Lipophilic Polyamines As Promising Components of Liposomal Gene Delivery Systems

pharmaceutics

Review Lipophilic Polyamines as Promising Components of Liposomal Gene Delivery Systems

Pavel A. Puchkov and Michael A. Maslov *

Lomonosov Institute of Fine Chemical Technologies, MIREA—Russian Technological University, Vernadsky Ave. 86, 119571 Moscow, Russia; [email protected] * Correspondence: [email protected]; Tel.: +7-499-600-8202

Abstract: Gene therapy requires an effective and safe delivery vehicle for nucleic acids. In the case of non-viral vehicles, including cationic liposomes, the structure of compounds composing them determines the efficiency a lot. Currently, cationic amphiphiles are the most frequently used compounds in liposomal formulations. In their structure, which is a combination of hydrophobic and cationic domains and includes spacer groups, each component contributes to the resulting delivery efficiency. This review focuses on polycationic and disulfide amphiphiles as prospective cationic amphiphiles for gene therapy and includes a discussion of the mutual influence of structural components.

Keywords: polyamines; spermine; cationic amphiphiles; cationic liposomes; disulﬁde groups; gemini amphiphiles; gene delivery

1. Introduction Citation: Puchkov, P.A.; Maslov, M.A. Gene therapy is a modern and promising method for treating severe hereditary and Lipophilic Polyamines as Promising acquired diseases, including COVID-19 immunization, through the delivery of therapeutic Components of Liposomal Gene nucleic acids (NAs) that can replace a damaged gene (pDNA), provide a new one, or block Delivery Systems. Pharmaceutics 2021, the expression of an unwanted protein (antisense oligonucleotides, siRNA) [1,2]. The direct 13, 920. https://doi.org/10.3390/ administration of therapeutic NAs is an inefﬁcient process due to multiple external and pharmaceutics13060920 internal limiting factors [3]. External factors lead to the instability of NAs in biological

Academic Editors: María Luisa Moyá ﬂuids (degradation by nucleases or interaction with albumin or low-density lipoproteins, and Manuel López-López causing the aggregation and rapid clearance of NAs) and a low degree of interaction with target cells. Internal factors are determined by the presence of membrane barriers (plasma,

Received: 18 May 2021 endosomal, and nuclear membranes) that present a challenge to NAs as they attempt to Accepted: 17 June 2021 reach the cytosol and nucleus [4]. Published: 21 June 2021 Overcoming these factors requires the development of special delivery vehicles. At present, viruses [5,6], which are highly effective but present some serious disadvantages, Publisher’s Note: MDPI stays neutral primarily associated with the induction of inflammatory and immune responses in the with regard to jurisdictional claims in body, fill this role. However, alternative non-viral delivery vehicles, such as cationic published maps and institutional affil- liposomes (CLs) based on cationic amphiphiles (CAs) [6–9], are being developed. The most iations. recent success is development of an mRNA vaccine [10] against COVID-19, where lipid nanoparticles deliver nucleoside-modified mRNA encoding a mutated form of the spike protein of SARS-CoV-2 [11]. Generally, CA structure is a combination of hydrophobic and cationic domains linked together by various spacer groups [12]. The positive charge of Copyright: © 2021 by the authors. CAs enables the “packing” of NAs due to electrostatic interactions with the formation Licensee MDPI, Basel, Switzerland. of lipoplexes (complexes of NAs with liposomes) and facilitates their interaction with a This article is an open access article negatively charged plasma membrane. distributed under the terms and In addition to CAs, liposomes can include helper lipids (for example, 1,2-dioleoyl-sn- conditions of the Creative Commons glycero-3-phosphoethanolamine, DOPE) [13–15], which promote the formation of a certain Attribution (CC BY) license (https:// lipid phase and favor cell transfection. Liposomes may also contain additional lipophilic creativecommons.org/licenses/by/ molecules that permit them to target certain cells [16] or increase their circulation time in 4.0/).

Pharmaceutics 2021, 13, 920. https://doi.org/10.3390/pharmaceutics13060920 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 920 2 of 27

Pharmaceutics 2021, 13, 920 certain lipid phase and favor cell transfection. Liposomes may also contain additional2 oflip- 27 ophilic molecules that permit them to target certain cells [16] or increase their circulation time in the bloodstream (for example, lipophilic derivatives of polyethylene glycol, PEG) [17]. If it is not stated below, CAs were used without additional components. the bloodstreamThe CA structure (for example, significantl lipophilicy affects derivatives the efficiency of polyethylene of NA delivery glycol, PEG)to eukaryotic [17]. If it cells.is not In stated recent below, years, CAs many were monocationic used without amphiphiles additional components. have been obtained [18–28] that form Theliposomes CA structure for NA significantly delivery. affectsIn this the review, efficiency we ofwill NA consider delivery topolycationic eukaryotic cells.amphiphiles,In recent years,which, many compared monocationic to monocationic amphiphiles analogs, have enable been the obtained more efficient [18–28] thattransport form ofliposomes NAs into forcells NA due delivery. to the formation In this review, of a system we will of distributed consider polycationic charges in the amphiphiles, polyamine which, compared to monocationic analogs, enable the more efficient transport of NAs into matrix and their ability to facilitate NA release from endosomes. This ability is strongly cells due to the formation of a system of distributed charges in the polyamine matrix and affected by the high H+ buffer capacity of polyamines containing titratable amines results their ability to facilitate NA release from endosomes. This ability is strongly affected by the in endosomal Cl− accumulation during acidification with presumed osmotic endosome high H+ buffer capacity of polyamines containing titratable amines results in endosomal disruption and enhanced lipoplex escape [29]. Cl− accumulation during acidification with presumed osmotic endosome disruption and enhanced lipoplex escape [29]. 2. Cationic Amphiphiles Based on Polyamines or Amino Acids 2.1.2. Cationic Cationic AmphiphilesAmphiphiles Ba Basedsed on onLinear Polyamines Polyamines or Amino Acids 2.1. CationicEnhancement Amphiphiles of NA Based transport on Linear by polycationic Polyamines amphiphiles may be related not only to distributedEnhancement charges. of NA On transportthe cell surface, by polycationic polyamine amphiphiles recognition may sites—for be related example, not only PAT to [30],distributed a polyamine charges. transporter—selectively On the cell surface, polyamine transport recognition both polyamines sites—for example,and their PAT deriva- [30], tives.a polyamine Moreover, transporter—selectively cancer cells have more transport such sites both on polyaminestheir surfaces, and which their derivatives.means that amphiphilesMoreover, cancer based cells on havepolyamines more such can sites transfect on their cancer surfaces, cells which more meansefficiently. that amphiphilesParticularly importantbased on polyaminesfactors in NA can delivery transfect are cancer the number cells more and efficiently.distribution Particularly of positive charges important in thefactors polyamine in NA delivery molecule. are theTransfection number and activity distribution (TA) has of positive been shown charges to in increase the polyamine as the numbermolecule. of Transfectionamino groups activity in the (TA)polyamine has been structure shown increases. to increase Compound as the number 1e (Figure of amino 1) exhibitedgroups in the the highest polyamine transfection structure increases.efficiency Compoundamong the synthesized1e (Figure1) lipopolyamines exhibited the highest 1a–g [31],transfection which suggests efficiency that among 1e can the use synthesized PAT and compete lipopolyamines with other1a– polyamines,g [31], which for suggests example,that spermine,1e can use PATfor binding and compete to certain with otherrecognition polyamines, sites foron example,the cell surface spermine, (in forparticular, binding withto certain the same recognition PAT). sites on the cell surface (in particular, with the same PAT).

Figure 1. LipopolyaminesLipopolyamines with with benzyl benzyl linker.

AnotherAnother factor factor affecting affecting the the efficiency efficiency of transfection is the hydrophobicity of the amphiphilicamphiphilic molecule. molecule. A A study study of of compounds compounds 22 andand 3a3a––dd,, which which contain contain sterols sterols (cortisol (cortisol andand its derivatives) asas hydrophobichydrophobic domainsdomains (Figure (Figure2), 2), revealed revealed that that TA TA increases increases with with an anincrease increase in thein the hydrophobicity hydrophobicity of theof the molecule molecule [32 [32].]. While While liposomes liposomes with with compound compound3d 3dwere were shown shown to beto be incapable incapable of deliveringof delivering NAs, NAs, possibly possibly due due to theto the lower lower hydrophobicity hydrophobi- cityof the of amphiphilethe amphiphile3d and 3d and ineffective ineffective formation formation of lipoplexes, of lipoplexes, compounds compounds3b and 3b and3c had 3c hadthe highestthe highest transfection transfection efficiencies. efficiencies. Notably, Notably, the contributionthe contribution of hydrophobicity of hydrophobicity to the to efficiency of NA delivery also depends on other parameters, primarily the CA/DOPE (the last was a helper lipid) ratio and N/P ratio (the ratio of the number of CA amino groups to the number of NA phosphate groups).

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thethe efficiencyefficiency ofof NANA deliverydelivery alsoalso dependsdepends onon otherother parameters,parameters, primarilyprimarily thethe CA/DOPECA/DOPE Pharmaceutics 2021, 13, 920 3 of 27 (the(the lastlast waswas aa helperhelper lipid)lipid) ratioratio andand N/PN/P ratioratio (the(the ratioratio ofof thethe numbernumber ofof CACA aminoamino groupsgroups toto thethe numbernumber ofof NANA phosphatephosphate groups).groups).

FigureFigure 2. 2. Cationic CationicCationic amphiphiles amphiphilesamphiphiles (CAs) (CAs) based based on on cortisol cortisol and and its its derivatives. derivatives.

SubsequentSubsequent studiesstudies have havehave shown shownshown [ 33[33][33]] that thatthat compounds compoundscompounds4b 4b4band andand4c 4c4c, which,, whichwhich contain containcontain double dou-dou- blebondsble bonds bonds in thein in the the polycyclic polycyclic polycyclic hydrophobic hydrophobic hydrophobic domain domain domain (Figure (Figure (Figure3), 3), 3), were were were the the the most most most effective. effective. effective. Three-Three- Three- componentcomponent liposomesliposomes formedformedformed from fromfrom compound compoundcompound4d 4d4d,, its, itsits dimeric dimericdimeric analog analoganalog4e 4e4e, and,, andand DOPE DOPEDOPE deliv- de-de- liverederedlivered plasmid plasmid plasmid DNA DNA DNA (pDNA) (pDNA) (pDNA) more more more efﬁciently effi efficientlyciently than than than two-component two-component two-component liposomes liposomes liposomes4e /DOPE.4e 4e/DOPE./DOPE.

FigureFigure 3. 3. CAs CAs with with different different polycyclic polycyclic hydrophobichydrophobic domains.domains.

HydrophobicHydrophobic substituentssubstituents inin thethe CACA structurestructure areare mainlymainly attached attached toto primaryprimary aminoamino groupsgroups of of the the polyamine.polyamine. A A different differentdifferent approach approach to to the the synthesissynthesis of of CAsCAs waswas proposedproposed byby 4 9 N 4 N 9 BlagbroughBlagbrough et et al.al. [[34–37],34[34–37],–37], whowhowho obtained obtainedobtained NN,4,,NN9-disubstituted-disubstituted spermine spermine derivativesderivatives 5a5a––jj withwith acylacyl oror alkylalkyl residuesresidues ofof variousvarious lengthslengths andand degreesdegrees ofof unsaturationunsaturation (Figure(Figure4 4).).4). All AllAll lipoplexes formed from acyl-substituted polyamines 5a–h exhibited high TA, excluding lipoplexeslipoplexes formedformed fromfrom acylacyl-substituted-substituted polyaminespolyamines 5a5a––hh exhibitedexhibited highhigh TA,TA, excludingexcluding amphiphiles 5b and 5f. However, only compounds 5f and 5g with stearoyl and oleoyl amphiphilesamphiphiles 5b5b andand 5f5f.. However,However, onlyonly compoundscompounds 5f5f andand 5g5g withwith stearoylstearoyl andand oleoyloleoyl residues had low toxicity toward FEK4 and HtTA cells. Notably, an increase in the degree residuesresidues had had low low toxicity toxicity toward toward FEK4 FEK4 and and HtTA HtTA cells. cells. Notably, Notably, an an increase increase in in the the degree degree of unsaturation of hydrocarbon chains increased both the efﬁciency of transfection and the cytotoxicity of the compounds. Alkyl derivatives of spermine 5i and 5j had comparable or slightly higher transfection efﬁciencies but were much more toxic than their acyl analogs [35]. Pharmaceutics 2021, 13, 920 4 of 27

of unsaturation of hydrocarbon chains increased both the efficiency of transfection and the cytotoxicity of the compounds. Alkyl derivatives of spermine 5i and 5j had compara- Pharmaceutics 2021, 13, 920 4 of 27 ble or slightly higher transfection efficiencies but were much more toxic than their acyl analogs [35].

