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Cooperativity of the SUMO and Ubiquitin Pathways in Genome Stability

Covalent attachment of ubiquitin (Ub) or SUMO to DNA repair proteins plays critical roles in maintaining genome stability. These structurally related polypeptides can be viewed as distinct road signs, with each being read by specific protein interaction motifs. Therefore, via their interactions with selective readers in the proteome, ubiquitin and SUMO can elicit distinct cellular responses, such as directing DNA lesions into different repair pathways. On the other hand, through the action of the SUMO-targeted ubiquitin ligase (STUbL) family proteins, ubiquitin and SUMO can cooperate in the form of a hybrid signal. These mixed SUMO-ubiquitin chains recruit “effector” proteins such as the AAA+ ATPase Cdc48/p97-Ufd1-Npl4 complex that contain both ubiquitin and SUMO interaction motifs. This review will summarize recent key findings on collaborative and distinct roles that ubiquitin and SUMO play in orchestrating DNA damage responses.

Introduction
The integrity of the cell's genome is under constant threat from various external and internal genotoxic agents, such as UV and oxidative stress. Unrepaired damage can lead to cell death or gene mutations that alter cellular behavior, potentially leading to the demise of the whole organism [1]. DNA damage can range from subtle changes in nucleotide base structure to breaks in both strands of the DNA double helix, and can occur in all phases of the cell cycle. Cells diligently scan their genome with damage sensor proteins and bring in specialized repair enzymes once a lesion is detected. Small lesions, such as simple nucleotide alterations on one strand of DNA can be repaired by base excision repair (BER) or nucleotide excision repair (NER). Extensive and difficult-to-repair DNA damage, such as double-strand breaks (DSBs) or replication forks stalled at DNA lesions, elicits a more extensive response called the DNA damage response (DDR), leading to increased production of repair enzymes and cell-cycle arrest until repair is complete [2].
ATM and ATR are key kinases that drive the DDR by phosphorylating chromatin, DNA repair and cell cycle checkpoint proteins at sites of DNA damage. In addition to phosphorylation, a plethora of other post-translational modifications (PTMs) are critical to the DDR. PTMs alter the chromatin environment as well as the interactions or activity of some repair enzymes to promote signal amplification, sequential recruitment, and removal of factors to allow for downstream repair steps. Using PTMs to modulate protein interactions and activity is a common and effective strategy to induce rapid and reversible responses. Proteins can be a modified by a small chemical moiety, such as the phospho-or acetyl-groups; or by another protein, such as members of the ubiquitin family [2][3][4].
Two structurally related small protein modifiers, ubiquitin and SUMO, are conjugated to target proteins via a similar cascade of biochemical reactions. Both SUMO and ubiquitin play important, often interconnected, and even interdependent roles in DNA damage repair. There are a number of excellent reviews discussing the roles of ubiquitin and SUMO in DNA repair published in the past few years [4][5][6][7][8][9][10][11]. This review will summarize some of the most recent advances in understanding the roles of ubiquitin and SUMO in DNA damage repair, with emphasis on the collaborative roles of these modifications.

Ubiquitin and SUMO Pathways, and Their Crosstalk
Covalent attachment of the carboxyl terminus of ubiquitin or SUMO (ubiquitylation or sumoylation) to a lysine residue on a substrate protein involves a similar relay of E1 activating, E2 conjugating, and E3 ligase actions [3]. Ubiquitylation and sumoylation are readily reversible, and deconjugation is carried out by ubiquitin-or SUMO-specific iso-peptidases, such as DUBs or Ulp/SENPs. Thus, just like phosphorylation, SUMO and ubiquitin are highly dynamic modifiers of protein behavior.
Crosstalk between the SUMO and ubiquitin pathways had been documented, and in general was of an antagonistic nature [12]. This made the discovery of the STUbL (SUMO-Targeted Ubiquitin Ligase) family of proteins all the more intriguing. STUbLs, including mammalian RNF4, budding yeast Slx5-Slx8, and fission yeast Rfp1/2-Slx8 heterodimers, can direct SUMO conjugates for degradation at the proteasome [13][14][15][16][17]. In this mechanism, a tandem array of SUMO interaction motifs (SIMs) in STUbLs enables them to selectively ubiquitylate target proteins that are modified by polymeric SUMO chains ( Figure 1). Thus, in addition to their distinct signaling roles, SUMO and ubiquitin cooperate in the processing of certain target proteins. Additional examples of crosstalk between SUMO and ubiquitin pathways will be given throughout this review. DNA damage promotes recruitment of sumoylation enzymes and crosstalk between the SUMO and ubiquitin pathways. In the process of repairing a DSB, resection generates ssDNA, which is coated with RPA. Through interaction with RPA, SUMO E3 ligase Siz2 is recruited to the damage site to promote the sumoylation of protein groups involved in DNA repair. SUMO-SIM contacts create a glue effect to enhance protein-protein interactions and the recruitment of additional factors. SUMO chains can form, assisted by multiple SIM-containing E4 elongases. SUMO chains may then attract the tandem SIM-containing ubiquitin E3 ligase, STUbL, which adds ubiquitin to the SUMO chain to produce ubiquitin-SUMO hybrid chains, a signal that is "read" by proteins containing both a ubiquitin interacting motif (UIM) and a SIM. These hybrid chains can either recruit additional repair factors, or promote protein extraction from complexes/chromatin through the activity of the AAA + ATPase complex Cdc48/p97-Ufd1-Npl4. STUbL can also curb SUMO chain elongation by capping the SUMO chain with ubiquitin. SUMO chains can also be actively antagonized by SUMO isopeptidases, such as SENP6. Genotoxins such as CPT promote the dissociation of SENP6 from chromatin, therefore shifting the balance toward more sumoylation at the damage site. Crosstalk between the SUMO and ubiquitin pathways has been demonstrated on Top1, ID complex, Rad52, and XPC extraction.

DNA Damage-Induced Recruitment of SUMO and Ubiquitin E3 Ligases
Ubiquitin pathway enzymes far out-number those found in the SUMO pathway, even though both are involved in diverse cellular activities. It is thought that the SUMO pathway achieves substrate specificity in part by regulating the subcellular localization of sumoylation/desumoylation enzymes. For example, the PIAS family and Nse2/Mms21 SUMO E3 ligases can associate with the genome by interacting with DNA and/or chromatin-bound proteins. The PIAS family proteins, including mammalian PIAS1-4, budding yeast Siz1/2, and fission yeast Pli1, contain a SAP (Scaffold attachment factor, Acinus and PIAS) domain, which directly binds DNA. The DNA repair process can further promote interaction between the SUMO E3 ligase and damaged chromatin ( Figure 1). For example, RPA-coated ssDNA generated by resection is required for Siz2 accumulation at DNA DSBs and local Siz2-mediated sumoylation of DNA repair factors [18]. Mechanistically, the SIZ2 SAP domain extension (eSAP) interacts directly with the middle subunit of the RPA complex, Rfa2. Interestingly, neither DNA binding by the core SAP domain, nor SUMO interaction via the SIM of SIZ2, is required for its recruitment to DSBs.
The Nse2/Mms21 SUMO E3 ligase does not contain a SAP domain, but is part of the chromatin-associated Smc5-Smc6 complex [19][20][21][22]. How Smc5-Smc6, and thereby Nse2, is recruited to chromatin and/or DNA lesions is generally poorly defined. Fission yeast Smc5-Smc6 accumulates during S phase at the heterochromatic centromeres, and redistributes to subtelomeres in an Nse2 SUMO E3 ligase-dependent manner following DNA alkylation (MMS) damage [23]. This indicates a role for Smc5-Smc6 mediated sumoylation in its own accrual at DNA damage sites. Moreover, the Smc5-Smc6 subunit Nse1 contains an E3 ubiquitin ligase RING domain that also supports Smc5-Smc6 accumulation at MMS-induced DNA lesions [24,25]. It should be noted that an initial analysis of Nse1 indicated that by itself it did not support in vitro E3 ubiquitin ligase activity [24]. However, a more recent study revealed that Nse1 must be in complex with a MAGE domain cofactor (MAGE-G1/Nse3) to exhibit E3 ubiquitin ligase activity in standard in vitro assays [26].
Therefore, cooperativity between sumoylation and ubiquitylation exists within the same complex, potentially acting as a switch for relocalization of Smc5-Smc6 in response to genotoxic insults (such cooperativity will be further discussed below). Despite these correlative results, the targets and mechanisms of Smc5-Smc6-mediated sumoylation or ubiquitylation in its localization to DNA lesions remain undefined. Notably, human Smc5-Smc6 was recently shown to localize at certain DNA lesions via an interaction network that is based on RNF168-catalyzed ubiquitin chains [27]. These ubiquitin chains are bound by the E3 ubiquitin ligase RAD18, which is proposed to act structurally and not catalytically in this role due to the absence of its cofactor Rad6 ( Figure 2A). Finally, Rad18 recruits Smc5-Smc6 via a phospho-dependent interaction with the SLF1 subunit of the SLF1/SLF2 loading factor ([27]; Figure 2A). A role for human Nse2 or Nse1 E3 ligase activities in loading remains to be tested.
Once at DNA damage sites SUMO E3 ligases locally sumoylate DDR proteins, which promotes interactions amongst them via SUMO-SIM contacts and creates a "protein glue" effect. For example, the checkpoint protein ATRIP is recruited to RPA-coated ssDNA where it becomes SUMO-2/3 modified. ATRIP sumoylation promotes interaction of multiple ATRIP-associated proteins, including ATR, RPA70, TopBP1, and the MRN complex, which all have affinity for SUMO2 chains [28]. Certain lesions such as ICLs induce the recruitment of Smc5-Smc6 to DNA damage sites via the interaction of ubiquitin chains, generated by RNF168, with RAD18 and the Smc5-Smc6 subunits SLF1 and SLF2. "P" indicates phosphorylation of DDR factors, including RAD18, that is also required for Smc5-Smc6 recruitment; (B) DNA at stalled replication forks may directly bind the SUMO mimic Esc2, which via its integral SUMO-like domains (SLDs) is suggested to attract STUbL to facilitate the proteolytic removal of anti-recombinase Srs2; (C) SUMO chains are added to a Top1-DNA adduct and/or nearby factors to signal STUbL-mediated ubiquitylation and subsequent extraction/degradation facilitated by the Cdc48/p97-Ufd1-Npl4 complex. In addition, in budding yeast the SUMO chain can recruit a SIM-containing metalloprotease Wss1, which is activated by ssDNA. Wss1 also cooperates with a Cdc48 complex, containing another cofactor Doa1.
In addition to local SUMO E3 ligase recruitment, regulated SUMO deconjugation enhances the sumoylation of certain DDR targets. The SUMO deconjugating enzyme SENP6 dissociates from RPA70 in the presence of camptothecin (CPT) causing RPA SUMO´2{3 to accumulate, which facilitates Rad51 recruitment to CPT-induced damage sites [35].

