pharmaceutics
Review Evolution from Covalent to Self-Assembled PAMAM-Based Dendrimers as Nanovectors for siRNA Delivery in Cancer by Coupled in Silico-Experimental Studies. Part II: Self-Assembled siRNA Nanocarriers
Erik Laurini, Domenico Marson * , Suzana Aulic, Maurizio Fermeglia and Sabrina Pricl
Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTS), Department of Engineering and Architecture, University of Trieste, 34127 Trieste, Italy * Correspondence: [email protected]; Tel.: +39-040-558-3750
Received: 21 June 2019; Accepted: 8 July 2019; Published: 10 July 2019
Abstract: In part I of this review, the authors showed how poly(amidoamine) (PAMAM)-based dendrimers can be considered as promising delivering platforms for siRNA therapeutics. This is by virtue of their precise and unique multivalent molecular architecture, characterized by uniform branching units and a plethora of surface groups amenable to effective siRNA binding and delivery to e.g., cancer cells. However, the successful clinical translation of dendrimer-based nanovectors requires considerable amounts of good manufacturing practice (GMP) compounds in order to conform to the guidelines recommended by the relevant authorizing agencies. Large-scale GMP-standard high-generation dendrimer production is technically very challenging. Therefore, in this second part of the review, the authors present the development of PAMAM-based amphiphilic dendrons, that are able to auto-organize themselves into nanosized micelles which ultimately outperform their covalent dendrimer counterparts in in vitro and in vivo gene silencing.
Keywords: RNAi therapeutics; siRNA delivery; amphiphilic dendrons; PAMAM dendrimers; self-assembling; nanovectors; gene silencing
1. Self-Assembling PAMAM-Based Amphiphilic Dendrons: A New Paradigm for siRNA Delivery In its broadest sense, self-assembly describes the natural tendency of physical systems to exchange energy with their surroundings and assume patterns or structures of low free energy. Random thermal motions bring constituent particles together in various configurations, and only those with significantly favorable interaction energy forming, tend to persist, and eventually become predominant. The information on the shape and size of the ultimate self-assembled entity is embodied in the structures of the individual components. A system slowly approaching equilibrium assumes a simple repetitive structure, while a dynamic system may generate structures of great complexity. For example, molecules in a cooling glass of water self-assemble as simple ice crystals, while the same molecules in a turbulent cloud with temperature and humidity gradients self-assemble as complex snowflakes of enormous variety. In relation to chemistry, self-assembly constitutes the quintessence of nanotechnology-based techniques leading to the design of novel materials [1–4]. It relies on the cumulative effects of multiple non-covalent interactions to assemble molecular building blocks into supramolecular entities in a reversible, controllable, and specific way, yet with relatively little synthetic effort. In particular is the ability of self-assembled structures to behave as more than the sum of their individual parts, and exhibit completely new properties [5].
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Pharmaceutics 2019, 11, x FOR PEER REVIEW 2 of 26 With these concepts in mind, the authors envisaged the idea of creating small amphiphilic poly(amidoamine)With these (PAMAM)-based concepts in mind, dendrons the authors which, envi uponsaged auto-organizationthe idea of creating intosmall nanosized amphiphilic micelles, could mimicpoly(amidoamine) the covalent, (PAMAM)-based high-generation dendrons dendrimer which, upon counterparts auto-organization in size, into shape nanosized and function, in particularmicelles, for could in vitro mimic and the in vivocovalent, siRNA high-generat deliveryion [6 –dendrimer8], as set outcounterparts below. in size, shape and function, in particular for in vitro and in vivo siRNA delivery [6–8], as set out below. 2. siRNA Delivery by Single-Tail Self-Assembling Amphiphilic Dendrons 2. siRNA Delivery by Single-Tail Self-Assembling Amphiphilic Dendrons
2.1. Design,2.1. Design, Optimization Optimization and and Chemico-Physical Chemico-Physical Characterization Characterization of ofSingle-Tail Single-Tail Self-Assembling Self-Assembling AmphiphilicAmphiphilic Dendrons Dendrons Our workOur in work this fieldin this started field started with the with design, the design optimization, optimization andsynthesis and synthesis of a seriesof a series of amphiphilic of dendronsamphiphilic (1–6) characterized dendrons (1– by6) characterized a single hydrophobic by a single hydrophobic alkyl chain alkyl of variable chain of lengthvariable and length a hydrophilic and PAMAMa hydrophilic head with PAMAM 8 primary head aminewith 8 primary terminal amine groups terminal [7], groups as shown [7], as inshown Scheme in Scheme1. In 1. addition,In addition, two non-amphiphilic dendrons characterized by a hydrophilic pentaethylene glycol (PEG) two non-amphiphilic dendrons characterized by a hydrophilic pentaethylene glycol (PEG) chain (7) chain (7) and by the presence of the sole PAMAM head (8) were also produced as negative reference and by thecompounds. presence of the sole PAMAM head (8) were also produced as negative reference compounds.
SchemeScheme 1. The chemical1. The chemical structure structure of the of PAMAM-based the PAMAM-based self-assembling self-assembling amphiphilic amphiphilic dendrons dendrons bearing a singlebearing alkyl chaina single of alkyl variable chain of length variable ( 1length–6). ( The1–6). twoThe two non-amphiphilic non-amphiphilic dendrons characterized characterized by by a hydrophilic pentaethylene glycol (PEG) chain (7) and by the presence of the sole PAMAM head a hydrophilic pentaethylene glycol (PEG) chain (7) and by the presence of the sole PAMAM head (8) (8) used as negative reference compounds are also shown (see text for details). Adapted from [7] with used asthe negative permission reference of John compounds Wiley and Sons, are 2016. also shown (see text for details). Adapted from [7] with the permission of John Wiley and Sons, 2016. Mesoscale simulations (see Figure S1, Table S1, and text in Supporting Information for details) Mesoscalewere initially simulations employed (seeto predict Figure the S1, self-assembly Table S1, andof all textdendrons in Supporting 1–8. According Information to the in silico for details) were initiallyresults, employedthe amphiphilic to predict dendrons the 1– self-assembly6 were able to auto-organize of all dendrons into spherical1–8. According nanosized tomicelles the in silico results,(shown the amphiphilic in Figure 1 dendrons for dendrons1–6 4were, 3 and able 2 as to an auto-organize example), with into an average spherical diameter nanosized D and micelles aggregation number Nagg ranging from 5.6 nm and 5 for micelles. This was generated by dendron 1 (shown in Figure1 for dendrons 4, 3 and 2 as an example), with an average diameter D and aggregation to 8.1 nm and 13 for those originated by the self-assembling of dendron 6 (Table 1). Contextually, no numberstable Nagg nanostructureranging from formation 5.6 nm was and ob 5served for micelles. for the negative This wascontrols generated 7 and 8, as by shown dendron in the1 leftto 8.1 nm and 13 forpanel those of Figure originated 1d for dendron by the 8.self-assembling of dendron 6 (Table1). Contextually, no stable nanostructure formation was observed for the negative controls 7 and 8, as shown in the left panel of Figure1d for dendron 8.