4 9 FigureFigure 4.4. NN44,N9-disubstituted-disubstituted spermine spermine derivatives. derivatives.

4 Asymmetric analogsanalogs6a –6af (Figure–f (Figure4) were 4) subsequently were subsequently developed developed [ 38]. N4-myristoleoyl- [38]. N4- 9 4 9 myristoleoyl-N9-myristoylspermineN9-myristoylspermine (6c) and N4-oleoyl- (6c) andN 9N-stearoylspermine4-oleoyl-N9-stearoylspermine (6d) showed (6d the) showed highest theefficiency highest of efficiency siRNA delivery of siRNA into delivery FEK4 and into HtTA FEK4 cells, and comparable HtTA cells, tocomparable the efficiency to the of theef- ficiencycommercial of the transfectant commercial TransIT-TKO transfectant (Mirus TransIT-TKO Bio, Madison, (Mirus WI, Bio, USA). Madison, Amphiphile WI, USA).6f withAm- phiphilea lithocholoyl 6f with residue a lithocholoyl effectively residue delivered effectively NAs but delivered caused cell NAs death. but Thecaused least cell effective death. 4 9 4 9 wasThe leastN -cholesteryl- effective wasN -oleoylspermine N4-cholesteryl-N (96e-oleoylspermine). (6e). 11 1212 When NN1,N,N12-substituted-substituted spermine spermine derivatives derivatives 7a7a–d– d(Figure(Figure 5),5 ),structural structural isomers isomers of amphiphilesof amphiphiles 5c, 5c5d,,5d 5f,, 5f5g,,5g were, were synthesized synthesized and and studied studied [39], [39 the], theefficiency efficiency of pDNA of pDNA deliverydelivery into into FEK4 FEK4 and and HtTA HtTA cells cells by by complexes complexes with with amphiphiles amphiphiles 7a7a––dd waswas lower, lower, while the toxicity was higher than with amphiphile 5g. siRNA delivery efficiency efficiency mediated by compounds 7a and 7c was comparable to the efficiencyefficiency of delivery usingusing amphiphileamphiphile 5g5g..

Figure 5. Spermine derivatives with acyl-substitutedacyl-substituted terminal amino groups.

Multiple monosubstituted polyamine derivatives derivatives 8a8a––gg (Figure(Figure 55)) werewere obtainedobtained byby modifying spermine with fatty acid residues of various lengths and degrees of unsaturation [40]. [40]. Although Although an an increase increase in in the the length length of of the the fatty fatty acid acid residue residue increased increased toxicity, toxicity, it positivelyit positively affected affected the the penetration penetration of lipoplex of lipoplexeses through through the cell the cellmembrane membrane in vitro.in vitro Ex-. Experiments in vivo showed that the efﬁciency of NA delivery with N-butanoylspermine periments in vivo showed that the efficiency of NA delivery with N-butanoylspermine (8f) was higher than with N-decanoylspermine (8e). (8f) was higher than with N-decanoylspermine (8e). Mono- and disubstituted polycationic amphiphiles were developed based on spermine as a hydrophilic domain and cholesterol or 1,2-di-O-tetradecylglycerol as hydrophobic domains (Figure6)[ 41–43]. The amphiphiles had different spacer lengths and linker types. CLs were prepared using these amphiphiles and DOPE (1:1 mol.). Among monosubsti-

tuted amphiphiles 9a–c, compound 9b showed the highest TA. While transfecting the same percentage of cells as their monomeric analogs 9a–c, however, dimeric polycationic Pharmaceutics 2021, 13, 920 5 of 27

Mono- and disubstituted polycationic amphiphiles were developed based on spermine as a hydrophilic domain and cholesterol or 1,2-di-O-tetradecylglycerol as hydrophobic Pharmaceutics 2021, 13, 920 domains (Figure 6) [41–43]. The amphiphiles had different spacer lengths5 ofand 27 linker types. CLs were prepared using these amphiphiles and DOPE (1:1 mol.). Among monosubstituted amphiphiles 9a–c, compound 9b showed the highest TA. While transfecting the same percentage of cells as their monomeric analogs 9a–c, however, dimeric polycationic amphiphiles 10aamphiphiles–c provided 10a better–c provided expression better of the expression green fluorescent of the green protein. fluorescent The highest protein. The high- transfection efficiencyest transfection was exhibited efficiency by liposomeswas exhibi basedted by on liposomes amphiphile based10c on, which amphiphile were 10c, which superior to the efficiencywere superior of the to commercial the efficiency transfectant of the Lipofectamine commercial transfectant 2000 (Thermo Lipofectamine Fisher 2000 Scientific, Waltham,(Thermo MA, Fisher USA) forScientific, any type Waltham, of NAs transferredMA, USA) [for42 ,any43]. Targetedtype of NAs liposomes transferred [42,43]. based on CA 10cTargetedwere also liposomes successfully based employed on CA 10c in were vivo also [44 –successfully49]. employed in vivo [44–49].

Figure 6. Mono-Mono- and and dimeric dimeric polycationic polycationic amphiphiles amphiphiles ba basedsed on on spermine spermine and and triethylenetetramine. triethylenetetramine.

Analogs 10e–gAnalogswith ethoxypropylene, 10e–g with ethoxypropylene, octamethylene, octamethylene, and ethoxyethoxyethylene and ethoxyethoxyethylene spacers (Figurespacers6) permitted (Figure a greater6) permitted TA increase a greater than TA usingincrease10c thanin vitro using[50 10c]. Inin vitro contrast, [50]. In contrast, the replacementthe of replacement spermine with of spermine triethylenetetramine with triethylenetetramine (TETA, 11a,b )(TETA, led to a11a signiﬁcant,b) led to a significant decrease in TAdecrease [51]. in TA [51]. Extensive screeningExtensive of CAs screening [52] revealed of CAs that[52] revealed in the structure that in the of compoundsstructure of 12acompounds–j 12a–j through 29a–j,through both the 29a polyamine–j, both the matrix polyamine and the matrix hydrophobic and the hydrophobic components components changed changed (Figure7). Among(Figure CLs 7). formed Among from CLs these formed amphiphiles from these and amphiphiles DOPE (1:2 and weight DOPE ratio), (1:2 weight the ratio), the effective transfectioneffective of transfection HEK293 cells of wasHEK293 achieved cells was only ac byhieved liposomes only by with liposomes amphiphiles with amphiphiles 12a–j through 20a12a––jj containingthrough 20a an–j acyl containing substituent an acyl at the substituent terminal aminoat the terminal group. Moreover, amino group. Moreo- only eight compoundsver, only (eight12c, 12e compounds, 13d, 14c ,(12c16d, ,12e16g, 13d, 17h, 14c, and, 16d17j, 16g) were, 17h superior, and 17j) in were TA tosuperior in TA the commercialto transfectant the commercial Effectene transfectant (Qiagen, Effectene Hilton, Germany).(Qiagen, Hilton, Subsequent Germany). transfection Subsequent transfec- studies on HEK293,tion studies COLO on 205, HEK293, D17, HeLa, COLO and 205, PC3 D17, cells HeLa, showed and that PC3 these cells compoundsshowed that these com- mediated morepounds effective mediated NA transport more effective than did NA the transport commercial than transfectantsdid the commercial Effectene, transfectants Ef- DOTAP, and DC-Chol,fectene, whileDOTAP, their and toxicity DC-Chol, was lowerwhile thantheirthat toxicity of commercial was lower transfectants. than that of commercial transfectants.

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Figure 7. CAs with asymmetric acyl hydrophobic tails.

pH-SensitivepH-Sensitive polycationic polycationic amphiphiles amphiphiles 3030––3333 (Figure(Figure 88)) werewere obtainedobtained byby subse-subsequently coupling coupling amino amino acids acids (L (-histidineL-histidine and and L-cysteine)L-cysteine) and and fatty fatty acids acids (lauric, (lauric, oleic, oleic, and stearic)and stearic) to polyamines to polyamines [53,54]. [53 The,54]. size The of size complexes of complexes of amphiphiles of amphiphiles with withsiRNA siRNA was 160– was 210160–210 nm, and nm, the and maximum the maximum TA on TA U87 on cells U87 was cells achieved was achieved using using amphiphiles amphiphiles 30b–33b30b –with33b oleicwith residues. oleic residues. Among Among them, them,TA decreased TA decreased in the inseries the series30b > 30b 32b > > 32b 31b > > 31b 33b.> A33b corre-.A lationcorrelation was also was established also established between between the TA the and TA the and ability the ability of compounds of compounds 30b–30b33b– to33b dis-to ruptdisrupt the theintegrity integrity of erythrocyte of erythrocyte membranes. membranes. Leader Leader compound compound 30b30b basedbased on onethylenedi- ethylene- aminediamine exhibited exhibited the the highest highest hemolytic hemolytic activity activity at at pH pH 5.4, 5.4, which which corresponds corresponds to to the the onset ofof endosomal acidiﬁcation.acidification. Therefore,Therefore, when when using using this this amphiphile, amphiphile, one one can can expect expect effective effec- tiveNA NA release release inside inside cells cells due due to the to disruptionthe disruption of endosomal of endosomal membranes. membranes.

Figure 8. First generation of pH-sensitive polycationic amphiphiles.

TheThe second generation of pH-sensitive amphiphiles 34a34a––hh based on spermine was subsequentlysubsequently obtained (Figure 9 9).). InIn biologicalbiological teststests conductedconducted onon HeLaHeLa andand U87U87 cells,cells, thethe presence of anan LL-histidine-histidine inin the the amphiphile amphiphile structure structure did did not not improve improve TA. TA. In addition,In addition, no norelationship relationship was was found found between between the the efﬁciency efficiency of CAs of CAs and and the the distance distance between between hydropho- hydro- phobicbic domains. domains. Compound Compound34e 34eexhibited exhibited the highestthe highest activity activity in the in deliverythe delivery of pDNA of pDNA [55], [55],while while amphiphile amphiphile34f exhibited 34f exhibited the highest the highest activity activity in the indelivery the delivery of siRNA of siRNA [56,57 [56,57].]. The Theauthors authors also also noted noted that that they they did notdid utilizenot utilize helper helper lipids lipids in complex in complex formation formation since since the synthesized compounds were able to initiate a pH-dependent phase transition, which led the synthesized compounds were able to initiate a pH-dependent phase transition, which to the destabilization of the complexes and the release of NAs. led to the destabilization of the complexes and the release of NAs.

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Figure 9. Second generation of pH-sensitive polycationic amphiphiles.