Potential New SUMO E3 Ligases in DNA Repair
Besides their catalytic SP-RING domains, PIAS SUMO E3 ligases contain a single SIM that may help fuel target sumoylation in vivo by recruiting conjugation competent SUMO~Ubc9. Recently, RING-independent SUMO E3 ligases that utilize multiple SIMs to catalyze sumoylation have been described [36][37][38][39][40][41]. DNA repair proteins SLX4 and Wss1 both have multiple SIMs [38,39,42]. SLX4 is a large multi-domain protein that scaffolds and localizes key DNA endonucleases to support a number of DNA repair pathways. As with other multi-SIM containing proteins, SLX4 SIMs interact with SUMO chains. Distinguishingly however, SLX4 SIMs also mediate selective interaction with the charged E2 enzyme Ubc9~SUMO, thereby promoting autosumoylation and the sumoylation of the SLX4-interacting endonuclease subunit, XPF [39]. SLX4's SIMs are also required for its localization to certain DNA structures/lesions [39,42,43], where it may cooperate with PIAS-type ligases such as PIAS3 in sumoylating key DDR factors [32].
Wss1 is important for UV damage repair [44], and was recently found to proteolytically remove bulky DNA adducts such as topoisomerase I stalled in its cleavage complex by CPT [38,45] (Figure 2C). Interestingly, as determined for STUbL in fission yeast (see #6 below, [46]), Wss1 also cooperates with the Cdc48/p97 molecular segregase in Top1 lesion repair [38,45]. Wss1 protease activity is activated in vitro by ssDNA, and in vivo by genotoxic stress, in part due to its oligomerization. Like SLX4, Wss1 may exert SUMO E3 ligase activity via its tandem SIM and promote SUMO chain formation [38]. While SLX4 has been shown to interact preferentially with Ubc9~SUMO [39], such evidence is currently lacking for Wss1. Both factors may also/instead facilitate SUMO chain formation by providing a structural scaffold to nucleate and activate the sumoylation machinery; an E4 elongase activity. Binding of Wss1 to these self-catalyzed SUMO chains might, like ssDNA [38], promote its oligomerization and local activation.
The vertebrate ZNF451 protein was recently defined both structurally and biochemically as a novel factor with both E3 ligase and E4 elongase activities [40,41]. Importantly, the tandem SIMs of ZNF451 elongate SUMO2/3 chains catalytically (E4), in contrast to the SUMO chain-binding SIMs present in the STUbL RNF4 [41]. This indicated the presence of unique features in ZNF451 SIMs. Indeed, the ZNF451 inter-SIM linker domain contains a PLRP motif that is catalytically critical, and together with the first SIM binds Ubc9~SUMO2 [40,41]. The SUMO2 E3 ligase activity of ZNF451 also requires a zinc finger, which likely recruits the first SUMO2 (substrate) for subsequent SIM-dependent elongation. These analyses of ZNF451 underscore the need for caution previously noted about SIM-containing factors masquerading as SUMO ligases in vitro [47]. Whether ZNF451 is a key player in the DDR is currently unknown; however, genotoxin-induced SUMO2/3 conjugation is globally reduced in ZNF451 deleted cells [41]. Given the apparently pivotal role played by SUMO2/3 chains in the DDR, it appears likely that ZNF451 activity will impact DNA repair.

SUMO Mimicry Integrates SUMO and Ubiquitin Signaling in DNA Repair
Rad60 of fission yeast is the founding member of a conserved protein family with two integral SUMO-like domains (SLDs) that plays key roles in genome stability [48,49]. These Rad60 family roles include mimicking SUMO to interact with SIM-containing factors such as STUbLs, and the SUMO conjugating enzyme Ubc9 [50][51][52][53]. The latter interaction promotes target sumoylation by the Smc5-Smc6-associated SUMO ligase Nse2/Mms21 in the highly evolutionarily diverged fission and budding yeasts [51,54].
Notably, fission yeast STUbL, Nse2 and Rad60 cooperate to repair stalled Topoisomerase I cleavage complexes (Top1cc) in cells lacking the Top1cc-resolving enzyme Tdp1 [55]. This was the first example of STUbL, Smc5-Smc6 and Rad60 acting in concert to promote DNA repair, and recent analyses are supporting broad conservation of this phenomenon. In budding yeast, the Slx5-Slx8 STUbL was shown to antagonize Nse2/Mms21-dependent SUMO conjugates, indicating they also have overlapping targets [54,56]. In addition, the budding yeast Rad60 family protein Esc2 binds DNA at stalled replication forks to regulate the proteolysis of the helicase and anti-recombinase Srs2, which it appears to achieve by recruiting Slx5-Slx8 to ubiquitylate Srs2 ( Figure 2B) [57]. Moreover, Smc5-Smc6 and Esc2 share overlapping roles in regulating replication stress-associated recombination [58,59].
Esc2-dependent Slx5-Slx8 localization to replication forks may be an example of SUMO-independent STUbL recruitment. There is a precedent for such STUbL recruitment, in the recognition and ubiquitylation of unmodified budding yeast Matα2 by Slx5-Slx8 [60,61]. However, Rad60 is modified by SUMO [62], and so a similar modification of Esc2 could enhance STUbL recruitment at replication forks.
A recent striking example of cooperativity between STUbL, Smc5-Smc6 and Rad60 is the relocalization of DNA breaks within Drosophila heterochromatin to the nuclear periphery to undergo repair, which requires all three factors [34]. Compromising this relocalization pathway renders cells sensitive to genotoxic stress, such as ionizing radiation [34]. As these studies were done with knockdowns of each factor, which of the spectrum of functions of dRad60, STUbL and Smc5-Smc6 involved awaits a more detailed analysis of hypomorphic alleles.
Of note, relocalization of at risk genomic loci/lesions to the nuclear periphery in budding yeast also depends on the Slx5-Slx8 STUbL [63,64]. Based on the cooperativity of Smc5-Smc6, STUbL and Rad60 family proteins in DNA repair in fission yeast and Drosophila, it will be interesting to determine the role for budding yeast Esc2 and Smc5-Smc6 in this phenomenon. In this regard, Smc5-Smc6 was shown to promote relocalization of a DNA DSB in the rDNA locus of budding yeast to an extranucleolar site for repair [65]. Thus, although currently piecemeal, aggregate data in budding yeast supports the conservation and key nature of a cooperative Smc5-Smc6, STUbL and Rad60 (Esc2) DNA repair axis.
The only other example of SUMO mimicry is found in vertebrate UAF1, which also contains tandem SLDs [66]. The USP1/UAF1 complex deubiquitylates the Fanconi anemia protein FANCD2, which promotes homologous recombination (HR) and DNA cross-link repair. UAF1 SLD2 binds directly to a SIM in FANCI, bridging the interaction between USP1/UAF1 and FANCD2/FANCI. It is not known if UAF1 SLDs make additional contacts with the SUMO machinery, or are restricted to SIM:SUMO-like interactions.

Recruitment Mechanisms and Functions of STUbL on Chromatin
There are a number of excellent recent reviews covering many of the E3 ubiquitin ligases that are recruited to chromatin/DNA (e.g., [4,10]), so here we focus largely on the recruitment mechanisms and functions of the STUbL family. Examples of ubiquitin E3 ligases that directly contact DNA/chromatin are RNF168, RNF4 and BMI1-RING1B, which bind DNA and/or the nucleosome, and the UV-DDB complex that recognizes UV damaged DNA [10,67,68]. Several RING finger domain-containing ubiquitin E3s also belong to the SF2-helicase superfamily that translocate on DNA in an ATP-driven manner. For example, the activity of yeast Rad5 (human HLTF and SHPRH) and STUbL Uls1 (also known as Ris1) lead to ubiquitylation (and removal in the case of Uls1) of chromatin-associated proteins, such as PCNA and Rap1, and as such act like a "molecular sweeper" [10,69]. Notably, most of these E3 ligases also contain interaction motifs for SUMO, ubiquitin or other PTMs, supporting combinatorial and thus more selective recognition of their targets (see below and [4]).
The mammalian STUbL RNF4 binds to various DNA structures as well as nucleosomes [67,68]. RNF4 targets the nucleosome via a basic cluster in its RING domain, similar to RNF168 and RING1b-BMI1 [67]. Nucleosome targeting by RNF4 is important for repairing TRF2-depleted telomeres via 53BP1 recruitment and NHEJ [67]. Moreover, as for its recruitment to DNA DSBs, RNF4's SIMs are critical for telomere repair, suggesting that interactions of RNF4 with both SUMO and nucleosomes cooperatively promote DNA repair [67,[70][71][72].
STUbLs appear to have multiple functions at DNA damage sites ( Figure 1). As SUMO E3s are brought to the damage site to catalyze local sumoylation, heightened local accumulation of SUMO could lead to uncontrolled growth of SUMO chains. While SUMO chains may promote interaction of repair proteins at the damage site, unrestricted growth could also hinder the downstream repair process. By attaching ubiquitin to the SUMO amino terminus [73], STUbL can both stop the growth of SUMO chains and target them for proteasome-mediated protein degradation. Notably, fission yeast STUbL mutant's genotoxin sensitivity is fully suppressed in cells that are unable to form SUMO chains, indicating that a major genome protective function of STUbL is to prevent SUMO chain accumulation [46,51].
STUbL activities also generate hybrid ubiquitin-and SUMO-chains, which function as a unique signal to attract proteins that contain recognition motifs for both modifications [46,74]. Interaction between a hybrid Ub-SUMO chain and its corresponding recognition motifs can lead to further protein recruitment. For example, RAP80 contains both a UIM and SIM that bind hybrid chains to stabilize RAP80's residence at DSBs and facilitate the recruitment of BRCA1 [74]. On the other hand, Ub-SUMO chains can also promote protein extraction from complexes/chromatin and/or degradation, as in the case of Cdc48/p97-Ufd1-Npl4 segregase recruitment (Figure 1) [4,46,[75][76][77]. For both RAP80 and Ufd1, integral ubiquitin-and SUMO-recognition motifs synergize in protein interaction as they bind hybrid ubiquitin/SUMO chains more avidly than homogenous chains of either modifier ( [74] and personal observation).