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(a) (b)
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FigureFigure 1. The1. The computer computer simulation simulation ofof thethe self-assembledself-assembled micelles micelles generated generated by the by theamphiphilic amphiphilic dendronsdendrons4 (a 4), (3a),( b3), (b and), and2 (2c ).(c). In In all all panels, panels, the hydrophilic hydrophilic PAMAM PAMAM head head is portrayed is portrayed as purple as purple andand lilac lilac beads, beads, the the hydrophobic hydrophobic tail is is represente representedd by bylight light gray gray beads beads while whilethe linker the between linker betweenthe hydrophilic and the hydrophobic portion of the molecule is shown as green beads. Water and the hydrophilic and the hydrophobic portion of the molecule is shown as green beads. Water and counterions are portrayed as a gray field for clarity. The right images in each panel show only the counterions are portrayed as a gray field for clarity. The right images in each panel show only the micellar cores. (d, left) Zoomed view of the micelles formed by dendron 4. The micellar cores are d left 4 micellarhighlighted cores. by ( , a light) Zoomedgray surface. view (d, of right the) micellesComputer formed simulation by dendronof the non-amphiphilic. The micellar dendron cores are highlighted8 selected by as a negative light gray control, surface. showing (d, right the non-) Computerself-assembling simulation characteri of thestics non-amphiphilic of this system. dendron 8 selected as negative control, showing the non-self-assembling characteristics of this system. σ 2 Table 1. Micelle aggregation number (Nagg), surface charge density ( m, e/nm ), diameter2 (D, nm), Table 1. Micelle aggregation number (Nagg), surface charge density (σm, e/nm ), diameter (D, nm), critical micelle concentration (CMC, μM) and free energy of micellization (ΔGm, kJ/mol) for dendrons µ critical1–6 micelle as predicted concentration by computer (CMC, simulations.M) and The free co energyrresponding of micellization experimental (∆ Gvaluesm, kJ /mol)of thefor micelle dendrons 1–6 asdiameter predicted (Dexp by, nm) computer and critical simulations. micelle concentration The corresponding (CMCexp, μM) experimental are also reported. values Adapted of the from micelle diameter[7] with (D expthe, permission nm) and critical of John micelle Wiley and concentration Sons. (CMCexp, µM) are also reported. Adapted from [7] with the permission of John Wiley and Sons. ΔG Dendron Nagg D Dexp CMC CMCexp Dendron Nagg DDexp CMC CMCexp m ∆Gm 1 1 55 5.6 5.60.1 ± 0.1 - - 415 415 398 398 ± 22 22 −38.7 38.7 ± ± − 2 2 66 5.9 5.90.1 ± 0.1 - - 127 127 116 116 ± 11 11 −44.4 44.4 ± ± − 3 3 77 6.2 6.20.1 ± 0.1 6.6 6.6 ±0.1 0.1 37.2 37.2 49.949.9 ± 3.33.3 −50.5 50.5 ± ± ± − 4 4 88 6.4 6.40.1 ± 0.1 6.8 6.8 ±0.1 0.1 11.4 11.4 15.615.6 ± 1.11.1 −56.4 56.4 ± ± ± − 5 5 1010 7.1 7.10.2 ± 0.2 7.3 7.3 ±0.1 0.1 9.13 9.13 11.211.2 ± 0.60.6 −57.5 57.5 ± ± ± − 6 6 1313 8.1 8.10.2 ± 0.2 7.8 7.8 ±0.2 0.2 8.50 8.50 8.248.24 ± 3.83.8 −57.8 57.8 ± ± ± −
The values of the critical micelle concentration (CMC) for the amphiphilic dendrons 1–6 (see The values of the critical micelle concentration (CMC) for the amphiphilic dendrons 1–6 Supporting Information for details) were further investigated from which a clear inverse relationship (seebetween Supporting the hydrophobic Information tail for length details) and werethe CMC further was investigatedobtained (Table from 1). In which other awords, clear the inverse relationshipamphiphilic between dendrons the bearing hydrophobic a longer tailhydrophobic length and component the CMC are was able obtainedto self-assemble (Table and1). pack In other words,more the efficiently amphiphilic than dendronsthose characterized bearing aby longer shorter hydrophobic alkyl tails. In componentaddition, the areassociated able to values self-assemble of and packthe free more energy efficiently of micellization than those (ΔG characterizedm) predicted by by simulation shorter alkyl (see tails. Supporting In addition, Information the associated for valuesdetails) of the were free energyall largely of micellizationnegative, supporting (∆Gm) predictedthe spontaneous by simulation and thermodynamically (see Supporting favored Information for details)micelle wereformation all largely for all negative,dendrons 1 supporting–6 (Table 1). the As spontaneousthe hydrophilic and portion thermodynamically was the same in favoredall Δ micelleamphiphilic formation dendrons, for all the dendrons differential1–6 contribution(Table1). As to theGm hydrophilic(and hence to portionCMC) is obviously was the samerelated in all to the length of the hydrophobic chain. amphiphilic dendrons, the differential contribution to ∆Gm (and hence to CMC) is obviously related to Experimental confirmation of in silico predictions were first performed by dynamic light the length of the hydrophobic chain. scattering (DLS) and transmission electron microscopy (TEM). Both these techniques confirmed that theExperimental amphiphilic dendrons confirmation predominantly of in silico formed predictions nanosized werespherical first micelles, performed as shown by in dynamic Figure 2 light scattering (DLS) and transmission electron microscopy (TEM). Both these techniques confirmed that the amphiphilic dendrons predominantly formed nanosized spherical micelles, as shown in Figure2 for dendrons 3–6. In particular, data analysis confirmed that an increase of the hydrophobic tail Pharmaceutics 2019, 11,, 324x FOR PEER REVIEW 44 of of 26 for dendrons 3–6. In particular, data analysis confirmed that an increase of the hydrophobic tail length from CC1414 ((3)) to C 22 ((66)) was was paralleled paralleled by by an an increase of the micellarmicellar diameterdiameter DDexpexp fromfrom 6.6 nm Pharmaceutics 2019, 11, x FOR PEER REVIEW 4 of 26 to 7.8 7.8 nm, nm, in in full full agreement agreement with with the the computer-bas computer-baseded predictions predictions (Table (Table 1).1 Quite). Quite interestingly, interestingly, the overallthe overallfor dimensionsdendrons dimensions 3– 6of. In these ofparticular, these micellar micellar data nano-objects analysis nano-objects confirmed are indeed are that indeed anvery increase very close close toof thethose to hydrophobic those characterizing characterizing tail the high-generationthe high-generationlength from C structurally14 (3 structurally) to C22 (6 flexible) was flexible paralleled triethanolam triethanolamine by an increaseine (TEA)-core (TEA)-coreof the micellar covalent covalent diameter dendrimers dendrimers Dexp from discussed 6.6 discussed nm in detailin detailto in 7.8 inthe nm, the companion in companion full agreement paper paper with [9] [ 9(e.g.,the] (e.g., computer-bas the the average averageed molecular predictions molecular diameters (Table diameters 1). Quite for for generation interestingly, generation 4, the 4,5 and 5 and 6 TEA-core6 TEA-coreoverall dendrimers dimensions dendrimers ofare arethese equal equal micellar to to5.2, 5.2,nano-objects 7.7 7.7 and and 10.1 are 10.1 nm, indeed nm, respectively). respectively).very close to Therefore, those Therefore, characterizing at least at least from the from the standpointthe standpointhigh-generation of structure of structure structurally and and flexibledimensions, dimensions, triethanolam the the self-assembledine self-assembled (TEA)-core covalent nanoparticles nanoparticles dendrimers obtained obtained discussed from from in the detail in the companion paper [9] (e.g., the average molecular diameters for generation 4, 5 and 6 amphiphilic dendrons can be considered as supramolecularsupramolecular dendrimerdendrimer mimics.mimics. TEA-core dendrimers are equal to 5.2, 7.7 and 10.1 nm, respectively). Therefore, at least from the standpoint of structure and dimensions, the self-assembled nanoparticles obtained from the amphiphilic dendrons can be considered as supramolecular dendrimer mimics.