Multiple phosphamidephosphamide derivatives derivatives containing containing long-chain long-chain alkyl alkyl substituents substituents (dodecyl, (dodecyl,tetradecyl, tetradecyl, and hexadecyl) and hexadecyl) were were obtained obtain ased hydrophobic as hydrophobic fragments fragments [58]. [58]. The The transfec- trans- fectiontion of of COS-1 COS-1 cells cells with with lipoplexes lipoplexes formed formed by by pDNA pDNA and and micelles micelles oror liposomesliposomes basedbased on amphiphiles 35a–d (Figure 10)10) showed that complexes based on micelles were only half as as effective effective as as complexes complexes based based on onCA CAliposomes/lipid liposomes/lipid helper/Chol helper/Chol (1:1:1 mol.). (1:1:1 DOPE mol.). andDOPE dipalmitoyl and dipalmitoyl phosphatidylcholine phosphatidylcholine (DPPC) (DPPC) have been have used been as used helper as lipids, helper but lipids, DPPC- but containingDPPC-containing liposomes liposomes have proven have provento be an to ineffective be an ineffective delivery deliveryvehicle. TA vehicle. on LLC TA and on B16BL6LLC and cells B16BL6 increased cells increased with an increase with an in increase the length in the of lengththe alkyl of thechains alkyl and chains the number and the ofnumber amino of groups amino in groups the polyamine. in the polyamine. For LLC cells, For LLC the cells,best compound the best compound was 35f based was 35fon spermine,based onspermine, and for B16BL6 and for cells, B16BL6 the best cells, compound the best compound was amphiphile was amphiphile 35c based on35c spermi-based dine.on spermidine. An analog of compounds 35c and 35f was obtained based on a synthetic polyamine– tetraethylenepentamine ( (35g35g,, FigureFigure 1010))[ 59[59].]. Liposomes Liposomes35g 35g/DOPE/DPPC/Chol/DOPE/DPPC/Chol (0.25:1:0.75:1 mol)mol) efﬁciently efficiently delivered delivered antisense antisense oligonucleotides oligonucleotides to eukaryotic to eukaryotic cells. Here,cells. Here,the introduction the introduction of lipophilic of lipophilic derivatives derivatives of polyethylene of polyethylene glycol (PEG)glycol and(PEG) a cyclic and a analog cyclic analogof the peptide of the peptide RGD ensured RGD ensured active targeting active targ of liposomeseting of liposomes to target cellsto target and increasedcells and thein- creasedefﬁciency the of efficiency NA delivery of NA [60 ,delivery61]. [60,61].

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Figure 10. Phosphamide derivatives of polyamines.

Cationic nucleoside amphiphiles maymay also be used for gene delivery. Thus,Thus, low-toxiclow-toxic uridine derivativesderivatives ofof various various polyamines polyamines (36a (36a–c–,c Figure, Figure 11 )11) were were synthesized synthesized and and used used for siRNAfor siRNA delivery. delivery. Their Their TA onTAHeLa on HeLa cells cells is almost is almost equal equal to that to that of Lipofectamine of Lipofectamine 2000 2000 but wasbut was not affectednot affected by polyamine by polyamine residue residue [62]. [6 Notably,2]. Notably, replacement replacement of polyamine of polyamine residue resi- withdue with L-arginine L-arginine gave gave the same the same results, results, while whileL-lysine L-lysine decreased decreased TA [ 63TA]. [63].

O O R= H H NH H2 H2 N N R O a: N NH3 4Cl N O H 3 N O H2 O H H O H2 H2 N N b: N N NH O O O O NH3 3Cl H H2 NH HN N (9Z)-C H NH HN (9Z)-C H c: N NH (9Z)-C18H35 (9Z)-C18H35 c: NH3 2Cl 36a c 36a c Figure 11. Cationic nucleoside amphiphiles. Amphiphiles 37a–d (Figure 12), in which the polyamine was bound to the hydrophobic Amphiphiles 37a–d (Figure 12), in which the polyamine was bound to the hydropho- domain via carbamoyl or amide linkers, formed liposomes with DOPE or compound 35h bic domain via carbamoyl or amide linkers, formed liposomes with DOPE or compound (Figure 11) and were used to deliver pDNA [64]. Protonation of the imidazolium residue 35h (Figure 11) and were used to deliver pDNA [64]. Protonation of the imidazolium res- of amphiphile 35h during endosomal acidiﬁcation can induce rupture of the endosomal idue of amphiphile 35h during endosomal acidification can induce rupture of the endo- membrane and favors NA release [65,66]. Transfection of OVCAR-3, IGROV-1, and HeLa somal membrane and favors NA release [65,66]. Transfection of OVCAR-3, IGROV-1, and cells with complexes formed at different N/P ratios (4:1–12:1) showed that 37c/DOPE HeLa cells with complexes formed at different N/P ratios (4:1–12:1) showed that liposomes provided efﬁcient pDNA delivery exceeding that of the commercial transfectant 37c/DOPE liposomes provided efficient pDNA delivery exceeding that of the commercial Lipofectamine 2000. Relative TA decreased in the series 37c > 37b > 37a 37d. It should transfectant Lipofectamine 2000. Relative TA decreased in the series 37c ˃ 37b ˃ 37a ˃˃ also be noted that the use of amphiphile 36 as a helper lipid did not increase TA but did 37d. It should also be noted that the use of amphiphile 36 as a helper lipid did not increase increase37d. It should the cytotoxicity also be noted of lipoplexes. that the use of amphiphile 36 as a helper lipid did not increase TA but did increase the cytotoxicity of lipoplexes. TA butIn vivodid increasedelivery the of cytotoxicity pDNA by sterically of lipoplexes. stabilized liposomes 37c/DOPE/PEG4600- Chol (43:43:14 mol.) in a 4:1 (wt) ratio with pDNA led to a 33-fold increase in protein expression relative to unprotected DNA [67].

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Figure 12. Amphiphiles based on polyamines and cholesterol.

InIn vivovivo deliverydelivery ofof pDNApDNA byby stericallysterically stabilizedstabilized liposomesliposomes 37c/DOPE/PEG4600-/DOPE/PEG4600- FigureCholFigure (43:43:14 12.12. AmphiphilesAmphiphiles mol.) in basedbased a 4:1 on on(wt) polyaminespolyamines ratio with andand pDNA cholesterol.cholesterol. led to a 33-fold increase in protein expression relative to unprotected DNA [67].. NewNewIn vivo CAsCAs delivery 38a38a––ccc(Figure (Figure(Figure of pDNA 13 13),13),), in byinin which whichwhichsterically the thethe cationic stabilizedcationiccationic domain domaindomain liposomes was waswas linked linked37clinked/DOPE/PEG4600- to the toto the cholesterolthe choles-choles- residueterolterolChol residueresidue(43:43:14 via an viavia ether mol.) anan bond inetherether a [4:168 bondbond], (wt) formed [68],[68],ratio liposomes formedformedwith pDNA liposomesliposomes with led DOPE to withawith and33-fold DOPEDOPE were increase used andand for werewere in transfection protein usedused forex-for oftransfectiontransfectionpression AGS and relative Huh-7ofof AGSAGS to cells. unprotectedandand Huh-7 TheHuh-738a cells.cells. /DOPEDNA TheThe [67] liposomes 38a. /DOPE/DOPE more liposomesliposomes efﬁciently moremore delivered efficientlyefficiently pDNA delivereddelivered into AGSpDNANew cells, into CAs while AGS 38a thecells,–c38b (Figure while/DOPE the13), liposomes 38b in which/DOPE/DOPE providedthe liposomesliposomes cationic effective providedprovideddomain transfection was effectiveeffective linked transfection oftransfection to Huh-7 the choles- cells. ofof InHuh-7terol both residue cells. cases, In via their both an TA cases,ether exceeded theirbond TA [68], that exceeded offormed commercial that thatliposomes ofof commercialcommercial transfectants with DOPE transfectantstransfectants [69 ].and Liposomes were [69].[69]. used Lipo-Lipo- with for dimericsomessomestransfection withwith gemini-amphiphile dimericdimericof AGS andgemini-amphiphile Huh-738c cells.also outperformed The 38c 38a alsoalso/DOPE outperformedoutperformed commercial liposomes moreagentscommercialcommercial efficiently in the agentsagents transfection delivered inin thethe oftransfectiontransfectionpDNA COS-7 into and AGSofof Huh-7 COS-7COS-7 cells, cells andand while [ Huh-770Huh-7]. the cells38bcells/DOPE [70].[70]. liposomes provided effective transfection of Huh-7 cells. In both cases, their TA exceeded that of commercial transfectants [69]. Lipo- somes with dimeric gemini-amphiphile 38c also outperformed commercial agents in the transfection of COS-7 and Huh-7 cells [70].

FigureFigure 13.13. Ether-linked cationiccationic amphiphiles.amphiphiles.

CAsCAs 39a,b39a,b withwithwith differentdifferentdifferent dicationicdicationicdicationic domainsdomainsdomains (Figure(Figure(Figure 1414)14)) formedformedformed liposomes,liposomes,liposomes, whichwhichwhich facilitated the transport of siRNA into MB49 and K562 cells, while amphiphile 39a was facilitatedfacilitatedFigure 13. Ether-linked thethe transporttransport cationic ofof siRNAsiRNA amphiphiles. intointo MB49 and K562 cells, while amphiphile 39a waswas superiorsuperior inin TATA tototo amphiphileamphiphileamphiphile 39b39b [[71].[71].71]. CAs 39a,b with different dicationic domains (Figure 14) formed liposomes, which H H facilitated(9(9Z)-C)-C1717H3333 theN transport of siRNA(9(9Z )-C)-Cinto1717H MB493333 N and K562 cells,NH22 while amphiphile 39a was superior in TA to amphiphile 39b [71]. O NH O N 22 H H H H (9(9Z)-C)-C1717H3333 N (9(9Z)-C)-C1717H3333 N NH22 (9Z)-C17H33 N (9Z)-C17H33 N NH2 O O O NH O 39a39aN 2 39b 39b H H (9Z)-C H N (9Z)-C H N NH FigureFigure 17 14.14. 33Branched cationic amphiphiles.amphiphiles.17 33 2 O O ComparingComparing39a the the TA TA of of CAs CAs that that contained contained 39bvariousrious various polyaminespolyamines polyamines inin theirtheir intheir structurestructure structure (Fig-(Fig- (Figure 15) revealed that CLs composed of both phosphatidylcholine (Phospholipon 90G) ureFigure 15) 14. revealed Branched that cationic CLs composed amphiphiles. of both phosphatidylcholine (Phospholipon 90G) and andcompoundscompounds compounds 40d–40dg containingcontaining–g containing sperminespermine spermine (5:1(5:1 (5:1 mol.)mol.) mol.) couldcould could deliverdeliver deliver pDNApDNA pDNA toto to HeLaHeLa HeLa cells, cells, whileComparing other CLs showed the TA noof CAs transfection that contained [72]. Moreover, various polyamines CAs with a shorterin their chain structure length (Fig- of acylure 15) substituents revealed that exhibited CLs composed lower TA. of both phosphatidylcholine (Phospholipon 90G) and compounds 40d–g containing spermine (5:1 mol.) could deliver pDNA to HeLa cells,

Pharmaceutics 2021, 13, 920 10 of 27 Pharmaceutics 2021, 13, 920 10 of 27

Pharmaceutics 2021, 13, 920 while other CLs showed no transfection [72]. Moreover, CAs with a shorter chain length10 of 27 while other CLs showed no transfection [72]. Moreover, CAs with a shorter chain length of acyl substituents exhibited lower TA. of acyl substituents exhibited lower TA.

Figure 15. CAs based on various polyamines. Figure 15. CAs based on various polyamines. Analogs of compounds 40d–f based on spermine (compounds 41a–c, 42a–c, 43a–c) Analogs of compounds 40d–f based on spermine (compounds 41a–c,, 42a–c, 43a–c) were obtained to study the influence of the structure core on TA (Figure 16) [73]. The were obtained to study the influenceinfluence of the structurestructure corecore onon TATA (Figure(Figure 1616))[ [73].73]. TheThe efficiency of pDNA delivery mediated by liposomes based on DOPE and amphiphiles efficiencyefficiency of pDNA delivery mediated by liposomes based on DOPEDOPE andand amphiphilesamphiphiles 42b,c, and 43a (1:1, weight ratio) was higher or comparable to that of Lipofectamine 2000. 42b,c, and 43a (1:1, weight ratio) was higher or or comparable comparable to to that that of of Lipofectamine Lipofectamine 2000. 2000. Moreover, unlike the other formulations, liposomes based on 43a with a core of 2-amino- Moreover, unlike the other formulations, liposomes based on 43a with a core of 2-amino- 1,3-propanediol retained their efficiency in the presence of serum. Investigation of the ef- 1,3-propanediol retained retained their their efficiency efficiency in in th thee presence presence of of serum. serum. Investigation Investigation of the of theef- fect of hydrophobic domains on transfection revealed that myristoyl residues provided fecteffect of of hydrophobic hydrophobic domains domains on on transfection transfection revealed revealed that that myristoyl myristoyl residues provided more effective TA. more effective TA.

