Aminoglycosides: from Antibiotics to Building Blocks for the Synthesis and Development of Gene † Delivery Vehicles

Review Aminoglycosides: From Antibiotics to Building Blocks for the Synthesis and Development of Gene † Delivery Vehicles

Maria Cristina Bellucci 1 and Alessandro Volonterio 2,*

1 Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy; [email protected] 2 Department of Chemistry, Material and Chemical Engineer “Giulio Natta”, Politecnico di Milano, via Mancinelli 7, 20131 Milano, Italy * Correspondence: [email protected]; Tel.: +39-02-2399-3139 This review is dedicated to the memory of Leonardo Manzoni. † Received: 30 June 2020; Accepted: 7 August 2020; Published: 11 August 2020

Abstract: Aminoglycosides are a class of naturally occurring and semi synthetic antibiotics that have been used for a long time in fighting bacterial infections. Due to acquired antibiotic resistance and inherent toxicity, aminoglycosides have experienced a decrease in interest over time. However, in the last decade, we are seeing a renaissance of aminoglycosides thanks to a better understanding of their chemistry and mode of action, which had led to new trends of application. The purpose of this comprehensive review is to highlight one of these new fields of application: the use of aminoglycosides as building blocks for the development of liposomal and polymeric vectors for gene delivery. The design, synthetic strategies, ability to condensate the genetic material, the efficiency in transfection, and cytotoxicity as well as when available, the antibacterial activity of aminoglycoside-based cationic lipids and polymers are covered and critically analyzed.

Keywords: aminoglycosides; gene delivery; non-viral vectors

1. Introduction “The future of aminoglycosides: the end or renaissance?”. This is the title of an excellent review by Prof. Sylvie Garneau-Tsodikova et al. published exactly ten years ago [1]. In this paper, the authors interrogated the future of aminoglycosides (AGs) as antibiotics. Indeed, AGs, displacing broad-spectrum activity against both Gram-negative and Gram-positive bacteria, have been used to fight pathogenic bacteria for a long time, actually since 1943 when streptomycin was discovered [2]. The mode of action of AGs has been widely investigated, showing that AGs are able to bind to the decoding A-site of the 16S ribosomal RNA of bacteria, resulting in the interference of protein synthesis that eventually leads to cell death and to helix 69 in the 50S ribosomal subunit hampering mRNA/tRNA translocation and ribosome recycling [3–5]. These recognition events depend mainly on electrostatic interactions between the positively charged ammonium groups on the AG scaffold and the negatively charged phosphates present on the target, and, secondary, on less important contributions coming from hydrogen bond networking. However, the heavy use (or abuse) of AGs during the time resulted in the development of antibiotic resistance through different mechanisms encompassing reduced uptake and increased efflux, modification of the ribosomal target, and AG regioselective modification (namely acetylation, phosphorylation, and nucleoside transferring) by AG-modifying enzymes [6]. Moreover, the clinical efficiency of AGs has been also hindered by their nephrotoxicity [7] and ototoxicity [8]. In order to fight the acquired resistance and/or toxicity, different

Antibiotics 2020, 9, 504; doi:10.3390/antibiotics9080504 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, x 2 of 28 Antibiotics 2020, 9, 504 2 of 29 different strategies for the selective functionalization of AGs have been developed and recently reviewed [9,10]. However, although many structure–activity relationship (SAR) studies have been strategiesaddressed, for resistance the selective against functionalization different microorgani of AGs havesms been as well developed as inherent and recently toxicity reviewed remains [a9 ,big10]. However,challenge althoughto be faced. many Ten structure–activity years later from relationshipthe above-mentioned (SAR) studies review, have the been research addressed, on the resistance AGs is againststill ongoing, different and microorganisms although the failure as well of different as inherent projects toxicity could remains have aelicited big challenge the end of to AGs, be faced. the Tenbetter years understanding later from the of above-mentioned the mode of action review, and the ch researchemistry, on which the AGs triggered is still ongoing,an extension and although of their theapplications, failure of di hasfferent led projectsto a renaissance could have of elicitedthese drugs. the end Indeed, of AGs, AGs the betterare currently understanding being investigated of the mode ofnot action only andas chemistry,building blocks which for triggered the synthesis an extension of new of theirantibacterial applications, agents, has ledbut toalso a renaissance as potential of theseantifungals, drugs. Indeed,antiprotozoals, AGs are genetic currently regulating being investigated agents, and not as only cationic as building heads blocksin the fordevelopment the synthesis of ofmultifunctional new antibacterial drug agents, delivery but vectors also as potential[11]. antifungals, antiprotozoals, genetic regulating agents, and asIn cationic this review, heads we in will the developmentfocus the reader’s of multifunctional attention on the drug use delivery of AGs vectors as scaffolds [11]. for building molecularIn this or review, polymeric we willvehicles focus to the facilitate reader’s the attention introduction on the of genes use of into AGs ce aslls sca(geneffolds delivery). for building After moleculara brief introduction or polymeric on AG vehicles classi tofication facilitate and thethe introductiongene delivery of field, genes we into will cells provide (gene an delivery). overall Afterdescription a brief introductionof AG-based on cationic AG classification lipids and and thepolymers gene delivery projected field, as we gene will providedelivery an vectors, overall descriptionencompassing of AG-based the chemistry, cationic the lipids ability and to polymers condensate projected DNA/RNA, as gene the delivery efficiency vectors, on delivering encompassing the thegenetic chemistry, material the to ability the target to condensate cell organelles, DNA/RNA, cytotoxicity, the efficiency and, onwhen delivering available, the the genetic antibacterial material toactivity. the target Although cell organelles, a certain cytotoxicity,amount of work and, has when been available, done in the this antibacterial field, a real activity.definition Although of which a certainparameters amount should of work be taken has been into doneconsideration in this field, to help a real in definition the design of of which new parametersAG-based liposomes should be takenand/or into polymers consideration for efficient to help gene in the delivery design application of new AG-based is still liposomesmissing. The and scope/or polymers of this review for effi cientis to genecollect, delivery for the application first time, isall still the missing.information The scopeavailabl ofe this on reviewthe literature is to collect, and to for try the to firstdefine time, a sort all the of information“rule of thumb”, available which on will the be literature useful for and the to tryfutu tore definedevelopment a sort of of “rule more of efficient thumb”, AG-based which will gene be usefuldelivery for vectors. the future We development also believe of that more this effi cient“relatively AG-based unexplored” gene delivery field vectors. of application We also believeof AG thatantibiotics this “relatively could further unexplored” help in field the offuture application of the ofrenaissance AG antibiotics of this could class further of fascinating help in the natural future ofmolecules. the renaissance of this class of fascinating natural molecules.

2. Classiﬁcation and Chemistry of Aminoglycosides (AGs) 2. Classification and Chemistry of Aminoglycosides (AGs) AGsAGs areare aa classclass of naturally occurring and semi-synthetic polyamino sugars that can be classified classiﬁed basedbased onon thethe substitutionsubstitution ofof thethe 2-deoxystreptamine2-deoxystreptamine (2-DOS) ring, a common building block present onon mostmost AGsAGs (Figure(Figure1 1).). Accordingly,Accordingly, there there are are three three main main classes classes of of AGs: AGs: 4,5-disubstituted 4,5-disubstituted 2-DOS2-DOS AGs;AGs; 4,6-disubstituted 2-DOS 2-DOS AGs; AGs; and and a third a third class class encompassing encompassing monosubstituted monosubstituted 2-DOS 2-DOS AGs AGs and andstreptomycin. streptomycin.

FigureFigure 1.1. Structures of some aminoglycosides (AGs) discussed in this review.

AtAt ﬁrstfirst sight, the selective selective functionalization functionalization of of AGs AGs could could seem seem an an insurmountable insurmountable task task due due to tothe the presence presence of ofmany many hydroxy hydroxy and and amino amino function functionalal groups, groups, each each one one havi havingng similar similar reactivity. reactivity. However,However, thethe extensiveextensive workwork donedone onon thethe searchsearch ofof AGAG derivativesderivatives in orderorder toto overcomeovercome antibiotic Antibiotics 2020,, 99,, 504x 3 of 2928

resistance and/or toxicity has paved the way to solving this challenging task. In general, some resistancefunctional and groups/or toxicity are much has pavedmore available the way to to solving functionalization this challenging being task. more In general,accessible some because functional they groupsare less aresterically much hindered. more available More tospecifically, functionalization some AGs being such more as Neo, accessible Kan A becauseand B, and they Tob are bear less stericallyonly one primary hindered. hydroxy More group specifically, that can some be expl AGsoited such for as selective Neo, Kan functionalization A and B, and (Figure Tob bear 2). onlyActually, one after primary the protection hydroxy group of the thatamino can function be exploiteds with Boc for or selective Cbz, this functionalization primary hydroxy (Figure group2 ).is Actually,selectively after transformed the protection in a good of theleaving amino group functions by reaction with with Boc an or excess Cbz, this of sterically primary hindered hydroxy grouptriisopropylbenzensulfonyl is selectively transformed chloride in a(TIBS-Cl). good leaving The group resulting by reaction sulfate withis displaced an excess by of stericallydifferent hinderednucleophiles triisopropylbenzensulfonyl such as amines, azide, and chloride thiols, (TIBS-Cl). in order Theto obtain resulting the sulfatefinal adducts is displaced or intermediates by different nucleophilesready for further such aschemoselective amines, azide, fu andnctionalization. thiols, in order Analogously, to obtain the th finale presence adducts of or only intermediates one, less readysterically for furtherhindered chemoselective methyleneamino functionalization. function in ot Analogously,her AGs such the as presence Nea, Par, of Kan only A one, and less B, stericallyand Tob hinderedcould be exploited methyleneamino to discern function the reactivity in other of AGsthe amino such asfunctional Nea, Par, groups. Kan A A andccordingly, B, and Tob by couldreacting be exploitedNea, Par, Kan to discern A and theB, and reactivity Tob with of theCbz- aminoN-hydroxy-5-norbornene-2,3-dicarboxylic functional groups. Accordingly, by acid reacting imide, Nea, or Par,more Kan in general, A and B, with and N Tob-hydroxysuccinimide with Cbz-N-hydroxy-5-norbornene-2,3-dicarboxylic (NHS) esters, chemoselective protection acid imide, or acylation or more of inthe general, methyleneamino with N-hydroxysuccinimide moiety can be achieved (NHS) esters,respectively. chemoselective However, protection the latter or acylationstrategy is of less the methyleneaminopursued, mainly for moiety two reasons: can be achieved (1) the yields respectively. are not very However, high, and the (2) latter one strategyamino functional is less pursued, group mainlyis sacrificed, for two the reasons: amino (1)groups the yields being are generally not very high,important and (2) for one the amino function functional of AGs. group Along is sacrificed, with the theformer amino groupsstrategies, being generallyother importantsynthetic for the functionpathways of AGs. Alongrelaying with theon former strategies,multistep otherprotection/functionalization/deprot synthetic pathways relaying onection multistep approaches protection have/functionalization been developed./deprotection However, approaches since the havechemistry been exploited developed. for However,the synthesi sinces of theAG-based chemistry gene exploited delivery forvectors the synthesis reviewed of herein AG-based pertains gene to deliverythe strategies vectors depicted reviewed in Figure herein 2, pertains we will to not the go strategies through depicted these strategies, in Figure su2,ggesting we will not that go interested through thesereaders strategies, refer to the suggesting comprehensive that interested review readerscited in refer[9,10]. to the comprehensive review cited in [9,10].

