acids) such as the urocanic acid. [START_REF] Ihm | Improved Biocompatibility of Polyethylenimine (PEI) as a Gene Carrier by Conjugating Urocanic Acid: In Vitro and In Vivo[END_REF] The conjugation of other amino acids or their analogs similarly demonstrated enhanced transfection efficiency, particularly the grafting of glycolic acid to PEI chains, although the degree of substitution seems always to be a critical parameter. [START_REF] Zhang | Amino Acid-Modified Polyethylenimines with Enhanced Gene Delivery Efficiency and Biocompatibility[END_REF] The peptidization of PEI chains was hypothesized to reduce cytotoxicity and enhance the cellular uptake of non-viral gene delivery vectors. The approach can be additionally used to improve the intracellular performance of gene carriers, such as by modifying PEI chains with pH triggered peptides. In this framework, the PEI functionalization with the C6M3 peptide enabled membrane lysis and efficient endosome release of the supramolecular assemblies, as well as high gene transfer capability along with biocompatibility to red blood cells at neutral pH. [START_REF] Chen | Peptide Modified Polycations with pH Triggered Lytic Activity for Efficient Gene Delivery[END_REF] The enhanced cellular uptake of peptide-modified PEI chains was also demonstrated for PEI decorated with Arg-Gly-Asp (RGD) which can target integrin receptors overexpressed in cancer cells. [START_REF] Park | A Review of RGD-Functionalized Nonviral Gene Delivery Vectors for Cancer Therapy[END_REF][START_REF] Fu | RGD Peptide-Based Non-Viral Gene Delivery Vectors Targeting Integrin αvβ3 for Cancer Therapy[END_REF] The coating of PEI-DNA polyplexes with RGD or HIV-1 TAT peptides increased the cellular uptake of the therapeutic carriers and the transfection efficiency in neuronal cells. The experimental data additionally suggested augmented endosomal escape, pointing out that the modifications assist different steps of the delivery pathway. [START_REF] Suk | Gene Delivery to Differentiated Neurotypic Cells with RGD and HIV Tat Peptide Functionalized Polymeric Nanoparticles[END_REF] The targeting capability has been similarly observed for PEI conjugate to the RPM peptide that targets preferably invasive colorectal cancer.

[163]