Figure 2. TEM images of spherical micelles formed by the amphiphilic dendrons 3–6. Adapted from Figure 2. TEM images of spherical micelles formed by the amphiphilic dendrons 3–6. Adapted from [7] with[7] with the the permission permission of John of John Wiley Wiley and and Sons, Sons, 2016. 2016. Figure 2. TEM images of spherical micelles formed by the amphiphilic dendrons 3–6. Adapted from [7] with the permission of John Wiley and Sons, 2016. The predicted CMC values were also confirmed confirmed by experiments using py pyrenerene as a hydrophobic fluorescentfluorescentThe probeprobe predicted (Table (Table CMC1). 1). The values The progressive wereprogressive also increaseconfirmed increase in by alkyl experimentsin alkyl chain chain length using length resulted pyrene resulted as in a a hydrophobic drop in of a CMCdrop expof CMCfromfluorescent 398exp µfromM for 398probe dendron μ (TableM 1forto 1). 8.24dendron TheµM progressive for dendron1 to 8.24increase6. TheμM in highfor alkyl valuesdendron chain of length the 6. computational The resulted high in valuesa/ experimentaldrop ofof the computational/experimentalCMCCMC forexp systems from 1398and μM2 arefor CMC thedendron likely for systems 1 reason to 8.24 1 why and μM micelle2 forare dendronthe formation likely 6 .reason The by thesehigh why two valuesmicelle dendrons of formation the was bynot these observedcomputational/experimental two dendrons under the was conditions not CMC observed adoptedfor systems under for 1 DLS thande /conditions2TEM are the experiments. likely adopted reason Further,fo whyr DLS/TEM micelle in agreement formation experiments. with Further,the simulation,by these in agreement two nodendrons CMC with was value the not simulation, could observed be experimentallyunderno CMC the conditionsvalue could determined adopted be experimentally fo forr DLS/TEM7 and 8 experiments.,determined confirming for the 7 andnon-self-assembling 8Further,, confirming in agreement the properties non-self-asse with the of simulation, thesembling two properties no negative-control CMC value of these could systems. two be experimentally negative-control determined systems. for 7 and 8, confirming the non-self-assembling properties of these two negative-control systems. 2.2. In In Silico/Experimental Silico/Experimental InteractionInteraction of Single Tail Self-AssemblingSelf-Assembling DendronsDendrons andand siRNAsiRNA 2.2. In Silico/Experimental Interaction of Single Tail Self-Assembling Dendrons and siRNA Mesoscale simulations were again exploited for predicting the interactions between the amphiphilic MesoscaleMesoscale simulations simulations were were againagain exploitedexploited forfor predictingpredicting the the interactions interactions between between the the amphiphilicdendronsamphiphilic1– 8dendronsand dendrons siRNA 1– 81 molecules, –and8 and siRNA siRNA as molecules, molecules, illustrated asas in illustratedillustrated Figure3. in in Figure Figure 3. 3.
(a) (b) (a) (b)
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(c) (d)
(e) (f)
Figure 3. Cont. (e) (f)
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(g) (h)
FigureFigure 3. Morphologies 3. Morphologies of of the the systemssystems formed formed at ata dend a dendrimer-to-siRNArimer-to-siRNA charge charge ratio (N/P) ratio = (N10 /byP) the= 10 by the siRNAsiRNA molecules and and the the amphiphilic amphiphilic dendrons dendrons 1 (a),1 2( (ab),),2 3 ((bc), 43 ((dc), 54 ((ed),), 65 (f(),e ),7 6(g()f and), 7 (8g ()h and) as 8 (h) as obtainedobtained from from molecular simulations. simulations. Left Left panels panels are zoomed are zoomed images images of the right of the panels. right Colors panels. as in Colors Figure 2, except for the siRNA molecules, shown as red sticks and the water and counterions, as in Figure2, except for the siRNA molecules, shown as red sticks and the water and counterions, portrayed in the left panels as light blue field for clarity. Adapted from [7] with the permission of John portrayed in the left panels as light blue field for clarity. Adapted from [7] with the permission of John Wiley and Sons, 2016. Wiley and Sons, 2016. The computer images showed that, at the same dendrimer-to-siRNA charge ratio (N/P) of 10, theThe nucleic computer acid fragments images showed are well encased that, at within the same the micellar dendrimer-to-siRNA network formed by charge all self-assembling ratio (N/P) of 10, the nucleicamphiphilic acid fragmentsdendrimers are 1–6 welland so encased are efficiently within protected the micellar from network the surrounding formed byenvironment. all self-assembling For amphiphilicthe non-self-assembling dendrimers 1–6 andsystems so are 7 eandfficiently 8, the protected siRNA frommolecules the surrounding are interspersed environment. within an For the non-self-assemblingunorganized solution systems of single7 and dendrons8, the siRNAand water, molecules and therefore are interspersed in principle withinmore susceptible an unorganized to solutionultimate of singledegradation dendrons by e.g., and RNAses. water, and therefore in principle more susceptible to ultimate degradationThe byin silico e.g., RNAses.quantitative characterization of the siRNA/amphiphilic dendron interactions was nextThe carried in silico out quantitative by atomistic characterizationmolecular dynamics of (MD) the siRNA simulations/amphiphilic performed dendron in the framework interactions of was the so-called molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) methodology [9–15] next carried out by atomistic molecular dynamics (MD) simulations performed in the framework of (the methodology is described in detail in the Supporting Information in the companion paper the so-called molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) methodology [9–15] [9]).The values of free energy of binding (ΔGbind) between the different self-assembling dendrons 1–6 (the methodologytowards the siRNA is described sequence in directed detail inagainst the Supporting the mRNA Informationcoding for the in heat the companionshock protein paper 27 [9]). The values(Hsp27)—a of free small energy molecular of binding chaperone (∆Gbind which) between is a vital the regulator different of self-assembling cell survival and dendrons a major 1player–6 towards the siRNAin drug sequence resistance—were directed initially against calculated, the mRNA as coding listed in for the the first heat column shock proteinof Table 272. (Hsp27)—aHowever, in small molecularorder chaperoneto perform whicha rigorous is a vitaldiscussion regulator about of cellthese survival aspects, and the a concept major player of effective in drug free resistance—were energy of initiallybinding calculated, (ΔGbind, aseff) listedwas introduced, in the first as column follows. of TableFrom2 .an However, extensive inanalysis order toof performeach dendron a rigorous micelle/siRNA complex MD trajectory, the number Neff of dendron branches (each bearing a positive discussion about these aspects, the concept of effective free energy of binding (∆Gbind,eff) was introduced, as follows.charge) From in permanent an extensive contact analysis with ofthe each siRNA dendron was determined, micelle/siRNA leading complex to the MD values trajectory, listed thein the number second columns of Table 2. The precise identification of each dendrimer branch involved in the N of dendron branches (each bearing a positive charge) in permanent contact with the siRNA was eff siRNA binding, and the corresponding individual contribution afforded to the overall binding determined, leading to the values listed in the second columns of Table2. The precise identification of energy, was then carried out by a per-residue free energy decomposition technique (described in the eachSupporting dendrimer Information branch involved in the in companion the siRNA paper binding, [9]). andThe thecumulative corresponding results of individual this analysis contribution are affordedreported to the in overallthe third binding columnenergy, of Table was 2, showing then carried the effective out by contribution a per-residue to freethe total energy free decompositionenergy of techniquebinding (described (ΔGbind,eff). inHowever, the Supporting to be able Information to compare simulation in the companion data among paper themselves [9]). The (and cumulative with resultsexperimental of this analysis data, see are below) reported the in ef thefective-charge-normalized third column of Table values2, showing of ΔGbind,eff the effective, i.e., ΔGbind,eff contribution⁄Neff, to thewere total considered, free energy as ofshown binding in the (∆ fifthGbind,eff column). However, of Table 2. to be able to compare simulation data among themselves (and with experimental data, see below) the effective-charge-normalized values of ∆Gbind,eff, i.e., ∆Gbind,eff/Neff, were considered, as shown in the fifth column of Table2.