H2N BocHN BocHN OH 1) Boc2O, TEA OTIBS Nu Nu AG AG AG 2) TIBS-Cl, py HO HO HO

SO2Cl - AG = Neo, Kan A, Kan B, Tob TIBS-Cl = Nu = N3 , RSH, RNH2

O O O O O N N H N H N H2N O O Ph 2 O R 2 NHCbz NH2 N R O O H AG AG AG Route A Route B HO HO HO

AG = Nea, Par, Kan A, Kan B, Tob Figure 2. General strategies for the chemoselective functionalization of AGs. 3. Gene Delivery 3. Gene Delivery Gene delivery is a fundamental step of gene therapy that wants to prevent or treat not only Gene delivery is a fundamental step of gene therapy that wants to prevent or treat not only genetic diseases, but also other disorders including vascular diseases, neurodegenerative and infectious genetic diseases, but also other disorders including vascular diseases, neurodegenerative and disorders, and many types of cancers by introducing genetic material into specific cells in order to infectious disorders, and many types of cancers by introducing genetic material into specific cells in replace a defective or missing gene, to inactivate a gene that is functioning improperly, or to add order to replace a defective or missing gene, to inactivate a gene that is functioning improperly, or to a gene/protein function [12–14]. There exist two major categories of gene therapy, encompassing add a gene/protein function [12–14]. There exist two major categories of gene therapy, encompassing germline and somatic gene therapy (Figure3)[ 15]. While germline gene therapy, although very germline and somatic gene therapy (Figure 3) [15]. While germline gene therapy, although very promising, suffers from ethical concerns and is prohibited in many countries [16,17], somatic gene promising, suffers from ethical concerns and is prohibited in many countries [16,17], somatic gene delivery systems have finally reached the clinical and market after facing quite numerous obstacles delivery systems have finally reached the clinical and market after facing quite numerous obstacles and failures [18]. Ex vivo somatic gene therapy relies on the manipulation in vitro of selected cells Antibiotics 2020, 9, 504 4 of 29 Antibiotics 2020, 9, x 4 of 28 and failures [18]. Ex vivo somatic gene therapy relies on the manipulation in vitro of selected cells that are first explanted from the body and, after the manipulation, re-implanted into the target tissues. that are first explanted from the body and, after the manipulation, re-implanted into the target tissues. On the other hand, in in vivo somatic gene delivery, the delivering of the genetic material occurs in On the other hand, in in vivo somatic gene delivery, the delivering of the genetic material occurs in situ [19]. However, two major barriers must be overcome for this technology to be efficient: both situ [19]. However, two major barriers must be overcome for this technology to be efficient: both DNA DNA and RNA must first be protected from the action of hydrolyzing enzymes present in the and RNA must first be protected from the action of hydrolyzing enzymes present in the extracellular extracellular matrix and, second, must be helped to cross the cell membrane since they are not able matrixto enter and, the second, cell spontaneously must be helped mainly to cross due the cellto membraneelectrostatic since repulsion they are with not ableglycoproteins to enter the and cell spontaneouslyphospholipids. mainly In order due to to surmount electrostatic such repulsion barriers, with many glycoproteins physical methods and phospholipids. have been developed, In order to surmountencompassing, such barriers,among others, many physicalmechanical, methods electrical, have beenhydrodynamic, developed, and encompassing, ultrasonic among[20]. Another others, mechanical,way to achieve electrical, these goals hydrodynamic, is to develop and a biological ultrasonic or [20 chemical]. Another helper, way referred to achieve to as these a vector, goals that is to developshould be a biological able to pack or chemical and protect helper, the referred gene outs toide as a the vector, cell, that cross should the cell be ablemembrane, to pack and protectfinally thedeliver gene the outside gene the into cell, the cross target the organelle. cell membrane, Such andvehicles finally are deliver classified the geneas viral into and the targetnon-viral organelle. gene Suchdelivery vehicles vectors are [21,22]. classified Viruses as viral are and so far non-viral the most gene efficient delivery gene vectors delivery [21 vectors,,22]. Viruses having are reached so far the in mostsome e casesfficient clinical gene deliveryapproval vectors, by the U.S. having Food reached and Drug in some Administration cases clinical (FDA) approval and bythe the market U.S. Food[23]. andHowever, Drug Administration the use of viruses (FDA) is hampered and the market by thei [r23 marked]. However, immunogenicity, the use of viruses limited is hamperedcarrying capacity, by their markedand insertional immunogenicity, mutagenesis. limited In carryingorder to capacity,overcome and the insertional drawbacks mutagenesis. associated with In order the touse overcome of viral thevectors, drawbacks scientists associated are focusing with theattention use of on viral develo vectors,ping scientists efficient areand focusing non-cytotoxic attention non-viral on developing vectors, ewhichfficient are and generally non-cytotoxic cationic non-viralliposomes/micelles vectors, which or polymers are generally that are cationicable to condensate liposomes/ micellesthe genetic or polymersmaterial thatthrough are able electrostatic to condensate interactions. the genetic Liposomes material through are composed electrostatic of amphiphilic interactions. Liposomesmolecules aresubstantially composed made of amphiphilic of a cationic molecules head and substantiallylipophilic tail(s) made tethered of a cationic by a linker head [24]. and The lipophilic cationic tail(s)head tetheredis important by a linkerfor the [24 condensation]. The cationic of head the isgenetic important material for the through condensation electrostatic of the interactions, genetic material the throughnumber electrostaticof cationic charges, interactions, the nature, the number and tridimensional of cationic charges, disposition the of nature, the cationic and tridimensional head groups dispositionhaving a great of theinfluence cationic on head the transfection groups having efficiency a great [25]. influence Lipophilic onthe tails transfection can be classified efficiency as linear [25]. Lipophilichydrocarbon tails alkyl can chains, be classified encompassing as linear saturated hydrocarbon and alkylunsaturated chains, chains, encompassing (per)fluorinated saturated alkyl and unsaturatedchains, and cholesterol chains, (per)fluorinated derivatives. These alkyl are chains, important and cholesterol for the stability derivatives. of the liposomes, These are membrane important forcrossing, the stability and influence of the liposomes, the transfection membrane efficiency crossing, [26]. and Finally, influence the linker, the transfection although eprobablyfficiency less [26]. Finally,considered the linker, if not althoughfor chemical probably purposes, less considered is important if not because for chemical its length, purposes, tridimensional is important geometry, because itsand length, chemical tridimensional composition geometry, might affect and the chemical transfecti compositionon efficiency, might biodegradability, affect the transfection and cytotoxicity efficiency, biodegradability,of the vectors [27]. and cytotoxicity of the vectors [27].

FigureFigure 3.3. DiDifferentﬀerent gene delivery systems. Antibiotics 2020, 9, 504 5 of 29

There is a great variety of polymeric gene delivery vectors, which renders their classification quite difficult. Polymers used in gene delivery encompass linear, branched, or dendrimeric structures and their monomers range from simple molecules such as ethyleneamine, ethylenamidamine, and acrylate to more sophisticated carbohydrates, amino acids, carbosilane, and phosphorous containing monomers [28]. Natural polymers such as chitosan or commercially available polymers like linear and branched polyethyleimine (lPEI or bPEI) or polyethylenaminoamine (PAMAM) dendrimers of different generations are probably the most widely used polymers in this field, with bPEI 25 KDa considered the gold standard [29]. Polymeric cationic gene delivery vectors often suffer from the transfection efficiency/cytotoxicity paradox: the more cationic moieties in the structure, the more efficient and the more cytotoxic the polymer. For this reason, as well as for their polyplex instability, poor selectivity, insufficient endosomal escape, and difficult DNA release, there is great interest in the synthesis of new polymers and in the functionalization of non-viral polymeric vectors with different moieties, encompassing lipidic chains, cationic moieties, amino acids, peptides, and saccharides [30]. In this review, we will cover the use of AGs as building-blocks for the synthesis of liposomes, for the functionalization of commercially available polymers, and for the synthesis of hyperbranched polymers.

4. AG-Based Lipid Gene Delivery Vectors In the quest for efficient and safe non-viral gene delivery vectors, cationic lipids are very attractive because they are relatively easy to design, synthesize, and characterize, being made of tethering three modules: a cationic moiety, a lipophilic tail(s), and a linker to connect them. Among the cationic moieties, AGs have reached a certain interest in the last twenty years. The rational beyond the use of AGs is manifold: (1) they are natural binders of a great variety of RNAs through electrostatic interactions [31]; (2) although they have lower affinity for DNA, some AGs are able to tightly interact with DNA and to protect it from nucleases [32]; (3) they possess a broad spectrum antibacterial activity that could be exploited for the preparation of multifunctional vectors [1,6]; (4) there is a good variety of commercial available AGs with a different number of amines with different spatial dispositions that could be exploited to fine tune the efficiency/cytotoxicity of the corresponding liposomes; and (5) the chemistry of AGs is well documented [9,10]. Aside from the AG moiety, the performances of these vectors strongly depend on the lipophilic tail(s) tethered and on the nucleic acid delivered. For this reason, we decided to organize this section in different sub-sections describing the behavior of AG-based lipidic vectors built with “conventional lipidic tails” such as cholesterol or linear hydrocarbon chains for DNA delivery and RNAs delivery. A third section will be dedicated to AG-based lipidic vectors bearing “non-conventional” lipidic tails.

4.1. AG-Based Lipid Vectors for DNA Delivery The first paper dealing with the use of AGs to build gene delivery vectors appeared in 2002 where the synthesis of two kanamycin-cholesterol conjugates, namely kanamycin-cholesterol KanaChol 1 and the corresponding triguanidinium-kanamycin-cholesterol TGKanaChol 2, was described (Figure4)[ 33]. Kanamycin A was tethered to cholesterol via a carbamoyl bond, a functional group that is considered both chemically stable and biodegradable. According to route A depicted in Figure2, thus sacrificing one out of the four amino groups on the kanamycin scaffold, the unique reactivity of the only methyleneammino group has been exploited [34]. Liposomes made with KanaChol 1 and TGKanaChol 2 in Hepes buffer solution (pH 7.4) were able to deliver in vitro plasmid pCMV-Luc, a luciferase-expressing plasmid DNA, in an efficient way, showing almost the same results both in terms of efficiency and in terms of cytotoxicity, whereas kanamycin itself was ineffective. In more detail, the transfection in three cell lines, namely HeLa, HEK293, and COS cell lines, was most successful at a N/P ratio (nitrogen to phosphate groups) around 5, resulting in a decreasing efficiency for higher value of N/P ratios, probably due to an increase of toxic effects. This result was quite unforeseen since TGKanaChol 2 was expected to be more efficient than KanaChol 1. Indeed, guanidinoglycosides are known to have both higher RNA affinity and much greater ability Antibiotics 2020, 9, 504 6 of 29 to cross cell membranes compared to their unguanidinylated counterparts [35–40]. Interestingly, co-formulation with dioleoylphosphatidylthanolamine (DOPE), a neutral phospholipid that is known to help endosomal escape of the internalized lipoplexes [41], did not affect the in vitro transfection efficiency of KanaChol 1, demonstrating that KanaChol 1 is an efficient non-viral gene delivery vector that does not need liposomal formulation. However, co-formulation with DOPE was useful in order to deliver cholesterol-poly(ethylene glycol) (Chol-PEG) stabilized DNA in vivo through intranasal

Antibioticsadministration 2020, 9, x into mouse airways. 6 of 28

Figure 4. StructuresStructures of Kanamycin A-based lipids 1–8.