Coordinated and Distinct Roles of Ubiquitin and SUMO in DNA Repair
As discussed above, SUMO and ubiquitin modifications can cooperatively facilitate both DNA repair factor recruitment and removal at lesions (see also [4]). In both scenarios, the presence of both modifications improves binding affinity, specificity, or both. Below are examples of recently characterized coordinated and distinct roles of ubiquitin and SUMO in DNA repair.
Two central components of the Fanconi anemia (FA) repair pathway, FANCI and FANCD2 (the ID complex), are sumoylated in response to DNA damage [78]. FANCI sumoylation has also been observed in several proteomics studies [71,79,80]. ID complex sumoylation is mediated by PIAS1/4, and antagonized by SENP6. ID complex sumoylation at DNA damage sites again requires the concerted action of PTMs, in this case phosphorylation by ATR and monoubiquitylation, which together license the chromatin loading of ID. Subsequently, ID sumoylation promotes its polyubiquitylation by RNF4 and extraction from chromatin by the p97/Cdc48 segregase complex. In contrast, a sumoylation defective ID complex is refractory to RNF4, p97, and SENP6 regulation. Thus, the cooperative role of STUbL and Cdc48/p97 initially defined in fission yeast [46], also has critical functions in human genome stability [78]. Overall, multiple PTMs and members of the SUMO pathway (PIAS1/4, SENP6, and STUbL) work in concert to fine-tune the balance of activated ID complex at DNA lesions.
Using SUMO to fine-tune the balance between repair factor recruitment and removal likely represents a common strategy used in multiple DNA repair pathways. HR is a key process used in DNA DSB repair, interstrand crosslink (ICL) removal, and Post-Replicative Repair (PRR). During HR, invasion of homologous double-stranded DNA (dsDNA) by a 3'-single stranded DNA (ssDNA) requires the coating of ssDNA with recombinase Rad51 to form a nucleoprotein filament. The loading of Rad51 is promoted by Rad52, which displaces RPA from ssDNA while assisting Rad51 binding.
In budding yeast, Rad52 is sumoylated upon DNA damage by Siz2 [81]. Rad52 sumoylation is enhanced by ssDNA [82], likely due to recruitment of Siz2 by RPA coated ssDNA generated during DSB resection [18]. A Rad52 mutant refractory to sumoylation appears impaired in its interaction with SIM-containing cofactor Rad51 [83]; although Rad52 sumoylation has only subtle effects on HR in vivo [81,82,84]. This is a recurrent theme in SUMO biology, where multiple components within a pathway are decorated by SUMO, possibly providing signaling redundancy.
In addition to, or instead of promoting HR, Rad52 sumoylation can curb Rad51 loading by attracting the Cdc48/p97-Ufd1-Npl4 molecular segregase complex through a SIM in Ufd1 [75,83]. The Cdc48 complex then drives the dissociation of Rad52-SUMO and Rad51 from DNA [83]. Notably, genotoxic stress such as HU or MMS can also induce the Slx8-dependent degradation of Rad52-SUMO, suggesting that Rad52-SUMO dissociated by Cdc48 might also be subject to STUbL-mediated ubiquitylation [64].
UV induced DNA damage is detected by the sensor proteins DDB2 and XPC. Following UV radiation, DDB2 and XPC are ubiquitylated by the Cul4A/B E3 ligase [85]. Whereas ubiquitylated DDB2 is rapidly degraded via p97/Cdc48-Ufd1-Npl4 and UBXD7-mediated mechanisms, XPC ubiquitylation enhances its interaction with DNA [86]. XPC is also sumoylated, and XPC sumoylation promotes both global genome NER (ggNER) and UV-induced ubiquitylation of XPC [87]. Although RNF4 is recruited to laser-induced DSBs through SUMO-SIM interactions and is important for DSB repair, it is dispensable for UV-induced ubiquitylation of XPC. Therefore, SUMO-SIM interaction is not the sole factor in STUbL target selection. Instead, another SIM-containing STUbL called RNF111/Arkadia mediates the UV-induced ubiquitylation of sumoylated XPC [88]. XPC ubiquitylation (K63 chain topology) by RNF111 promotes XPC's release from UV-lesions, and thus more efficient recruitment of downstream NER factors such as XPG and XPF/ERCC1 [89].
In contrast to the SUMO and ubiquitin recognition motifs of RAP80 and Ufd1 that synergize to interact with Ub-SUMO hybrid chains, those in SLX4 appear to direct it into distinct DNA repair pathways. SLX4 is a platform that interacts with multiple classes of repair proteins, including SLX1, XPF Rad1 /ERCC1 Rad10 , the MUS81-EME1 Holliday Junction (HJ) resolvase, the MSH2-MSH3 mismatch repair complex, the telomeric protein TRF2, and the mitotic control kinase PLK1 [90]. As anticipated from this scaffolding role, SLX4 is key for multiple DNA repair pathways, including ICL repair and proper chromosome segregation in the absence of the RecQ helicase BLM [91][92][93][94]. SLX4's ubiquitin and SUMO interacting motifs direct SLX4 to ICL and replicative stress at Common Fragile Sites (CFS), respectively [39,42]. Therefore, the coincidence of SUMO and ubiquitin interaction motifs does not necessarily indicate affinity for Ub-SUMO hybrid chains. It is likely that the particular juxtaposition of these motifs in a protein, together with other PTMs and protein interaction domains, dictates their functionality.

Conclusions and Future Directions
SUMO and ubiquitin modifications critically orchestrate the DDR in both time and nuclear space. At DNA damage sites, both modifiers promote the rapid recruitment of DDR proteins. Just as crucial for DNA repair and genome stability, recent discoveries have revealed direct cooperation between SUMO and ubiquitin in chromatin remodeling at DNA lesions. Certain SUMO targets are ubiquitylated by STUbLs, thereby attracting Cdc48/p97-Ufd1-Npl4 or other effectors with affinity for Ub-SUMO hybrid chains. This chromatin remodeling machinery, coupled to recycling or proteasomal degradation of extracted targets, is likely to be a broadly used and key mechanism in the dynamic control of chromatin constituents.
Key questions remaining include, how is selectivity generated in the STUbL pathway? That is, RNF4 and RNF111 STUbLs both preferentially bind SUMO chains, but nevertheless have distinct SUMO conjugated targets. Perhaps subnuclear compartmentalization of STUbLs plays a role, as it does in generating target specificity of the desumoylating enzymes ULP1/2 (human SENP1/2). Regulation of STUbLs by additional PTMs such as phosphorylation is also likely, to license their activities on particular targets.
To derive general "rules" for STUbL and other E3 ligase targeting to DNA lesions, chromatin mass spectrometry (CHROMASS; [27]) in combination with comprehensive PTM analysis to identify repair factors and their modifications at various stages of a DNA repair process would be informative.


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Materials Science
Medicine

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Biology
Materials Science
Chemistry

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Soft Interaction in Liposome Nanocarriers for Therapeutic Drug Delivery

The development of smart nanocarriers for the delivery of therapeutic drugs has experienced considerable expansion in recent decades, with the development of new medicines devoted to cancer treatment. In this respect a wide range of strategies can be developed by employing liposome nanocarriers with desired physico-chemical properties that, by exploiting a combination of a number of suitable soft interactions, can facilitate the transit through the biological barriers from the point of administration up to the site of drug action. As a result, the materials engineer has generated through the bottom up approach a variety of supramolecular nanocarriers for the encapsulation and controlled delivery of therapeutics which have revealed beneficial developments for stabilizing drug compounds, overcoming impediments to cellular and tissue uptake, and improving biodistribution of therapeutic compounds to target sites. Herein we present recent advances in liposome drug delivery by analyzing the main structural features of liposome nanocarriers which strongly influence their interaction in solution. More specifically, we will focus on the analysis of the relevant soft interactions involved in drug delivery processes which are responsible of main behaviour of soft nanocarriers in complex physiological fluids. Investigation of the interaction between liposomes at the molecular level can be considered an important platform for the modeling of the molecular recognition processes occurring between cells. Some relevant strategies to overcome the biological barriers during the drug delivery of the nanocarriers are presented which outline the main structure-properties relationships as well as their advantages (and drawbacks) in therapeutic and biomedical applications.

Introduction
Liposomes composed of surfactants, natural or synthetic (phospho)lipids, and block copolymer vesicles represent a versatile platform for the development of improved drug delivery therapies in a wide range of biomedical applications [1]. The ability of liposomal systems to entrap both lipophilic and hydrophilic compounds enables a large variety of drugs to be encapsulated by these vesicles, including deoxyribonucleic acids (DNA), proteins, or imaging agents [2,3]. The use of liposomes based nanocarriers in drug delivery technologies offers many benefits connected with their capacity for a versatile self-assembly, governed by a number of soft interactions that regulate colloidal stability of CMC) and temperature (critical micelle temperature, CMT) [6]. As the amphiphilic self-assembly is a dynamic process, the micellar aggregates present a dynamic structure in which the unimers are rapidly exchanged between micelles and the bulk solution (with lifetimes between 10 −5 and 10 −3 s) [6]. The shape and size of a given nanocarrier aggregate depends on the molecular geometry of its component surfactant molecules and the solution conditions, such as surfactant concentration, temperature, pH, and ionic strength [6,14]. Control over the shapes allows the possibility to develop and manipulate nanostructure architectures. According to Israelachvili et al. [6], a preliminary estimate of shape and size can be made with the analysis of the critical packing parameter Cpp = V0/A0·lc, where V0 is the effective volume occupied in the aggregate core region by hydrophobic chains, lc is the maximum effective length (critical chain length), and A0 is the effective hydrophilic head group surface area at the aggregate-solution interface. As schematically reported in Figure 1, with the increment of the Cpp value the structure of aggregates can range from spherical micelles (Cpp ≤ 1/3), to cylindrical micelles (1/3 ≤ Cpp ≤ 1/2), vesicles (1/2 ≤ Cpp ≤ 1), and lamellar structures (Cpp = 1) [6]. For larger values of the critical packing parameter Cpp the amphiphiles will assemble into "inverted" phases [6,14].

Soft Interactions Involved in Liposome Nanocarriers
Interactions and forces between colloidal nanocarriers in suspensions play an important role in determining the properties of the materials, such as their stability, shelf life, and transport properties. Despite the weakness of the forces involved, the relevant number of these soft interactions will produce an overall effect that is strong enough to hold different molecular subunits together as well as ensure their stability in solution. Moreover, the weakness of the involved interactions makes the structure more flexible, thereby enabling the system to withstand minor perturbation while preserving the reversibility of the self-assembled structure. The main weak (non-covalent) forces acting on soft nanocarriers are hydrogen bonding, hydrophobic effects, electrostatic interactions, Van der Waals forces, and steric interactions. A summary of the strength and characteristic ranges of the main soft interactions are reported in Table 1.