Table 2. The free energy of micellization (∆Gbind, kcal/mol), number of effective charges (Neff), effective free energy of binding (∆Gbind,eff, kcal/mol), and effective-charge-normalized free energy of binding (∆Gbind,eff/Neff) between siRNA and dendrons 1–6 as derived from molecular simulations. The last column reports the siRNA/dendron binding data (C50, µM) obtained from ethidium bromide (EB) displacement assays. Adapted from [7] with the permission of John Wiley and Sons, 2016.
Dendron ∆Gbind Neff ∆Gbind,eff ∆Gbind,eff/Neff C50 1 8.23 0.91 13 1 2.47 0.32 0.19 0.03 - − ± ± − ± − ± 2 13.4 1.1 18 1 4.30 0.45 0.24 0.03 42.0 2.0 − ± ± − ± − ± ± 3 17.1 1.3 22 1 6.00 0.36 0.27 0.02 16.9 0.5 − ± ± − ± − ± ± 4 22.6 1.2 31 1 12.7 0.41 0.41 0.04 7.0 0.3 − ± ± − ± − ± ± 5 46.6 1.6 49 1 28.9 0.70 0.59 0.04 5.3 0.4 − ± ± − ± − ± ± 6 57.8 2.3 62 1 34.7 0.62 0.56 0.05 4.8 0.1 − ± ± − ± − ± ± Pharmaceutics 2019, 11, x FOR PEER REVIEW 6 of 26
Table 2. The free energy of micellization (ΔGbind, kcal/mol), number of effective charges (Neff), effective free energy of binding (ΔGbind,eff, kcal/mol), and effective-charge-normalized free energy of binding
(ΔGbind,eff/Neff) between siRNA and dendrons 1–6 as derived from molecular simulations. The last
column reports the siRNA/dendron binding data (C50, μM) obtained from ethidium bromide (EB) displacement assays. Adapted from [7] with the permission of John Wiley and Sons, 2016.
ΔGbind,eff/Ne Dendron ΔGbind Neff ΔGbind,eff C50 Pharmaceutics 2019, 11, 324 ff 6 of 26 1 −8.23 ± 0.91 13 ± 1 −2.47 ± 0.32 −0.19 ± 0.03 - 2 −13.4 ± 1.1 18 ± 1 −4.30 ± 0.45 −0.24 ± 0.03 42.0 ± 2.0 From the values3 reported−17.1 in ± Table 1.3 2, 22 the ± 1 e ffective−6.00 ± interaction0.36 −0.27 between ± 0.02 the 16.9 micelles ± 0.5 formed by the different dendrons with4 siRNA−22.6 ( ∆± G1.2bind,e 31ff) ± increases 1 −12.7 with± 0.41 increasing−0.41 ± 0.04 alkyl chain 7.0 ± length, 0.3 the last three dendrons definitely being5 the−46.6 strongest ± 1.6 siRNA 49 ± 1 binders−28.9 ± 0.70 in the order:−0.59 ±4 0.04< 5 < 6. 5.3 Limiting ± 0.4 the discussion to these three self-assembling6 −57.8 systems, ± 2.3 the 62 micelles± 1 −34.7 formed ± 0.62 by dendron−0.56 ± 0.054 were 4.8 characterized ± 0.1 by a total number of charges equal to +64, the 48% of which (Neff = 31 are involved in productive siRNA binding. This translatesFrom the values into the reported corresponding in Table∆ G2, the effect/N ivevalue interaction of 0.41 between kcal/mol. the In micelles contrast, formed the other by two the bind,eff eff − edifferentfficient self-assembled dendrons with micelles siRNA generated(ΔGbind,eff) byincreases dendrons with5 andincreasing6 were notalkyl only chain both length, able to the exploit last three 61% ofdendrons their positive definitely charges being (49 the/80 stro andngest 62/102 siRNA for 5 and binders6, respectively) in the order: but 4 < they 5 < 6 also. Limiting did it more the discussion efficiently (to∆ Gthese three/N self-assembling= 0.59 kcal/mol systems, for 5 and the0.56 micelles kcal/ molformed for 6 by, Table dendron1), ultimately 4 were characterized characterizing by the a bind,eff eff − − correspondingtotal number of micelles charges as equal the best to +64, siRNA the binders48% of which of the whole(Neff = 31 series. are involved in productive siRNA binding.In silico This data translates were experimentally into the corresponding confirmed Δ byGbind,eff fluorescent/Neff value ethidium of −0.41 bromide kcal/mol. (EB) In displacement contrast, the assaysother two (see efficient Figure self-assembledA1 in Appendix micellesA). According generated to theseby dendrons tests, the 5 and value 6 were of C not50 (i.e., only the both dendron able to concentrationexploit 61% of at their which positive the fluorescence charges (49/80 of siRNA-intercalated and 62/102 for 5 and EB is6, reducedrespectively) by 50%), but isthey an also indication did it ofmore siRNA efficiently binding (Δ effiGciency:bind,eff/Neff The = lower−0.59 kcal/mol the C50 value, for 5the and stronger −0.56 kcal/mol the siRNA for/micelle 6, Table binding. 1), ultimately As seen incharacterizing Figure A1, the the negative-control corresponding (i.e.,micelles non-self-assembling) as the best siRNA dendrons binders 7ofand the8 wholewere notseries. able to displace EB fromIn thesilico nucleic data acid. were In line,experimentally also for the self-assemblingconfirmed by dendronfluorescent1, bearing ethidium the shortest bromide alkyl (EB) tail (Cdisplacement14), the C50 valueassays was (see not Figure determined, A1 in Appendix due to the A). high According CMC value to these required tests, the for itsvalue self-assembly. of C50 (i.e., Onthe dendron the other concentration hand, for the at dendron which the series fluorescence2–6, progressively of siRNA-intercalated decreasing C EB50 valuesis reduced were by obtained 50%), is (Figurean indication A1 and of TablesiRNA2, lastbinding column), efficiency: with dendron The lower6 beingthe C50 the value, most the eff ectivestronger siRNA the siRNA/micelle binder, in full agreementbinding. As with seen computer-based in Figure A1, the predictions. negative-control (i.e., non-self-assembling) dendrons 7 and 8 were not able to displace EB from the nucleic acid. In line, also for the self-assembling dendron 1, bearing 2.3.the shortest In Vitro siRNAalkyl tail Delivery (C14), Performancethe C50 value of was Single-Tail not determined, Self-Assembling due Dendronsto the high CMC value required for itsBased self-assembly. on the results On describedthe other above,hand, dendronsfor the dendron4–6 were series selected 2–6, forprogressivelyin vitro delivery decreasing of siRNA C50 moleculesvalues were targeting obtained both (Figure the Hsp27 A1 and and Table the 2, translationally last column), controlledwith dendron tumor 6 being protein the (TCTP, most effective a highly conservedsiRNA binder, protein in full present agreement in all eukaryoticwith computer-based organisms predictions. that regulate cell survival in human tumors) in human castration-resistant prostate cancer (HCPC) PC-3 and C4-2 cell lines (Figure4a,b). In these 2.3. In Vitro siRNA Delivery Performance of Single-Tail Self-Assembling Dendrons experiments, all other dendrons (1, 3, 7 and 8) were also tested for comparison and/or negative controls.