KanamycinIn order to check A was the tethered influence to of thecholesterol linker and via lipophilic a carbamoyl tails, abond, small a library functional of kanamycin-based group that is consideredliposomes wereboth chemically synthetized stable using and 6-aminocapronic biodegradable. acid According (Cap), lysineto route (Lys), A depicted and succinic in Figure acid 2, (Suc)thus sacrificinglinkers, and one cholesterol, out of the dioleyl four amino amine, groups and disteraryl on the aminekanamycin lipophilic scaffold, tails (Figure the unique4)[ 42 reactivity]. In more of detail, the onlyfollowing methyleneammino the same synthetic group strategy has been (Path exploi A, Figureted [34].2) three Liposomes new derivatives made with with KanaChol cholesterol 1 were and prepared,TGKanaChol namely 2 in KanaCapCholHepes buffer solution3 having (pH a 6-aminocapronic 7.4) were able to acid deliver linker, in and vitro two plasmid derivatives pCMV-Luc, having a luciferase-expressinglysine linker, one of them, plasmid KanaLysChol DNA, in 4an, bearing efficien onet way, cholesterol, showing while almost the the other, same KanaLysDiChol results both in5, termsbears twoof efficiency cholesterol and units. in terms Moreover, of cytotoxicity, two derivatives whereas with kanamycin a lipophilic itself dioctadecylamine was ineffective. and In succinic more detail,acid linker, the transfection namely KanaSucDODA in three cell 6lines,and thenamely corresponding HeLa, HEK293, triguanidinylated and COS cell TGKanaSucDODA lines, was most successful7, and one at bearing a N/P ratio dioleylamine (nitrogen instead, to phosphate KanaSucDOLA groups) around8 has 5, also resulting been synthetized in a decreasing [42]. efficiency The new forcholesterol higher derivativesvalue of N/3P– 5ratios,were notprobably fully soluble due to in an water, increase thus lipolexesof toxic effects. were prepared This result in the was presence quite unforeseenof DOPE and since tested TGKanaChol at different N2 /Pwasratios expected on five to mammalian be more efficient cell lines. than In four KanaChol out of the1. fiveIndeed, cell guanidinoglycosideslines tested, new compounds are known showed to have better both transfectionhigher RNA e affinityfficiencies and compared much greater to KanaChol ability to1 cross, and cell membranes compared to their unguanidinylated counterparts [35–40]. Interestingly, co- formulation with dioleoylphosphatidylthanolamine (DOPE), a neutral phospholipid that is known to help endosomal escape of the internalized lipoplexes [41], did not affect the in vitro transfection efficiency of KanaChol 1, demonstrating that KanaChol 1 is an efficient non-viral gene delivery vector that does not need liposomal formulation. However, co-formulation with DOPE was useful in order to deliver cholesterol-poly(ethylene glycol) (Chol-PEG) stabilized DNA in vivo through intranasal administration into mouse airways. In order to check the influence of the linker and lipophilic tails, a small library of kanamycin- based liposomes were synthetized using 6-aminocapronic acid (Cap), lysine (Lys), and succinic acid (Suc) linkers, and cholesterol, dioleyl amine, and disteraryl amine lipophilic tails (Figure 4) [42]. In Antibiotics 2020, 9, 504 7 of 29 the best efficiencies were reached at N/P ratios around 5 and 10, showing that the use of longer spacers between the cationic head group and the lipophilic moiety is favorable and provides better physico-chemical properties to the lipoplexes. Notably, the presence of a further amino function in KanaLysChol 4 compared to KanaCapChol 3 did not markedly affect the transfection efficiency. In addition, the substitution of cholesterol with aliphatic long chains such as in KanaSucDODA 6 and KanaSucDOLA 8 resulted in better efficiencies when administrated without DOPE, probably because the fluidity of the lipid layers benefited from this substitution. Co-formulation with DOPE produced a different effect, slightly increasing the efficiency of KanaSucDODA 6 and slightly decreasing that of KanaSucDOLA 8. As already observed for KanaChol 1 and TGKanaChol 2, guanidinylation of KanaSucDODA 6 did not enhance the transfection properties of the vector, TGKanaSucDADA 7, being much less efficient than 6. All these data confirmed that AG-based gene delivery vectors are efficient and non-cytotoxic gene delivery vectors when administrated at low values of the N/P ratio, as the vectors have a long spacer and long alkyl chains as lipophilic tails are much more powerful. However, the beneficial effect of the longer linker was not observed in vivo, since only KanaLysChol 4 resulted in being more efficient than KanaChol 1 when co-administrated with DOPE and Chol-PEG to BALB/c mice through intranasal administration. The impact of the cationic moiety of AG-based liposomes has been addressed through the synthesis of different derivatives starting from neomycin, paromomycin, and neamine. Following the synthetic strategy depicted in Path A of Figure2, four paromomycin derivatives were prepared, namely ParoChol 9 (the analog of KanaChol 1 where paromomycin is linked to cholesterol through a carbamate moiety), ParoSucDOLA 10 (the analogue of KanaSucDOLA 8), and two derivatives, ParoCapSucDOLA 11 and ParoLysSucDOLA 12, bearing dioleyl lipidic chains and longer linkers made by coupling 6-aminocaproic acid or lysine to succinic acid, respectively (Figure5)[ 43]. Path B of Figure2 was exploited in order to synthetize the derivatives of neomycin B. Accordingly, after the protection of all the amino functional groups with ditertbutyldicarbonate, the only primary hydroxyl group was selectively functionalized as triisopropylbenzensulfate and substituted by thioethanolamine, giving rise to the formation of intermediate 13, which was reacted with cholesterylchloroformiate and N-succinyl-dioleylamine, giving rise to the formation, after Boc deprotection, of liposomes NeoChol 14 and NeoSucDOLA 15, respectively (Figure6). Finally, in order to study the importance of the number of the amino moieties and the size of the cationic head group, dimeric paromomycin conjugate DiParoLinkerDiDOLA 17 was prepared using an ad hoc designed long glutamic acid-lysine-succinic acid linker (Figure7). The transfection efficiencies of these paromomycin- and neomycin-based liposomes have been evaluated at different values of N/P ratios (between 5 and 19) in two mammalian cell lines, namely HeLa and HEK293. All of these derivatives showed very good transfection efficiencies at their optimal charge ratio, with the dioleyl derivatives the best performing luciferase-expressing plasmid DNA vectors. Moreover, ParoSucDOLA 10 and NeoSucDOLA 15 resulted in being superior in gene transfection and were less cytotoxic than Lipofectamine, a commercially available transfection agent. Co-formulation with lipid DOPE once again increased the performance of the cholesteryl derivatives ParoChol 9 and NeoChol 14, while it did not affect, if not decreased, the efficiencies of the dioleyl derivatives. These results can be explained considering the physico-chemical characteristics of the vectors, in particular their assembly properties. Indeed, as demonstrated by measuring the surface pressure-area isotherms, dioleyl derivatives can form monolayers at the water–air interface, probably because the presence of long alkyl chains makes the liposomes less soluble in water. However, co-assembly with DOPE likely facilitates the formation of the hydrophobic layers, resulting in better performances. Noteworthy, the presence of two AGs did not improve the efficiency of the liposome, with dimeric DiParoLinkerDiDOLA 16 as efficient as monoparomomycin derivatives. Another interesting observation coming from this study was that paromomycin derivative ParoSucDOLA 10 resulted in being more performant than the corresponding NeoSucDOLA 15 in both cell lines, even if the latter bore more amino groups of six versus four. This unexpected observation Antibiotics 2020, 9, 504 8 of 29 could be caused by a structural reason (the linker is anchored in different positions) or/and by the stronger DNA complexation that can lead to a lower tendency to release the cargo. Analogously to kanamycin derivatives, ParoLysSucDOLA 12 and NeoChol 14 were also able to form Chol-PEG stabilized lipoplexes when co-formulated with DOPE and to deliver DNA in vivo into mouse airways, rendering these vectors promising for further in vivo evaluations. The simplest AG is neamine, with 2-DOS singularly substituted at position 4 with the 6-amino-glucosaminyl group. Actually, neamine is the core scaffold for the synthesis of a selected library of cationic lipids [44]. To not sacrifice an amino function, a route to selectively functionalize hydroxyl groups at position 5 and 40 has been developed [45]. Accordingly, after protection of the amines with the trityl group, reaction with p-metoxybenzylchloride (PMB-Cl) promoted by an excess of NaH led to the formation of a mixture of dibenzylated and tribenzylated derivatives 17 and 18, respectively, which were separated by flash chromatography (Figure8a). Alkylation of 17 with a slight excess of 1,4-dibromobutane produced monoalkylbromide derivative 19 in good yield and alkylation of 18 with an excess of 1,6-dibromohexane yielded intermediate 20. Bromine of the resulting monoalkylbromides was displaced with different amines producing, after full deprotection, the target cationic lipids 21–26 bearing one neamine scaffold. Besides compound 21 where neamine was conjugated to an alkyl ammonium group, five neamine-based lipids were synthetized by tethering the AG to two C12 alkyl chains or two stearyl alkyl chains (C18), three of them, namely 22–24, through the C4 linker at the 40 position and two of them, 25 and 26, through the C6 linker at position 5 (Figure8b). A seventh derivative 27 bearing one neamine moiety and two stearyl chains was prepared by a slightly different strategy starting from bromide 20 [44]. Moreover, other five cationic lipids 29–33 bearing two neamine units linked to a single or double alkyl chain through different tripodal linkers (Figure9b) were prepared by classical coupling chemistry starting from monoalkyl bromide 20, which was converted to amineAntibiotics28 2020by,displacing 9, x the bromine atom with an azide followed by Staudinger reduction (Figure8 of9a). 28

Figure 5. Structures of paromomycin-based lipids 9–12.

Figure 6. Synthesis of neomycin-based lipids 14, 15: (i) Boc2O, TEA, DMF/H2O, (ii) TIBS-Cl, pyridine, (iii) 2-aminoethanethiol, EtONa/EtOH, (iv) cholesteryl chloroformate, TEA, THF/DMF, (v) TFA/DCM, (vi) N-succinyl-dioleylamine, HOAt/EDC, DFM/DCM, (vii) TFA/DCM. Antibiotics 2020, 9, x 8 of 28

Antibiotics 2020, 9, 504 9 of 29 Figure 5. Structures of paromomycin-based lipids 9–12.

Figure 6. Synthesis of neo neomycin-basedmycin-based lipids 14, 15:: (i) Boc 2O,O, TEA, TEA, DMF/H DMF/H2O,O, (ii) (ii) TIBS-Cl, TIBS-Cl, pyridine, pyridine, (iii) 2-aminoethanethiol, EtONa/EtOH, EtONa/EtOH, (iv) cholesteryl cholesteryl chloroformate, chloroformate, TEA, TEA, THF/DMF, THF/DMF, (v) (v) TFA/DCM, TFA/DCM, Antibiotics(vi) N 2020-succinyl-dioleylamine,, 9, x HOAtHOAt/EDC,/EDC, DFMDFM/DCM,/DCM, (vii)(vii) TFATFA/DCM./DCM. 9 of 28

FigureFigure 7.7. Structure of diparomomycin lipidlipid 1616..

The transfection efficiencies of these paromomycin- and neomycin-based liposomes have been evaluated at different values of N/P ratios (between 5 and 19) in two mammalian cell lines, namely HeLa and HEK293. All of these derivatives showed very good transfection efficiencies at their optimal charge ratio, with the dioleyl derivatives the best performing luciferase-expressing plasmid DNA vectors. Moreover, ParoSucDOLA 10 and NeoSucDOLA 15 resulted in being superior in gene transfection and were less cytotoxic than Lipofectamine, a commercially available transfection agent. Co-formulation with lipid DOPE once again increased the performance of the cholesteryl derivatives ParoChol 9 and NeoChol 14, while it did not affect, if not decreased, the efficiencies of the dioleyl derivatives. These results can be explained considering the physico-chemical characteristics of the vectors, in particular their assembly properties. Indeed, as demonstrated by measuring the surface pressure-area isotherms, dioleyl derivatives can form monolayers at the water–air interface, probably because the presence of long alkyl chains makes the liposomes less soluble in water. However, co- assembly with DOPE likely facilitates the formation of the hydrophobic layers, resulting in better performances. Noteworthy, the presence of two AGs did not improve the efficiency of the liposome, with dimeric DiParoLinkerDiDOLA 16 as efficient as monoparomomycin derivatives. Another interesting observation coming from this study was that paromomycin derivative ParoSucDOLA 10 resulted in being more performant than the corresponding NeoSucDOLA 15 in both cell lines, even if the latter bore more amino groups of six versus four. This unexpected observation could be caused by a structural reason (the linker is anchored in different positions) or/and by the stronger DNA complexation that can lead to a lower tendency to release the cargo. Analogously to kanamycin derivatives, ParoLysSucDOLA 12 and NeoChol 14 were also able to form Chol-PEG stabilized lipoplexes when co-formulated with DOPE and to deliver DNA in vivo into mouse airways, rendering these vectors promising for further in vivo evaluations. The simplest AG is neamine, with 2-DOS singularly substituted at position 4 with the 6-amino- glucosaminyl group. Actually, neamine is the core scaffold for the synthesis of a selected library of cationic lipids [44]. To not sacrifice an amino function, a route to selectively functionalize hydroxyl groups at position 5 and 4′ has been developed [45]. Accordingly, after protection of the amines with the trityl group, reaction with p-metoxybenzylchloride (PMB-Cl) promoted by an excess of NaH led to the formation of a mixture of dibenzylated and tribenzylated derivatives 17 and 18, respectively, which were separated by flash chromatography (Figure 8a). Alkylation of 17 with a slight excess of 1,4-dibromobutane produced monoalkylbromide derivative 19 in good yield and alkylation of 18 with an excess of 1,6-dibromohexane yielded intermediate 20. Bromine of the resulting monoalkylbromides was displaced with different amines producing, after full deprotection, the Antibiotics 2020, 9, x 10 of 28

target cationic lipids 21–26 bearing one neamine scaffold. Besides compound 21 where neamine was conjugated to an alkyl ammonium group, five neamine-based lipids were synthetized by tethering the AG to two C12 alkyl chains or two stearyl alkyl chains (C18), three of them, namely 22–24, through the C4 linker at the 4′ position and two of them, 25 and 26, through the C6 linker at position 5 (Figure 8b). A seventh derivative 27 bearing one neamine moiety and two stearyl chains was prepared by a slightly different strategy starting from bromide 20 [44]. Moreover, other five cationic lipids 29–33 bearing two neamine units linked to a single or double alkyl chain through different tripodal linkers (Figure 9b) were prepared by classical coupling chemistry starting from monoalkyl bromide 20, Antibioticswhich was2020 , converted9, 504 to amine 28 by displacing the bromine atom with an azide followed10 ofby 29 Staudinger reduction (Figure 9a).

FigureFigure 8.8. (a) Synthesis and ( (b)) structures structures of of lipids lipids 2121––2727 bearingbearing one one neamine neamine unit. unit. Antibiotics 2020, 9, 504 11 of 29 Antibiotics 2020, 9, x 11 of 28

FigureFigure 9.9. ((aa)) SynthesisSynthesis andand ((b) structures of lipids 29–33 bearingbearing two two neamine neamine units. units.