Sugar Decoration of PEI Chains

The engineering of bioactive glyconanostructures whose outer shell is decorated with sugar residues potentially enhances the cellular uptake of polyplexes via cellsurface reception, which can therefore augment the bioavailability of nucleic acids. The cell-specific targeting can be used with carbohydrates due to their specificity to lectins that can be present in the cell surfaces. [START_REF] Yan | Glycotargeting to Improve Cellular Delivery Efficiency of Nucleic Acids[END_REF] Along these lines, enhanced cellular uptake of bioactive molecules was evidenced using oligosaccharide-shelled hyperbranched PEI complexed to ATP molecules, [START_REF] Appelhans | Hyperbranched PEI with Various Oligosaccharide Architectures: Synthesis, Characterization, ATP Complexation, and Cellular Uptake Properties[END_REF] and the strategy was considered to enhance the biocompatibility and in vivo gene delivery efficacy of polyplexes, for instance, by grafting maltose or maltotriose moieties. [START_REF] Gutsch | Biocompatibility and Efficacy of Oligomaltose-grafted Poly(ethylene imine)s (OM-PEIs) for in Vivo Gene Delivery[END_REF] Moreover, the sugar-decoration may provide shielding to the polyplexes making them less susceptible to opsonization, and offering more robust serum stability as required for systemic delivery systems. [START_REF] Erbacher | Transfection and Physical Properties of Various Saccharide, Poly(ethylene glycol), and Antibody-Derivatized Polyethylenimines (PEI)[END_REF] The PEI chains were also galactosylated by reductive amination to target hepatocytes. [START_REF] Kunath | Galactose-PEI-DNA Complexes for Targeted Gene Delivery: Degree of Substitution Affects Complex Size and Transfection Efficiency[END_REF] The sugar provided a shielding effect to the supramolecular aggregates, and the cytotoxicity was reduced by increasing the degree of galactosylation however, the transfection efficiency was evidenced to be highly affected by the degree of substitution, which indeed seems to be critical, regardless of the chemical nature of the substituent. The use of glycosylated PEI was demonstrated to provide enhanced gene transfer, although the efficacy is reduced in differentiated cells compared to undifferentiated counterparts. [START_REF] Fajac | Uptake of Plasmid/Glycosylated Polymer Complexes and Gene Transfer Efficiency in Differentiated Airway Epithelial Cells[END_REF] The behavior was suggested to be related to impaired intracellular trafficking of the distinct polyplexes. The substitution of primary amine groups in bPEI chains with lactose residues was demonstrated by us to lead to a substantial reduction in cytotoxicity with a balanced effect in gene expression. [START_REF] Albuquerque | Sweet Vector for Gene Delivery: the Sugar Decoration of Polyplexes Reduces Cytotoxicity with a Balanced Effect on Gene Expression[END_REF] The simultaneous alkylcarboxylation and galactose conjugation to 25 kDa PEI is effective in the delivering of DNA to hepatocyte cells. The alkylcarboxylated chains presumably improve the hydrophilic-hydrophobic balance in the polycations which are uptaken in high yields due to the abundance of asialoglycoprotein receptors (ASGPRs) in liver parenchymal cells. [START_REF] Dehshahri | Plasmid DNA Delivery into Hepatocytes Using a Multifunctional Nanocarrier Based on Sugarconjugated Polyethylenimine[END_REF] Enhanced uptake of galactosylated PEI by cells overexpressing ASGPRs was also evidenced for other derivatives. [START_REF] Dehshahri | Targeted double domain nanoplex based on galactosylated polyethylenimine enhanced the delivery of IL-12 plasmid[END_REF] PEI chains were likewise functionalized with β-glucan and the derivative demonstrated enhanced cellular uptake and higher transfection efficiency compared to bPEI in RAW264.7 cells. The evidences suggest that the derivatives are able to recognize and bind to membrane receptors of such particular cell type. [START_REF] Lee | Polyethylenimine-Functionalized Cationic Barley β-Glucan Derivatives for Macrophage RAW264.7 Cell-Targeted Gene Delivery Systems[END_REF] The mannosylation of PEI chains was demonstrated to be useful for targeting specific cell types for self-amplifying RNA (saRNA) delivery and expression. [START_REF] Blakney | Mannosylated Poly(ethylene imine) Copolymers Enhance saRNA Uptake and Expression in Human Skin Explants[END_REF] Overall, sugar decoration of PEI is able to enhance the cellular uptake of polyplexes due to the suitable affinity of the moieties to components present in the cell membranes, and the approach typically enhances serum stability and biocompatibility (increased cell viability is normally monitored).