Figure 16. Spermine-based CAs with different cores. Figure 16. Spermine-based CAs with different cores. A library of CAs (more thanthan 12001200 compounds)compounds) waswas developeddeveloped usingusing combinatorialcombinatorial A library of CAs (more than 1200 compounds) was developed using combinatorial chemistry methodology, methodology, in in which which both both the the hydr hydrophobicophobic (the (the length length of the of thealkyl alkyl chain, chain, the chemistry methodology, in which both the hydrophobic (the length of the alkyl chain, the typethe type of linker, of linker, and and the the presence presence of of addition additionalal functional functional groups) groups) and and the the cationic (the type of linker, and the presence of additional functional groups) and the cationic (the number of amino groups, the presence of cycles,cycles, and otherother functionalfunctional groups)groups) domainsdomains number of amino groups, the presence of cycles, and other functional groups) domains varied [74]. [74]. The The results results of ofin invitro vitro experimentexperimentss on onHeLa HeLa cells cells revealed revealed the following the following rela- varied [74]. The results of in vitro experiments on HeLa cells revealed the following relationships:relationships: (1) TA (1) increased TA increased in the in presence the presence of either of either two long-chain two long-chain or several or several shorter shorter alkyl tionships: (1) TA increased in the presence of either two long-chain or several shorter alkyl substituentsalkyl substituents linked linked by an by amide an amide bond bond to th toe cationic the cationic domain domain (the (the optimal optimal length length was was 8– substituents linked by an amide bond to the cationic domain (the optimal length was 8– 128–12 carbon carbon atoms); atoms); (2) (2) high high TA TA was was achiev achieveded by by compounds compounds with with two two or more aminoamino 12 carbon atoms); (2) high TA was achieved by compounds with two or more amino groups in the cationic domain, with TETA offeringoffering the best option; (3) the presence of a groups in the cationic domain, with TETA offering the best option; (3) the presence of a secondary amino group in the cationic domain positively affectedaffected TATA (Figure(Figure 1717).). secondary amino group in the cationic domain positively affected TA (Figure 17). Based on these findings, ﬁndings, multiple CAs were selected for extended biological studies, Based on these findings, multiple CAs were selected for extended biological studies, which showed that the efﬁciencyefficiency of NANA deliverydelivery toto primaryprimary macrophagesmacrophages exceededexceeded that which showed that the efficiency of NA delivery to primary macrophages exceeded that of commercial transfectants. In In contrast, the transfection of HeLa or HepG2 cells by the of commercial transfectants. In contrast, the transfection of HeLa or HepG2 cells by the selected amphiphiles was poor. selected amphiphiles was poor.

PharmaceuticsPharmaceutics2021 2021,,13 13,, 920 920 1111 of 2727

FigureFigure 17.17. AA combinatorialcombinatorial librarylibrary ofof CAs.CAs.

AccordingAccording toto thethe resultsresults ofof inin vitrovitro tests,tests, thethe 1717 mostmost effectiveeffective compoundscompounds forforin in vivovivo siRNAsiRNA deliverydelivery (siFVII(siFVII andand siApoB,siApoB, whichwhich suppresssuppress thethe expressionexpression ofof bloodblood coagulationcoagulation factorfactor VIIVII andand apolipoprotein apolipoprotein B, B, respectively), respectively), were were selected. selected. For For this, this, liposomes liposomes containing contain- CA/PEG-lipiding CA/PEG-lipid (mPEG2000-palmitoylceramide (mPEG2000-palmitoylceramide or mPEG2000-dimyristoylglycerol)/Chol or mPEG2000-dimyristoylglyc- (42:10:48erol)/Chol mol.) (42:10:48 were mol.) formed. were These formed. CAs Th commonlyese CAs commonly contained contained various diamines various diamines or TETA. Theor TETA. most The effective most formulationeffective formulation (achieving (achie moreving than more 90% than suppression 90% suppression of target of target gene expression)gene expression) was based was based on compound on compound44 with 44 with TETA TETA (Figure (Figure 17). The17). deliveryThe delivery of siRNAs of siR- inNAs the in lungs the lungs and macrophages and macrophages of mice of mice and macaque and macaque liver cellsliver usingcells using these these liposomes liposomes also significantly suppressed the target genes [74]. also significantly suppressed the target genes [74]. Subsequently, the library of compounds was expanded by synthesizing amphiphiles Subsequently, the library of compounds was expanded by synthesizing amphiphiles 45a–d and 46a–d with various hydrophobic domains (TETA and propylenediamine were 45а–d and 46а–d with various hydrophobic domains (TETA and propylenediamine were chosen as cationic domains). Of these, the most effective were amphiphiles with a hydroxyl chosen as cationic domains). Of these, the most effective were amphiphiles with a hy- group in the hydrophobic domain, while the domain itself was bound to the polyamine droxyl group in the hydrophobic domain, while the domain itself was bound to the poly- with an ether or amide linker [75]. The hydrophobic domains based on oligoethylene glycol amine with an ether or amide linker [75]. The hydrophobic domains based on oligoeth- or octadecyl substituents led to an absence of TA. It should be noted that CAs 45c, 46a, ylene glycol or octadecyl substituents led to an absence of TA. It should be noted that CAs and 46d were more effective than amphiphile 44 in vitro; however, they were inferior to 45c, 46a, and 46d were more effective than amphiphile 44 in vitro; however, they were amphiphile 44 in siRNA delivery in vivo. inferior to amphiphile 44 in siRNA delivery in vivo. In vitro screening of amphiphilic derivatives of spermine, spermidine, putrescine, and In vitro screening of amphiphilic derivatives of spermine, spermidine, putrescine, cadaverine showed that spermine derivative 47e facilitated the more efficient delivery of siRNAand cadaverine into human showed hepatocellular that spermine carcinoma derivative Huh-7 47e facilitated cells [76]. the Furthermore, more efficient liposomes delivery 47eof siRNA/Chol/DSPC/mPEG2000-palmitoylceramide/galactosylceramide into human hepatocellular carcinoma Huh-7 cells [76]. Furthermore, delivered liposomes siRNA in47e vivo/Chol/DSPC/mPEG2000-palmitoylceramide/galactosylceramide, which led to a significant decrease in hepatitis C virus replication delivered in the hepatocytesiRNA in cellsvivo, of which mice led [76]. to a significant decrease in hepatitis C virus replication in the hepatocyte cells of mice [76]. 2.2. Cationic Amphiphiles Based on Cyclic Polyamines 2.2. CationicLipophilic Amphiphiles derivatives Based of macrocyclicon Cyclic Polyamines polyamines can be used as liposomal components.Lipophilic Such compounds derivatives are of less macrocyclic prone to self-packing, polyamines can which be improvesused as liposomal binding tocompo- NAs. Twonents. new Such cyclen-based compounds amphiphiles are less prone48a,b to self(Figure-packing, 18), whichwhich differedimproves in binding the type to of NAs. hy- drophobicTwo new cyclen-based domain (cholesterol amphiphiles or diosgenin), 48a,b (Figure were synthesized 18), which [differed77]. The in results the type of in of vitro hy- biologicaldrophobic studies domain showed (cholesterol that liposomesor diosgenin), formed were by synthesized amphiphiles [77].48a,b Theand results DOPE of in had vitro a lowbiological cytotoxicity studies but, showed because that they liposomes bound easilyformed to by serum amphiphiles proteins, 48a,b were and inferior DOPE to Lipo-had a fectaminelow cytotoxicity 2000 in deliveringbut, because NAs they tocells. bound The ea introductionsily to serum of theproteins, quaternary were ammonium inferior to groupLipofectamine into the structure2000 in delivering of the amphiphile NAs to cells. (amphiphiles The introduction49a,b) did of not the increase quaternary the TA;ammo- the liposomesnium group remained into the inferiorstructure to of Lipofectamine the amphiphile 2000 (amphiphiles [78]. However, 49a,b amphiphile) did not increase49b, which the containedTA; the liposomes diosgenin remained as a hydrophobic inferior domain,to Lipofectamine was more effective2000 [78]. but However, also more amphiphile toxic than amphiphile 49a, which contained cholesterol.

Pharmaceutics 2021, 13, 920 12 of 27

Pharmaceutics 2021, 13, 920 49b, which contained diosgenin as a hydrophobic domain, was more effective but12 also of 27 more49b, which toxic than contained amphiphile diosgenin 49a, whichas a hydropho containedbic cholesterol. domain, was more effective but also more toxic than amphiphile 49a, which contained cholesterol.

Figure 18. Cyclen-based amphiphiles. Figure 18. Cyclen-based amphiphiles. In order to increase the TA of liposomes, compounds 50a–c were synthesized (Figure 18), inIn which orderorder totothe increaseincrease cyclene the the and TA TA the of of liposomes, hydrophobicliposomes, compounds compounds domain50a were 50a–c were –arrangedc were synthesized synthesized by a peptide–nu-(Figure (Figure 18), cleosidein18), which in which spacer the cyclene the [79]. cyclene Amphiphile and the and hydrophobic the 50b hydrophobic with domain a diosgenin domain were residue arranged were arranged (in by liposomal a peptide–nucleoside by a compositionpeptide–nu- withspacercleoside DOPE) [79 spacer]. demonstrated Amphiphile [79]. Amphiphile50b thewith highest 50b a diosgeninwith efficien a diosgenincy, residue exceeding residue (in liposomalthat (in of liposomal Lipofectamine composition composition 2000. with DOPE)with InDOPE) the demonstrated structure demonstrated of the CAs highest the 51a highest–e efﬁciency,, hydrophobic efficien exceedingcy, domains exceeding that were of that Lipofectamine linked of Lipofectamine to the cyclen 2000. 2000. using the L-histidineIn the structure residue ofof (Figure CAsCAs 51a 19),–e,, as hydrophobichydrophobic well as additional domainsdomains linkers werewere linked(amide,linked toto ether, thethe cyclencyclen carbamoyl, usingusing andthe Lester)-histidine [80]. residue All lipoplexes (Figure formed1919),), as well by amphiphiles as additional 51a linkers –e, DOPE (amide, and ether, NAs carbamoyl, were less toxicand ester)ester) than [ Lipofectamine80[80].]. All All lipoplexes lipoplexes 2000, formed formedand amphiphile by by amphiphiles amphiphiles 51a with51a –51ae hexadecyl, DOPE–e, DOPE and substituents and NAs NAs were were less demon- toxic less stratedthantoxic Lipofectaminethan the Lipofectamine lowest toxicity. 2000, and2000, The amphiphile and results amphiphile of51a transfectionwith 51a hexadecyl with studies hexadecyl substituents revealed substituents that demonstrated only demon- am- phiphilesthestrated lowest the 51c toxicity.lowest–e, containing toxicity. The results aThe tocopherol ofresults transfection ofresidue transfection studies as a hydrophobic revealedstudies revealed that domain, only that amphiphiles were only more am- effective51cphiphiles–e, containing than 51c –Lipofectaminee, containing a tocopherol a2000. residuetocopherol Compound as a residue hydrophobic 51e withas a hydrophobican domain, ester linker were domain, moreexhibited effective were the high-more than estLipofectamineeffective TA. than Lipofectamine 2000. Compound 2000.51e Compoundwith an ester 51e linkerwith an exhibited ester linker the highestexhibited TA. the highest TA.