TheseThese twelvetwelve derivatives were were submitted submitted to toSARs SARs in several in several cell celllines lines giving giving interesting, interesting, and andsomewhat somewhat unexpected, unexpected, insights. insights. The results The resultsobtained, obtained, although although somewhat somewhat variable variablewithin the within cell theline cell observed, line observed, can be can summarized be summarized in the in thefollowing following points: points: (1) (1)liposomes liposomes made made by by cationic cationic lipids lipids bearingbearing onlyonly oneone neamineneamine moietymoiety werewere moremore eefficientfficient than those bearing two two neamine neamine units, units, those those bearingbearing twotwo fattyfatty acidacid chainschains performedperformed more than those bearing only only one one chain; chain; (2) (2) stearyl stearyl chains chains conferredconferred betterbetter propertiesproperties to the the vector vector compared compared to to dodecyl dodecyl alkyl alkyl chains; chains; (3) (3)functionalization functionalization at 4′ at- 4neamine0-neamine position position is ispreferable, preferable, prob probablyably because, because, being being at at the the end end of of the the neamine neamine core, core, it it facilitates facilitates thethe interactions interactions withwith the DNA; DNA; (4) (4) the the presence presence of of a diethylamino a diethylamino moiety moiety between between the linker the linker and the and fatty acid chains made the vector more performant, probably increasing the flexibility and the Antibiotics 2020, 9, 504 12 of 29 Antibiotics 2020, 9, x 12 of 28 theendosomal fatty acid buffering chains made capacity the vectorof the more vector; performant, and (5) probablyco-assembly increasing with DOPE the flexibility facilitated and thethe endosomaltransfection bu abilityffering of capacity the vectors, of the as vector; already and observed (5) co-assembly in some cases with DOPEwith kanamycin, facilitatedthe paromomycin, transfection abilityand neomycin of the vectors, derivatives. as already In general, observed the in most some performant cases with kanamycin, vector resulted paromomycin, in being compound and neomycin 24, derivatives.whose efficiency In general, and negligible the most performantcytotoxicity, vector when resultedco-formulated in being with compound DOPE, were24, whose comparable efficiency to andthose negligible of Lipofectamine cytotoxicity, and when ParoSucDOLA/DOPE co-formulated with 10 DOPE,. The efficiency were comparable of compound to those 24 of was Lipofectamine also tested andin vivo ParoSucDOLA by intravenous/DOPE administration10. The effi ciencyof 24/DOPE of compound formulations24 was in alsomice. tested However,in vivo theby performances intravenous administrationresulted in being of significantly24/DOPE formulations lower than those in mice. obtained However, with PEI the 22 performances kDa and were resulted similar into beingthose significantlyobtained with lower ParoSucDOLA than those 10 obtained/DOPE, witheven PEIif no 22 lethality kDa and was were observed similar regardless to those obtained of the doses with ParoSucDOLAadministrated. 10/DOPE, even if no lethality was observed regardless of the doses administrated.

4.2. AG-Based Lipid Vectors for RNA Delivery Other thanthan DNA, DNA, RNAs RNAs can can also also be usedbe used as therapeutic as therapeutic agents agents for gene for therapygene therapy in order in to order interact to orinteract produce or specificproduce functions specific functions within the within cell [22 ].the In cell particular, [22]. In thereparticular, is great there interest is great in the interest development in the ofdevelopment cationic lipids of thatcationic can belipids exploited that can as be effi exploitecient deliveryd as efficient systems delivery for double systems stranded for double small interfering stranded RNAsmall (siRNA),interfering which RNA promotes (siRNA), gene which silencing promotes (protein gene inhibition) silencing in (protein the cytosol inhibition) [46], and in for the messenger cytosol RNA[46], and (mRNA), for messenger which expresses RNA (mRNA), functional which proteins expresse [47s ].functional However, proteins non-viral [47]. gene However, delivery non-viral vectors thatgene are delivery efficient vectors in the deliverythat are ofefficient DNA are in notthe consequentlydelivery of DNA efficient are for not siRNA consequently or mRNA efficient delivery for as thesiRNA structural or mRNA characteristics delivery as of the such structural genes quite characteri different.stics Thus, of such soon genes after thequite application different. of Thus, AG-based soon vectorsafter the was application successfully of AG-based demonstrated vectors for was DNA successfully delivery, four demonstrated AG-succinyl-dioleyl for DNA delivery, derivatives, four three AG- ofsuccinyl-dioleyl them KanaSucDOLA derivatives,8, ParoSucDOLA three of them10 ,KanaSucDOLA and NeoSucDOLA 8, ParoSucDOLA15 being the best 10, performersand NeoSucDOLA in DNA delivery,15 beingalong the withbest aperformers new tobramycin in DNA analogue, delivery, TobraSucDOLA along with 34a, werenew testedtobramycin to measure analogue, their siRNATobraSucDOLA transfection 34, ewerefficiencies tested (Figure to measure 10)[48 th].eir siRNA transfection efficiencies (Figure 10) [48].

Figure 10. Aminoglycoside-based lipids tested as siRNA delivery vectors.vectors.

The four vectors resulted in being good siRNA transfectants at various N/P ratios, the ability of them depending on the specific geometries. The structural features of the corresponding liposomes were determined by cryogenic transmission electron microscopy (cryo-TEM) and small-angle X-ray Antibiotics 2020, 9, 504 13 of 29

The four vectors resulted in being good siRNA transfectants at various N/P ratios, the ability of them depending on the specific geometries. The structural features of the corresponding liposomes were Antibiotics 2020, 9, x 13 of 28 determined by cryogenic transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering scattering(SAXS) experiments: (SAXS) experiments: 4,6-disubstituted-2-DOS 4,6-disubstituted-2-DOS lipids 8 and lipids34 formed 8 and 34 liposomes formed liposomes having concentric having concentricmultilamellar multilamellar “onion like” “onion structures like” exhibitingstructures regularexhibiting spacing regular of circa spacing 70 Å of between circa 70 theÅ between consecutive the consecutiverepeats, with repeats, the siRNA with molecules the siRNA well-ordered molecules in well-ordered the lipid membrane, in the whilelipid 4,5-disusbituted-2-DOSmembrane, while 4,5- disusbituted-2-DOSderivatives 10 and 15 derivativesformed smaller 10 and liposomes15 formed withsmaller a much liposomes more with disordered a much structure. more disordered Indeed, structure.lipid/siRNA Indeed, complexes lipid/siRNA obtained complexes with ParoSucDOLA obtained with10 and ParoSucDOLA NeoSucDOLA 10 15andwere NeoSucDOLA more active 15 in weregene more silencing active in in GFP-expressing gene silencing d2GFPin GFP-expressing cells, probably d2GFP thanks cells, to probably their liposome thanks smaller to their diametersliposome smaller(around diameters 50 nm in positive (around complexes, 50 nm in i.e., positive high N complexes,/P ratios), higher i.e., high colloidal N/P stability,ratios), higher but, at thecolloidal same stability,time, better but, propensity at the same to time, endosomal better propensity escape into to theendosomal cytosol. escape In particular, into the thecytosol. better In endosomalparticular, theescape better likely endosomal arises from escape the greaterlikely arises flexibility from ofthe the greater lamellar flexibility microdomains of the lamellar in the siRNA microdomains complexes, in thefavoring siRNA the complexes, so-called flip-flopfavoring mechanism the so-called [49 flip-flop]. Afterward, mechanism a fully SAR[49]. studyAfterward, was performed a fully SAR in study order wasto assess performed the importance in order to of assess the hydrophobic the importance domains, of the the hydrophobic spacers between domains, the cationic the spacers head between and the thelipophilic cationic tails, head and and the the behavior lipophilic of tails, stimuli and responsivethe behavior linkers of stimuli in delivering responsive di linkersfferent nucleicin delivering acids differentencompassing nucleic DNA, acids siRNA, encompassing and mRNA. DNA, Starting siRNA, from and tobramycin mRNA. Starting and exploiting from tobramycin the reactivity and of exploitingthe only methylenamino the reactivity of moiety the only (route methylenamin A, Figure2),o a moiety set of eight (route new A, tobramycin-basedFigure 2), a set of lipidseight wasnew tobramycin-basedprepared (Figure 11 lipids)[50]. was prepared (Figure 11) [50].

OH HO OH O H OH N N n =12 35 O O n n =14 36 HO HO NH2 O O n n =16 37

H2N O OH NH2

OH HO OH H X = -(CH2)2- 38 OH N N X = -(CH ) - 39 O O X 2 4 X = -(CH2)6- 40 HO HO NH O O O 2 X = -S-S- 41

H2N O OH NH2 O OH HO OH O O H OH N O O N 42 H HO HO NH2 O O O O H2N O OH NH2

FigureFigure 11. 11. Tobramycin-basedTobramycin-based lipids lipids 3535––4242 testedtested as as mRNA, mRNA, DNA, DNA, and siRNA delivery vectors vectors..

InIn particular, particular, vectors 35–37 differentiatedifferentiate from the length of the lipophilic lipophilic alkyl alkyl chains, chains, C14, C14, C16, C16, and C18, C18, respectively, respectively, while while compounds compounds 3838–41–41 differentiatedifferentiate for for the the length length of the of linker the linker (C6, (C6,C8, and C8, C10)and C10)between between tobramycin tobramycin and dioleyl and dioleyl alkyl chains, alkyl chains, compound compound 41 bearing41 bearing a linear a linker linear of linker six atoms of six withatoms a withstimuli a stimuli responsive responsive disulfide disulfide functional functional group groupin the in middle. the middle. Finally, Finally, compound compound 42 was42 designedwas designed to have to havea similar a similar structure structure to a towell-known a well-known commercially commercially available available cationic cationic lipid lipid1,2- dioleoyl-3-trimethylammonium-propane1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (DOTAP) with witha hydrolizable a hydrolizable diester diester function function [51]. [The51]. morphologiesThe morphologies of the of liposomes the liposomes made made by se bylected selected candidates, candidates, namely namely TobraSucDOLA TobraSucDOLA 34, the34, conjugatethe conjugate with with the adipic the adipic linker linker 38, and38, and the thediester diester derivative derivative 42 were42 were determined determined by CryoTEM. by CryoTEM. In general, liposomes made with siRNA with a size between 80 and 150 nm resulted in being smaller than those obtained with mRNA and DNA, both having sizes between 200 and 500 nm. All vectors were assessed in transfection experiments in three cell lines, two of them being mammalian primary cell lines that better mimic the in vivo environment. Regardless of the nature of the gene (DNA, mRNA, or siRNA), three of the new synthetized vectors, namely the derivative with adipic linker 38, Antibiotics 2020, 9, 504 14 of 29

In general, liposomes made with siRNA with a size between 80 and 150 nm resulted in being smaller than those obtained with mRNA and DNA, both having sizes between 200 and 500 nm. All vectors were assessed in transfection experiments in three cell lines, two of them being mammalian primary cell lines that better mimic the in vivo environment. Regardless of the nature of the gene (DNA, mRNA, or siRNA), three of the new synthetized vectors, namely the derivative with adipic linker 38, that with palmityl linker 39, and the diester derivative 42, resulted in having similar, if not slightly superior transfection efficiency than TobraSucDOLA 34 in easily tranfectable HeLa cells, with compound 42 being the most efficient. Interestingly, compound 42 showed increased performance, up to 2–3 fold with mRNA, when used to transfect aorta smooth muscle cells and human fibroblast cells, being a better mRNA and DNA, and comparable siRNA vector than Lipofectamine 3000. However, the better efficiency of 42 did not seem to be correlated with the biodegradability of the ester functions, but more likely with the smaller dimension of the lipoplexes and with an easier capacity to release the nucleic acid once inside the cell. Moreover, all the vectors showed negligible cytotoxicity. On the other hand, quite surprisingly, the vector with stimuli responsive disulfide bridge 36 showed very poor transfection efficiency. Indeed, it was previously demonstrated that other amphiphilic vectors containing a disulfide bridge resulted in more efficient DNA vectors likely because the reduction of the internalized lipid facilitates the release of the cargo into the cytosol [52,53]. For liposome 41, however, this was not the case because the reduction of the linker inhibited in some way the endosomal escape, as demonstrated by the increased efficiency observed when 41 was co-formulated with neutral fusogenic DOPE. These SAR studies confirmed that the length of the linker is an important parameter to be considered in building efficient AG-based cationic liposomes as well as the nature of the lipophilic tails, with the dioleyl chain a better choice compared to shorter or saturated alkyl chains. As evidenced also for the AG-based liposomes, the efficiency of a vector to deliver the genetic material into the appropriate organelle does not depend only on the capacity to cross cell membrane. Other mechanisms such as endosomal escape and the ability to release the nucleic acid once inside the cell, among others, are very important and should be taken into consideration for the development of new universal non-viral vectors [54]. The performance of AG-based liposomes in endosomal escaping and gene complexation/decomplexation can be tuned by co-formulation with lipids, referred to as helpers, with which the lamellar phase of the lipoplexes can be modulated. In particular, the DNA transfection efficiency of ParoSucDOLA 10 when co-formulated with the lipid helpers HLN or HLP (Figure 12a), bearing a pH-buffering imidazole ring tethered to dioleyl alkyl chains through an amido- or phosphoramide-based linker, respectively, was increased most likely because the endosomal escape/DNA release mechanisms were favored [55]. Interestingly, the behavior of the two helpers was different, since the best performance was obtained when ParoSucDOLA 10 was co-formulated with HLP, the helper having a phosphoramide-based linker. In order to study in more depth the effect of the supramolecular assemblies in the delivery of mRNA, DNA, and siRNA, and in particular the effect of the combination of linkers HLN and HLP with paromomycin-based cationic lipids with the same lipophilic tails (dioleyl alkyl chains), ParoPhosDOLA 43 (Figure 12b) was ad hoc synthesized exploiting the same synthetic strategy seen for ParoSucDOLA 10 [56]. Four supramolecular assemblies were tested, namely homogeneous ParoSucDOLA 10/HLN and ParoPhosDOLA 43/HLP, where the cationic lipids and the helpers have the same amide-based or phosphoramide-based linker, respectively, and hybrid ParoSucDOLA 10/HLP and ParoPhosDOLA 43/HLN, where the nature of the linkers is different. Regardless of the genetic material delivered (mRNA, DNA, siRNA) and the cell lines used (HeLA, C2C12, MEF, rat aorta muscle cells, and human fibroblasts), the hybrid combinations were much more efficient than the homogeneous ones. Cryo-TEM images of the lipoplexes with DNA showed that in homogeneous assemblies, the nucleic acid is less tightly bounded because the cationic moieties are well organized in the structure, thus less available to interact with the negatively charged phosphates of the genetic material. Indeed, while hybrid liposomes showed cationic external surfaces that facilitate the interaction with the cell membrane, likely favoring internalization, homogeneous liposomes showed external corona with negatively charged DNA. This is Antibiotics 2020, 9, x 14 of 28