Environmentally-Responsive PEI Chains

Polyethylenimine is known to be an efficient gene delivery vector however, it is accepted that chemical modifications are useful to augment transfection rates and decrease reactions of the immune system. The strategy of using PEI derivatives with environmentally-responsive properties usually targets non-viral gene delivery vectors with high transfection efficiency, but lower cytotoxicity. The method can be used to shield the positively charged surface of PEI during systemic circulation, thereby avoiding the non-specific adsorption of negatively charged proteins (abundant in the bloodstream) which typically induces immune responses. Nevertheless, residual positive charges at the surface of polyplexes are desired to enhance cellular uptake via favorable electrostatic interactions. Thus, charge recovery can be reached at the interface with targeting cells by using environmentally-responsive PEI chains that respond to slightly acid microenvironments commonly found in tumor sites, for example. In this framework, shielded PEI-based polyplexes were constructed using aldehyde-containing PEG chains able to react with the amino groups forming stable Schiff bases in physiological environment. The shielding provided decreased cytotoxicity, improved stability and prolonged circulation time. The PEG de-shielding was achieved by the cleavage of the chemical bonds at slightly acidic milieu, then allowing the recovery of positive charge with beneficial outputs with respect to cellular uptake and transfection activity. [START_REF] Guan | Ultrasensitive pH Triggered Charge/Size Dual-Rebound Gene Delivery System[END_REF] The shielding of positively charged PEI/pDNA polyplexes was also reached by using zwitterionic polymers with charge conversion features. [START_REF] Chen | Poly(l-glutamic acid)-Based Zwitterionic Polymer in a Charge Conversional Shielding System for Gene Therapy of Malignant Tumors[END_REF] Concerning the intercellular compartment, approaches to targeted PEI chains of high molar mass that can be degraded in the intracellular environment may conduct to gene vectors of lower toxicity. [START_REF] Islam | Major Degradable Polycations as Carriers for DNA and siRNA[END_REF] In this context, biodegradable PEI derivatives were constructed by using different functional groups such as disulfide bonds, [START_REF] Breunig | Breaking up the Correlation Between Efficacy and Toxicity for Nonviral Gene Delivery[END_REF][START_REF] Zhu | Dual-Responsive Polyplexes with Enhanced Disassembly and Endosomal Escape for Efficient Delivery of siRNA[END_REF][START_REF] Lei | Glutathione-Sensitive RGD-Poly(ethylene glycol)-SS-Polyethylenimine for Intracranial Glioblastoma Targeted Gene Delivery[END_REF] and ketal, [START_REF] Knorr | Acetal Linked Oligoethylenimines for use as pH-Sensitive Gene Carriers[END_REF] imine, [START_REF] Che | Biologically Responsive Carrier-Mediated Anti-Angiogenesis shRNA Delivery for Tumor Treatment[END_REF][START_REF] Jiang | Chitosan-Graft-Polyethylenimine as a Gene Carrier[END_REF][START_REF] Kim | Polyethylenimine with Acid-Labile Linkages as a Biodegradable Gene Carrier[END_REF] amide, [START_REF] Petersen | Poly(ethylenimine-col-lactamide-co-succinamide): A Biodegradable Polyethylenimine Derivative with an Advantageous pH-Dependent Hydrolytic Degradation for Gene Delivery[END_REF] ester, [START_REF] Forrest | A Degradable Polyethylenimine Derivative with Low Toxicity for Highly Efficient Gene Delivery[END_REF][START_REF] Zhang | Low Molecular Weight PEI-Based Vectors via Acid-Labile Ortho Ester Linkage for Improved Gene Delivery[END_REF] hydrazone [START_REF] Fang | Low Molecular Weight PEIs Modified by Hydrazone-Based Crosslinker and Betaine as Improved Gene Carriers[END_REF] and carbamate [START_REF] Xu | Novel Poly(ethylene imine) Biscarbamate Conjugate as an Efficient and Nontoxic Gene Delivery System[END_REF] linkers. These investigations overall reported reduced cytotoxicity and enhanced transfection rates regardless of the functional group providing degradability. Disulfide bonds were for instance used to link short PEI chains to produce longer ones. The intracellular degradation was induced by the presence of glutathione (GSH), and the release of less toxic PEI fragments was identified. [START_REF] Breunig | Breaking up the Correlation Between Efficacy and Toxicity for Nonviral Gene Delivery[END_REF] The reductive degradation is, therefore, manifested to be an innovative strategy in the design of polymeric non-viral vectors with improved efficacy and reduced levels of hypoxia. [START_REF] Zhang | Reactive Oxygen Species (ROS)-Degradable Polymeric Nanoplatform for Hypoxia-Targeted Gene Delivery: Unpacking DNA and Reducing Toxicity[END_REF] Truthfully, the use of environmentally-responsive and degradable chains is possibly one of the best strategies to balance benefits and drawbacks of polymeric gene delivery vectors. [START_REF] Chen | Biodegradable Polymers for Gene-Delivery Applications[END_REF]

Shielding of PEI Chains

The use of shielding agents is intended to provide extracellular stability to polyplexes, thus reducing aggregation in highly complex media such as plasma and accordingly, increasing the blood circulation time. Indeed, bare PEI polyplexes are susceptible to protein adsorption whenever swimming in biological fluids. The PEGylation approach is overall used in nanomedicine to provide stealth properties and colloidal stability, thus reducing the recognition of nanocarriers by the immune system, [START_REF] Kolate | PEG -A Versatile Conjugating Ligand for Drugs and Drug Delivery Systems[END_REF] as well as their cytotoxicity depending on the final composition. [START_REF] Shuai | Novel Biodegradable Ternary Copolymers hy-PEI-g -PCL-b-PEG: Synthesis, Characterization, and Potential as Efficient Nonviral Gene Delivery Vectors[END_REF][START_REF] Petersen | Polyethylenimine-graft-Poly(ethylene glycol) Copolymers: Influence of Copolymer Block Structure on DNA Complexation and Biological Activities as Gene Delivery System[END_REF] Despite the outstanding advantage, it is widely reported in the literature that such a strategy reduces the cellular uptake mainly due to the reduction in the zeta potential of the assemblies, then decreasing the strength of cell adhesion. The presence of PEG also interferes in the nuclei acid condensation process. [START_REF] Erbacher | Transfection and Physical Properties of Various Saccharide, Poly(ethylene glycol), and Antibody-Derivatized Polyethylenimines (PEI)[END_REF] Ultimately, it inevitably reduces the desired high transfection levels of gene delivery systems. [START_REF] Nguyen | Evaluation of Polyether-Polyethyleneimine Graft Copolymers as Gene Transfer Agents[END_REF] This can be to some extent balanced by precisely selecting PEG molar mass and degree of conjugation. [START_REF] Smith | Impact of Molecular Weight and Degree of Conjugation on the Thermodynamics of DNA Complexation and Stability of Polyethylenimine-graft-Poly(ethylene glycol) Copolymers[END_REF][START_REF] Zhang | Poly(ethylene glycol)-block-Polyethylenimine Copolymers as Carriers for Gene Delivery: Effects of PEG Molecular Weight and PEGylation Degree[END_REF] The advantages of optimized PEGylation have been demonstrated in vivo. Increased circulation time and gene transfer without significant toxicity in tumor bearing mice were observed for PEI-based PEGylated assemblies in comparison with non-PEGylated counterparts. [START_REF] Ogris | PEGylated DNA/Transferrin-PEI Complexes: Reduced Interaction with Blood Components, Extended Circulation in Blood and Potential for Systemic Gene Delivery[END_REF] Yet, the searching for PEG substitutes is essential because it is widely accepted that the PEGylation is not able to completely prevent protein adsorption, [START_REF] Schöttler | Protein Adsorption is Required for Stealth Effect of Poly(ethylene glycol)-and Poly(phosphoester)-Coated Nanocarriers[END_REF] and that PEG triggers the activation of the complement system. [START_REF] Hamad | Poly(ethylene glycol)s Generate Complement Activation Products in Human Serum through Increased Alternative Pathway Turnover and a MASP-2-Dependent Process[END_REF] Additionally, the therapeutic efficacy of PEG-based nanomedicines is usually compromised by the presence of PEG antibodies (anti-PEG)