Figure 19. Cyclen-based amphiphiles with L-histidine backbone. Figure 19. Cyclen-based amphiphiles with L-histidine backbone. Cyclen derivatives containing oleoyl residues as a hydrophobic domain and amino acidsCyclen ( L-phenylalanine,-phenylalanine, derivatives LcontainingL-tyrosine,-tyrosine, LoleoylL-serine,-serine, residues or or glycine) glycine) as a hydrophobicas as a a backbone domain (Figure and 2020) )amino were developedacids (L-phenylalanine, [81]. [81]. Transfection Transfection L-tyrosine, of of HEK293 HEK293 L-serine, cells cells orshowed showedglycine) that thatas liposomes a liposomesbackbone based (Figure based on DOPE on 20) DOPE were and anddeveloped compounds [81]. Transfection52a–d with of one HEK293 fatty acid cells residue showed (1:1 that mol.) liposomes exhibited based no on TA. DOPE Among and amphiphiles with two long-chain substituents, the compounds with phenyl (53) or pheny- lalanyl (55a) spacers demonstrated the highest activity (which was nevertheless lower than that of Lipofectamine 2000). Moreover, analogs with saturated hydrocarbon residues of

Pharmaceutics 2021, 13, 920 13 of 27

compounds 52a–d with one fatty acid residue (1:1 mol.) exhibited no TA. Among am- Pharmaceutics 2021, 13, 920 phiphilescompounds with 52a two–d withlong-chain one fatty substituents, acid residue the (1:1 compounds mol.) exhibited with phenyl no TA. ( 53Among) or phenyl- 13am- of 27 alanylphiphiles (55a with) spacers two long-chaindemonstrated substituents, the highest the activity compounds (which with was phenyl nevertheless (53) or lower phenyl- than thatalanyl of (Lipofectamine55a) spacers demonstrated 2000). Moreover, the highest analogs activity with (which saturated was neverthelesshydrocarbon lower residues than of variousthat of Lipofectaminelengths were obtained2000). Moreover, based on analogs amphiphile with saturated 53 (structures hydrocarbon not shown). residues Among of them,variousvarious an lengths amphiphile were withobtained tetradecyl based based onsubstitu on amphiphile amphiphileents was 5353 the(structures(structures most effective, not not shown). shown). comparable Among Among to them, an amphiphile with tetradecyl substituents was the most effective, comparable to Lipofectaminethem, an amphiphile 2000. with tetradecyl substituents was the most effective, comparable to LipofectamineLipofectamine 2000.

Figure 20. Cyclen-based amphiphiles with different amino acid backbones. FigureFigure 20. Cyclen-based amphiphiles amphiphiles with with different different amino amino acid acid backbones. backbones.

2.3.2.3.2.3. Cationic Cationic Amphiphiles Amphiphiles BasedBased Based onon on AminoAmino Amino Acids Acids Acids AminoAmino acids areare oftenoften employed employedemployed asas as thethe the hydrophilichydrophilic hydrophilic domainsdomains domains ofof of CAs,CAs, CAs, as as theyas they they are are are natural nat- nat- uralcompoundsural compoundscompounds and convenientand convenient building buildingbuilding blocks blocks blocks for creating for for creating creating a positive a apositive positive charge charge incharge molecules. in inmole- mole- To cules.identifycules. ToTo new identify compounds new compoundscompounds for CL formulation, forfor CLCL formulation, formulation, a number a ofanumber number amphiphiles of of amphiphiles amphiphiles56–58 (Figure 56 56–58– 2158 ) (Figurebased(Figure on 21)21)L-lysine, based onL-histidine, L-lysine,-lysine, LL and-histidine,-histidine,L-arginine and and L wasL-arginine-arginine synthesized was was synthesi synthesi and furtherzedzed and and used further further with used DOPE used with(1:1with mol.) DOPEDOPE for (1:1 liposomal mol.) for preparations. liposomalliposomal preparations.preparations. Their efﬁciency Their Their ofefficiency efficiency NA delivery of of NA NA in delivery thedelivery presence in inthe the of presenceserumpresence was of studiedserum was [82 ].studied [82].[82].

FigureFigure 21.21. CAsCAs based on amino acidsacids andand chol chol cholesterolesterolesterol containing containing no no spacers. spacers.

TATA is is known to decrease decrease inin thethe presence presence of of serum serum due due to to the the the interaction interaction interaction of of negatively negatively negatively chargedchargedcharged proteins proteins with with CAs,CAs, whichwhich inhibitsinhibits NA NA delivery. delivery. The The average average size size of oflipoplexes lipoplexes formedformedformed by by amphiphilesamphiphiles 565656–––585858 increasedincreasedincreased in in in the the the presence presence presence of of of serum serum from from 180 180 nm nm to to 828 828 nm nm forforfor amphiphile amphiphile 5656,, upup tototo 171017101710 nm nmnm for forfor amphiphile amphiphileamphiphile 5757 57,, and,and and upup up toto to 23452345 2345 nm nm nm for for for amphiphile amphiphile amphiphile58 . 58. Amphiphile 56 was able to transfect eukaryotic cells at serum concentrations up to 58Amphiphile. Amphiphile56 was56 was able able to transfect to transfect eukaryotic eukaryotic cells cells at serum at serum concentrations concentrations up to up 40%. to 40%. The TA of lipoplexes containing amphiphiles 56–58 was 2.8–3.5-fold higher than that 40%.The TA The of TA lipoplexes of lipoplexes containing containing amphiphiles amphiphiles56–58 56was–58 2.8–3.5-fold was 2.8–3.5-fold higher higher than that than of that the of the commercial transfectant DOTAP (Roche Life Technologies, Switzerland), while am- ofcommercial the commercial transfectant transfectant DOTAP DOTAP (Roche (Roche Life Technologies, Life Technologies, Switzerland), Switzerland), while amphiphile while amphiphile 57, with an L-histidine residue, exhibited the lowest activity. phiphile57, with an57, L-histidinewith an L-histidine residue, exhibitedresidue, exhibited the lowest the activity. lowest activity. In the structure of of CAs CAs 59a59a,b,b andand 60a60a,b,b (Figure(Figure 22), 22), cholesterol cholesterol was was bound bound to toL-lysineL-lysine In the structure of CAs 59a,b and 60a,b (Figure 22), cholesterol was bound to L-lysine oror L-histidine-histidine by by various various linker linker groups groups [83]. [83 Th].e Thebest besttransfection transfection of COS-7 of COS-7 cells was cells pro- was or L-histidine by various linker groups [83]. The best transfection of COS-7 cells was pro- providedvided by byamphiphiles amphiphiles 59b59b andand 60b60b withwith L-lysine,L-lysine, which, which, however, however, demonstrated demonstrated greater greater vided by amphiphiles 59b and 60b with L-lysine, which, however, demonstrated greater cytotoxicitycytotoxicity than amphiphiles amphiphiles 59a59a andand 60a60a containingcontaining LL-histidine.-histidine. The The authors authors proposed proposed cytotoxicity than amphiphiles 59a and 60a containing L-histidine. The authors proposed that this greater toxicity was induced by the damaging electrostatic interaction of L-lysine amino groups with the negatively charged plasma membrane. Replacing the ester linker with an amide one (amphiphiles 61a,b) reduced the toxicity of the compounds. The L-lysine

derivative 61b was still more effective than amphiphile 61a based on L-histidine [84]. PharmaceuticsPharmaceutics 20212021,, 1313,, 920920 1414 ofof 2727

thatthat thisthis greatergreater toxicitytoxicity waswas inducedinduced byby thethe damagingdamaging electrostaticelectrostatic interactioninteraction ofof LL-lysine-lysine amino groups with the negatively charged plasma membrane. Replacing the ester linker Pharmaceutics 2021, 13, 920 amino groups with the negatively charged plasma membrane. Replacing the ester 14linker of 27 withwith anan amideamide oneone (amphiphiles(amphiphiles 61a61a,,bb)) reducedreduced thethe toxicitytoxicity ofof thethe compounds.compounds. TheThe LL-ly--ly- sinesine derivativederivative 61b61b waswas stillstill moremore effectiveeffective thanthan amphiphileamphiphile 61a61a basedbased onon LL-histidine-histidine [84].[84].

FigureFigure 22.22. CAsCAs based based onon aminoamino acidsacids andand cholesterolcholesterol withwith variousvarious linkers.linkers.

AmphiphilesAmphiphiles 62a62a––dd areare basedbased onon LL-arginine-arginine andand certaincertain sterolssterols linkedlinkedlinked bybyby amideamideamide andandand esterester bondsbonds (Figure(Figure 2323)23))[ [85].[85].85]. Cholesterol-based Cholesterol-based compound 6262a62аа exhibitedexhibited the the highest highest TA, TA, formingforming lipoplexeslipoplexes withwith aa particleparticleparticle sizesizesize ofofof approximatelyaapproximatelypproximately 100100100 nm.nm.nm. TheThe sizesizesize ofofof lipoplexeslipoplexeslipoplexes formedformed byby amphiphilesamphiphiles 62c62c,,,dd waswas 200–300200–300 nm,nm, andand theythey poorlypoorly penetratedpenetrated cells. cells.cells. ItIt shouldshould bebe notednoted thatthat nonenone ofof thethe amphiphilesamphiphiles 62a62a––dd waswas ableable tototo outperformoutperformoutperform LipofectamineLipofectamineLipofectamine 200020002000 inin thethe presencepresence ofof serum.serum.

FigureFigure 23.23. CAsCAs based based on on LL-arginine-arginine and and various various sterols. sterols.

CAsCAs 63a63a––cc andand 63e63e––jj (Figure(Figure 2424)24)) werewerewere synthesizedsynthesizedsynthesized basedbasedbased ononon LLL-arginine,-arginine,-arginine, LLL-lysine,-lysine,-lysine, andandand LL-histidine-histidine [86]. [[86].86]. The The efficiency efficiencyefficiency of of cell cell transfection transfection decreaseddecreased withwithwith anan increaseincrease ininin thethethe lengthlengthlength ofof thethe alkylalkyl chains,chains, whilewhile thethe maximummaximum efficiencyefficiencyefficiency waswas providedprovided byby amphiphilesamphiphilesamphiphiles 63a63a andandand 63e63e withwith tetradecyl tetradecyl substituents.substituents. The The type type of of th thethee cationic cationic group group insignificantly insignificantlyinsignificantly affected affected TA TA inin thethe absenceabsence ofof serum.serum. However, However, amphiphilesamphiphilesamphiphiles 63a63a––cc basedbased onon L-arginineL-arginine demonstrateddemonstrateddemonstrated thethe mostmost efficientefficientefficient NANANA deliverydeliverydelivery ininin thethethe presencepresencepresence ofofof serum.serum.serum. AnAn increaseincreaseincrease in inin the thethe number numbernumber of ofof positively positivelypositively charged chargedcharged groups groupsgroups in amphiphiles inin amphiphilesamphiphiles63k, l 63k63kprovided,,ll pro-pro- anvidedvided increase anan increaseincrease in the efficiencyinin thethe efficiencyefficiency of pDNA ofof pDNApDNA delivery deliverydelivery to HepG2 toto HepG2HepG2 cells. Moreover, cells.cells. Moreover,Moreover, the resulting thethe result-result- TA wasinging TATA superior waswas superiorsuperior to that of toto both thatthat Lipofectamine ofof bothboth LipofectamineLipofectamine 2000 and 2000 polyethyleneimine2000 andand polyethyleneiminepolyethyleneimine [87]. For HEK293T [87].[87]. ForFor cells,HEK293THEK293T amphiphile cells,cells, amphiphileamphiphile63k based 63k63k on basedLbased-arginine onon LL-arginine-arginine was comparable waswas comparablecomparable to commercial toto commercialcommercial transfectants, trans-trans- whilefectants,fectants, the whilewhileL-lysine thethe derivativeLL-lysine-lysine derivativederivative63l demonstrated 63l63l demonstrateddemonstrated inferior inferior efficiency.inferior efefficiency.ficiency. The results TheThe of resultsresults pDNA ofof deliverypDNApDNA deliverydeliveryin vivo ininshowed vivovivo showedshowed that CA thatthat63l CACAwas 63l63l 2.5-fold waswas 2.5-fold2.5-fold more effectivemoremore effectiveeffective thanpolyethyleneimine, thanthan polyethylene-polyethylene- whileimine,imine,63k whilewhilewas 63k63k almost waswas almost 10-foldalmost 10-fold10-fold more effective. moremore effective.effective. Low-toxicity lysine-containing CAs 63d, 64a–h (Figure 24) were synthesized to study the effect of spacer length and the type of counterion on TA [88]. CLs formed by DOPE and trifluoroacetates 64g and 64h delivered NAs more efficiently than other CAs, while CLs containing uncharged amphiphiles 64a,b demonstrated the lowest activity. An increase in the spacer length in CAs from 0 to 7 methylene units increased TA in both the presence and absence of serum, and all amphiphiles studied were more effective than Lipofectamine 2000 under both conditions. Pharmaceutics 2021, 13, 920 15 of 27

No R1 R2 R3 n 63a С14Н29 - 63b С16Н33 -

63c С18Н37 -

63d С14Н29 -

63e С14Н29 - 63f С16Н33 -

63g С18Н37 - 63h С14Н29 - 63i С16Н33 -

63j С18Н37 -

63k (9Z)-C17H33 -

63l (9Z)-C17H33 -

64a С14Н29 3 64b С14Н29 5

64c С14Н29 7 64d С14Н29 3 64e С14Н29 5

64f С14Н29 7 64g С14Н29 3 64h С14Н29 5

64i С14Н29 3

64j С14Н29 3

64k С16Н33 3 64l С16Н33 5 64m С16Н33 7

64n С16Н33 11 65 С16Н33 -

Figure 24. CAs based on L-arginine, L-lysine, and L-histidine.