Soft Interactions Involved in Liposome Nanocarriers
Interactions and forces between colloidal nanocarriers in suspensions play an important role in determining the properties of the materials, such as their stability, shelf life, and transport properties. Despite the weakness of the forces involved, the relevant number of these soft interactions will produce an overall effect that is strong enough to hold different molecular subunits together as well as ensure their stability in solution. Moreover, the weakness of the involved interactions makes the structure more flexible, thereby enabling the system to withstand minor perturbation while preserving the reversibility of the self-assembled structure. The main weak (non-covalent) forces acting on soft nanocarriers are hydrogen bonding, hydrophobic effects, electrostatic interactions, Van der Waals forces, and steric interactions. A summary of the strength and characteristic ranges of the main soft interactions are reported in Table 1.

Hydrogen Bonding and Hydrophobic Forces
Hydrogen bonds are fundamental interactions for many relevant functions in bio-systems, as they are strong enough to bind biomolecules together but weak enough to be broken inside living organisms and cells when necessary. For example, water hydrogen bonding has many important implications on the properties of water and its relevant functions in biological systems [15]. In water the nonbonding electrons are the H-bond acceptors and the hydrogen atoms are the H-bond donors. This polarity within water molecules results in an attraction force between the negative portion (of oxygen) and positive partial charges (of hydrogen). H-bond acceptors and donors in biomolecules are due to the presence of molecular dipoles of hydrogen's atoms H (H-bond donors) bonded with electronegative atoms like oxygen O, nitrogen N, or fluorine F (H-bond acceptors). Hydrogen bonding is an important component of the major macromolecules in biochemistry (i.e., proteins, nucleic acids, and carbohydrates). The hydrogen bonding of proteins provides the organization for distinct folds and also provides the selectivity in protein-ligand interactions in molecular recognition processes. Apart from protein-ligand interactions, hydrogen bonds are implicated in many intermolecular interactions, including those involving protein-liposome, protein-protein, and protein-nucleic acid complexes [16]. Hydrogen bonds between complementary nucleotides favour the connection of two strands of a DNA helix together, where the complementary base pairs of guanine and cytosine (or adenine and thymine) are connected together by means of hydrogen bonding. Moreover, hydrogen bonding between phosphate groups on DNA nucleotides favours a helical structure conformation for two strands of DNA. Each base can also form hydrogen bonds with the external water mediated environment. Despite the softness/weakness of the (internal/external) hydrogen bond interactions, the presence of a large number of hydrogen bonds in biological macromolecules make them highly stable systems in different water environments. Another important interaction connected with the hydrogen bond is the hydration (or solvation) force [16][17][18]. The peculiar polarisation of water molecules near the hydrophilic solute interfaces sensitively alters the liquid structure with respect to the structure usually found in bulk water. [18,19].
The hydrophobic effect describes the unfavourable interaction of non-polar substances with water, and plays an important role in many soft nanocarrier systems as it regulates the tendency of non-polar (hydrophobic) molecules to self-aggregate [20]. When a hydrophobic compound is inserted in water the disruption of the H-bonding water network favourites a rearrangement of the water molecules around the non-polar molecules, with the creation of larger cavities to accommodate an assembly of non-polar (solute) molecules. In this case water molecules, distorted by the insertion of the hydrophobic component, will create new hydrogen bonds thus inducing an ice-like cage structure (called a clathrate) around the hydrophobic molecules. The hydrophobic interaction can be traced back to an effective (mutual) attractive hydrophobic force, between the surfaces of non-polar molecules in water. The effect of this interaction is formation of entropically more favourable aggregated structures generated to minimize the disruption of the water structure of water molecules migrating from the bulk water. Hydrophobic interactions play important roles in the intramolecular and intermolecular associations of biomolecules in various biological phenomena including protein folding and stabilization of macromolecules such as proteins and bio-membranes [16]. For example proteins, with both polar and non-polar side-chains, fold in water with the non-polar groups largely turned inward in order to avoid water [21,22].

Van der Waals Forces
The Van der Waals interaction is generated by permanent (or induced) dipoles within the nanocarriers that may result in net attractive forces between biological nano-components. This interaction is given by short-range repulsive forces and a long-range attractive London dispersion force generated by a temporary (short-lived) dipole induced by a polarisation of the electron distribution of an adjacent atom. For example, uncharged hydrophobic alkyl chains of phospholipids which have no permanent dipole are dominated by Van der Waals interactions which drive the packing of hydrophobic tails inside the bio-membrane bilayers [21].

Elctrostatic Interaction: Electrical Double Layer (EDL)
As the lipid bilayer represents the backbone structure of cell membranes, the functioning of cells is mainly related to electrostatic and electrochemical processes that occur at (or across) their lipid membranes. For example, liposomes' surface electrostatic potential has been shown to have a strong influence on the binding constant and uptake of active compounds, such as hematoporphyrin (a charged photosensitizer in photodynamic therapy of tumours) [9]. As the binding of active ionic species strongly affects the aggregation behaviour of cells, electrostatic interaction in charged liposomes represents a fundamental force for the investigation of bio-membrane/ion interactions.
Most liposome nanocarriers carry some surface charge in aqueous environments due to the ionization/dissociation of surface groups, or adsorption of charged molecules/ions to the nanocarrier surface. The nanocarrier surface charge is balanced by a cloud of counterions around the particle ( Figure 2). This cloud of charges, called an electrical double layer (EDL), is composed of a(n) (immobile) layer of ions strongly bound to the charged surface (Stern layer) and an adjacent region of loosely associated mobile ions (diffuse layer) [22]. Overlapping EDLs of two like-nanocarriers induces a repulsive force caused by the osmotic interactions between counterions. In a first approximation the thickness of the diffuse electric double layer is represented as the inverse of the Debye-Huckel screening constant κ which is determined, at a given temperature T, by the ionic strength I of the solvent (in mol/L) (where e is the unit of electron charge, K B the Boltzmann constant, and N a the Avogadro number) [22].

Steric Stabilization Forces
Nanocarriers in a biological environment very often undergo aggregation in specific solution conditions [11,22]. Nanocarrier stability can be enhanced through the addition of small amounts of polymer to the solution medium. Both synthetic polymers and biopolymers are widely used as additives against aggregation, thus leading to the steric stabilisation of a nanocarrier in a wide range The delivery mechanism and liposome efficiency in drug delivery processes are strongly influenced by the typology and density of the charge of the liposome surface through its ζ-potential [14,22]. A high electrostatic repulsion on the surface of charged liposomes prevents their aggregation and flocculation and could promote their interaction with cells [7][8][9][10][11]22]. Characterization of nanoparticle surface charge is usually done by measuring the particle zeta potential, which corresponds to the electrostatic potential of the nanocarriers measured at the so-called shear plane, (i.e., from the surface where ions are not bound to the particle) [22].

Steric Stabilization Forces
Nanocarriers in a biological environment very often undergo aggregation in specific solution conditions [11,22]. Nanocarrier stability can be enhanced through the addition of small amounts of polymer to the solution medium. Both synthetic polymers and biopolymers are widely used as additives against aggregation, thus leading to the steric stabilisation of a nanocarrier in a wide range of solvents conditions. In general, the magnitude of the force between (polymers coated) surfaces depends on whether the polymer is simply adsorbed or irreversibly grafted (stealth liposomes) onto the nanocarrier surface, on the coverage regime of polymer (grafting regime on each surface), and finally, on the solvent quality (poor or theta solvent conditions) and charging regime (ionic strength) [23]. Due to the different conditions of specific biological systems encountered, steric interaction is not straightforward to describe. The force law between two flat surfaces covered with so-called polymer brushes can be derived by theoretical approaches based on polymer-scaling concepts. More specifically, the steric repulsive interaction (as a function of the distance h) between two liposomes with polyethylene glycol (PEG) polymer layers of thickness L brush and radius R can be expressed through an exponentially decaying force law, according to the following equation [23]: for 0.4 L brush < h < 1.8 L brush , where L brush = (N EO¨l 5/3 /s 2/3 ). N EO is the number of monomers (-OCH 2 CH 2 -) in the PEG chain, l is the effective segment length, and s is the distance between the grafting points [23].

Structural Characterization of Liposome Nanocarriers
The biological behaviour of liposomes is strongly influenced by control parameters such as shape, size, morphology, surface characteristics, and lamellarity. Those features play a crucial role [8,14] as they influence both their biodistribution and passive targeting abilities upon drug administration. For these reasons, liposome size, colloidal stability, and aggregation properties (as well as their time evolution) should be probed (in situ) in solution conditions that reproduce standard administration conditions. Among the experimental methods used, the small-angle neutron scattering (SANS) and small angle X-rays scattering (SAXS) are the most important and widely utilized non-destructive experimental approaches for the structural investigation of liposome-based complex nanocarriers [24,25]. More specifically, the formation of different structures can be efficiently evidenced by scattering experiments, thus highlighting the important role of relevant molecular conformations in many different processes of drug delivery approach. Scattering methods provide the basic items to examine the structure and interactions of nanoparticles in solution and to study the growth process and conformational changes that are relevant for the understanding (and prediction) of functional processes in bio-nanotechnology [26,27]. Electron microscopy techniques, such as scanning electron microscope (SEM) and cryo-TEM (transmission electron microscope) (or TEM using freeze-fracturing approach) provide an accurate determination of liposome size, since they allow for the visualization of individual liposomes and can resolve particles of varying size, morphology, and lamellarity [28,29]. Another important microscopic technique utilized to analyze liposome morphology and size is the atomic force microscopy (AFM); a technique which provides high resolution information on the three-dimensional profile of liposomes without the necessity of removing them from their native solution environment [29]. Different techniques are available for the determination of size and size distribution of liposome nanocarriers, among which the most widely applied include dynamic light scattering and size-exclusion chromatography. In dynamic (or quasielestic) light scattering (DLS or QELS, respectively) measurements of the laser scattered intensity-intensity time correlation function g 2 (t) allow information to be obtained about the hydrodynamic radius R H of diffusing nanoparticles in solution from the analysis of translational diffusion coefficient D = K B T/6πηR H (where k B is the Boltzmann constant, T the absolute temperature, and η the viscosity of the solvent) [30]. Detailed information about the profile of the liposome population over the whole range of sizes is also acquired by means of the size-exclusion chromatography technique (SEC) [31]. This technique is able to separate and quantify liposome populations by exploiting the time-based resolution of hydrodynamic size, where the separation process is based on the elution velocity from a column equipped with a porous substrate. Alternatively, by means of the field-flow fractionation (FFF) separation techniques, a field (thermal-gradient, centrifugal, electrical, or magnetic) is applied perpendicularly to the flow of a fluid colloidal suspension pumped through a narrow channel, which causes the separation of the particles within the fluid, depending on their differing mobilities under the force field exerted. Finally, Zeta (ζ) potential measurements can be useful to address the electrostatic effects in charged nanocarriers by measuring the electrophoretic mobility µE using principles of phase analysis light scattering (PALS) [22,32]. By means of the measurement of the migration rate (i.e., velocity v) of charged nanoparticles in solution under the effect of an electric field E, an estimation of the electrophoretic mobility µE of the particles (i.e., µE = v/E) can be obtained, which allows for the calculation of the particles' zeta potential, ζ, and the nanocarriers' surface charge [32].