produced by the immune system, [START_REF] Zhang | Anti-PEG Antibodies in the Clinic: Current Issues and Beyond PEGylation[END_REF] thus inducing the so-called accelerated blood clearance (ABC). [START_REF] Ishida | Accelerated Blood Clearance (ABC) Phenomenon Upon Repeated Injection of PEGylated Liposomes[END_REF] Hence, PEG alternatives are claimed and amongst them, one finds different biocompatible polymers such as poly (2-oxazoline)s, polyglycerols, polyvinylpyrrolidone, polyacrylamides and zwitterionic polymers, particularly those based on sulfobetaine. [START_REF] Abbina | PEGylation and its Alternatives[END_REF] The use of copolymers containing PEtOx and PEI is useful to control the charge density of the polymer chains via partial acid hydrolysis with significant biological consequences. [START_REF] Bauer | Rethinking the Impact of the Protonable Amine density on Cationic Polymers for Gene Delivery: A Comparative Study of Partially Hydrolyzed Poly(2-ethyl-2-oxazoline)s and Linear Poly(ethylene imine)s[END_REF][START_REF] Blakney | One Size Does Not Fit All: The Effect of Chain Length and Charge Density of Poly(ethylene imine) Based Copolymers on Delivery of pDNA, mRNA, and RepRNA Polyplexes[END_REF] In the same lines, the use of block copolymers PEI-b-PEtOx as gene vectors provided enhanced cell viability without reducing transfection rates compared to PEI equivalents, [START_REF] Hsiue | Nonviral Gene Carriers Based on Diblock Copolymers of Poly(2-ethyl-2-oxazoline) and Linear Polyethylenimine[END_REF] and lPEI-comb-PEtOx polymers with different molar masses and grafting densities have been also proposed as vectors for gene delivery. [START_REF] Haladjova | Polyplex Particles Based on Comb-Like Polyethylenimine/Poly(2-ethyl-2oxazoline) Copolymers: Relating Biological Performance with Morphology and Structure[END_REF] In such a case, the transfection efficiency was evidenced to be dependent on the morphology of the manufactured assemblies. The grafting strategy has been explored by Gwak et al. who demonstrated enhanced stability of polyplexes in the presence of competing polyanions, and nuclease protection by using the cationic amphiphilic copolymer poly(lactide-co-glycolide)-graft-polyethylenimine (PgP) as transfecting agent, [START_REF] Gwak | Physicochemical Stability and Transfection Efficiency of Cationic Amphiphilic Copolymer/pDNA Polyplexes for Spinal Cord Injury Repair[END_REF] and copolymers made by poly(5-methyl-5allyloxycarbonyl-trimethylene carbonate) and PEI (PMAC-g-PEI) were evidenced to provide enhanced transfection efficiency and lower cytotoxicity to 293T cells compared to PEI. [START_REF] Wang | Polyethylenimine-Grafted Polycarbonates as Biodegradable Polycations for Gene Delivery[END_REF] The further introduction of 5,5-dimethyl-trimethylene carbonate (DTC) enabled the control over charge density and hydrophobicity, notably affecting endosomal escape and nucleic acid unpacking. [START_REF] He | Poly[(5-methyl-5allyloxycarbonyl-trimethylene carbonate)-co-(5,5-dimethyl-trimethylene carbonate)] with Grafted Polyethylenimine as Biodegradable Polycations for Efficient Gene Delivery[END_REF] The control over charge density on PEI-based polyplexes was similarly achieved via the physical adsorption of anionic polyelectrolyte layers. [START_REF] Chakraborty | Serum-Independent Nonviral Gene Delivery to Innate and Adaptive Immune Cells Using Immunoplexes[END_REF] Besides the use of polymeric materials to shield PEI-based gene carriers, the goal can be also targeted by using small molecules such as via the succinylation of the polycation. [START_REF] Warriner | Succinylated Polyethylenimine Derivatives Greatly Enhance Polyplex Serum Stability and Gene Delivery In Vitro[END_REF] Zwitterion-like derivatives were reported to reduce the aggregation in serum media and increase transfection rates. The enhanced activity was also attributed to reduced polymer-nucleic acid interaction strength therefore enabling a smoother unpacking of the genetic material in the intracellular environment. Nevertheless, a required balanced with regard to nucleic acid condensation was highlighted, which is tuned by the neutralization of primary and secondary amines and accordingly, once again dependent on the degree of substitution of the amine groups. The succinylation of PEI chains also conduct to remarkably lower polymer toxicity compared to unmodified chains.