Pharmaceutics 2021,, 1313,, 920920 16 of 27

and trifluoroacetates 64g andand 64h delivereddelivered NAsNAs moremore efficientlyefficiently thanthan otherother CAs,CAs, whilewhile CLs containing uncharged amphiphiles 64a,,b demonstrateddemonstrated thethe lowestlowest activity.activity. AnAn in-in- Pharmaceutics 2021, 13, 920 16 of 27 crease in the spacer length in CAs from 0 to 7 methylene units increased TA in both the presence and absence of serum, and all amphiphiles studied were more effective than Lipofectamine 2000 under both conditions. A similar effect of spacer length on TA was observed in the amphiphile series 63f, A similar effect of spacer length on TA was observed in the amphiphile series 63f, 64k–m, and 65 based on L-lysine (Figure 24) [89]. Lipoplexes with amphiphile 63f, the 64k–m, andand 6565based based on onL-lysine L-lysine (Figure (Figure 24)[ 24)89 ].[89]. Lipoplexes Lipoplexes with with amphiphile amphiphile63f, the 63f struc-, the structureture of which of which lacks alacks spacer, a spacer, were unable were unable to deliver to NA,deliver but NA, as the but spacer as the length spacer increased length increasedincreasedfrom 3 to 7fromfrom methylene 33 toto 77 methylenemethylene units, TA increased. units,units, TATA increased.increased. Notably, amphiphiles Notably,Notably, amphiphilesamphiphiles64k–m with 64k a hydropho-–m withwith aa hydrophobicbic oligomethylene oligomethylene spacer functioned spacer functioned as more active asas moremore transfectants activeactive transfectantstransfectants than amphiphiles thanthan am-am-65 phiphilescontaining 65 a containingcontaining hydrophilic aa oxyethylenehydrophilichydrophilic oxyethylene spacer.oxyethylene spacer.spacer. Amphiphiles 64d64d,,,gg andandand 64i 64i,,j,jjbased basedbased on ononL L-lysine-lysine-lysine and andandL -arginineL-arginine-arginine (Figure (Figure(Figure 24 24)24)) were werewere studied stud-stud- iediedto evaluate toto evaluateevaluate TA in TATA PC-12, inin PC-12,PC-12, HeLa, HeLa,HeLa, and neuronal andand neurneur cellsonal [90 cells]. Lipoplexes [90]. Lipopl containingexes containing amphiphiles am- phiphiles64i,j were 64i more,,jj werewere effective moremorethan effectiveeffective both thanthan lipoplexes bothboth lipoplexeslipoplexes containing containingcontaining amphiphiles amphiphilesamphiphiles64d,g and 64d the,,g com- andand thethemercial commercialcommercial transfectant transfectatransfecta Lipofectaminent Lipofectamine 2000. 2000. Cholesterol-containing CAs CAs 66a66a,,b, b (Figure(Figure(Figure 25)25) 25 werewere) were recentlyrecently recently developeddeveloped developed basedbased based onon L-ly--ly- on sineL-lysine and anddiamines diamines [91]. When [91]. When transfecting transfecting HEK293, HEK293, HeLa, PC-3, HeLa, and PC-3, HC-04 and cells, HC-04 the cells, effi- cacythe efﬁcacy of liposomes of liposomes 66b/DOPE 66b/DOPE (1:1,:1, mol.)mol.) (1:1, waswas mol.) comparablecomparable was comparable toto thatthat to ofof that LipofectamineLipofectamine of Lipofectamine 2000,2000, even2000, in even the inpresence the presence of serum. of serum. Moreover, Moreover, the toxicitytoxicity the toxicity ofof 66b/DOPE66b/DOPE of 66b/DOPE waswas significantlysignificantly was signiﬁcantly lowerlower thanthanlower thatthat than ofof that LipofectamineLipofectamine of Lipofectamine 2000.2000. 2000.

CF3COO O 3 O H3N H3N N ()n N O N ()n N O H H 66a, n=0 NH2 66b, n=1 2 66b, n=1 L Figure 25. CAsCAs based based on on L-lysine-lysine-lysine andand and diamines.diamines. diamines.

Polycationic amphiphilesamphiphiles67a 67а,b,,b(Figure (Figure(Figure 26 26)26)) consisting consistingconsisting of of theof thethe dipeptides dipeptidesdipeptides glycylhistidine glycylhisti-glycylhisti- dineand glycyllysineand glycyllysine as well as well as a as tocopherol a tocopherol residue residueresidue were werewere synthesized synthesizedsynthesized [92 ].[92].[92]. While WhileWhile the thethe TA TATA of of67b 67b/DOPE/DOPE/DOPE liposomes liposomesliposomes containing containingcontaining a aa lysine lysinelysine residue residueresidue was waswas higher higherhigher on onon HEK293 HEK293HEK293 andandand HeLaHeLa cells thanthan that that of of 67a/DOPE/DOPE liposomesliposomes liposomes containingcontaining a a histidine histidine residue, thethe 67b67b/DOPE/DOPE/DOPE liposomesliposomes did not achieve the results ofof LipofectamineLipofectamine 2000.2000. However, the complexes were more resistantresistant to serum activity than was the commercial transfectant.transfectant.

Figure 26. CAsFigure based on 26. dipeptides.CAs based on dipeptides. Polycationic amphiphiles 68a–d and 69d (Figure 27) were synthesized based on Polycationic amphiphiles 68a–d andand 69d (Figure(Figure 27)27) werewere synthesizedsynthesized basedbased onon L -or--or- L-ornithine [93,94]. Liposomes CA/DOPC and complexes with pDNA were formed using nithine [93,94]. Liposomes CA/DOPC and complexes with pDNA were formed using dif- different component ratios. The resulting efﬁciency of cell transfection, which was higher ferentferent componentcomponent ratios.ratios. TheThe resultingresulting efficiefficiency of cell transfection, which was higher than for the commercial transfectant DOTAP (Avanti Polar Lipids, Alabaster, AL, USA), thanthan forfor thethe commercialcommercial transfectanttransfectant DOTAPDOTAP (Avanti(Avanti PolarPolar Lipids,Lipids, Alabaster,Alabaster, AL,AL, USA),USA), depended on the ratio of lipids used in the complex. Amphiphile 68d with three aminoethyl depended on the ratio of lipids used in thethe complex.complex. AmphiphileAmphiphile 68d68d withwith threethree ami-ami- substituents exhibited the highest activity and was also used to deliver siRNA, which led noethyl substituents exhibited the highest activity and was also used to deliver siRNA, whichto the speciﬁcled to the suppression specific suppre of thession target of genethe target [95]. gene [95]. whichAmino led to acidsthe specific are used suppre to developssion of not the only target classical gene [95]. head-to-tail amphiphiles but also symmetric gemini-amphiphiles, as seen in the L -histidine derivatives 70a–d (Figure 28), which contain two alkyl substituents 10 to 16 carbon atoms in length [96,97]. CLs were formed with derivatives obtained and DOPE. With the exception of 70a with decyl residues, all CLs bound pDNA well. However, their TA was comparable to or lower than that of

Lipofectamine 2000 [96]. Pharmaceutics 2021, 13, 920 17 of 27 Pharmaceutics 2021, 13, 920 17 of 27

1 R =(9Z)-C18H35 R1 =(9Z)-C H R1 O 18 35 R1 O O H O Pharmaceutics 20211 , 13, 920 HN H2N NH3 17 of 27 PharmaceuticsR 2021O, 13, 920 N R2 NH3 H N NH 17 of 27 1 N 2 2 H32 R O NH R 2 NH3 2 a: R = NH3 c: R = H2N NH3 O H 2 2 68a d a: R = NH c: R = N NH3 O 68a d 3 2Cl or 2CF3COO 4Cl or 4CF3COO O O 1 2Cl or 2CF3COO 4Cl or 4CF3COO R =(9Z)-C18H35 NH 1 O O 3 R1 O O NH3 RR1 OO N O N R2 NH N O2 N R2 H N NH H N NH 3 HH 2 H 2 3 2 3 1 H H 2 H2N NH3 2 H2N R1 O N b: R = NH d: R = H2N NHNH3 NH R 1 2 2 NH3 3 2 3 R OO 69d N R b: R = NH3 d: R = NHH2 NH3 69d H 2 2 O a: R = 3Cl or 3CF COONH3 c: R = 5Cl or 5CFN COO NH3 68a d 3Cl or 3CF COO3 3 3 5Cl or 5CF3COO Figure 27. CAs2Cl based or 2CFon L-ornithine.3COO 4Cl or 4CF3COO O OFigure 27. CAs based on L-ornithine. NH 1 3 R O O 2 N Amino2 N acidsR are used to develop not only classical head-to-tail amphiphilesNH3 but also H AminoH acids are used toH 2developN notNH only3 classical Hhead-to-tail2N amphiphiles but also R1 O symmetric gemini-amphiphiles,gemini-amphiphiles,b: R2 = as as seen seenNH in in the the d:L L-histidine -histidineR2 = derivatives derivativesNH 70a 70a–d– (FigureNHd (Figure 28), 28), 69d 3 3 which containcontain twotwo alkylalkyl substituents substituents 10 10 to to 16 16 carbon carbon atoms atoms in inlength length [96,97]. [96,97]. CLs CLs were were 3Cl or 3CF COO 5Cl or 5CF COO formed withwith derivativesderivatives obtained obtained and and3 DOPE. DOPE. With With the the exception exception of of70a3 70a with with decyl decyl resi- resi- dues, allall CLsCLs boundFigure bound 27.pDNA pDNA CAs well. based well. However, onHowever, L-ornithine. their their TA TA was was comparable comparable to toor orlower lower than than that that of Lipofectamine 2000 2000 [96]. [96]. Amino acids are used to develop not only classical head-to-tail amphiphiles but also symmetric gemini-amphiphiles, as seen in the L -histidine derivatives 70a–d (Figure 28), which contain two alkyl substituents 10 to 16 carbon atoms in length [96,97]. CLs were formed with derivatives obtained and DOPE. With the exception of 70a with decyl residues, all CLs bound pDNA well. However, their TA was comparable to or lower than that of Lipofectamine 2000 [96].

Figure 28. 28. Gemini-amphiphilesGemini-amphiphiles based based on on L-histidine. L-histidine.