that with palmityl linker 39, and the diester derivative 42, resulted in having similar, if not slightly superior transfection efficiency than TobraSucDOLA 34 in easily tranfectable HeLa cells, with compound 42 being the most efficient. Interestingly, compound 42 showed increased performance, up to 2–3 fold with mRNA, when used to transfect aorta smooth muscle cells and human fibroblast cells, being a better mRNA and DNA, and comparable siRNA vector than Lipofectamine 3000. However, the better efficiency of 42 did not seem to be correlated with the biodegradability of the ester functions, but more likely with the smaller dimension of the lipoplexes and with an easier capacity to release the nucleic acid once inside the cell. Moreover, all the vectors showed negligible cytotoxicity. On the other hand, quite surprisingly, the vector with stimuli responsive disulfide bridge 36 showed very poor transfection efficiency. Indeed, it was previously demonstrated that other amphiphilic vectors containing a disulfide bridge resulted in more efficient DNA vectors likely because the reduction of the internalized lipid facilitates the release of the cargo into the cytosol [52,53]. For liposome 41, however, this was not the case because the reduction of the linker inhibited in some way the endosomal escape, as demonstrated by the increased efficiency observed when 41 was co-formulated with neutral fusogenic DOPE. These SAR studies confirmed that the length of the linker is an important parameter to be considered in building efficient AG-based cationic liposomes as well as the nature of the lipophilic tails, with the dioleyl chain a better choice compared to shorter or saturated alkyl chains. As evidenced also for the AG-based liposomes, the efficiency of a vector to deliver the genetic material into the appropriate organelle does not depend only on the capacity to cross cell membrane. Other mechanisms such as endosomal escape and the ability to release the nucleic acid once inside the cell, among others, are very important and should be taken into consideration for the development of new universal non-viral vectors [54]. The performance of AG-based liposomes in endosomal escaping and gene complexation/decomplexation can be tuned by co-formulation with lipids, referred to as helpers, with which the lamellar phase of the lipoplexes can be modulated. In particular, the DNA transfection efficiency of ParoSucDOLA 10 when co-formulated with the lipid helpers HLN or HLP (Figure 12a), bearing a pH-buffering imidazole ring tethered to dioleyl alkyl chains through an amido- or phosphoramide-based linker, respectively, was increased most likely because the endosomal escape/DNA release mechanisms were favored [55]. Interestingly, the Antibioticsbehavior2020 of, 9,the 504 two helpers was different, since the best performance was obtained 15when of 29 ParoSucDOLA 10 was co-formulated with HLP, the helper having a phosphoramide-based linker. In order to study in more depth the effect of the supramolecular assemblies in the delivery of mRNA, probablyDNA, and the siRNA, major, and if not in the particular only, reason the effect for the of di thefferent combination efficiencies of inlinkers gene transfectionHLN and HLP between with homogeneousparomomycin-based and hybrid cationic systems, lipids since with intracellular the same mechanisms lipophilic such tails as endosomal (dioleyl escapealkyl andchains), gene releaseParoPhosDOLA in the diff erent43 (Figure organelles 12b) was (cytosol ad hoc for synthesized RNAs and nucleus exploiting for DNA)the same are synthetic likely to notstrategy be aff ectedseen byfor the ParoSucDOLA different composition, 10 [56]. with the cationic heads and the lipophilic tails identical.

Figure 12. Structures of (a) nitrogen-based helper lipid HLN and phosphorous-based helper lipid HLP, and (b) ParoPhosDOLA lipid 43.

Although the aforementioned studies outlined a sort of guideline for the design of efficient AG-based gene delivery vectors, and even though it is known that efficient gene delivery vehicles could be identified through high throughput screening of libraries of liposomes [57,58], a real combinatorial approach for the SARs on AG-based cationic lipids has been exploited only in two studies, the first performed by the group of Prof. Anderson at Massachusetts Institute of Technology (MIT) and appeared in 2013 [59], and the second published this year by the group of Prof. Siegwar at University of Texas (UT) Southwestern [60]. In both studies, a one-step simple functionalization of different unprotected AGs with lipophilic epoxides and, in the second case, also with lipophilic α,β-unsaturated ester Michael acceptors, led to the preparation of libraries of AG-modified lipids that were tested for siRNA and mRNA delivery in vivo, respectively. In the first study, eight different AGs, namely neomycin, amikacin, paromomycin, ribostamycin, kanamycin, hygromycin, geneticin, and gentamicin, along with 2-DOS, were reacted with a stoichiometric amount of terminal epoxides bearing linear alkyl chain lengths of 10–16 carbons in order to fully functionalize the AG amines (Figure 13a) [59]. All the cationic lipids obtained were able to form stable nanoparticles with siRNA (up to 70% entrapment ratio) when co-formulated with cholesterol, DSPC, and PEG2000-DMG, showing a hydrodynamic diameter in the range of 60–200 nm and a slightly negative surface charge, with the PEG200 chain occupying the outer layer. The transfection efficiency of the synthetized AG cationic lipids was first evaluated in vitro by measuring knockdown of luciferase in HeLa cells using Lipofectamine 2000 as the control. Interestingly, lipids bearing aliphatic chains longer that C13 were inefficient, probably due to solubility concerns, and those with a length of C11 or C12 were the most performant. In addition, the cationic AG was found to be important, the most efficient liposomes being those bearing hygromycin, compound 45 being the best performer and 44 the second best (Figure 13b), and gentamycin (the third and fourth), probably due to their geometrical and topological features. The cationic liposomes that gave the best in vitro results were selected to study the transfection in vivo through intravenous injections in mice at 1 mg/Kg siRNA dose. The results obtained were consistent with those obtained in vitro, with hygromycin derivatives 46, 45, 47, and 44, in this order, the most performing vectors, reaching high levels (>90%) of gene silencing. Moreover, these liposomes also did not show significant toxicity at higher doses, demonstrating that they possessed all the right features to be considered between the most promising delivery vectors for in vivo gene silencing. Antibiotics 2020, 9, 504 16 of 29 Antibiotics 2020, 9, x 16 of 28

FigureFigure 13. 13.( (aa)) CombinatorialCombinatorial synthesissynthesis ofof lipidiclipidic modiﬁedmodified AGs. ( (bb)) Structures Structures of of the the most most performant performant lipidslipids developed developed at at MIT MIT [ [59]59] andand ((cc)) structurestructure ofof thethe mostmost performantperformant lipid developed at at UT UT [60]. [60].

TheThe successsuccess ofof the former study study inspired inspired the the design design of of a second a second library library of AG-based of AG-based lipids lipids to be to betested tested as asmRNA mRNA delivery delivery vectors vectors inin vivo vivo toto the the liver. liver. Four Four different different AGs, AGs, namely namely hygromycin, hygromycin, gentamycin,gentamycin, amikacin,amikacin, andand geneticingeneticin were reacted with seven epoxides epoxides bearing bearing lipophilic lipophilic tails, tails, analogouslyanalogously to to the the former former study study (Figure (Figure 13 13a),a), and and with with five five lipophilic lipophilicα ,αβ,-unsaturatedβ-unsaturated ester ester in in order order to fullyto fully functionalize functionalize the aminothe amino functional functional groups groups on the on AG the sca AGffold scaffold through through the nucleophilic the nucleophilic epoxide openingepoxide andopening Michael and addition, Michael respectivelyaddition, respectively [60]. All the [60]. lipids All werethe lipids able to were encapsulate able to encapsulate mRNA very effimRNAciently, very forming efficiently, liposomes forming with liposomes sizes between with si 100zes and between 200 nm, 100 low and dispersity, 200 nm, low and dispersity, a surface chargeand a closesurface to zero. charge In aclose preliminary to zero. inIn vitroa preliminaryscreening, in gentamycin- vitro screening, andamikacin-based gentamycin- and liposomes amikacin-based gave the bestliposomes results gave in terms the best of mRNA results transfection in terms of mRNA on IGROV1 transfection ovarian on cancer IGROV1 cells ovarian when co-formulated cancer cells when with cholesterol,co-formulated DOPE, with and cholesterol, PEG-DMG DOPE, at 20 andN/P PEG-DMGratio, with at those 20 N/P arising ratio, from with the those nucleophilic arising from epoxide the openingnucleophilic the most epoxide performant. opening Inthe order most to performant. measure the Inin vivoorderdelivery to measure efficiencies the in andvivo to delivery find the bestefficiencies content formulations,and to find the the best best content candidates formulat wereions, administered the best candidates to C57BL/6 were mice throughadministered parental to systemicC57BL/6 administration mice through parental by tail vain. systemic Gentamycin administration derivative by bearingtail vain. a C10Gentamycin lipophilic derivative tail 48 (Figure bearing 13c) resulteda C10 lipophilic in being thetail most 48 (Figure promising 13c) candidate resulted in to bebeing further the evaluatedmost promising in future. candidate In general, to be in contrastfurther withevaluated that previously in future. reportedIn general, for in siRNA contrast delivery with that [59 previously], the derivatives reported with for gentamicin siRNA delivery as the [59], cationic the moietyderivatives were morewith activegentamicin than those as the containing cationic hygromycin, moiety were confirming more active that thethan transfection those containing ability of ahygromycin, particular vector confirming also depends that the on transfection the delivered abi geneticlity of material a particular it isdesigned vector also for. depends on the delivered genetic material it is designed for. 4.3. AG-Based Lipid Vectors with “Non-Conventional” Lipidic Tails 4.3. AG-Based Lipid Vectors with “Non-Conventional” Lipidic Tails AGs have also been coupled to “non-conventional” lipidic tails. Three neomycin-based lipids 49–51AGshave have been also synthetized been coupled for e ffitocient “non-conventiona delivery intol” the lipidic liver tails. by exploiting Three neomycin-based lipophilic vitamin lipids E as49 the–51 lipophilichave been tailsynthetized and targeting for efficient moiety delivery for the intoα-tocopherol the liver by transport exploiting protein lipophilic (Figure vitamin 14)[ E 61as]. Afterthe lipophilic the ability tail of and cationic targeting lipids moiety to interact for the withα-tocopherol the RNA transport duplex protei was assessedn (Figure by 14) UV [61]. melting After analysis,the ability Cy3-modified-siRNA of cationic lipids to interact was complexed with the withRNA lipidsduplex49 was–51 assessedand the transfectionby UV melting effi analysis,ciency of theCy3-modified-siRNA corresponding lipoplexes was complexed was measured with in lipids mouse 49 hepatocellular–51 and the carcinomatransfection (Hepa efficiency 1–6) cell of lines.the corresponding lipoplexes was measured in mouse hepatocellular carcinoma (Hepa 1–6) cell lines. The The cells treated with naked Cy3-siRNA did not show any fluorescence, while those transfected with cells treated with naked Cy3-siRNA did not show any fluorescence, while those transfected with the the lipoplexes showed intense fluorescence, the liposome made with 51 being the most performant. lipoplexes showed intense fluorescence, the liposome made with 51 being the most performant. Moreover, the effective uptake was corroborated by measuring RNAi activity in the liver cancer cells. Moreover, the effective uptake was corroborated by measuring RNAi activity in the liver cancer cells. Indeed, the liposome made with 51, and, to a lesser extent, the liposome made with 50, were able to Indeed, the liposome made with 51, and, to a lesser extent, the liposome made with 50, were able to induce RNA silencing in mouse LDLR-overexpressing McA-TTP cells. induce RNA silencing in mouse LDLR-overexpressing McA-TTP cells. Antibiotics 2020, 9, 504 17 of 29 Antibiotics 2020, 9, x 17 of 28

Figure 14. Structures of neomycin-vitamin E lipids 49–51.