Passive Targeting in Nanocarrier Drug Delivery: Beyond the Biological Barriers
Passive targeting (i.e., when no specific targeting ligands are used) represents the major mechanism for many intravenously administered long-circulating liposome nanocarriers that satisfy the size constraints (i.e., 100-200 nm in diameter) and consequently are able to extravasate from vessels and translocate in tumour tissues, thus concentrating into the target site. Tumour (or inflammatory) tissues are characterized by increased vascular permeability. This effect is associated with malignant tissues that develop a discontinuous endothelium with large vascular fenestrations (pores) that allow liposomes of less than about 500 nm in size to diffuse outside the blood vessels (extravasation), thus entering the tumour interstitial space. The presence of pores (with diameters between 100 and 500 nm) induces extravasation of most drug-loaded liposomes of small dimensions [33,34]. After their administration, liposome nanocarriers are then sequestered from the circulation through fenestrations in their microvasculature, and will accumulate in the organs of the mononuclear phagocyte system (MPS) (also known as reticulo endothelial system, RES). Although the liver and the spleen exhibit the largest capacity for liposomal uptake (as they present sensitive blood irroration), liposome nanocarriers will also accumulate in minor quantity in the lungs, kidney, bone marrow, and lymph nodes. Liposomes undergo successive clearing in the MPS by resident macrophages via direct interactions with the phagocytic cells [33][34][35]. In addition, the interaction of liposomes with blood proteins plays a crucial role in the clearance process and tissue distribution of intravenously injected liposomes. During their circulation in the bloodstream, liposomes interact with plasma proteins, such as opsonins, high-density lipoproteins (HDLs), and low-density lipoproteins (LDLs) [36]. Opsonins include various protein types (such as immunoglobulin, fibronectin, lipoproteins) which help MPS to recognize and eliminate liposomes through the vesicle opsonization process, consisting of the adsorption of plasma proteins onto the phospholipid membrane. Elimination of liposomes takes place in different ways. A first way involves absorption of plasma proteins on the liposome surface followed by their recognition by the MPS. This results in the excretion of the drug cargo at the hepatic level and its successive metabolism by Kupffer cells. In a second way, liposomes are metabolized by the splenic macrophages. Finally, after their accumulation, liposomes are metabolized and eliminated by the target tissues. The clearance process by the MPS via serum proteins' opsonization depends on a variety of factors including size, surface charge, and colloidal stability [37]. Generally, large negatively charged liposomes are eliminated more rapidly than small, neutral or positively charged liposomes [37]. Concerning the high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs) contained in the blood, they interact with liposomes giving rise to a reduction of their colloidal stability. More specifically, the interaction with lipoproteins causes lipid transfers (and consequent lipid depletion) and structural changes on the surface of liposomes, followed by the liposome destruction and rapid release of the cargo to the plasma [36,37]. Interaction of liposomes with key blood proteins-such as immunoglobulins, complement proteins, apolipoproteins, fetuin, and thrombospondin-has been shown to play an important role in liposome recognition by specific phagocytes and (non-macrophage) hepatic cells [8]. In this case, the binding of blood proteins to liposomes strongly depends on the vesicles' properties, such as their morphology, surface curvature and charge, lipid bilayer composition, and packing [8]. Alternatively, non-opsonic blood proteins can be adsorbed onto particle surfaces and experience conformational changes that probably expose chemical groups that could either be recognized directly by certain phagocyte cell surface receptors or could act as ligands for subsequent recognition by blood opsonins. A number of specific plasma membrane receptors (such as lectin receptors, CD14 receptor), or various classes of scavenger receptors (e.g., classes A, B, and D), has been identified in this specific phospholipid recognition process [8].

Drug Delivery Mechanism
When a liposome interacts with a cell, the delivery of the drug and its distribution in the target cell can occur in several ways [33][34][35][36][37]. A first mechanism consists of liposome's adsorption into the cell membrane, where the lipid bilayer of the nanocarrier is degraded by mechanical strain or the action of enzymes. The active drugs will be released into the extracellular fluid, where they will diffuse through the cell membrane and the cytoplasm. A second way consists of the fusion of the liposomal membrane with the plasma membrane of the target cell, with the release of liposomal content directly into the cytoplasm [38]. Finally, the third way is the receptor-mediated endocytosis [39]. This frequent process only regards vesicles with a maximum diameter of 150 nm and the active ingredients that can endure the acidic environment of lysosomes (where liposomes undergo the enzymes' action) [40]. Phagocytosis can also occur, but involves liposomes larger than 150 nm that are phagocytosed by specialized cells of the immune system, (such as macrophages, monocytes, and Kupffer cells) [33][34][35][36][37].

Strategies to Prolong the Circulation Time
When injected into blood circulation liposomes rapidly interact with the biological system. The clearance of circulating liposomes nanocarriers from the bloodstream, coupled with their high uptake by the MPS, represent an obstacle to any attempt at targeting to tumours. In order to preserve the optimal liposome nanocarrier efficiency, the organism's defenses must be circumvented by avoiding the liposome nanocarrier recognition and the consequent neutralization and elimination of the invading active drugs. The physico-chemical properties of nanocarriers, such as size (and morphology) and surface functionality (charge typology, stiffness), are the main parameters that can affect their biological clearance. Different strategies can be adopted to prolong the circulation time, while the relevant mechanisms of stabilization can be exploited to improve colloidal stability in biological media [41,42].
A first approach to prolong the release rate of entrapped active substances consists of the choice of drugs with enhanced hydrophobic character. This characteristic promotes a slow release and prolongation in the liposome nanocarrier for up to several days. Moreover, prolongation in the circulation time (of several hours) in neutral liposomes has been obtained by incorporating cholesterol lipids [41,42]. Due to its hydrophobic character, cholesterol preferentially interacts with the core region of the membrane, thus inducing a dense packing of phospholipids. This reduces their permeability (bilayer-tightening effect) and increases their in vivo and in vitro stability, thus inhibiting their transfer Nanomaterials 2016, 6, 125 9 of 26 to high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs). Favourable results were also obtained with liposomes containing a small molar fraction of a negatively charged glycolipid, such as phosphatidylinositol. The bio-distribution of such nanocarriers evidenced therapeutic potential in cancer treatment for increasing the concentration of cytotoxic agents in tumour tissues while minimizing the likelihood of toxicity to the MPS [42].

Electrostatic Stabilization
One approach to stabilize liposome nanocarriers in solution consists of the inclusion of electrostatic repulsive forces by including charged components that confer a net (positive or negative) charge. Charged liposome have many advantages compared with neutral ones. For example, the presence of a surface charge induces electrostatic repulsion among liposomes by creating a sensitive ζ-potential that prevents aggregation or flocculation processes in solution. Moreover, a high surface charge could promote the interaction of liposomes with the cells [43]. For example, it has been shown that modification of the nanocarrier surface charge can influence the electrostatic interaction of the nanocarriers with components in the gastrointestinal tract following oral administration, thus conferring selectivity to diseased tissue [44]. Some investigations indicate that negatively charged liposomes are less stable than neutral and positively charged liposomes when injected into blood circulation, as they rapidly interact with the biological system following their opsonization with circulating proteins, thus inducing toxic effects and a rapid uptake by the MPS [45].
On the contrary, cationic liposomes are mainly preferred for gene delivery, based on the electrostatic interaction between phospholipids such as the lipid dioleoylphosphatidylethanolamine (DOPE) which have a positive charge from the amine head group (NH 3+ ) and negatively charged nucleic acids [46]. Generally, neutral phospholipids are, in most cases, also required for the stabilization of the liposome/DNA complex [47,48]. Moreover, they showed enhanced anti-tumour efficiency when associated with anti-tumoural drugs, such as Doxorubicin [49]. Cationic liposomes may also favour the interactions with the glycoproteins of the endothelial cell membranes [50] and the achievement of longer circulation half-lives [51]. Together with the number of advantages provided by the employment of cationic nanocarriers, some drawbacks should also be pointed out. The enhancement of the positive charge may favour, in fact, cationic liposome aggregation with anionic species present in the blood, thus inducing an unwanted enhanced uptake by the MPS and a diminished accumulation in tumour tissues [52]. Finally, it is worth noting that together with size-and coating-dependent uptake of nanocarriers by microvascular endothelial cells [53][54][55] that physical forces such as cyclic shear stress and cyclic stretch (under the physiological conditions of the pulsatile blood flow) may also affect cell-nanoparticle interactions resulting in reduced uptake into endothelial cells [56].
It is worth pointing out that in some specific cases the supramolecular self-assembly processes combined with cooperative charge effects may enhance the stability to the nanocarrier during their heterotopic aggregate formation. Investigation of a novel heterotopic nanoparticle formed through the electrostatic interaction between the anionic porphyrin 5,10,15,20-tetrakis(4-sulfonatophenyl)-21H,23H-porphine (TPPS) entangled in cationic modified amphiphilic cyclodextrins indicated the important role of nanocarrier charges in the modulation of colloidal stability in solution [57]. As reported in Figure 3, the morphological evolution of the heterotopic nanoparticle formation indicates that sensitive structural rearrangements, essentially driven by electrostatic interaction, are responsible for a significant increase of vesicle dimensions connected with a charge equilibration within the system. Further investigation in anti-cancer drug delivery experiments evidenced that, in the range of porphyrin/CD molar ratios between 1:10 and 1:50, the porphyrin is solubilized in monomeric form and photosensitizes the production of singlet oxygen ( 1 O 2 ) for cancer therapy treatments [58]. Moreover, at the same molar ratio the ability of the investigated amphiphilic cyclodextrin to transport porphyrins into tumour cells indicated specificity at the nuclear compartment level [58]. These findings are of paramount importance for the development of a new generation of biocompatible cyclodextrin-based smart liposome nanocarriers for the photodynamic therapy of tumours. It is worth stressing that combined steric and electrostatic interaction generated by drugs inclusion may induce structural transitions in liposomes that strongly influence the structural stability of the nanocarrier, as demonstrated by many studies performed in bio-membranes [57][58][59][60][61][62].
the range of porphyrin/CD molar ratios between 1:10 and 1:50, the porphyrin is solubilized in monomeric form and photosensitizes the production of singlet oxygen ( 1 O2) for cancer therapy treatments [58]. Moreover, at the same molar ratio the ability of the investigated amphiphilic cyclodextrin to transport porphyrins into tumour cells indicated specificity at the nuclear compartment level [58]. These findings are of paramount importance for the development of a new generation of biocompatible cyclodextrin-based smart liposome nanocarriers for the photodynamic therapy of tumours. It is worth stressing that combined steric and electrostatic interaction generated by drugs inclusion may induce structural transitions in liposomes that strongly influence the structural stability of the nanocarrier, as demonstrated by many studies performed in bio-membranes [57][58][59][60][61][62].