(a) (b)
Figure 4. Cont.
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(c)
Figure 4. Self-assembled dendron-mediateddendron-mediated siRNA siRNA delivery delivery and and gene gene silencing silencing of Hsp27 of Hsp27 (a) and (a) TCTP and TCTPproteins proteins (b) in human(b) in human prostate prostate cancer cancer PC-3 (solidPC-3 (solid bars) andbars C4-2) and (patternedC4-2 (patterned bars) cellbars) lines cell (Nlines/P =(N/P10). =Control: 10). Control: Untreated Untreated cells. The cells. results The obtainedresults obtained from non-self-assembling from non-self-assembling dendrons 1dendrons, 7 and 8 are 1, 7 also and shown 8 are alsofor negative-control shown for negative-control purposes. (c) Expressionpurposes. of(c) Hsp27 Expression in PC-3 of cells Hsp27 treated in PC-3 with 50cells nM treated siRNA with delivered 50 nM by siRNAself-assembled delivered dendron by self-assembled4 (N/P = 10), by Lipofectamine,dendron 4 (N/P and = by 10), the high-generationby Lipofectamine, covalent and triethanolamine by the high- generation(TEA)-core PAMAM-basedcovalent triethanolamine dendrimer (TEA)-core G7. Control: PAMAM-based Untreated cells. dendrimer Negative controls:G7. Control: Self-assembled Untreated cells.dendron Negative4 alone controls: and a scrambled Self-assembled (i.e., non-silencing) dendron 4 siRNAalone and sequence. a scrambled The expression (i.e., non-silencing) levels of Hsp27 siRNA and sequence.TCTP proteins The wereexpression quantified levels by of western Hsp27 blots and 72 TCTP h post proteins treatment were using quantified vinculin asby the western control. blots Adapted 72 h postfrom treatment [7] with the using permission vinculin of as John the Wiley control. and Adapte Sons, 2016.d from [7] with the permission of John Wiley and Sons, 2016. As shown in this Figure, the best siRNA delivery capacity was obtained with the nanovector based on theAs self-assembled shown in this form Figure, of dendron the best4 siRNA, whose delivery related genecapacity silencing was eobtainedffect was with even the superior nanovector to that basedobtained on the both self-assembled with the gold form standard of dendron commercial 4, whose vector related Lipofectamine gene silencing and witheffect the was high-generation even superior to(G 7that) covalent obtained PAMAM-based both with the dendrimer.gold standard This commercial featured anvector extended Lipofectamine triethanolamine and with (TEA) the high- core generationdeveloped by(G our7) covalent group and PAMAM-based discussed in detail dendrimer. in the companion This featured paper an [9 ].extended As anticipated triethanolamine by in silico (TEA)predictions, core developed nanocarriers by formed our group by dendrons and discussed bearing shorterin detail aliphatic in the (companion1–3), hydrophilic paper (7[9].) or As no anticipatedchains (8) fail by toin elicitsilico meaningfulpredictions, genenanocarriers silencing formed in all cases. by dendrons Indeed, bearing while 7 shorterand 8 are aliphatic intrinsically (1–3), hydrophilicunable to self-assembly (7) or no chains (negative (8) fail controls), to elicit the meaningful former dendron gene silencing set (1–3) in likely all cases. cannot Indeed, generate while stable 7 andmicelles 8 areand intrinsically/or siRNA unable complexes to self-assembly at the experimental (negative conditionscontrols), the employed former dendron for transfection, set (1–3)due likely to cannottheir high generate CMC andstable C50 micellesvalues (Tablesand/or1 siRNAand2). Oncomp thelexes other at hand, the experimental nanomicelles conditions from dendrons employed with forlonger transfection, alkyl tails (due5 and to6 )their were high also ableCMC to achieveand C50 significant values (Tables siRNA 1 delivery and 2). and On silenced the other the hand, target nanomicellesHsp27 and TCTP from genes, dendrons yet with with performance longer alkyl lower tails than(5 and that 6) obtainedwere also with able dendron to achieve4. significant siRNAThese delivery lastresults and silenced seem to the be target in slight Hsp27 contradiction and TCTP withgenes, the yet computer-based with performance prediction lower than reported that obtainedin Table2 with, according dendron to 4 which. the best (i.e., most favorable) values of ∆Gbind,eff/Neff and C50 are indeedThese obtained last results for self-assembled seem to be in slight dendrons contradi5 andction6. with This the ranks computer-based these nanovectors prediction as the reported tightest insiRNA Table binders. 2, according To investigate to which the whether best (i.e., this most disagreement favorable) was values real of or Δ onlyGbind,eff apparent,/Neff and cellular C50 are indeed uptake obtainedexperiments for basedself-assembled on flow cytometry dendrons were5 and performed 6. This ranks using these siRNA nanovectors in complex as with the micellestightest siRNA from 4, binders.5, and 6 (FigureTo investigate5a). These whether data clearly this show disagreement that this step was is notreal governing or only theapparent, di fferential cellular behavior uptake of experimentsthese nanovector based/siRNA on flow performance cytometry alongwere performed the in vitro usinggene silencingsiRNA in pathway,complex aswith all micelles three systems from 4are, 5, characterizedand 6 (Figure by5a). comparable These data cellularclearly show uptake that effi thisciency. step is not governing the differential behavior of these nanovector/siRNA performance along the in vitro gene silencing pathway, as all three systems are characterized by comparable cellular uptake efficiency.