[213]

PEI Chains with Specific Targeting Ligands

One widely investigated avenue towards the development of optimized non-viral vectors is the derivatization of polycations by using ligands to target specific cells. The use of molecules that are recognized by receptors present at the cell surfaces may intensify cell binding thereby enhancing cellular uptake and further gene expression.

The investigations concerning active targeting rely mostly on genetic approaches for cancer treatment. The strategy has been exploited for instance using transferrin (Tf), kDa) was determined by other authors using a combination of sedimentation velocity analysis and scanning force microscopy. [START_REF] Perevyazko | Polyelectrolyte Complexes of DNA and Linear PEI: Formation, Composition and Properties[END_REF] Recently, it has been highlighted that the number of loaded DNA copies can be precisely tuned by kinetically controlling the PEI/DNA assembly process, underscoring that charge neutralization is not a ratelimiting step, and that the assembly time is chiefly governed by chain folding and compaction of polyelectrolyte units. The control over the mixing conditions using flash nanocomplexation enabled the tuning over pDNA (~ 6 kbp) payload from 1.3 to 21.8 copies per particle with average hydrodynamic size ranging from 35 to 130 nm. The assemblies with intermediate payloads (6 to 10 pDNA copies per particle) evidenced to be more effective in mediating gene expression. [START_REF] Hu | Kinetic Control in Assembly of Plasmid DNA/Polycation Complex Nanoparticles[END_REF] Indeed, the delivered number of copies is particularly relevant concerning the applicability of polyplex formulations. For instance, the number of copies to be delivered in large mammals is expected to be in the range of 10 13 copies/kg [START_REF] Wang | Adeno-Associated Virus Serotype 8 Efficiently Delivers Genes to Muscle and Heart[END_REF] thus corresponding to roughly 2 x 10 -11 mol/kg or 100 g/kg of 10 kb pDNA. These numbers have to be considered only as rough estimations since they depend on many variables, including the size of the plasmid. Nevertheless, the use of non-viral vectors is a clever strategy also in this regard since it can carry more than one DNA copy, which is the loading capacity of a viral vector (one copy per capsid).