AG-lipophilic peptide conjugates could be designed in order to build multifunctional liposomes with the the ability ability to to deliver deliver genetic genetic materials materials and/or and /lipophilicor lipophilic drugs drugs into cells, into cells,maintaining, maintaining, if not ifincreasing, not increasing, the ability the ability of AGs of AGs to tofight fight bacteria. bacteria. Accordingly, Accordingly, NN-Boc-Boc protected protected dipeptidedipeptide phenylalanine-dihydrophenylalanine linked to 6-amino capronic acid (BocNH-Phe-(BocNH-Phe-∆ΔPhe-Cap-OH) was activated as a NHS-ester and reacted with unprotectedunprotected neomycin yielding NeoCapPhe∆ΔPhe 52 (Figure 1515))[ [62,63].62,63]. NeoCapPhe Δ∆Phe 52 is able to to self-assemble self-assemble in in spherical spherical core-shell core-shell nanostructures of around 50–70 nm with a hydrophobic hydrophobic core core surrou surroundednded by by exterior exterior hydrophilic hydrophilic neomycin neomycin moieties. moieties. Such aa supramolecularsupramolecular structure structure is is responsible responsible for for the the ability ability of NeoCapPhe of NeoCapPhe∆PheΔPhe52 to 52 encapsulate to encapsulate both hydrophobicboth hydrophobic drugs drugs such as such curcumin as curcumin and eosin and through eosin hydrophobicthrough hydrophobic and π–π staking and π interactions,–π staking andinteractions, pDNA throughand pDNA electrostatic through interactions,electrostatic asinte evidencedractions, as by evidenced TEM (curcumin), by TEM UV(curcumin), fluorescence UV quenchingfluorescence (eosin), quenching and gel (eosin), retardation and gel assay retardat (pDNA).ion assay In particular, (pDNA). complete In particular, GFP-encoding complete pDNA GFP- complexationencoding pDNA arises complexation at 3.33 weight arises/weight at 3.33 (w /weight/weightw) ratio. The transfection (w/w) ratio. ability The transfection of NeoCapPhe ability∆Phe of 52NeoCapPhewas assessedΔPhe in 52 two was cell assessed lines, namelyin two cell MCF-7 lines, and namely N2a, MCF-7 showing and good N2a, e showingfficiency good to deliver efficiency GFP expressingto deliver GFP plasmid expressing at a 66.6 plasmidw/w ratio, at a 66.6 being w/w a betterratio, being transfectant a better than transfectant Lipofectamine. than Lipofectamine. The showed transfectionThe showed etransfectionfficiency mainly efficiency depends mainly on thedepends small on size the of small the lipoplexes size of the (around lipoplexes 40–60 (around nm), on40–60 the abilitynm), on of the the ability liposome of tothe protected liposome the to plasmidprotecte fromd the nucleases, plasmid from and onnucleases, the easy disassemblyand on the easy into thedisassembly cytoplasm into for the nuclear cytoplasm localization, for nuclear as localizati demonstratedon, as demonstrated through pH studies.through pH Besides studies. negligible Besides cytotoxicity,negligible cytotoxicity, these liposomes these resultedliposomes in havingresulted good in having antibacterial good antibacterial activity in both activity Gram-positive in both Gram- and Gram-negativepositive and Gram-negative bacterial strains. bacterial strains. Multivalency is a well-known methodology in order to amplify a molecular recognition process involving carbohydrates such as ligand/cell surface interactions [64]. One possible strategy to promote multivalency is to tether multiple ligand units to a flexible scaffold, either a molecular entity or a polymer. We thought to exploit this strategy in order to amplify the ability of AGs to interact with the cell surface, and eventually cell internalization, by tethering AGs to the upper rim of lipophilic calix[4]arenes, a macrocyclic oligomer successfully used to build efficient gene delivery vectors by anchoring guanidinylated moieties [65,66]. Accordingly, after functionalization of neamine, paromomycin, and neomycin with a linker bearing an isothiocyanate moiety, we used tetraminocalix[4]arene to build three AGs-calix[4]arene cationic lipids, namely NeaCalix 53, NeoCalix 54, and ParoCalix 55,

Antibiotics 2020, 9, x 17 of 28

Figure 14. Structures of neomycin-vitamin E lipids 49–51.

AG-lipophilic peptide conjugates could be designed in order to build multifunctional liposomes with the ability to deliver genetic materials and/or lipophilic drugs into cells, maintaining, if not increasing, the ability of AGs to fight bacteria. Accordingly, N-Boc protected dipeptide

Antibioticsphenylalanine-dihydrophenylalanine 2020, 9, 504x linked to 6-amino capronic acid (BocNH-Phe-ΔPhe-Cap-OH)18 of 28 29 was activated as a NHS-ester and reacted with unprotected neomycin yielding NeoCapPheΔPhe 52 (Figure 15) [62,63]. NeoCapPheFigureΔPhe 15.52 isSynthesis able to ofself-assemble NeoCapPheΔ inPhe spherical 52. core-shell nanostructures ofthrough around isothiocyanate-amine 50–70 nm with a hydrophobic click chemistry core surrou (Figurended 16)[ 67by]. exterior The ability hydrophilic to complex neomycin pDNA encodingmoieties. SuchmodifiedMultivalency a supramolecular firefly luciferase is a well-known structure was assessed is methodologyresponsible through for in the theorder fluorophore-exclusion ability to amplify of NeoCapPhe a molecular titrationΔPhe recognition 52 assayto encapsulate showing process involvingboththat AGs-Calixhydrophobic carbohydrates53–55 drugswere such ablesuch to asas completely ligand/cellcurcumin pack andsurface DNA eosin interactions around through the 1.5–2hydrophobic[64]. NOne/P ratio, possible and exhibiting π strategy–π staking greater to promoteinteractions,DNA complexation multivalency and pDNA ability is tothrough than tether the multiple electrostatic gold standard ligand inte transfectantunitsractions, to a flexible as bPEI evidenced 25scaffold, kDA. by Moreover,either TEM a molecular(curcumin), having at entity their UV orfluorescenceoptimal a polymer.N/P ratioquenching We athought hydrodynamic (eosin), to exploit and diameter thisgel strategyretardat around ionin 150 orderassay nm to(pDNA). with amplify an overall Inthe particular, ability positive of AGscomplete surface to interact charge, GFP- withencodingthese the lipoplexes cellpDNA surface, displayed complexation and suitable eventu arises featuresally atcell 3.33 forinternalization, weight/weight gene delivery. by Indeed, (w/w tethering) ratio. AGs-Calix TheAGs transfection53to –the55 at upper their ability optimalrim of lipophilicNeoCapPheN/P charge calix[4]arenes, ratiosΔPhe 52 exhibited was assesseda macrocyclic transfection in two oligomer cell efficiencies lines, su namelyccessfully in two MCF-7 cell used lines and to higher N2a,build showing efficient than those good gene of efficiency thedelivery gold vectorstostandard deliver by bPEI,GFP anchoring expressing with ParoCalix guanidinylated plasmid55 as at the a 66.6 bestmoieties w/w performant, ratio, [65,66]. being even Accordingly, a better in the transfectant presence after of functionalizationthan serum, Lipofectamine. which better of neamine,Themimics showed the paromomycin, intransfection vivo environment, efficiencyand neomycin showingmainly with depends at a the link sameoner thebearing time small negligible ansize isothiocyanate of the cytotoxicity. lipoplexes moiety, (around Remarkably, we 40–60used AGs-Calixtetraminocalix[4]arenenm), on the sca abilityffolds 53of– 55tothe and buildliposome their three lipoplexes to AGs-calix[4]areneprotecte withd pDNAthe plasmid werecationic ablefrom tolipids, inhibitnucleases, namely Gram-negative and NeaCalix on theE. easy coli53,. NeoCalixdisassemblygrowth, with 54 ,into and the the lipoplexesParoCalix cytoplasm being55 ,for through morenucleare isothiocyanate-amineff localizatiective. Moreover,on, as demonstrated it isclick known chemistry through that AGs (Figure pH are studies. most 16) [67]. e Besidesffective The abilitynegligibleagainst to Gram-negative complex cytotoxicity, pDNA these bacteria, encoding liposomes AGs-Calix modified resulted firefly53– 55in, havingluciferase and their good was pDNA antibacterial assessed complexes through activity also the in showed fluorophore- both Gram- good exclusionpositiveantibacterial and titration activityGram-negative assay against showing bacterial Gram-positive that strains. AGs-CalixS. lutea 53, even–55 were if to aable lesser to extent.completely pack DNA around the 1.5–2 N/P ratio, exhibiting greater DNA complexation ability than the gold standard transfectant bPEI 25 kDA. Moreover, having at their optimal N/P ratio a hydrodynamic diameter around 150 nm with an overall positive surface charge, these lipoplexes displayed suitable features for gene delivery. Indeed, AGs-Calix 53–55 at their optimal N/P charge ratios exhibited transfection efficiencies in two cell lines higher than those of the gold standard bPEI, with ParoCalix 55 as the best performant, even in the presence of serum, which better mimics the in vivo environment, showing at the same time negligible cytotoxicity. Remarkably, AGs-Calix scaffolds 53–55 and their lipoplexes with pDNA were able to inhibit Gram-negative E. coli. growth, with the lipoplexes being more effective. Moreover, it is known that AGs are most effective against Gram-negative bacteria, AGs-Calix 53–55, and their pDNA complexes also showed good antibacterial activity against Gram-positive S. lutea, even if to a lesser extent. Figure 15. Synthesis of NeoCapPhe∆Phe 52.

Figure 16. StructuresStructures of AG-calix[4]arene conjugates 53–55. Antibiotics 2020, 9, 504 19 of 29

5. Polymeric AG-Based Gene Delivery Vectors Along with cationic liposomes, natural and synthetic polymers have been widely studied in order to seek non-viral vectors able to surmount all the physiological barriers for an ideal gene delivery vector [68–70]. Cationic polymers can be basically grouped in two classes: cationic polymers that are commercially available, encompassing gold standard bPEI, lPEI, poly-lysins, PAMAM dendrimers, and chitosan, which are probably the most studied as they are easy to functionalize, and the cationic polymers that are ad hoc designed and synthetized. In this section, we will outline the chemical and biological properties of polymers bearing AGs, either introduced to functionalize commercially available cationic polymers or used as building blocks to build new hyperbranched structures.