Pharmaceutics 2019, 11, 324 8 of 26 Pharmaceutics 2019, 11, x FOR PEER REVIEW 8 of 26
Pharmaceutics 2019, 11, x FOR PEER REVIEW 8 of 26
(a) (b)
Figure 5. ((aa)) The The mean mean fluorescence fluorescence(a) intensity intensity of of PC-3 PC-3 cells cells treated treated(b ) with with siRNA labeled with the fluorescentfluorescentFigure dye5. (a Cy-3)Cy-3 The ininmeancomplex complex fluorescence with with self-assembled self-assembledintensity of PC-3 nanovectors nanovectors cells treated4, 45 with,, and5, and siRNA6 (50 6 (50 nM labeled nM siRNA, siRNA, with N the/P N/P= 10). = 10).Non-treated Non-treatedfluorescent cells dye cells and Cy-3 cellsand in complex treatedcells treated withwith labeledwithself-assembled labeled siRNA siRNAnanovectors alone werealone 4 used, were5, and as used 6 control. (50 asnM control. (siRNA,b) siRNA (N/Pb) siRNArelease = releasefrom10). their from Non-treated complexes their complexes cells with and micelles withcells treatedmicelles of dendrons with of ladendronsbeled4 (red), siRNA5 4(black), (red), alone 5 andwere(black),6 used(yellow) and as 6control. as(yellow) assessed (b) assiRNA usingassessed EB usingdisplacementrelease EB displacement from assay their performed complexes assay performedwith in the micelles presence in of the dendrons of pres heparin.ence 4 (red), Adaptedof heparin.5 (black), from andAdapted [ 76] (yellow) with from the as permission assessed[7] with the of permissionJohnusing Wiley EB andof Johndisplacement Sons, Wiley 2016. and assay Sons, performed 2016. in the presence of heparin. Adapted from [7] with the permission of John Wiley and Sons, 2016. With thisthis aspectaspect ruled ruled out, out, it it was was reasoned reasoned that that the the siRNA siRNA release release of its of nanovector its nanovector could could be the be factor the With this aspect ruled out, it was reasoned that the siRNA release of its nanovector could be the factorunderlying underlying the best the performance best perfor in genemance silencing in gene of thesilencing4/siRNA of complex. the 4/siRNA Thus, complex. it was determined Thus, it that was it factor underlying the best performance in gene silencing of the 4/siRNA complex. Thus, it was determinedwas the dissociation that it ofwas the the nucleic dissociation acid fragments of the from nucleic their 4acid, 5, and fragments6 self-assembled from their nanovectors 4, 5, and via 6 the self- EB determined that it was the dissociation of the nucleic acid fragments from their 4, 5, and 6 self- displacement assay in the presence of heparin, a highly negatively charged polysaccharide that can compete assembledassembled nanovectors nanovectors via via the the EB EB displacement displacement assayassay inin the the presence presence of ofheparin, heparin, a highly a highly negatively negatively chargedwith siRNAcharged polysaccharide in polysaccharide nanocarrier that binding. that can can compete Ascompete can be withwith seen siRNAsiRNA in Figure inin 5 nanocarrierb,nanocarrier by increasing binding. binding. heparin As Ascan concentration canbe seenbe seenin the in FiguresiRNAFigure 5b, molecules by 5b, increasing by wereincreasing more heparin heparin efficiently concentration concentration displaced the fromthe siRNA their complexmoleculesmolecules with were were4 morethan more efficiently from efficiently the twodisplaced alternativedisplaced fromdendron fromtheir micelles. their complex complex This with led with us 4 to than4 thethan conclusionfrom from the the thattwotwo the alternativealternative siRNA release denddendron processron micelles. micelles. might This be This moreled ledus effective tous the to fromthe conclusionthe self-assembledconclusion that thatthe dendron thesiRNA siRNA 4releasethan release from process process the 5 might mightand 6 becomplexes,be more effective effective respectively. from from the the self-assembled self-assembled dendron dendron 4 thanCollectively,4 thanfrom from the 5the and these 5 and 6 complexes, results 6 complexes, led torespectively. respectively. the conclusion that, in agreement with computer predictions, the supermolecularCollectively,Collectively, these assemblies these results results formedled led to to the by the 5conclusion conclusionand 6 were that, actually inin agreement agreement too stable with towith dissemblecomputer computer predictions, in physiologicalpredictions, the supermolecular assemblies formed by 5 and 6 were actually too stable to dissemble in theconditions. supermolecular This enhanced assemblies stability formed negatively by 5 affectsand 6 the were siRNA actually release too in thestable cellular to dissemble environment, in physiological conditions. This enhanced stability negatively affects the siRNA release in the cellular physiologicalthereby impairing conditions. the relevant This gene enhanced silencing stability effect. Atnegatively the same affects time, the the complexes siRNA release of micelles in the originated cellular environment, thereby impairing the relevant gene silencing effect. At the same time, the complexes by dendron 4 and siRNA represent the best compromise in term of nucleic acid fragment binding and environment,of micelles thereby originated impairing by dendron the 4 relevantand siRNA gene represent silencing the best effect. compromise At the same in term time, of nucleic the complexes acid ofrelease micellesfragment efficiency, originated binding and these byand dendron propertiesrelease efficiency,4 and ultimately siRNA and translate representthese properties into the the best mostultimately compromise potent translate biological in term into performance. of the nucleic most acid fragmentThepotent finalbinding biological step ofand performance. the releasein vitro efficiency,characterization and these concerned properties the investigation ultimately translate of the cellular into toxicitythe most of potentour single-tail biologicalThe final self-assembled performance. step of the in vitro dendrons. characterization As shown concerned in Figure the6 investigationfor the PC-3 of cell the linecellular as an toxicity example, noneTheof of our the final single-tail self- step and of non-self-assemblingself-assembled the in vitro characterization dendrons. dendrons As shown wereconcerned in endowed Figure the 6 for withinvestigation the cytotoxicPC-3 cell lineof properties. the as ancellular example, Analogous toxicity ofresults ournone single-tail were of obtainedthe self-assembledself- withand non-self-assembling the alternative dendrons. prostateAs dendroshown cancerns in wereFigure cell endowed line, 6 for C4-2. the with PC-3 cytotoxic cell line asproperties. an example, none Analogousof the self- results and were non-self-assembling obtained with the al ternativedendrons prostate were cancerendowed cell line, with C4-2. cytotoxic properties. Analogous results were obtained with the alternative prostate cancer cell line, C4-2.
(a) (b)
FigureFigure 6. Metabolite 6. Metabolite toxicity toxicity (a )(a and) and membrane membrane damaging activity activity (b ()b of) ofself- self- and and non-self-assembling non-self-assembling dendronsdendrons1–8 and1–8 theirand their complexes complexes(a) with with a scrambled a scrambled (i.e., (i.e., non-silencing) non-silencing)( siRNAb) siRNA sequence sequence (50 (50 nM nM siRNA, N/P =siRNA,10) in PC-3N/P = cells 10) usingin PC-3 the cells 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium tetrazolium bromide (MTT) Figurebromide 6. Metabolite (MTT) assay toxicity and ( thea) and lactat membranee dehydrogenase damaging (LDH) activity assay, respectively.(b) of self- and Untreated non-self-assembling cells were assay and the lactate dehydrogenase (LDH) assay, respectively. Untreated cells were used for control. dendronsused for1– 8control. and their Adapted complexes from [7] withwith thea scrambled permission (i.e.,of John non-silencing) Wiley and Sons, siRNA 2016. sequence (50 nM Adapted from [7] with the permission of John Wiley and Sons, 2016. siRNA, N/P = 10) in PC-3 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay and the lactate dehydrogenase (LDH) assay, respectively. Untreated cells were used for control. Adapted from [7] with the permission of John Wiley and Sons, 2016.