2.4. Disulfide DisulfideDisulfide Cationic Cationic Amphiphiles Amphiphiles SeveralSeveral special special cell cell proteins proteins and and peptides peptidespeptides (for (for(for example, example,example, glutathione glutathioneglutathione and and reductases) reductases)reductases) can reduce reduce the thethe disulfide disulfidedisulfide bond bondbond [98]. [[98].98]. Disruption Disruption of of the the disulfide disulfidedisulfide bond bond in inthe the CA CACA molecule, molecule,molecule, inin turn, turn, can cancan increase increaseincrease the thethe degree degreedegree of ofof NA NANA release releaserelease from fromfrom the thethe lipoplex lipoplexlipoplex in ininthe thethe cell cellcell and andand thereby therebythereby increase TA. Figureincreaseincrease 28. TA.TA. Gemini-amphiphiles based on L-histidine. DisulfideDisulfide amphiphile amphiphile 7171, ,based based on on LL-lysine-lysine and and L-arginineL-arginine (Figure (Figure 29), 29 formed), formed CLs CLs Disulfide amphiphile 71, based on L-lysine and L-arginine (Figure 29), formed CLs capable of transfecting HeLa and B16 cells in the absence or presence of serum, and its 2.4.capable Disulfide ofof transfectingtransfecting Cationic Amphiphiles HeLa HeLa and and B16 B16 cells cells in in the the absence absence or or presence presence of ofserum, serum, and and its its efficiencyefficiency exceeded that that of of its its analog analog lacking lacking a adisulfide disulfide bond bond [99]. [99 ].InIn vivo vivo studiesstudies on on the the efficiencySeveral exceeded special cellthat proteinsof its analog and lackingpeptides a (fordisulfide example, bond glutathione [99]. In vivo and studies reductases) on the delivery of the the luciferase luciferase gene gene to to xenograft xenograft mice mice showed showed a high a high expression expression of ofthe the reporter reporter candelivery reduce of the luciferasedisulfide bond gene to[98]. xenograft Disruption mice of showed the disulfide a high bondexpression in the ofCA the molecule, reporter protein in isolated tumors tumors in in the the case case of of CA CA 7171, ,which which exceeded exceeded that that of of the the amphiphile amphiphile protein in isolated tumors in the case of CA 71, which exceeded that of the amphiphile withoutin turn, cana disulfide disulfide increase bond bond the (structure (structuredegree of not notNA shown) shown)release or orfrom polyethyleneimine. polyethyleneimine. the lipoplex in the cell and thereby increasewithout TA.a disulfide bond (structure not shown) or polyethyleneimine. Disulfide amphiphile 71, based on L-lysine and L-arginine (Figure 29), formed CLs capable of transfecting HeLa and B16 cells in the absence or presence of serum, and its efficiency exceeded that of its analog lacking a disulfide bond [99]. In vivo studies on the delivery of the luciferase gene to xenograft mice showed a high expression of the reporter protein in isolated tumors in the case of CA 71, which exceeded that of the amphiphile

withoutFigure 29. a Disulfide Disulfidedisulfide amphiphiles amphiphiles bond (structure based based on not on L-lysineL shown)-lysine and and or L-arginine. Lpolyethyleneimine.-arginine. Figure 29. Disulfide amphiphiles based on L-lysine and L-arginine. During the the oxidation oxidation of of thiol thiol groups groups to to di disulfidesulfide groups, groups, lipophilic lipophilic thio thio compounds compounds 72a–dDuring (Figure(Figure the 3030) )oxidation basedbased on on ofL -ornithineL thiol-ornithine groups or or spermine tospermine disulfide compacted compacted groups, pDNA lipophilic pDNA molecules molecules thio compounds into into small micellarsmall72a–d micellar (Figure complexes complexes 30) based [100]. [100]. Theiron L -ornithineTheir TA in TA 3T3 in cellsor 3T3 spermine increased cells increased compacted with increasingwith increasingpDNA alkyl molecules substituents.alkyl sub- into Spermine-basedstituents.small micellar Spermine-based complexes CA 72d was CA[100]. less72d Their effectivewas TAless ineffective than 3T3 its cells ornithine-basedthan increased its ornithine-based with analog increasing 72banalog. alkyl 72b sub-. stituents.Disulfide Spermine-based amphiphiles CA73a 72d,b (Figure was less 31 )effective containing than an itsL -histidineornithine-based or L-lysine analog residue 72b. wereFigure obtained 29. Disulfide based amphiphiles on tocopherol based on [101 L-lysine]. Liposomes and L-arginine. CA/DOPC (1:1 mol) formed complexes with pDNA at various N/P ratios (from 1 to 8) with sizes ranging from 150 to 190 nm.During The the TA oxidation of liposomes of thiol73b wasgroups lower to di thansulfide that groups, of liposomes lipophilic based thio on compoundsL-histidine 72aderivative–d (Figure73a and30) based the commercial on L-ornithine transfectant or spermine Lipofectamine compacted 2000. pDNA molecules into small micellar complexes [100]. Their TA in 3T3 cells increased with increasing alkyl substituents. Spermine-based CA 72d was less effective than its ornithine-based analog 72b.

Pharmaceutics 2021, 13, 920 18 of 27

O H H N N R1 R2 PharmaceuticsPharmaceutics 20212021,, 1313,, 920920 1818 of of 2727 O SH 72a d 2CF3COO 2CF COO NH NH 2CF3COO NH3 O NH3 H H NH NH3 a: R1 =,R3 2 N=C NH b: 1 3 2 a: R =,RR1 =C12H25 b:RR2 =,R=C14H29 O SH 2CF3COO NH3 72a d 3 H2N NH3 4CF3COO 2CF COO 3NH3 2CF3COO 1 NH3 2 1 NH3 2 c: R =,R=C16H33 d: R =,R=C14H29 H2 NH3 NH3 a: R1 =,R2 =C H 1 N NH2 3 12 25 b: R =,R=C3 14H29 Figure 30. Disulfide amphiphiles based on L-ornithine or spermine. 2CF COO NH 3 3 H2N NH3 4CF3COO Disulfide amphiphilesNH 73a,b (Figure 31) containing an L-histidine or L-lysine residue 1 3 2 1 2 c: R =,R=C16H33 d: R =,R=C14H29 were obtained based on tocopherol [101]. LiposomesH CA/DOPC2 (1:1 mol) formed complexes with pDNA at various N/P ratios (from 1 to 8)N with sizesNH ranging3 from 150 to 190 L nm.Figure The 30. TA DisulﬁdeDisulfide of liposomes amphiphiles 73b basedwas lower on L-ornithine than that or of spermine. liposomes based on L-histidine derivative 73a and the commercial transfectant Lipofectamine 2000. Disulfide amphiphiles 73a,b (Figure 31) containing an L-histidine or L-lysine residue were obtained based on tocopherol [101]. Liposomes CA/DOPC (1:1 mol) formed complexes with pDNA at various N/P ratios (from 1 to 8) with sizes ranging from 150 to 190 nm. The TA of liposomes 73b was lower than that of liposomes based on L-histidine derivative 73a and the commercial transfectant Lipofectamine 2000.

Figure 31. DisulfideDisulﬁde amphiphiles based on tocopherol.

The introduction introduction of of a adisulfide disulfide bond bond into into the the amphiphile amphiphile structure structure also alsomakes makes it pos- it siblepossible to significantly to significantly reduce reduce CAs CAs toxicity toxicity while while maintaining maintaining a acomparable comparable level level of of TA. TA. CA/DOPC liposomes, liposomes, based based on on biodegradable biodegradable CAs CAs 74a74a––dd (Figure(Figure 32)32) with a disulfidedisulfide bond,Figure differed 31. Disulfidein the number amphiphiles of amino based groups on tocopherol. in the polyaminepolyamine matrix had significantlysignificantly lower toxicity (except for amphiphile 74a)) than their analogs 68a–d,, which did not contain a disulfide disulfideThe introduction bond bond [102]. [102 ].of Moreover, Moreover,a disulfide compounds compoundsbond into the 74c74c amphiphile andand 74d74d demonstrated structuredemonstrated also comparable makes comparable it pos- or superiororsible superior to significantlyTA TAto amphiphile to amphiphile reduce 68d CAs68d. Thus, .toxicity Thus, an increase anwhile increase maintaining in the in number the number a comparableof amino of amino groups level groups inof CAsTA. in increasedCAsCA/DOPC increased TA, liposomes, while TA, while the based introduction the introductionon biodegradable of a disulfide of a disulfide CAs group 74a group– ddecreased (Figure decreased 32) the withtoxicity the a toxicity disulfide of lipo- of plexes,lipoplexes,bond, differed allowing allowing in for the the fornumber use the useof ofhigher of amino higher CA groups CAdosages. dosages. in the polyamine matrix had significantly lower toxicity (except for amphiphile 74a) than their analogs 68a–d, which did not contain a disulfide bond [102]. Moreover, compounds 74c and 74d demonstrated comparable or superior TA to amphiphile 68d. Thus, an increase in the number of amino groups in CAs increased TA, while the introduction of a disulfide group decreased the toxicity of lipoplexes, allowing for the use of higher CA dosages.

FigureFigure 32. 32. DisulfideDisulﬁde amphiphiles amphiphiles based based on on different different polyamines. polyamines.

Biological testing on COS-7 cells of disulfide disulﬁde amphiphiles 75a,,b (Figure 3333),), basedbased onon polyamines and amino acids [103], [103], revealed low toxicity and rather high TA comparablecomparable to monocationic analogs 75c,d. Moreover, only amphiphile 75a retained its activity in the presence of serum. Figure 32. Disulfide amphiphiles based on different polyamines.

Biological testing on COS-7 cells of disulfide amphiphiles 75a,b (Figure 33), based on polyamines and amino acids [103], revealed low toxicity and rather high TA comparable

Pharmaceutics 2021, 13, 920 19 of 27 Pharmaceutics 2021, 13, 920 19 of 27

Pharmaceutics 2021, 13, 920 19 of 27 to monocationic analogs 75c,d. Moreover, only amphiphile 75a retained its activity in the to monocationic analogs 75c,d. Moreover, only amphiphile 75a retained its activity in the presence of serum.serum.

FigureFigure 33.33. DisulfideDisulﬁde amphiphiles amphiphiles based basedbased on onon polyamines, polyamines,polyamines, amino aminoamino acids, acids,acids, and andand cholesterol. cholesterol.cholesterol.

SynthesizedSynthesized gemini-amphiphilesgemini-amphiphilesgemini-amphiphiles76a 76a76a–c––(Figurecc (Figure(Figure 34 ), 34), which34), whichwhich contained containedcontained disulfide disulfide disulfide groups betweengroups between the cationic thethe cationiccationic and hydrophobic and and hydrophobic hydrophobic domains, do domains,mains, differed differed differed in the in in the spacer the spacer spacer connecting connecting connecting the quaternarythe quaternary ammonium ammoniumammonium groups groupsgroups [104 [104]. [104].]. Liposomes Liposomes Liposomes76a 76a 76a/DOPE/DOPE/DOPE and and 76c 76c/DOPE/DOPE efficiently efficientlyefficiently transfectedtransfected HeLa HeLa andand HT1080HT1080 HT1080 cells.cells. cells. For For For PC3AR PC3AR PC3AR cells, cells, cells, liposomes liposomes liposomes 76b 76b76b/DOPE/DOPE/DOPE proved proved proved to to be tobe bethethe most most efficientefficient efficient andand wereandwere werealso also capable capable also capable of of delivering delivering of delivering NAs NAs to to NAsdifficult-to-transfect difficult-to-transfect to difficult-to-transfect HaCaT HaCaT HaCaTcells. cells.

Figure 34. Disulfide gemini-amphiphiles with different spacers. FigureFigure 34.34. DisulﬁdeDisulfide gemini-amphiphilesgemini-amphiphiles withwith differentdifferent spacers.spacers.