5.1. Functionalization of Polymers with AGs PAMAM dendrimers are certainly among the most studied cationic polymers in gene delivery [71]. Indeed, they possess several features that render their use very intriguing such as well-defined architecture, easy functionalization, the presence of tertiary amines able to act as proton sponges, and negligible cytotoxicity. However, undecorated PAMAM dendrimers, besides the generation considered, are not very efficient, being less efficient than the gold standard bPEI. For this reason, in order to increase their efficiencies while maintaining their negligible cytotoxicity, a lot of interest has been paid to the functionalization of the outer shell of PAMAM dendrimers with different moieties, ranking from cationic, anionic, and lipophilic to targeting moieties [30]. In this scenario, we decided to assess the potential of AG-based PAMAM vectors by linking three aminoglycosides, namely neamine, neomycin, and paromomycin, to PAMAM generation four (PAMAM G4), which is the most studied PAMAM dendrimer showing the best balance between efficiency and toxicity [72]. In order to exploit isothiocyanate-amine click chemistry, neamine, paromomycin, and neomycin were functionalized with a linker bearing the suitable isothiocyanate group through the synthetic pathways depicted in Figure2 of path A for neamine and paromomycin, and path B for neomycin, and successively clicked to the dendrimer outer primary amines. Due to steric constraints, full functionalization of the 64 outer PAMAM G4 amines was not possible and PAMAM G4-Nea 56, PAMAM G4-Paro 57, and PAMAM G4-Neo 58 bearing 46, 40, and 40 AGs, respectively, were obtained, as assessed by 1H-NMR and MALDI spectroscopy (Figure 17a). Interestingly, conjugates 56–58 were less able to complex DNA than undecorated PAMAM G4. However, they were able to fully complex pDNA at a low N/P ratio and form poliplexes with a hydrodinamic diameter between 150 and 200 nm. The transfection efficiency of poliplexes prepared with luciferase expression plasmid pGL3 at different N/P ratios was assessed in three cell lines, namely HeLa, Cos-7, and U87, in complete medium and in the presence of increasingly high concentration of serum. As expected, it was not possible to find an optimum value of N/P ratios for all the poliplexes, but PAMAM G4-Paro 57 and PAMAM G4-Neo 58 showed better transfection efficiencies and lower cytotoxicity than gold standard 25 KDa bPEI (and much better than undecorated PAMAM G4), each one at their optimal N/P ratio. In particular, PAMAM G4-Paro 57 resulted in being more performant than bPEI, even in the presence of 10% and 50% in volume of fetal bovine serum (FBS). Afterward, we decided to also investigate the role of the PAMAM generation [73]. Accordingly, with the same synthetic strategy and a slightly different linker, we prepared four new conjugates starting from PAMAM dendrimers of different generations, namely PAMAM G2-Neo 59, PAMAM G4-Neo 60, and PAMAM G7-Neo 62, bearing 13, 40, and 307 neomycin moieties, respectively (Figure 17b) [73]. Moreover, in order to provide further insights on the different behavior of AGs and guanidinoglycosides in gene delivery, we also prepared PAMAM G4-GNeo 61, to which only 15 guanidinoneomycins were possible to tether due to the higher steric hindrance of the guanidine group compared to the amino function. Interestingly, conversely to PAMAM with higher generations, the ability to complex DNA of PAMAM G2 was increased by conjugation with neomycin. Nevertheless, besides PAMAM G7, the transfection efficiencies of undecorated PAMAM dendrimers were raised by conjugation with neomycin at almost any N/P ratios, as assessed by treating two different cell lines, namely HeLa and COS-7 cells, with pGL3 poliplexes, which were more performant than the gold standard bPEI 25 KDa. Antibiotics 2020, 9, 504 20 of 29 Antibiotics 2020, 9, x 20 of 28

Interestingly, PAMAM G2-NeoG2-Neo 59 resultedresulted inin beingbeing thethe mostmost performantperformant vector for didifferentfferent values of NN/P/P ratios,ratios, maintainingmaintaining inin allall casescases negligiblenegligible cytotoxicity.cytotoxicity. This result is noteworthy because the production of PAMAM G2, which in general is less eefficientfficient in deliveringdelivering drugs and less cytotoxic compared to to PAMAM PAMAM G4, G4, is is much much less less costly costly and and time time consuming. consuming. Moreover, Moreover, PAMAM PAMAM G2-Neo G2-Neo 59 showed59 showed very very good good antibacterial antibacterial activity activity against against E. coliE.. coli in. a in concentration-dependent a concentration-dependent manner. manner. As Asalready already demonstrated demonstrated with with AG-based AG-based liposomes, liposomes, guanidinylation guanidinylation of the of theAG AGamines amines did didnot notfavor favor the overallthe overall activity activity of the of vector, the vector, with with PAMAM PAMAM G4-GNeo G4-GNeo 61 being61 being less lessefficient efficient than than both both PAMAM PAMAM G2- NeoG2-Neo 59 and59 and PAMAM PAMAM G4-Neo G4-Neo 60. 60.

Figure 17. (a) Structures of AG-based PAMAM G4 conjugates 56–58 and (b) structures of neomycin based PAMAM dendrimers ofof didifferentﬀerent generationsgenerations 5959––6262..

In general, polymer functionalization is intended not only to increase the transfection efficiency eﬃciency of the original polymer, but also, in some cases, to render the vector selective by tethering targeting moieties [30]. In this sense, bPEI 10 KDa and 25 KDa, which are their self-efficient polymeric non- viral vectors, have been functionalized with gentamycin and neomycin in order to enhance the Antibiotics 2020, 9, 504 21 of 29

moietiesAntibiotics 2020 [30]., 9 In, x this sense, bPEI 10 KDa and 25 KDa, which are their self-efficient polymeric non-viral21 of 28 vectors, have been functionalized with gentamycin and neomycin in order to enhance the transfection etransfectionfficiency in efficiency the kidney in the by kidney targeting by megalintargeting transmembranemegalin transmembrane protein [prot74].ein Indeed, [74]. Indeed, it has it been has demonstratedbeen demonstrated that megalin that megalin is a highly is a expressedhighly expressed receptor receptor in proximal in proximal tubular epithelial tubular epithelial cells, which cells, are thewhich most are populous the most populous cell type incell the type kidney. in the Alongkidney. with Along cubilin with protein,cubilin protein, megalin megalin is involved is involved in the endocyticin the endocytic uptake ofuptake many of ligands many andligands in the and retention in the of retention drugs including of drugs AGs including [75]. Accordingly, AGs [75]. bPEIAccordingly, 10 kDa and bPEI 25 10 kDa kDa were and reacted25 kDa withwere direactedfferent with quantities different of 6-bromohexanoicquantities of 6-bromohexanoic acid (around 10%,acid 30%,(around and 10%, 50% 30%, primary and amine50% primary substitution) amine substitution) in order to obtain in order carboxyalkyl-PEI10 to obtain carboxyalkyl-PEI10 and -PEI25 63 and–64 -, respectively,PEI25 63–64, having respectively, a different having degree a different of functionalization degree of functionalization (Figure 18). The resulting(Figure 18). polymers The resulting bearing freepolymers carboxylic bearing acid free functions carboxylic were coupledacid functions to unprotected were coupled gentamicin to andunprotected neomycin gentamicin AGs, leading and to theneomycin formation AGs, of leading PEI10-Gen, to the PEI25-Gen, formation PEI10-Neo,of PEI10-Gen, and PEI25-Gen, PEI25-Neo PEI10-Neo,65–68, respectively, and PEI25-Neo with di ff65erent–68, degreesrespectively, of grafting. with different degrees of grafting.

Figure 18. Synthesis of AG-PEI conjugates 65–68.68.

Interestingly, PEI-AG conjugates conjugates formed formed smaller smaller polyplexes polyplexes compared compared to toundecorated undecorated PEIs: PEIs: at atC/PC /=P 2,= PEI25-AGs2, PEI25-AGs 66 and66 and68 formed68 formed polyplexes polyplexes with withan average an average size of size about of about143–163 143–163 nm, while nm, whilePEI10-AGs PEI10-AGs 65 and65 67and had67 anhad average an average size sizeof about of about 166–170 166–170 nm. nm. The The transfection transfection efficiencies efficiencies of ofpolyplexes polyplexes formed formed by bycomplexati complexationon of pDNA of pDNA encoding encoding EGFP EGFP with with AG-based AG-based polymeric polymeric vectors vectors 65– 6568– were68 were tested tested in inmegalin-expressing megalin-expressing cell cell lines lines HK-2 HK-2 and and MDCK MDCK and and the the non-megalin-expressing HepG2 cell line as a comparison. Notably, Notably, AG-PEI vectors were able to transfect megalin-expressing cell lines with higher eefficiencyfficiency than undecorated PEIs and carboxyalkyl-PEIs 6363––6464 at C/PC/P ratios higher than 2 (around 7-fold increase), the performance of the the vectors vectors being being dependent dependent from from the the PEIs’ PEIs’ molecular weights (the higher, the better), the AG grafted,grafted, and the degreedegree ofof grafting.grafting. In general, gentamicin enhanced the performance of the vector to a greater extent than neomycin, where the higher the degree of grafting, the higherhigher thethe transfectiontransfection eefficiency.fficiency. The mostmost performantperformant polyplex was PEI25-GentPEI25-Gent66 66with with around around 10% 10% of gentamicinof gentamicin grafted grafted (calculated (calculated on the on original the original PEI free PEI amines) free whenamines) formulated when formulated at C/P = 4. at More C/P = interestingly, 4. More interestingly, regardless regardless of the C/P ratio, of the a C/P much ratio, higher a much transfection higher etransfectionfficiency was efficiency observed was in megalin-expressing observed in megalin-e cellxpressing lines compared cell lines with compared non-megalin with expressing non-megalin cell linesexpressing with the cell increase lines with of AGs the moietiesincrease tetheredof AGs tomoieties the vector, tethered reaching to the a 7-fold vector, increase reaching for a the 7-fold best increase for the best cationic polymer 66. Moreover, megalin specificity was corroborated by performing transfection studies in the megalin-expressing NK-2 cell line in the presence of increasing quantities of human serum albumin, a known targeting megalin competitor. Accordingly, the Antibiotics 2020, 9, 504 22 of 29 cationic polymer 66. Moreover, megalin specificity was corroborated by performing transfection studiesAntibiotics in 2020 the, 9 megalin-expressing, x NK-2 cell line in the presence of increasing quantities of human22 of 28 serum albumin, a known targeting megalin competitor. Accordingly, the transfection efficiency of vectorstransfection66 and efficiency68 was inhibited of vectors with 66 the and increase 68 was of albumin,inhibited wherewith completethe increase inhibition of albumin, was reached where withcomplete a dose inhibition of 20 mg /wasmL. reached with a dose of 20 mg/mL.

5.2.5.2. SynthesisSynthesis ofof HyperbranchedHyperbranched PolymersPolymers BasedBased onon AGAG ScaScaffoldsffolds TheThe paradoxparadox accordingaccording toto whichwhich thethe transfectiontransfection eefffificiencyciency andand cytotoxicitycytotoxicity ofof aa cationiccationic polymerpolymer areare oftenoften proportional proportional is probablyis probably one ofone the mostof the important most important issues to beissues challenged to be inchallenged the development in the ofdevelopment new polymeric of new gene polymeric delivery vectorsgene delivery [28]. Accordingly, vectors [28]. aAccordingly, possible alternative a possible of commerciallyalternative of availablecommercially polymeric available transfectants polymeric tr suchansfectants as PEI andsuch PAMAMas PEI and dendrimers PAMAM dendrimers is the synthesis is the synthesis of novel hyperbranchedof novel hyperbranched polymers polymers starting fromstarting natural, from biodegradablenatural, biodegradable building buil blocks.ding AGs, blocks. being AGs, natural being compoundsnatural compounds bearing manybearing functional many functional groups, and groups, in particular and in primary particular amines, primary can beamines, exploited can forbe theexploited preparation for the of preparation hyperbranched of hyperbranched polymeric structures polyme withric structures different with cross-linkers. different cross-linkers. The combination The ofcombination combinatorial of combinatorial synthesis, high synthesis, throughput high screening, throughput and chemoinformaticscreening, and chemoinformatic models in order tomodels predict in whichorder to are predict the key which physicochemical are the key physicochemical parameters to take parameters into account to take is often into aaccount winning is often strategy a winning to find leadstrategy candidates, to find as frequentlylead candidates, demonstrated as frequently in medicinal demonstrated chemistry [76 ].in Accordingly, medicinal thechemistry group of Prof.[76]. RageAccordingly, has developed the group a quantitative of Prof. Rage structure–activity has developed relationship a quantitative (QSAR) structure–activity modeling for relationship correlating the(QSAR) properties modeling of AG-based for correlating hyperbranched the properties polymers of AG-based with their abilityhyperbranched to deliver polymers pDNA into with the their cell. Inability a pioneering to deliver study, pDNA a library into the of cell. 56 AG-based In a pioneer hyperbrancheding study, a library polymers of 5669 AG-basedwas synthesized hyperbranched through epoxidepolymers ring 69 was opening synthesized polymerization through epoxide starting ring from openin seveng AGspolymerization and eight diglycidylstarting from ethers seven (DGE) AGs (Figureand eight 19)[ diglycidyl77]. ethers (DGE) (Figure 19) [77].

FigureFigure 19.19. SynthesisSynthesis ofof AG-DGEAG-DGE hyperbranchedhyperbranched polymerspolymers 6969..