Steric Stabilization
Another important approach for the improvement of circulation times consists of the conjugation of hydrophilic polymers to the surface of the liposome nanocarriers. This approach is generally accomplished by the conjugation on the nanocarrier surface of natural (e.g., dextran, alginate, chitosan) or synthetic (e.g., poly(ethylene glycol), PEG; poly(vinyl alcohol), PVA; poly(vinyl pyrrolidone), PVP) polymers [62]. This approach allows most of the challenges encountered by liposome nanocarriers in drug delivery processes (such as the low blood circulation half-life, toxicity, interception by the immune system, biocompatibility, and antigenicity issues) to be overcome. Among the hydrophilic polymers, PEG represents the most widely used polymer conjugate. PEGylation of the liposome surface creates a local surface concentration of highly hydrated polymer brushes (Figure 4). This hydration shell around the particle sterically inhibits both hydrophobic and electrostatic interactions with plasma proteins or cells, thus reducing the liposomal uptake process by the MPS [63] interception by the immune system, biocompatibility, and antigenicity issues) to be overcome. Among the hydrophilic polymers, PEG represents the most widely used polymer conjugate. PEGylation of the liposome surface creates a local surface concentration of highly hydrated polymer brushes (Figure 4). This hydration shell around the particle sterically inhibits both hydrophobic and electrostatic interactions with plasma proteins or cells, thus reducing the liposomal uptake process by the MPS [63]  As a result, PEGylated liposomes are not opsonized and are able to escape the capture by the cells' phagocytic systems by rendering the nanocarriers invisible to macrophages ("Stealth liposomes"). Many studies demonstrated that PEGylated liposomes were able to improve the stability and blood-circulation time, together with low plasma clearance, and low volume of distribution (with minimal interaction with non-tumoural tissues) [64][65][66]. Chen et al. [67] showed that plasma protein adsorption onto nanocarrier surfaces on PEG-coated nanocarriers strongly depends on the PEG chain length and density at the nanocarrier surface.
It is worth noting that the ability of PEG to increase the circulation lifetime of the vehicles strongly depends on both the amount of grafted PEG and the length or molecular weight of the polymer [64], while high molecular weight of entangled PEG chains may prefer aggregation in large clusters [68]. Interpenetration of long hydrophilic PEG chains belonging to different nanocarriers may cause, in fact, the depletion of the solvent in the outer layer of the nanocarrier, thus favouring the formation of larger clusters of entangled aggregates [69,70]. Temperature-dependent attractive interaction arising from the synergistic effects of different coexisting interactions in the system [71,72] can be exploited for triggering the assembly/disassembly process in PEG-based nano-aggregates. Interestingly, recent computer simulations and fluorescence experiments [73] evidenced that a model hydrophobic drug 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP), besides being As a result, PEGylated liposomes are not opsonized and are able to escape the capture by the cells' phagocytic systems by rendering the nanocarriers invisible to macrophages ("Stealth liposomes"). Many studies demonstrated that PEGylated liposomes were able to improve the stability and blood-circulation time, together with low plasma clearance, and low volume of distribution (with minimal interaction with non-tumoural tissues) [64][65][66]. Chen et al. [67] showed that plasma protein adsorption onto nanocarrier surfaces on PEG-coated nanocarriers strongly depends on the PEG chain length and density at the nanocarrier surface.
It is worth noting that the ability of PEG to increase the circulation lifetime of the vehicles strongly depends on both the amount of grafted PEG and the length or molecular weight of the polymer [64], while high molecular weight of entangled PEG chains may prefer aggregation in large clusters [68]. Interpenetration of long hydrophilic PEG chains belonging to different nanocarriers may cause, in fact, the depletion of the solvent in the outer layer of the nanocarrier, thus favouring the formation of larger clusters of entangled aggregates [69,70]. Temperature-dependent attractive interaction arising from the synergistic effects of different coexisting interactions in the system [71,72] can be exploited for triggering the assembly/disassembly process in PEG-based nano-aggregates. Interestingly, recent computer simulations and fluorescence experiments [73] evidenced that a model hydrophobic drug 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP), besides being embedded deep within the hydrophobic core of the liposome, is also able to be wrapped by the hydrophilic PEG layer surrounding the lipid molecules, thus improving the drug loading efficiency of the liposome [73]. It is worth noting that the enhanced steric stability of PEG-bearing liposomes can have the drawback of a reduced ability to interact with the intended targets or not being able to completely avoid cumulative uptake by cells, as evidenced by a recent published review in this area [74].

Liposome-Liposome Interaction and Molecular Recognition Processes
Fusion and fission of vesicles are particularly important phenomena since they are at the basis of biological membrane processes such as vesicle-cell membrane fusion, endocytosis, exocytosis, and cell fission (cell division) [75,76]. Adhesion and fusion of biological membranes are mediated by non-covalent protein-protein and protein-carbohydrate interactions that may involve a range of receptor-mediated structural modifications involving tissue integrity and cellular rearrangements.

Liposome Aggregation-Fusion
Depending on the biological solution condition encountered, mutual liposome interaction can give rise to aggregation (or clustering), fusion, and fission processes [75,76]. In aggregation processes vesicles aggregate into large clusters, where each vesicle structure remains intact while its drug contents do not mix or leak out. On the contrary, in fusion processes vesicles merge into large vesicles, while their components mix, sometimes with leakage. Finally, in fission processes small vesicles are formed from large ones. Aggregation and fusion can be considered spontaneous processes occurring at slow rates and may eventually lead to flocculation and precipitation of the liposome. These interaction processes can also be induced by a number of external/internal factors (stimuli-responsive liposomes) [77][78][79]. Stimuli-responsive liposomes regulate the stability/permeability of the liposome nanocarrier by the inclusion of a triggerable component which has the task of reacting when triggered by stimuli from target tissues (pH, redox potential) or applied from outside the organism (light, temperature, hyperthermia, ultrasound, and (electro)magnetic field) [77]. For example, a pH-sensitive liposome which is stable at the physiological pH encountered in regular tissues will typically acquire fusogenic properties under the acidic environment generally encountered in pathological tissues, thus releasing its active drugs to nearby tumours and infected tissues.

Governing the Colloidal Stability in Bio-Systems
Experimental assessment of the colloidal stability in the biological environment is not a straightforward task and a wide range of different components further contributes to this complexity. When nanocarriers are delivered into cell culture media their properties are strongly influenced by the high ionic content that, very often, gives rise to an aggregation processes due to the attenuation of the screened electrostatic interactions and consequent suppression of the stabilizing effect of the electric double layer (EDL). Consequently, the destabilized suspension will rapidly collapse and the nanocarriers will aggregate.
A classical theoretical approach to determine the stability of a colloidal nanocarrier in solution is given by the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory [78] in a pioneering work describing the stability of colloids in solution. According to this model, the interaction potential between two identical nanoparticles (i.e., spherical macroions of diameter σ = 2R) placed at a distance r (centre to centre distance) is determined by the balance between the Van der Waals attractive forces, V A , which is expressed as a function of the A which is the Hamaker constant (which depends on the dielectric constant mismatch between the particles and the surrounding fluid [22,78]).
and the screened Coulomb (repulsive) interaction V R described by the following equation: e is the unit of electron charge, K B the Botzmann constant, N a the Avogadro number, and κ is the Debye-Huckel constant (equation 1). A sketch of the DLVO interaction energy as a function of particle separation is reported in Figure 5. The presence of an energy barrier resulting from the repulsive force prevents two particles adhering together while approaching one another. Particles with a sufficiently high repulsion can avoid flocculation and the colloidal system will be stable. On the other hand, particles colliding with sufficient energy are able to overcome that barrier, thus favouring an aggregation process between particles. In certain solution conditions (e.g., at high ionic strength) there is a possibility of a "secondary minimum" where a weaker and reversible adhesion between particles may exist. These weak aggregates, although sufficiently stable against Brownian motion, may dissociate under an externally applied perturbation (i.e., due to a temperature increase).
force prevents two particles adhering together while approaching one another. Particles with a sufficiently high repulsion can avoid flocculation and the colloidal system will be stable. On the other hand, particles colliding with sufficient energy are able to overcome that barrier, thus favouring an aggregation process between particles. In certain solution conditions (e.g., at high ionic strength) there is a possibility of a "secondary minimum" where a weaker and reversible adhesion between particles may exist. These weak aggregates, although sufficiently stable against Brownian motion, may dissociate under an externally applied perturbation (i.e., due to a temperature increase). As previously stated, the adsorption of synthetic polymers or biopolymers onto the nanocarrier's surface can lead to a steric stabilization of the colloid that prevents two particles from forming attractive Van der Vaals interactions. It is worth pointing out that a Yukawa type potential is an adequate mathematical form to also properly describe the steric repulsion of polymer grafted nanocarriers. Moreover, the total interaction potential between nanocarriers in solution may be approximated by using two separate Yukawa potentials accounting for both the attraction and As previously stated, the adsorption of synthetic polymers or biopolymers onto the nanocarrier's surface can lead to a steric stabilization of the colloid that prevents two particles from forming attractive Van der Vaals interactions. It is worth pointing out that a Yukawa type potential is an adequate mathematical form to also properly describe the steric repulsion of polymer grafted nanocarriers. Moreover, the total interaction potential between nanocarriers in solution may be approximated by using two separate Yukawa potentials accounting for both the attraction and repulsion present in the system. This approach has been recently used to model, by small-angle neutron scattering (SANS) experiments, both the attractive and repulsive parts of the interaction between nanoparticles [79].
Despite the important information provided, the application of the DLVO theory for the analysis of the interactions in therapeutic nanocarrier systems in solution has the difficulty associated with having to rationalize the ionic strength parameters in the presence of charged components from a buffer solution. This is partially attributed to the difficulty of the theory to correctly describe the behaviour of the interface between particles and media, but also to the intrinsic experimental inaccuracy in the values of parameters, such as surface charge density, ligand density, and adsorption processes commonly encountered in drug delivery processes. SAXS and SANS structure factor S(q) analysis performed in the framework of liquid state theory [80] has been employed to investigate the interparticle interactions in different colloidal systems, including micelles, liposomes, dendrimers and protein [81][82][83][84].