Pharmaceutics 2019, 11, 324 9 of 26
Pharmaceutics 2019, 11, x FOR PEER REVIEW 9 of 26 In essence, all in silico and in vitro results concurred to indicate 4 as the amphiphilic dendron endowed withIn essence, the best allfeatures in silico and for einffi vitrocient results self-assembly, concurred andto indicate easy and 4 as stablethe amphiphilic formation dendron of complexes with theendowed siRNA with molecules the best for features delivery. for efficient Once inside self-assembly, the cell, and there easy is eandffective stable disassembly formation of during complexes with the siRNA molecules for delivery. Once inside the cell, there is effective disassembly endosomal release for subsequent potent gene silencing. during endosomal release for subsequent potent gene silencing. 2.4. In Vivo siRNA Delivery Performance of Single-Tail Self-Assembling Dendron 4 2.4. In Vivo siRNA Delivery Performance of Single-Tail Self-Assembling Dendron 4 The in vitro best performing nanomicelles generated by the dendron 4 were finally challenged for The in vitro best performing nanomicelles generated by the dendron 4 were finally challenged in vivo gene silencing in a prostate cancer xenografted nude mouse model. The results obtained are for in vivo gene silencing in a prostate cancer xenografted nude mouse model. The results obtained gatheredare in gathered Figure in7. Figure 7.
(a) (b)
(c)
FigureFigure 7. (a) 7.In (a vivo) In vivogene gene silencing silencing achieved achieved byby treating nude nude mice mice bearing bearing prostate prostate cancercancer (PC-3) (PC-3) 3 tumorstumors (30–50 (30–50 mm3 mm) with) with Hsp27 Hsp27 siRNA siRNA delivered delivered by by the the self-assembling self-assembling dendron dendron 4, buffer4, bu solutionffer solution (control), the dendron 4 alone, and a scrambled siRNA/4 complex (negative controls). Treatments (3 (control), the dendron 4 alone, and a scrambled siRNA/4 complex (negative controls). Treatments mg/kg siRNA, N/P = 5) were administered for a period of 4 weeks via intraperitoneal injections (twice (3 mg/kgper siRNA, week). N(b/)P In= vivo5) were inhibition administered of tumor growth for a period quantified of 4 weeksby tumor via size intraperitoneal and (c) antiproliferation injections (twice per week).activity (b )inIn tumors vivo assessedinhibition by immunohistochemistry of tumor growth quantified in mice treated by tumor as described size and for panel (c) antiproliferation (a). Panel activity(c in), from tumors left to assessed right: control, by immunohistochemistry 4 alone, 4/siRNA, 4/scrambled in mice siRNA. treated Adapted as described from [7] with for the panel (a). Panel (cpermission), from left of toJohn right: Wiley control, and Sons,4 alone, 2016. 4/siRNA, 4/scrambled siRNA. Adapted from [7] with the permission of John Wiley and Sons, 2016. Images in Figure 7 support the effective and specific in vivo gene silencing induced upon Imagesadministration in Figure of 7the support Hsp27-directed the e ff ectivesiRNA anddelivered specific by thein vivoself-assembledgene silencing nanovector induced 4. In upon particular, an impressive reduction of ~80% in Hsp27 expression and of ~65% in tumor size after 4 administration of the Hsp27-directed siRNA delivered by the self-assembled nanovector 4. In particular, weeks of treatment were achieved (Figure 7a,b). an impressiveThese reduction data, coupled of ~80% with no in discernable Hsp27 expression signs of in andvivo oftoxicity ~65% (Figure in tumor 8) and size the afterabsence 4 weeksof of treatmentmice were weight achieved loss, support (Figure the 7potentiala,b). utilization of the amphiphilic compound 4 as a siRNA vector Thesefor future data, therapeutic coupled withimplementations. no discernable signs of in vivo toxicity (Figure8) and the absence of mice weight loss, support the potential utilization of the amphiphilic compound 4 as a siRNA vector for future therapeutic implementations.
PharmaceuticsPharmaceutics 20192019,, 1111,, x 324 FOR PEER REVIEW 1010 of of 26 26
(a) (b) (c)
(d) (e) (f)
(g)
FigureFigure 8. InIn vivo vivo levels ofof inflammatoryinflammatory cytokines cytokines interleukine-6 interleukine-6 (IL-6, (IL-6,a), a interferon-gamma), interferon-gamma (INF- (INF-γ, bγ),, band), and tumor tumor necrosis necrosis factor-alpha factor-alpha (TNF- (TNF-α, c),α observed, c), observed in male in male C57BL C57BL/6/6 mice treatedmice treated with 4 withand its4 and siRNA its siRNAcomplex complex (3.0 mg (3.0/kg siRNA,mg/kg siRNA, N/P = 5) N/P by = intravenous 5) by intravenous injection. injection. Non-treated Non-treated mice and mice animals and animals treated treatedwith bu withffer solutionsbuffer solutions were used were for used control. for control. Mice were Mice sacrificed were sacrificed 24 h after 24 h treatment after treatment and the and ELISA the ELISAassay wasassay performed was performed on the collected on the serum. collected (d) Hepatic serum. enzyme (d) Hepatic levels (asparticenzyme aminotransferaselevels (aspartic aminotransferaseAST (solid bars),alanine AST (solid transferase bars), ALT alanine (patterned transferase bars), gamma-glutamineALT (patterned bars), transferase gamma-glutamineγ-GLT (empty transferasebars), (e) kidney γ-GLT functions(empty bars), (creatinine (e) kidney CRE functions (mmol/L, (creatinine solid bars), CRE blood (mmol/L, urea nitrogen solid bars), BUN blood (µmol urea/L, nitrogenpatterned BUN bars), (μmol/L, and (f )patterned cholesterol bars), level and in (f the) cholesterol blood measured level in inthe the blood treated measured mice afterin the sacrifice. treated mice(g) Pathology after sacrifice. of major (g) organsPathology from of treated major miceorgans (HES from staining). treated mice Columns (HES (from staining). left to Columns right): Control, (from left4, 4 to/siRNA, right): andControl,4/scrambled 4, 4/siRNA, siRNA. and 4 Rows/scrambled (from siRNA. top to bottom): Rows (from Heart, top kidney,to bottom): liver, Heart, lung, kidney, spleen. liver,Adapted lung, from spleen. [7] with Adapted the permission from [7] with of John the permission Wiley and Sons,of John 2016. Wiley and Sons, 2016. 3. siRNA Delivery by Double-Tail Self-Assembling Amphiphilic Dendrons 3. siRNA Delivery by Double-Tail Self-Assembling Amphiphilic Dendrons 3.1. Design, Optimization and Chemico-Physical Charactrization of Double-Tail Self-Assembling 3.1.Amphiphilic Design, Optimization Dendrons and Chemico-Physical Charactrization of Double-Tail Self-Assembling Amphiphilic Dendrons With the haste of further improving the siRNA delivery and related gene silencing performance of the leadWith self-assembling the haste of further amphiphilic improving dendron the siRNA4, it was delivery questioned and related whether gene increasing silencing its performance hydrophobic ofportion the lead would self-assembling render this molecule amphiphilic even more dendron efficient 4 and, it ewasffective. questioned According whether to the devised increasing strategy, its hydrophobic portion would render this molecule even more efficient and effective. According to the
Pharmaceutics 2019, 11, 324 11 of 26 Pharmaceutics 2019, 11, x FOR PEER REVIEW 11 of 26 itdevised was ultimately strategy, decidedit was ultimately to decorate decided4 with to a seconddecorate C 184 -longwith a alkyl second chain. C18-long In order alkyl to chain. do so, In a seriesorder ofto computer-baseddo so, a series of molecular computer-based design andmolecular optimization design studiesand optimization were carried studies out, finallywere carried leading out, to thefinally new leading double-tail to the amphiphilic new double-tail dendron amphiphilic structure AD dendron(where ADstructurestands AD for Amphiphilic(where AD stands Dendron) for shownAmphiphilic in Figure Dendron)9a [ 6]. In shown buffer solutionin Figure per 9a se, [6]. molecular In buffer simulations solution per predicted se, molecularAD to self-assemble simulations intopredicted large (~200AD nm)to self-assemble vesicle-like structures into large (Figure (~2009b) nm) called vesicle-like dendrimersomes structures [ 16 ].(Figure After synthesis 9b) called of ADdendrimersomesby click-chemistry, [16]. After dynamic synthesis light scatteringof AD by (DLS)click-chemistry, and transmission dynamic electron light scattering microscopy (DLS) (TEM) and indeedtransmission confirmed electron both microscopy the size and (TEM) the shape indeed of the confirmed supermolecular both the entities size and formed the byshapeAD ofupon the self-assemblysupermolecular (Figure entities9c). formed Notably, by the AD vesicle upon corona self-assembly structure (Figure closely matched9c). Notably, a bilayer, the vesicle characterized corona bystructure a thickness closely of 7matched nm—approximately a bilayer, characterized equal to twice by thea thickness molecular of length 7 nm—approximately of AD (~3.5 nm). equal to twice the molecular length of AD (~3.5 nm).