AA firstfirst screeningscreening waswas performedperformed inin twotwo cancercancer cellcell lines,lines, namelynamely PC3PC3 humanhuman prostateprostate cellscells andand MiaMia PaCa-2PaCa-2 pancreaticpancreatic cancercancer cells,cells, withwith polyplexespolyplexes formedformed atat 25:125:1 polymerpolymer/pGL3/pGL3 plasmidplasmid weightweight ratio.ratio. TheThe sevenseven mostmost performantperformant candidates,candidates, Apra-RDE,Apra-RDE, Paro-RDE,Paro-RDE, Paro-GDE,Paro-GDE, Sis-RDE,Sis-RDE, Sis-GDE,Sis-GDE, Ami-EGDE,Ami-EGDE, and and Neo-GDE Neo-GDE were were selected selected for further for physicochemicalfurther physicochemical characterizations characterizations and experiments. and Theirexperiments. transfection Their e ffitransfectionciencies were efficiencies evaluated were independently evaluated indepe withndently two plasmids with two (pGL3 plasmids and pEGFP) (pGL3 atand di ffpEGFP)erent N /atP ratiosdifferent and N/P correlated ratios and with correlated the gold standardwith the gold bPEI standard 25 kDa. ForbPEI most 25 kDa. of the For polymers, most of thethe bestpolymers, performances the best were performances observed betweenwere observed 50 and between 100 N/P ratio, 50 and with 100 the N/P AG-based ratio, with polymers the AG-based being morepolymers efficient being than more the efficient bPEI 25 kDa,than the probably bPEI 25 thanks kDa, toprobably their reduced thanks cytotoxicity. to their reduced Physicochemical cytotoxicity. parametersPhysicochemical of the parameters polymers areof the very polymers important are invery promoting important effi incient promoting transgene efficient expression transgene and wereexpression used to and develop were used QSAR to modelsdevelop withQSAR support models vector with support regression vector (SVR). regression Exploiting (SVR). five Exploiting different descriptorfive different series, descriptor the authors series, were the able authors to build were cheminformatic able to build modeling,cheminformatic which resultedmodeling, in which being highlyresulted predictable in being highly in a new predictable set of experiments. in a new set Since of experiments. one of the majorSince one contributions of the major for contributions the efficient pDNAfor the deliveryefficient ofpDNA AG-DGE delivery polymers of AG-DGE was an polyme enhancedrs was hydrophobic an enhanced character, hydrophobic the same character, group was the speculatedsame group to was further speculated increase to their further transfection increase abilitytheir transfection by functionalization ability by withfunctionalization lipophilic chains. with Accordingly,lipophilic chains. three Accordingly, of the most performingthree of the polymers,most performing namely polymers, Neo-GDE, namely Paro-GDE, Neo-GDE, and Apra-GDE, Paro-GDE, and Apra-GDE, were reacted with three fatty acid acyl chlorides with respectively six-, fourteen-, and eighteen-linear carbon length chains in three different molar ratios, producing a library of 27 lipopolymer candidates (Figure 20) [78]. Antibiotics 2020, 9, 504 23 of 29 were reacted with three fatty acid acyl chlorides with respectively six-, fourteen-, and eighteen-linear carbon length chains in three different molar ratios, producing a library of 27 lipopolymer candidates (FigureAntibiotics 20 2020)[78, 9]., x 23 of 28

FigureFigure 20.20. SynthesisSynthesis ofof AG-GDEAG-GDE lipophiliclipophilic polymerspolymers 7070––7272..

AfterAfter somesome physicochemicalphysicochemical propertiesproperties werewere successfullysuccessfully assessedassessed such as pDNA complexation, hydrodynamichydrodynamic diameterdiameter determination,determination, andand zeta-potentialzeta-potential measurement,measurement, whichwhich showedshowed thatthat lipidiclipidic polymerspolymers70 70–72–72possessed possessed the the suitable suitable requirements requirements for apolymeric for a polymeric vector, the vector, transfection the transfection efficiencies inefficiencies different cellin different lines, without cell lines, and without in the presence and in th ofe uppresence to 50% of of up serum, to 50% were of serum, measured were at measured different Nat/P differentratios. TheN/P resultsratios. The obtained, results and obtained, corroborated and corroborated by chemoinformatic by chemoinformatic modeling, modeling, showed thatshowed the presence,that the presence, in low percentage, in low percentage, of long stearyl of long chains stearyl was chains fundamental was fundamental in order toin increaseorder to theincrease delivery the performancedelivery performance of the polymers, of the polymers, being Neo-GDE-C17 being Neo-GDE-C1770c and Apra-GDE-C17 70c and Apra-GDE-C1772c obtained 72c by obtained mixing acyl by chloridemixing acyl/polymer chloride/polymer in 1:2 molar in ratio, 1:2 molar where ratio, the most where performant the most performant vectors displayed vectors higherdisplayed transgene higher expressiontransgene expression than both bPEI than 25both kDa bPEI and 25 the kDa undecorated and the undecorated polymer, even polymer, in the presenceeven in the of serum. presence of serum.In addition to constructing new biodegradable polymers, there is a general increasing interestIn addition in exploiting to constructing stimuli responsive new biodegradable linkers [54 poly]. Accordingly,mers, there is usinga general Michael increasing acceptor interest N,N’- in bismethylenebisacrylamideexploiting stimuli responsive73 as degradablelinkers [54]. linkers, Accordingly, two AG-based using hyperbranched Michael acceptor polymers 74N,N’-–75 werebismethylenebisacrylamide prepared starting from 73 unprotected as degradable kanamycin linkers, andtwo gentamicin,AG-based hyperbranched respectively (Figure polymers 21) [ 7974,–8075]. Thewere formed prepared polymers starting showedfrom unprotected several features kanamyci suitablen and forgentamicin, efficient respectively gene delivery (Figure vectors 21) such [79,80]. as greatThe formed ability topolymers fully complex showed pDNA several at veryfeatures low Nsuitable/P ratios, for polyplex efficient dimensions gene delivery lower vectors than 100such nm, as degradationgreat ability to in fully acidic complex medium, pDNA and at high very bu lowffering N/P ratios, capacity, polyplex which dimensions facilitates endosomallower than 100 escape nm, anddegradation negligible in cytotoxicity.acidic medium, The and delivery high buffering performances capacity, of poliplexes which facilitates formed endosomal by 74–75 and escape pDNA and encodingnegligible EGFPcytotoxicity. evaluated The in HeLadelivery and pe COS-7rformances cell lines of atpoliplexes different formedN/P ratios by showed 74–75 and an increase pDNA inencoding the transfection EGFP evaluated efficiencies in HeLa with and an increaseCOS-7 cell in lines the N at/P differentratios, reaching N/P ratios the showed plateau an at increaseN/P = 50, in whichthe transfection was much efficiencies more active with than an the increase naturally in the occurring N/P ratios, chitosan reaching polysaccharide the plateau at and N/P approached = 50, which thewas e ffimuchciency more of 25 active kDa bPEI.than Interestingly,the naturally theoccurring performance chitosan of polymerpolysaccharide75 was onlyand slightlyapproached affected the byefficiency the presence of 25 kDa of serum, bPEI. renderingInterestingly, such the a polymerperformance as a promisingof polymer gene 75 was delivery only slightly vectorto affected be tested by inthe vivo presence. Moreover, of serum, the samerendering gentamicin-based such a polymer branched as a promising polymer gene75 displayed delivery vector good antitumor to be tested and in antibacterialvivo. Moreover, activities, the same as assessed gentamicin-based by the MTT branched assay against polymer HeLa 75 cancer displayed cells and good by antitumor measuring and the inhibitionantibacterial ratio activities, against E.as coliassessed, respectively by the MTT [80]. assay against HeLa cancer cells and by measuring the inhibition ratio against E. coli, respectively [80]. Antibiotics 2020, 9, 504 24 of 29 Antibiotics 2020, 9, x 24 of 28

Figure 21.21. Synthesis of kanamycin- and gentamicin-basedgentamicin-based hyperbranchedhyperbranched polymerspolymers 7474––7575..

6. Conclusions 6. Conclusions AGsAGs havehave beenbeen intensivelyintensively usedused toto fightfight bacteriabacteria overover eighteight decades,decades, sincesince streptomycinstreptomycin waswas discovereddiscovered inin thethe 1940s.1940s. However, the developmentdevelopment of antibacterialantibacterial resistance,resistance, principallyprincipally due toto modificationmodification ofof the the AG AG sca scaffoldffold by by AG-modifying AG-modifying enzymes enzymes as wellas well as the as frequentlythe frequently occurring occurring toxic toxic side esideffects effects such assuch ototoxicity as ototoxicity and nephrotoxicity, and nephrotoxicity, could have could led tohave the endled ofto this the class end ofof antimicrobials. this class of However,antimicrobials. if we considerHowever, the scientificif we productivityconsider the with scientific the word productivity “aminoglycoside(s)” with the in the word title, we“aminoglycoside(s)” notice that more than in the one title, thousand we notice papers that has mo appearedre than one in the thousand literature papers (Web ofhas Science appeared source) in the in theliterature last five (Web years, of meaning Science source) that AGs in are the still last of five great years, appeal meaning to the scientific that AGs community. are still of Thegreat continuing appeal to (andthe scientific somewhat community. growing) interest The continuing in AGs arises (and from somewhat two main growing) accomplishments: interest in (1) AGs a better arises knowledge from two ofmain the accomplishments: chemistry of AGs, (1) which a better provides knowledge the scientist of the chemistry with a rather of AGs, wide which portfolio provides of possibilities,the scientist andwith (2) a rather a better wide understanding portfolio of possibilities, of the AG mechanisms and (2) a better of action, understanding which leads of the to aAG broader mechanisms range ofof applications.action, which Amongleads to the a broader new trends, range the of useapplic ofations. AGs as Among cationic the moieties new trends, to build the multifunctional use of AGs as non-viralcationic moieties gene delivery to build vectors multifunctional has attracted non-vira the attentionl gene delivery of the vectors scientific has community attracted the working attention in theof the field. scientific Different community libraries of working cationic lipidsin the havefield. been Different produced libraries by tethering of cationic lipophilic lipids tailshave to been AG cationicproduced heads by tethering through suitablelipophilic linkers, tails to encompassing AG cationic heads stimuli through responsive suitable linkers, linkers, and encompassing using them to promotestimuli responsive the delivery linkers, of di andfferent using genetic them materials to promot suche the asdelivery DNA, of mRNA, different and genetic siRNA materials to the target such organelles.as DNA, mRNA, Although and greatsiRNA eff ortto the has ta beenrget madeorganelles. in this Although area, an ideal great AG-based effort has lipidic been made vector in is this not availablearea, an ideal yet. AG-based However, theselipidic studies vector shedis not light availa onble some yet. importantHowever, insights:these studies (1) it shed is very light important on some toimportant maintain insights: a certain (1) hydrophilicity it is very important/lipophilicity to main balance;tain a certain (2) the hydrophilicity/lipophilicity AGs charge density and geometry balance; have(2) the great AGs influence charge density on the vector and geometry performance; have (3) great the designinfluence of AG-basedon the vector vectors performance; bearing two (3) long the alkyldesign chains of AG-based (stearyl vectors or oleyl) bearing is often two a winninglong alkyl choice; chains (4) (stearyl the use or ofoleyl) linkers is often that a are winning not too choice; short is(4) preferable; the use of andlinkers (5) althoughthat are not guanidinoglycosides too short is preferable; are known and (5) to although be stronger guanidinoglycosides RNA binders and are to facilitateknown to internalization be stronger ofRNA bio-macromolecules, binders and to guanidination facilitate internalization of AG-based geneof bio-macromolecules, delivery liposomes doesguanidination not contribute of AG-based to better transfectiongene delivery effi ciency.liposomes Along does with not AG-based contribute liposomes, to better AGs transfection have also beenefficiency. exploited Along to synthesizewith AG-based cationic liposomes, polymeric AGs non-viral have vectors,also been both exploited by functionalizing to synthesize pre-existing cationic commerciallypolymeric non-viral available vectors, polymers both such by functionalizing as bPEI and PAMAM pre-existing dendrimers, commercially and byusing available their polymers selves as scasuchffolds as bPEI in polymerization and PAMAM reactions. dendrimers, In the and former by using cases, their more selves performant as scaffolds polymers in polymerization than the gold reactions. In the former cases, more performant polymers than the gold standard bPEI 25 kDa were Antibiotics 2020, 9, 504 25 of 29 standard bPEI 25 kDa were obtained at their optimal N/P ratio with neomycin and paromomycin, the cytotoxicity being similar or lower. Wide libraries of AG-based polymers were prepared starting from AG scaffolds and Michael acceptor bis-acrylamides or terminal bis-epoxide linkers, and with the help of ad hoc developed QSAR modeling; in this case, very efficient vectors have also been found, showing promising potential for future applications. However, even if a general rule of thumb can be extrapolated, it is still very difficult to be able to foresee the behavior of an AG-based polymer, since multiple barriers in the gene delivery process should be taken into consideration. Most of the results presented in this review come from in vitro experiments carried out to measure the transfection efficiencies of AG-based liposomes and polymers in different cell lines, some of them in the presence of serum, which better mimics the in vivo environment. A few in vivo experiments have been performed through intranasal instillation of Chol/PEG stabilized DOPE/lipoplexes to BALB/c mice or parenteral systemic administrations of Chol/PEG/DOPE stabilized lipid-modified AGs to C57BL/6 mice, showing promising preliminary results to be pushed forward for the design of multimodular AG-based vectors for in vivo gene delivery. In general, although some AG-based vectors are efficient transfectants, there are still challenges to overcome in order to be able to find the “ideal” vector that would be able to approach the efficiency of some viral vectors. However, thanks to the renaissance that AGs are facing, we believe that the community should stay optimistic in this sense.

Author Contributions: A.V. conducted the literature study and wrote the manuscript; M.C.B. contributed to writing, editing, revisiting, and updating the references. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

References and Note

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