Hydrogen Bonding Mediated Interactions between Liposomes and Molecular Recognition Processes
Interactions between liposomes at the molecular level through recognition processes outline strong similarities to the analogous cells incorporating recognizable amphiphiles in bio-membranes, and thus represent an important strategy for modeling the molecular recognition processes occurring between cells [85].
Particularly interesting in this respect are the molecular recognition processes in bio-membranes through the hydrogen bonding interaction. Hydrogen bonding plays a key role in the molecular recognition of carbohydrates by membrane proteins. As an example, in the formation of ligand-protein complex, the binding of the ligand to its specific site will be favoured if the energy of the hydrogen bonds in the complex and the entropy gain in releasing some bound water molecules are more favourable than the free-energy contribution of the hydrogen bonds between the binding partners and these water molecules (in their free non-bonded state) ( Figure 6). The introduction to liposome systems of hydrogen bonds forming functional groups can promote liposome bridging, thus leading to the formation of large aggregates. This process is sometimes followed by the liposome fusion in which the liposomes merge, and thereby share a common inner compartment. This second stage gives rise to the mixing of the aqueous content of liposomes with (or without) rupturing or leakage of the fused vesicles. It is worth noting that while vesicles aggregated by nonspecific forces, such as Van der Waals, are deformed and stressed, producing unstable structures, site-specific ligand-receptor coupling (such as biotin-streptavidin) promotes a secondary self-assembly into higher order structures composed of tethered (rather than adhering) vesicles in their original, unstressed state, as demonstrated by a Cryoelectron microscopy investigation [86]. Vesicle association by site-specific binding provides then an interesting mechanism for the production of easily controllable, stable, and reversible nano-biomaterials. As described by S Chiruvolu, et al. [86], hydrogen bonding can be used to aggregate vesicles into higher order structures in a reversible manner by means of site-specific ligand-receptor (biotin-streptavidin) coupling (Figure 7). In that case, biotin and streptavidin formed a very strong non-covalent complex (Ka « 10 15 M´1) by multiple tight hydrogen bonds.  Mixed liposome formed by PEGylated distearoylphosphatidylethanolamine lipid (PEG-DSPE) and biotin-(CH2)n-hexadecylphosphatidylethanolamine amphiphile showed the ability to form extended clusters of interconnected liposomes [87]. More specifically, avidin-coated liposomes interacted through biotin bridges leading to liposome-liposome adhesion. In this case, the insertion of fixed amounts of PEG (with molecular weight 750) linked at the head group of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipids created a repulsive steric barrier in order to accomplish the important function of eliminating non-specific liposome adhesion (Figure 8). It was found that the rate of avidin absorption decreased with the increasing amount of PEG750 introduced, while a 10 mol % PEG750 completely suppressed the binding of avidin to biotin. Mixed liposome formed by PEGylated distearoylphosphatidylethanolamine lipid (PEG-DSPE) and biotin-(CH 2 ) n -hexadecylphosphatidylethanolamine amphiphile showed the ability to form extended clusters of interconnected liposomes [87]. More specifically, avidin-coated liposomes interacted through biotin bridges leading to liposome-liposome adhesion. In this case, the insertion of fixed amounts of PEG (with molecular weight 750) linked at the head group of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipids created a repulsive steric barrier in order to accomplish the important function of eliminating non-specific liposome adhesion (Figure 8). It was found that the rate of avidin absorption decreased with the increasing amount of PEG750 introduced, while a 10 mol % PEG750 completely suppressed the binding of avidin to biotin. An investigation evidenced how cyanuric acid (CA) and melamine (M) functionalized lipids promote the formation of bio-membranes that exhibit intense hydrogen-bond driven surface recognition in water, with variable hydration at the lipid-water interface. This system guided recognition at the lipid-water interface using cyanurate-melamine hydrogen bonding (incorporated at 0.1-5 mol percent) in fluid phospholipid membranes, and was able to induce both vesicle-vesicle An investigation evidenced how cyanuric acid (CA) and melamine (M) functionalized lipids promote the formation of bio-membranes that exhibit intense hydrogen-bond driven surface recognition in water, with variable hydration at the lipid-water interface. This system guided recognition at the lipid-water interface using cyanurate-melamine hydrogen bonding (incorporated at 0.1-5 mol percent) in fluid phospholipid membranes, and was able to induce both vesicle-vesicle binding (with multivalent surface clustering) and membrane fusion [88]. Further, an interesting example of multivalent and selective hydrogen bonding is provided by nucleic acids. For example, peptide nucleic acids (PNA) and deoxyribonucleic acids (DNA) may be anchored to liposomes by properly attaching hydrophobic blocks. This approach allows for the investigation of hybridization interactions with complementary strands of DNA by the binding of DNA to the PNA liposomes [89]. Stengel et al. reported fusion of liposomes induced by DNA hybridization [90], where some specific forced bilayer contacts are able to trigger vesicle fusion (Figure 9). This last example provides evidence once again for how receptors located at the surface of biological cells may facilitate adhesion or fusion with other cells. The prominent role of those functional groups, which render liposomes recognizable through hydrogen bonding, can be exploited to mimic analogous interactions occurring between cells. Morphological transition induced by molecular recognition is at the basis of intense investigations concerning protein-drug interactions and for understanding the mechanism behind versatile bioactivities of drugs [91][92][93][94].

Liposome-Drug Interaction: Encapsulation and Delivery Approaches
Liposomal nanocarriers are able to protect the drug cargos from undesired side effects or degradation before reaching target sites and reduce systemic toxicity of drugs and therapeutic agents. The delivery of liposome-encapsulated drugs depends on the structure and characteristics of the (host) liposomes, the size and nature of the (guest) drug molecules, and their mutual interactions. Many strategies can be adopted to improve both drug encapsulation and delivery efficiency to the target site in an effort to avoid undesired side effects. We will shortly review, in the following sections, the main strategies adopted to optimize the drug release with improved bioavailability in tumour tissues.

Hydrophilic/Hydrophobic Drug Encapsulation and Release Properties
The encapsulation of drugs into the bilayer of liposomes, while facilitating their solubilisation in aqueous media, also provides additional protection and control against drug degradation. These conditions implement a sensitive amelioration in the toxicity profiles with improved therapeutic efficacy with respect to the unencapsulated ones, as evidenced by several clinical studies [95][96][97]. Hydrophilic drugs are localized in the aqueous core region or near the liposome hydrophilic head groups (water-lipid interface) with an encapsulation efficiency that depends on the volume of the aqueous phase inserted during the liposome formation process. The encapsulation efficiency of

Liposome-Drug Interaction: Encapsulation and Delivery Approaches
Liposomal nanocarriers are able to protect the drug cargos from undesired side effects or degradation before reaching target sites and reduce systemic toxicity of drugs and therapeutic agents. The delivery of liposome-encapsulated drugs depends on the structure and characteristics of the (host) liposomes, the size and nature of the (guest) drug molecules, and their mutual interactions. Many strategies can be adopted to improve both drug encapsulation and delivery efficiency to the target site in an effort to avoid undesired side effects. We will shortly review, in the following sections, the main strategies adopted to optimize the drug release with improved bioavailability in tumour tissues.

Hydrophilic/Hydrophobic Drug Encapsulation and Release Properties
The encapsulation of drugs into the bilayer of liposomes, while facilitating their solubilisation in aqueous media, also provides additional protection and control against drug degradation. These conditions implement a sensitive amelioration in the toxicity profiles with improved therapeutic efficacy with respect to the unencapsulated ones, as evidenced by several clinical studies [95][96][97]. Hydrophilic drugs are localized in the aqueous core region or near the liposome hydrophilic head groups (water-lipid interface) with an encapsulation efficiency that depends on the volume of the aqueous phase inserted during the liposome formation process. The encapsulation efficiency of hydrophobic drugs strongly depends on the length and packing density of the liposome acyl chains that host them, while an increased loading ability requires an increase in the bilayer lipophilic volume by choosing, for example, longer alkyl chain lipids [96,97]. Many anti-cancer drugs are of intermediate solubility, they undergo then a partition between the hydrophobic interior of the bilayer and the exterior (or interior) liposome aqueous phase. Liposome permeability plays a significant role in the encapsulation efficiency of hydrophobic/hydrophilic drugs, with the liquid crystalline phase that generally supports a more permeable character to the encapsulated drugs than the corresponding gel state [97]. The inclusion of macromolecules may also reduce lipid ordering giving a higher fluidity to the membrane and, in some cases, also inducing a local perturbation/disruption of the bilayer structure [98]. It is possible to switch from a fluid-phase to a solid-phase bilayer by incorporating cholesterol into liposomes (bilayer-tightening effect). The incorporation of cholesterol into liposomes stabilizes the lipid bilayer by reducing the rotational freedom (by increasing the orientation order) of the phospholipid hydrocarbon chains, thus inducing a more compact structure (with a reduced permeability) that causes an increased retention of the drug cargo in the liposomes.

Triggered Drug Release from Stimuli Responsive Liposomes (SRL)
Triggered drug release in a specific target tissue can be obtained with the inclusion of a triggerable component that allows liposome stability (and permeability) to be regulated by perturbing the structural conformation of the liposome nanocarrier (stimuli-responsive liposome, SRL) [99]. In this case liposomes are able to react when activated by internal stimuli coming from target tissues (such as pH, ionic strength, redox potential, enzymes) or applied external stimuli coming from outside the organism (such as hyperthermia, ultrasound, and electro-magnetic fields). For example, pH-sensitive liposomes which are stable at the physiological pH of normal tissues, will be subjected to destabilization under acidic environment encountered generally in pathological tissues, thus facilitating the release of its active components nearby tumours or inflammation/infection regions, thus increasing the therapeutic efficacy [99].
An interesting class of triggerable liposomes are the thermosensitive liposomes (TSL), where the lipid structure can be modified by the presence of temperature-sensitive lipids [100][101][102]. Temperature-sensitive liposomes release their encapsulated drugs at the melting-phase transition temperature (T m ), where the liposome permeability is influenced by the transition from the gel to the liquid crystalline phase of the lipid bilayer. For example temperature-sensitive liposomes often include dipalmitoylphosphatidylcholine as key lipid component, which presents a gel-to-liquid crystalline phase transition occurring at T = 41˝C. An example of a low temperature triggered TSL (LTSL), which has been developed by Celsion Corporation, is given by ThermoDox, a thermosensitive liposomes formulation containing Doxorubicin drug, and composed of liposomes 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC): mono steroyl PC (MSPC): PEG2000-DSPE (86:10:4, molar ratio) [100]. ThermoDox in combination with hyperthermia (HT) treatment (involving the heating of tumours to temperature of up to 42˝C) has been tested in clinical trials against liver cancer [101]. A recent study evidenced that the use of high temperature triggered TSL (HTSL) formulation with less MSPC lipid content and a higher phase transition temperature of T m = 44˝C gives higher stability and less content leakage compared to conventional LTSL, thus suggesting that the T m of TSL should be increased to obtain improved drug delivery efficiency to tumour [102]. It is worth noticing that in most cases the employment of SRL raised critical issues concerning the potential toxicity of liposome synthetic components and therapeutic efficiency (e.g., difficulty of tissue location and remote triggering) [100].