DisulfideDisulfide polycationic polycationic amphiphiles amphiphiles 77a 77a,b,,b (Figure (Figure(Figure 35) 3535) )were werewere based basedbased on onon spermine sperminespermine and andand cholesterol.cholesterol. The The disulfide disulfidedisulfide group group was was located located ei eithereitherther in in the thethe spacer spacerspacer structure structurestructure or oror as asas a aalinker linkerlinker connectingconnecting thethe cationic cationic and and hydrophobic hydrophobichydrophobic doma domainsdomainsins [105,106]. [[105,106].105,106]. CLs CLsCLs were werewere formed formedformed with withwith these thesethese amphiphiles and DOPE. The position of the disulfide group in the CA molecule affected amphiphilesamphiphiles and DOPE.DOPE. TheThe positionposition ofof thethe disulfidedisulfide groupgroup inin thethe CACA moleculemolecule affectedaffected both the sensitivity of the obtained liposomes to the action of reducing agents and their bothboth thethe sensitivitysensitivity of of the the obtained obtained liposomes liposomes to to the the action action of reducingof reducing agents agents and and their their TA. TA. Thus, introducing a disulfide group into the spacer structure (77a) led to a higher Thus,TA. Thus, introducing introducing a disulfide a disulfide group intogroup the into spacer the structurespacer structure (77a) led ( to77a a) higher led to liposomea higher liposome sensitivity and more efficient siRNA delivery than that achieved by CA 10c (Fig- sensitivityliposome sensitivity and more and efficient more siRNAefficient delivery siRNA delivery than that than achieved that achieved by CA by10c CA(Figure 10c (Fig-6), ure 6), which was not sensitive to reduction. CA 77b with a disulfide group as a linker whichure 6), waswhich not was sensitive not sensitive to reduction. to reduction. CA 77b withCA 77b a disulfide with a disulfide group as group a linker as was a linker less was less sensitive to reduction and delivered pDNA more efficiently than did its analogs sensitivewas less sensitive to reduction to reduction and delivered and delivered pDNA more pDNA efficiently more efficiently than did itsthan analogs did its [107 analogs]. [107]. [107].A library of disulfide CAs 78–89 (Figure 36) with various cationic domains was created. The first series of compounds, 79a–84a, contained a hydrophobic hexadecyl chain, including sulfur atoms [108]. CLs composed of disulfide CAs, cholesterol, DOPE and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:4:1:1, weight ratio) were used to deliver ribonucleoprotein complexes to GFP-HEK cells. Compounds 80a, 81a, 83a, 84a had the highest efficacy, comparable to or exceeding that of Lipofectamine 2000. An increase in the number of hydrophobic domains to three (78b–81b) increased the delivery efficiency of ribonucleoprotein complexes to GFP-HEK cells [109] and human mesenchymal stem cells [110]. Notably, liposomes based on compound 79a with two hydrophobic domains had practically no TA, while the use of an analog (79b)

with three hydrophobic domains made it possible to transfect stem cells more efﬁciently Pharmaceutics 2021, 13, 920 20 of 27

Pharmaceutics 2021, 13, 920 20 of 27

than was possible with the commercial transfectant Lipofectamine 2000. An increase in the Figure 35. Disulfide polycationic amphiphiles based on spermine and cholesterol. number of hydrophobic domains to four negatively affected TA regardless of the cationic domain. A study of the effect of the length of the hydrophobic domain demonstrated A library of disulfide CAs 78–89 (Figure 36) with various cationic domains was cre- Pharmaceutics 2021, 13, 920 that shortening the chain length to 12 atoms significantly impaired TA, while including20 of 27 ated. The first series of compounds, 79a–84a, contained a hydrophobic hexadecyl chain, 14–18 atoms in the chain provided no efficiency difference [110]. including sulfur atoms [108]. CLs composed of disulfide CAs, cholesterol, DOPE and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:4:1:1, weight ratio) were used to deliver ribonucleoprotein complexes to GFP-HEK cells. Compounds 80a, 81a, 83a, 84a had the highest efficacy, comparable to or exceeding that of Lipofectamine 2000. An increase in the number of hydrophobic domains to three (78b–81b) increased the delivery efficiency of ribonucleoprotein complexes to GFP-HEK cells [109] and human mesenchymal stem cells [110]. Notably, liposomes based on compound 79a with two hydrophobic domains had practically no TA, while the use of an analog (79b) with three hydrophobic domains made it possible to transfect stem cells more efficiently than was possible with the commercial transfectant Lipofectamine 2000. An increase in the number of hydrophobic domains to four negatively affected TA regardless of the cationic domain. A study of the effect of the length of the hydrophobic domain demonstrated that shortening the chain length to 12 atoms significantly impaired TA, whileFigureFigure including 35.35. DisulfideDisulfide 14–18 polycationicpolycationic atoms in amphiphilesamphiphilesthe chain pr basedbaovidedsed onon no sperminespermine efficiency andand cholesterol.differencecholesterol. [110].

A library of disulfide CAs 78–89 (Figure 36) with various cationic domains was created. The first series of compounds, 79a–84a, contained a hydrophobic hexadecyl chain, including sulfur atoms [108]. CLs composed of disulfide CAs, cholesterol, DOPE and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:4:1:1, weight ratio) were used to deliver ribonucleoprotein complexes to GFP-HEK cells. Compounds 80a, 81a, 83a, 84a had the highest efficacy, comparable to or exceeding that of Lipofectamine 2000. An increase in the number of hydrophobic domains to three (78b–81b) increased the delivery efficiency of ribonucleoprotein complexes to GFP-HEK cells [109] and human mesenchymal stem cells [110]. Notably, liposomes based on compound 79a with two hydrophobic domains had practically no TA, while the use of an analog (79b) with three hydrophobic domains made it possible to transfect stem cells more efficiently than was possible with the commercial transfectant Lipofectamine 2000. An increase in the number of hydrophobic domains to four negatively affected TA regardless of the cationic domain. A study of the effect of the length of the hydrophobic domain demonstrated that shortening the chain length to 12 atoms significantly impaired TA, while including 14–18 atoms in the chain provided no efficiency difference [110].

FigureFigure 36. 36. AA library library of of disulfide disulﬁde CAs CAs with with various various cationic cationic domains. domains.

Subsequently, the library of disulﬁde derivatives was expanded using a cholesterol residue in the structure of the hydrophobic domain, which reduced cytotoxicity relative to

that of long-chain analogs 79a–84a [111]. Compounds 85c, 87c, 88c were highly efﬁcient in delivering mRNA to all four investigated cell lines (B16F10, HEK, HeLa, NIH 3T3). Compound 86c was effective on all cells except NIH 3T3. The results of all four compounds were comparable or slightly inferior to that of Lipofectamine 2000. Compounds 84c and 89c demonstrated extremely low aggregation stability and delivery efﬁciency.

Figure 36. A library of disulfide CAs with various cationic domains.

Pharmaceutics 2021, 13, 920 21 of 27

2.5. Influence of Structural Components of Cationic Amphiphiles on the Efficiency of Nucleic Acid Delivery Each element of the CA structure performs a specific function and influences the TA. Hydrophobic domains are involved in the protection of NAs and promote the fusion of lipoplexes with cell membranes. Aliphatic hydrocarbon substituents usually represent these domains with a length of 10 to 18 carbon atoms, tocopherol, or sterols. The type of hydrophobic domain determines both the structure of the vesicles that a CA forms in the aqueous phase and its subsequent interaction with biological membranes. Liposomal formulations promote more efficient NA delivery than that accomplished by micelles or other types of nanoparticles [58]. Mostly, CAs used for transfection of eukaryotic cells are classic head-to-tail amphiphiles. Based on polyamines and amino acids, CAs synthesized with two hydrophobic domains (gemini-amphiphiles) can deliver NAs more efficiently than can their monosubstituted analogs [39,42,94,95]. An increase in the length of aliphatic hydrocarbon chains usually increases TA [32,35,40,53,58,88,89]. Notably, however, it may also increase the toxicity of compounds [40]. In contrast, for some spermine-based CAs, TA decreased with an increase in the length of aliphatic substituents [70,84]. Analysis of published data reveals that the optimal length of aliphatic substituents is 14–18 carbon atoms, while high TA is most often noted for CAs with myristoyl or tetradecyl substituents [71,79,84]. The degree of unsaturation of substituents also affects the efficiency of NA delivery: with an increase in unsaturation, TA increases but so does toxicity [20]. Thus, it is necessary to search for an optimal CA variant that effectively delivers NAs, while its toxicity remains within acceptable limits. When sterol derivatives are used as hydrophobic domains, one should prefer natural compounds, which do not cause significant toxicity. In this case, it is optimal to use a common and widely available sterol such as cholesterol [38,78,85], although diosgenin derivatives are also capable of efficient NA delivery [75–77,83]. Tocopherol is also employed as a hydrophobic domain [80,92,101]. The positively charged CA domain is responsible for the electrostatic interactions of amphiphiles and/or liposomes with NAs, the formation of stable lipoplexes, and the interaction of complexes with cell membranes. An increase in the number of amino groups in the structure of the cationic domain leads to an increase in the TA [31,56,60,81,82,85]. The most effective are CAs with domains based on polyamines, with the number (more than two [74]) and distribution of amino groups in the polyamine chain [31,53,93,102] playing important roles. Many studies reveal that the most effective are polyamines with four amino groups, primarily a natural polyamine—spermine [58,72,76]. However, CAs based on synthetic polyamines (TETA, triethylenepentamine, tributylenepentamine) can be more efficient in delivering NAs than their natural counterparts [31,59,74]. The cationic domain can also be designed based on cyclen [77–81] or amino acids. In this case, it is advisable to use the residues of L-arginine or L-lysine [82–84,86,90,92] but not L-histidine, the presence of which in the CA structure did not improve the NA delivery [84]. Linkers, connecting hydrophobic and cationic domains, determine the stability and biocompatibility of the CAs and play a key role in the efficiency of NA delivery. The most commonly used are ether, ester, carbamate, amide, and disulfide linkers. Among the most effective linkers imparting low toxicity to CAs are carbamoyl ones [42,43,52,87]. Efficient delivery of NAs is also observed with the presence of ether bonds in the CA structure [68,82]. The introduction of a disulfide linker into a CA molecule makes it sensitive to the action of intracellular reducing agents, which can increase the efficiency of NA delivery and/or reduce the toxicity of compounds [101,102,104]. Multiple studies have proven that a close arrangement of the hydrophobic and cationic domains complicates both domain’s functioning and interferes with the formation of liposomes. The introduction of a spacer into the CA structure and an increase in its length increase NA delivery efficiency [42,50,88,89,91]. Pharmaceutics 2021, 13, 920 22 of 27

Low cytotoxicity is an important feature of CLs. First generation of CAs containing quaternary ammonium head was rather toxic, but numerous recently developed polycationic amphiphiles provided non-toxic transfection [8]. As mentioned above, toxicity may be increased with the length and degree of unsaturation of hydrophobic tails. In sterol derivatives use of diosgenin may cause toxicity [77,85] probably due to the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Linkers may also affect toxicity. Thus, the replacement of the ester linker with an amide one reduced the toxicity of the L-lysine based CAs [84]. Also, it should be noted that direct binding of both hydrophobic and cationic domains leads to a signiﬁcant increase in toxicity of the CAs [35].

3. Conclusions and Perspectives In this review, we focused on the cationic amphiphiles, which are the major but not sole component of CLs. Development of additional components, such as targeting [16] or stealth [17] lipids, is an actual trend in non-viral gene delivery. Positive charge of CLs provides an effective binding and protection of NAs during transfection but also attracts serum proteins and other components during in vivo administration. Interaction with serum components may cause fast blood clearance of lipoplexes. Therefore, searching for possibilities to mask excessive CLs positive charge is required. One of such ways may be a use of PEG-lipids, which, however, decrease both lipoplex and NAs internalization into the cell. Alternatively, a coating of CLs or lipoplexes with additional lipid (polymer) shell may be also considered. Sophistication of liposomal formulations turns its manufacturing procedure into novel challenges including scale-up and quality control stages. A possible solution to overcome existing difficulties might be a shift from the single large-scale to the multiple parallel small-scale production. For this purpose, microfluidic technologies become very attractive due to their ability to provide continuous and reproducible liposome preparation. Recent progress in microfluidics allows to use this technique for production of rather complex nanoparticles containing biopolymers and even whole cells [112]. In conclusion, the development of effective and safe CLs for the delivery of therapeutic NAs requires employment of the right combination of structural elements in the CA molecule to promote the formation of both the liposomes and their complexes with NAs while avoiding interference and overcoming biological barriers. Once within the target cell, the complexes must release NAs with a high efficiency to provide biological/therapeutic effect.

Author Contributions: Conceptualization, P.A.P., M.A.M.; literature review, P.A.P.; writing the manuscript, P.A.P.; manuscript revision, M.A.M., and supervision, M.A.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of Science and Higher Education of the Russian Federation, Grant No. 0706-2020-0019. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the study design, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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