Investigations concerning the dynamic behavior of PEI/DNA polyplexes were also performed. They are certainly relevant to better understand the levels of transfection efficiency reported for different assemblies. Lisitsyna et al. suggested recently that at N/P = 2 all phosphate groups of DNA chains are bound to the nitrogen atoms of PEI, therefore forming a PEI/DNA core. [START_REF] Lisitsyna | Time-Resolved Fluorescence Spectroscopy Reveals Fine Structure and Dynamics of Poly(l-lysine) and Polyethylenimine Based DNA Polyplexes[END_REF] Further PEI addition results in positively charged shells. They evidenced a dynamic behavior of the complexes, where chains in the core and in the shell can be exchanged (PEI chains present in the shell can replace the ones attached to DNA in the core, and vice-versa). The PEI polyplexes are presumably much more dynamic than PLL counterparts and this can, at least to some extent, explain the favorable transfection using PEI polyplexes. Along these lines, it has been demonstrated that DNA chains can be exchanged between previously manufactured PEI-based polyplexes depending on the polymer/DNA interaction strength. This behavior has been observed particularly for lPEI partially substituted with histidine residues, whereas no exchange was evidenced in PLL polyplexes. The results simultaneously revealed that PEI-based polyplexes contain several DNA copies, and that remarkably different assemblies can be produced depending on the chemical features of the polymeric vector. Possibly, highly organized and well-compacted systems may disfavor further nucleic acid unpacking, thereby leading to reduced transfection capability. [START_REF] Pigeon | Evidence for Plasmid DNA Exchange After Polyplex Mixing[END_REF] This assumption has been highlighted by us while investigating His-lPEI and lPEI polyplexes as previously remarked. [START_REF] Maury | Influence of pDNA Availability on Transfection Efficiency of Polyplexes in Non-Proliferative Cells[END_REF] Still concerning the internal structure of such type of assemblies, the presence of large amounts of solvent has been determined inside PEI/DNA polyplexes. These authors highlighted that, apart from the N/P ratio, the concentration of the polyelectrolytes is highly relevant and, even at a fixed N/P ratio, size and charge of the electrostatic complexes are increasing functions of their concentration. [START_REF] Hou | Formation and Structure of PEI/DNA Complexes: Quantitative Analysis[END_REF] Taking into account the mechanism of complex formation, the PEI/DNA interaction has been proposed to be associated with both groove binding and electrostatic forces, the later acting at the external phosphate backbone, leading to DNA condensation. The binding was evidenced to be pH-dependent, [START_REF] Utsuno | Thermodynamics of Polyethylenimine-DNA Binding and DNA Condensation[END_REF] similarly demonstrated by Ketola et al., who also evidenced complete DNA condensation at N/P ~ 2. [START_REF] Ketola | Independent versus Cooperative Binding in Polyethylenimine-DNA and Poly(l-lysine)-DNA Polyplexes[END_REF] The mechanism of complex formation changes from independent binding at N/P < 0.6 and pH 7.4 to cooperative binding at higher N/P ratios. On the other hand, the complex formation is cooperative at all N/P ratios at pH 5.2, therefore suggesting that possibly, the manufacturing of the complexes at lower pH can be beneficial, although the transfection is performed at biological pH. This is linked to a higher amount of protonated amine groups as pH decreases. produced at N/P > 10. [START_REF] Perevyazko | Polyelectrolyte Complexes of DNA and Linear PEI: Formation, Composition and Properties[END_REF] The interaction of DNA with hydrophobically-modified PEI chains was also evidenced to occur in three steps. The biomacromolecule is initially partially compacted, then micrometric aggregates appear and further, compacted and positively charged assemblies with RH ranging from 52-86 nm were observed. [START_REF] Bellettini | Properties of Polyplexes Formed Through Interaction Between Hydrophobically-Modified Poly(ethylene imine)s and Calf Thymus DNA in Aqueous Solution[END_REF] Overall, a number of investigations were devoted to the dynamics, thermodynamics and structural features of PEI-based polyplexes, although there is still a missing link between the findings and the transfection activity. Similar investigations to be performed using different PEI derivatives would be highly valuable to identify relevant differences that can potentially be connected to different degrees of transfection efficiency.

Current Status towards Marketed Products

The origins of gene therapy remount to roughly 50 years ago with expectations and setbacks. At the present time, gene-based therapies are realistic to some extent, yet uncommon. This is at least partially justified by its focus, typically in rare disorders, then targeting a reduced number of patients. Such a specific feature raises remarkably the cost of treatments. The few number of commercially available products are essentially based on genetically modified viruses due to their natural ability to infect cells. [START_REF] Qtaish | Niosome-Based Approach for in Situ Gene Delivery to Retina and Brain Cortex as Immune-Privileged Tissues[END_REF] To the best of our knowledge, Onpattro (approved in the USA in August 2018)

is the only formulation based on a non-viral vector with market authorization for in vivo human use. [START_REF] Akinc | The Onpattro Story and the Clinical Translation of Nanomedicines Containing Nucleic Acid-Based Drugs[END_REF] This nanomedicine is based on lipid nanoparticles carrying RNA

CONCLUSIONS AND CRITICAL OPINION

The PEI-based non-viral vectors are efficient in the delivery of nucleic acids inside the cells, and potentially capable to substitute viral agents. The gene delivery by using non-viral vectors is nevertheless a multi-step process that poses many challenges, either regarding the extracellular or intracellular media. The advances in polymer and conjugate chemistry enabled the creation of a large number of PEI derivatives that were demonstrated to overwhelm some of the main difficulties. Figure 6 reports representative examples of approaches reported in the literature. Importantly, the derivatization is usually beneficial to different steps of the transfection process, although we highlight the main advantage. particularly of PEI derivatives, would be valuable since these features can be further connected to the different levels of transfection efficiency hitherto evidenced. The gene delivery by using polymeric platforms is undoubtedly a highly complex process and presumably, one will find the most promising vectors among those able to respond to the inherent features of the extracellular and intracellular environments to face the typically incompatible correlations.