Drug Delivery with Targeted Liposome (Active Targeting)
A promising approach to increase the specificity of drug delivery with minimal accumulation in non-target sites is obtained by conjugating, to the liposome surface, a specific ligand which is able to bind selectively to a given receptor on the target cell (active targeting) [99,103]. As previously stated, the ligand-receptor bindings are driven by reversible intermolecular forces, such as ionic and Van der Waals interactions or hydrogen bonds that ensure the reversibility of the association/dissociation of docking. A large variety of ligands (such as proteins, antibodies, peptides, lectins, aptamers, and vitamins) which can bind to overexpressed receptors on tumour tissues, have been investigated as biomarkers for targeted drug delivery [103]. Folic acid (FA), one of the most investigated targeting ligands, has a high binding affinity to folate receptors (FR), which are overexpressed in a wide range of FR-positive tumour types, including ovarian, lung, breast, brain, kidney, and colon cancers. Targeted liposomes, incorporating FA, tend to bind selectively to the FR and, by triggering cellular uptake via endocytosis, provide then a highly selective drug delivery strategy [104].
Recently, targeting a long-circulating PEGylated liposomal prodrug formulation of anti-tumour Mitomycin C to the folate receptor has been shown to enable a high liposomal drug payload and result in a stable formulation with reduced toxicity [105]. Moreover, stable Baicalin loaded folate-receptor-targeted liposomes showed higher cytotoxicity and cell uptake compared with non-targeted liposomes [106]. Interestingly, by introducing sliding tethered ligands with self-regulating length, additional topological interactions to the toolbox of ligand-receptor design have been recently demonstrated by Marques et al. [107], thus paving the way for the development of versatile and reusable bio-adhesive substrates for drug delivery and tissue engineering applications. Targeted liposomes have recently been the basic platform for the development of multifunctional prototypes containing diagnostic and therapeutic agents entrapped within the same theranostic nanocarriers [108]. For example, a new (theranostic) nanocarrier for tumour-specific imaging and therapy based on folate-conjugated liposome incorporating Ru(II) polypyridyl complexes was recently proposed [109]. In that case, the interaction between the vehicle and target HeLa cells (that overexpressed the folate receptor) brought to cell death by being exposed to laser irradiation contributed to the effective delivery of [Ru(phen) 2 (dppz)] 2+ to the cytoplasm and subsequent photoinduced interaction. This approach demonstrates that prominent tumour selectivity and therapeutic ability can be achieved by the successful combination of tumour specificity of folate-conjugated liposomes with the imaging and therapeutic potential rendered by [Ru(phen) 2 (dppz)] 2+ . Although several innovative configurations for targeted delivery of drugs have been developed in nanomedicine at a preclinical level, up to now few have been admitted in clinical trials, while none of them have been approved for the market yet, as we will see later [110,111].

Market and Clinical Development of Liposome-Based Drugs Formulations
Research and application of innovative solutions of liposome formulations, which have progressed toward a new generation of liposomes, are devoted mainly to cancer treatment and are mainly administrated intravenously, due to the high degradation of lipids in the gastrointestinal tract [110,111]. A special focus of the research is devoted to the investigation of the correlation of the blood circulation time and drug accumulation in target tissues with the specific physico-chemical properties of liposomal formulations investigated. Anti-cancer drugs Doxorubicin, Daunorubicin, Cisplatin, Paclitaxel, and Vincristine ( Figure 10) are among the most extensively investigated agents for the liposome-based drugs formulations, and several liposomal formulations of these agents are currently in clinical use in cancer therapy. [110,111]. A special focus of the research is devoted to the investigation of the correlation of the blood circulation time and drug accumulation in target tissues with the specific physico-chemical properties of liposomal formulations investigated. Anti-cancer drugs Doxorubicin, Daunorubicin, Cisplatin, Paclitaxel, and Vincristine ( Figure 10) are among the most extensively investigated agents for the liposome-based drugs formulations, and several liposomal formulations of these agents are currently in clinical use in cancer therapy. Doxorubicin (or Anthracycline) is a potent chemotherapy drug largely employed in the treatment of a wide range of cancers, including blood malignant cancers (such as leukaemia and lymphoma), many types of solid (carcinoma), and soft (sarcomas) tissue tumours [110,111]. It slows (or stops) the growth of cancer cells by blocking isomerase 2, an enzyme that cancer cells need in order to divide and grow. In order to prolong the drug circulation time in the bloodstream and to avoid the MPS system, the doxorubicin is generally contained in PEGylated liposome nanocarriers. For example, a widely employed PEGylated liposomal doxorubicin formulation for the treatment of epithelial ovarian Kaposi's sarcoma is Doxil, composed of hydrogenated soy L-α- Doxorubicin (or Anthracycline) is a potent chemotherapy drug largely employed in the treatment of a wide range of cancers, including blood malignant cancers (such as leukaemia and lymphoma), many types of solid (carcinoma), and soft (sarcomas) tissue tumours [110,111]. It slows (or stops) the growth of cancer cells by blocking isomerase 2, an enzyme that cancer cells need in order to divide and grow. In order to prolong the drug circulation time in the bloodstream and to avoid the MPS system, the doxorubicin is generally contained in PEGylated liposome nanocarriers. . Moreover, DaunoXome is a liposomal Daunorubicin formulation employed for the treatment of blood tumours, composed of DSPC and cholesterol (2:1 molar ratio). Finally, the Marqibo formulation, which is a liposome-based nanocarrier for the Vincristine chemotherapy drug, is employed for the treatment of metastatic malignant uveal melanoma. The previous five liposome-based drug formulations have been approved for intravenous application by the US Food and Drug Administration (FDA), and are already present in the market [110,111].
Other anti-cancer drugs formulated with liposomes are approved for (different stages) clinical studies. For example, a temperature-responsive version of PEGylated formulation of liposomal doxorubicin is provided by the nanocarrier Thermodox (composed of DPPC, mono steroyl PC (MSPC), and PEG2000-DSPE). Thermodox represents a triggerable release formulation, in phase III clinical trials, which is able to release its contents inside the tumour upon heat transfer, using a radio frequency ablation (RFA) process.
Other important liposome nanocarriers include liposomal formulations for the Paclitaxel (or Taxol), a potent anti-cancer drug used to treat ovarian, breast, lung, and pancreatic cancers [110,111]. Two important Paclitaxel formulations, still in clinical trials, are LEP-ETU (composed of DOPC lipids, cholesterol, and cardiolipin) employed for treatment of ovarian, breast, and lung cancers and EndoTAG-1 (composed of N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) and DOPC lipids) employed in anti-angiogenesis, breast, and pancreatic cancers treatments. Finally, Lipoplatin and SPI-077, which are liposomal formulations for the Cisplatin chemotherapy drugs, are in clinical trials for the treatment of head/neck and lung cancers [110,111].

Future Perspectives: The Theranostic Approach for Cancer Treatment
Liposome-based nanocarriers have been the longest-studied platform and have attracted considerable attention for their extensive applications in biotechnology, drug, and gene delivery. Recent developments in nanoscience stimulated the combination of the simple approaches of liposome technology with the advanced concept of supramolecular self-assembly for the development of more complex, hierarchical nanostructures [108,112,113]. The introduction of multi-functional, multi-component formulations that perform (within the same liposome theranostic platform) different functions, such as the delivery of combinations of therapeutic drugs, site-specific targeting/imaging capabilities, and/or stimuli responsive assembly/disassembly processes to control drug release, allows for the development of a large variety of theranostic systems capable of performing a defined set of bio-medical tasks ( Figure 11). Owing to a facile modulation of their size, hydrophobic/hydrophilic character, low toxicity, and biocompatibility, liposomal nanocarriers still represent one of the best platforms that continues to attract the renewed interest of researchers who, through combining basic principles of soft-matter assembly with the more advanced concepts of supramolecular (bio-)chemistry, work towards designing and developing complex theranostic nanocarriers [108,114]. There is no doubt that, with additional understanding of the main soft interactions involved in basic processes of drug delivery, it will be possible to achieve a deeper insight into the structure-function relationship involved in relevant pharmacokinetic processes. Those circumstances will inspire the development of novel biomimetic strategies that can be translated into practical therapeutics for clinic and market development. Owing to a facile modulation of their size, hydrophobic/hydrophilic character, low toxicity, and biocompatibility, liposomal nanocarriers still represent one of the best platforms that continues to attract the renewed interest of researchers who, through combining basic principles of soft-matter assembly with the more advanced concepts of supramolecular (bio-)chemistry, work towards designing and developing complex theranostic nanocarriers [108,114]. There is no doubt that, with additional understanding of the main soft interactions involved in basic processes of drug delivery, it will be possible to achieve a deeper insight into the structure-function relationship involved in relevant pharmacokinetic processes. Those circumstances will inspire the development of novel biomimetic strategies that can be translated into practical therapeutics for clinic and market development.

Conclusions
We presented recent advances in liposome based drug nanocarriers, by analyzing the relevant soft interactions involved in drug delivery processes which are responsible for the main behaviour of soft nanocarriers in complex physiological fluids. Relevant strategies to overcome the biological barriers during nanocarrier drug delivery are presented, outlining the main structure-properties relationship as well as their advantages (and drawbacks) in therapeutic and biomedical applications. The investigation of the soft interactions between liposomes at the molecular level can be considered to be an important framework for the modeling of the molecular recognition processes occurring between cells. These relatively weak interactions can be amplified by multiple binding at the liposome surface in multivalent complexes. While considerable research effort on the theoretical side is certainly necessary, further investigations of physico-chemical properties are required with different/complementary experimental methods in order to critically evaluate the stability of the nanocarriers in simulated environmental solutions which are close to the physiological fluid conditions.