Figure 1 .

 1 Figure 1. Schematic representation of the main extracellular and intracellular barriers

Figure 3 .

 3 Figure 3. Molecular structure of the main dendrimers used in non-viral gene delivery

Figure 4 .

 4 Figure 4. Molecular structure of the main polymers used in the manufacturing of non-

Figure 5 .

 5 Figure 5. Chemical structures of linear (A) and branched (B) PEI.

  cytotoxicity. Zhang et al. demonstrated the use of reactive oxygen species (ROS)degradable polymeric gene delivery vectors in the same perspective. Stimuli-responsive cross-linked OEI-TKx were manufactured using oligoethylenimine (OEI) via thioketal (TK) linkages that are cleaved selectively in ROS-rich environments (another common feature of tumor sites). The vectors induce lower cytotoxicity and enhanced gene transfection efficiency compared to PEI/DNA polyplexes. The performance was attributed to the cleavage of thioketal linkages and OEI-TKx/DNA disassembly in the intracellular ambient as triggered by high ROS-concentration mediated by

6 . 4 .

 64 folate (FR), integrins and epidermal growth factor (EGF) receptors, which are known to be overexpressed at the surface of a variety of cancer cells. Xie et al. for instance, functionalized PEI chains with transferrin (Tf-PEI) to optimize the gene transfection in asthmatics (transferrin receptors are overexpressed on cells responsible for this disease).[START_REF] Xie | Targeted Delivery of siRNA to Activated T Cells via Transferrin-Polyethylenimine (Tf-PEI) as a Potential Therapy of Asthma[END_REF] In vitro studies demonstrated notably enhanced cellular uptake and gene knockdown mediated by Tf-PEI polyplexes in human primary Activated T cells (ATCs), and in vivo biodistribution of the polyplexes on a murine asthmatic model confirmed that Tf-PEI polyplexes can efficiently and selectively deliver siRNA to ATCs. The folate ligand is also widely used for cell targeting purposes. For example, folic acid was coupled to 1.8 kDa bPEI via two successive N-acylation reactions. The conjugates with relatively low substitution amounts (especially PEI-FA0.65) conducted to the smallest polyplexes at weight ratio = 3 and nearly 100-fold higher transfection efficiency compared to unsubstituted chains. The performance was attributed to folate receptor-mediated cellular uptake, better pDNA binding ability and favorable pH buffering capacity.[START_REF] Cao | Folic Acid Directly Modified Low Molecular Weight of Polyethyleneimine for Targeted pDNA Delivery[END_REF] The folic acid targeting function was also evidences in PEIbased ternary complexes manufactured for gene delivery purposes.[START_REF] Guo | Preparation and Evaluation of pH-Responsive Charge-Convertible Ternary Complex FA-PEI-CCA/PEI/DNA with Low Cytotoxicity and Efficient Gene Delivery[END_REF] Guo and Lee similarly investigated PEI vectors functionalized with folate (FA).[START_REF] Guo | Receptor-Targeted Gene Delivery via Folate-Conjugated Polyethylenimine[END_REF] PEI-FA vectors transfected KB cells at the same level compared to unmodified complexes. The efficiency was improved by using the PEGylation strategy conferring increased availability of the ligand thus optimizing cell recognition. Different ligands were evaluated to target EGF-overexpressing cells including different peptides and antibodies.[START_REF] Ren | Peptide GE11-Polyethylene Glycol-Polyethylenimine for targeted Gene Delivery in Laryngeal Cancer[END_REF][START_REF] Liu | An Oligopeptide Ligand-Mediated Therapeutic Gene Nanocomplex for Liver Cancer-Targeted Therapy[END_REF][START_REF] Wang | A Gene Nanocomplex Conjugated with Monoclonal Antibodies for Targeted Therapy of Hepatocellular Carcinoma[END_REF] Concerning particularly the overexpression of αvβ3 integrin receptors on cancer cells, one example of targeting approach was the conjugation of Lthyroxine to PEI chains for pDNA delivery. These receptors hold binding sites for Lthyroxine and two-fold higher transfection rate compared to unmodified PEI was evidenced in cell lines overexpressing integrin.[START_REF] Sadeghpour | Double Domain Polyethylenimine-Based Nanoparticles for Integrin Receptor Mediated Delivery of Plasmid DNA[END_REF] Insights on the Formation and Structural Features of PEIbased/DNA PolyplexesAlthough a lot of efforts have been dedicated to optimize PEI-based polyplexes via the chemical modification of the polymer chains, fewer investigations have been performed with regard to the formation and structural features of the electrostatic complexes, and the majority of them have been performed only by using unmodified chains. Significant discoveries have been shared, although the link with transfection activity has not been robustly identified so far. The presence of free PEI chains in optimized formulations for transfection seems to be well accepted as suggested theoretically[START_REF] Ziebarth | Structural Comparisons of PEI/DNA and PEI/siRNA Complexes Revealed with Molecular Dynamics Simulations[END_REF] and repeatedly shown experimentally. Clamme et al. reported that ~ 86% of the PEI chains are freely diffusing in solution and that PEI/DNA polyplexes are composed by an average of 3.5 plasmids (5.1 kbp) and 30 PEI 25 kDa chains at N/P = 6.[START_REF] Clamme | Monitoring of the Formation and Dissociation of Polyethylenimine/DNA Complexes by Two Photon Fluorescence Correlation Spectroscopy[END_REF] These numbers are variable and the presence of PEI/DNA polyplexes composed on average by 8 to 32 pDNA (Mw = 2.1 x 10 6 g.mol -1 ) and 70 ± 25 lPEI chains(13.4

  Concerning the structural features of PEI/DNA polyplexes at different N/P ratios, Mengarelli et al. reported a sequence of weak and strong complexation followed by charge inversion and further dissociation of large aggregates as the polycation concentration increases. Smaller and negatively charged complexes at low polymer concentrations are produced first. They are then condensed at higher polycation concentration, thereby forming large anionic aggregates at N/P ~ 1. They are further dissolved as linked to charge inversion at N/P > 1.[230] This has been similarly observed by Perevyazko et al. who evidenced incomplete DNA condensation and formation of primary PEI/DNA complexes at N/P < 1, their merging at N/P ~ 2 leading to the formation of large aggregates (~ 1 m), and finally their progressive dissolution as N/P increases. Stable electrostatic complexes with average size of 170 ± 65 nm were

  interference (RNAi) for the treatment of amyloid polyneuropathy. The majority of the gene therapies in clinical trials are indeed based on viral vectors (around 70%) and, within the class of non-viral vectors, they are produced mainly by using lipids.Although many of them were abandoned in the pathway from the clinical evaluation towards market authorization, various others are currently being tested. Concerning polymer-based formulations, they are typically built by using a polycation (for nucleic acid condensation), PEG (for shielding purposes) and specific ligands (for cell targeting). This includes, for instance, CALAA-01 (first targeted polymer-based nanoparticle-carrying siRNA administered in humans) consisting of four components (siRNA, cyclodextrin, PEG and transferrin).[START_REF] Zuckerman | Correlating Animal and Human Phase Ia/Ib Clinical Data with CALAA-01, a Targeted, Polymer-Based Nanoparticle Containing siRNA[END_REF][START_REF] Davis | Evidence of RNAi in Humans from Systemically Administered siRNA via Targeted Nanoparticles[END_REF]. PEI-based DNA vaccination against neuroblastoma and gene therapy for pancreatic ductal adenocarcinoma are currently listed in clinical trials (https://www.clinicaltrials.gov). PEI-based formulations in clinical evaluation can be also found with derivatives such as PEG-PEIcholesterol.[START_REF] Chen | Production and Clinical Development of Nanoparticles for Gene Delivery[END_REF] Despite the clinical progresses, there is no polymer-based gene therapeutics with marketed authorization at the present time, as far as we know.

Figure 6 .

 6 Figure 6. Examples of PEI derivatives designed to overcome extracellular and

ACKNOWLEDGMENTS

We acknowledge the sponsoring by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant 2019/20470-8) and by the French National Funding Agency (ANR-17-CE18-0015-01-VINP). L.J.C.A and F.A.O acknowledge the fellowships granted by FAPESP (grants 2016/23844-8 and 2019/12944-0), and F.C.G acknowledges the fellowship granted by FAPESP (grant 2018/11038-2) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant 303268/2020-4).

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.