(a)
(b) (c)
(d) (e)
FigureFigure 9.9. ((aa)) The The structure structure of of the the double-tail double-tail amphiphilic amphiphilic dendron dendron simulated simulated (b) and (b) experimentaland experimental TEM imagesTEM images (c) of the (c vesicle-like) of the vesicle-like dendrimersomes dendrimersomes formed by ADformedalone by upon AD self-assembling alone upon self-assembling in buffer solution. in Inbuffer panel solution. b, the hydrophilic In panel b, units the hydrophilic of AD are portrayed units of AD in light are portrayed green, the hydrophobicin light green, units the hydrophobic are depicted inunits dark are green, depicted while in light dark grey green, spheres while are light used grey to show spheres some are representative used to show water some molecules.representative Simulated water (molecules.d) and experimental Simulated TEM (d) images and experimental (e) of the nanosized TEM sphericalimages ( micellese) of the formed nanosized by AD sphericalin the presence micelles of siRNAformed molecules by AD in in the solution. presence In of panel siRNA (d), molecules colors as in in panel solution. (b), while In panel the nucleic(d), colors acid as fragments in panel are(b), portrayedwhile the asnucleic orange acid sticks. fragments Adapted are from portrayed [6] with as permission orange sticks. of John Adapted Wiley from and Sons,[6] with 2014. permission of John Wiley and Sons, 2014.
Pharmaceutics 2019, 11, 324 12 of 26
QuitePharmaceutics surprisingly, 2019, 11, x FOR however, PEER REVIEW when simulations of the self-assembly of AD were performed 12 of 26 in the presence of siRNA many highly ordered smaller spherical micelles of 6–8 nm diameter were obtained, Quite surprisingly, however, when simulations of the self-assembly of AD were performed in with the siRNA fragments nicely interspersed within them (Figure9d). The explanation for this the presence of siRNA many highly ordered smaller spherical micelles of 6–8 nm diameter were vesicularobtained, to micellar with the structural siRNA fragments transition nicely likely interspers residesed inwithin the greaterthem (Figure positively 9d). The charged explanation surface for area exposedthis byvesicular the micelles to micellar with structural respect to transition the dendrimersomes, likely resides in which the greater provides positively more echargedfficient surface electrostatic interactionsarea exposed with by the the negatively micelles with charged respect siRNA. to the This, dendrimersomes, in turn, leads which to the provides formation more of efficient more stable and compactelectrostatic complexes interactions between with the the negatively self-assembled charged siRNA. nanocarriers This, in andturn, theleads nucleic to the formation acids molecules. of The computermore stable predictions and compact were complexes supported between by TEM the self-assembled imaging (Figure nanocarriers9e), confirming and the that nucleicAD acidsis able to dynamicallymolecules. self-assemble The computer into predictions responsive were and supported adaptive by supramolecular TEM imaging (Figure assemblies 9e), confirming in the presence that of externalAD stimuli,is able to i.e.,dynamically negatively-charged self-assemble small into responsive polyanions. and adaptive supramolecular assemblies in the presence of external stimuli, i.e., negatively-charged small polyanions. 3.2. In Vitro siRNA Delivery Performance of the Double-Tail Self-Assembling Dendron AD 3.2. In Vitro siRNA Delivery Performance of the Double-Tail Self-Assembling Dendron AD The nanosized micelles formed by self-assembly of the double-tail amphiphilic dendron AD The nanosized micelles formed by self-assembly of the double-tail amphiphilic dendron AD were able to effectively protect siRNA from degradation (e.g., by RNAses) and to promote fast and were able to effectively protect siRNA from degradation (e.g., by RNAses) and to promote fast and quantitativequantitative uptake uptake by by castrate-resistant castrate-resistant prostateprostate cancer PC-3 PC-3 cells—almost cells—almost 100% 100% internalization internalization of of the nanovectorthe nanovector/siRNA/siRNA complexes complexes was wasreached reached within 30 30 min. min. As As concerns concerns the themechanism mechanism of cell of cell internalizationinternalization of the of ADthe /ADsiRNA/siRNA complexes, complexes, macropinocytosis macropinocytosis was was found found to to be be the the leading leading cellular cellular entry mechanismentry mechanism for this nanovector for this nanovector/siRNA/siRNA system, insystem, utter analogyin utter analogy with the with G5 covalentthe G5 covalent PAMAM PAMAM dendrimer counterpartdendrimer discussed counterpart in the discussed companion in the paper companion (Figure paper 15a in(Figure [9]). Indeed,15a in [9]). neither Indeed, chlorpromazine neither (an inhibitorchlorpromazine of clathrin-mediated (an inhibitor of clathrin-mediated endocytosis) nor endocytosis) genistein (anor caveolae-mediated genistein (a caveolae-mediated uptake specific blocker)uptake were specific effective blocker) while were cytochalasin effective while D (a cytochalasin specific macropinocytosis D (a specific macropinocytosis inhibitor that, inhibitor by binding that, by binding to actin filaments, interferes with actin polymerization and assembly) inhibited to actin filaments, interferes with actin polymerization and assembly) inhibited cellular uptake cellular uptake in a dose-dependent manner, as presented in Figure 10b. Again in analogy with what in a dose-dependent manner, as presented in Figure 10b. Again in analogy with what observed for the observed for the G5 TEA-core PAMAM covalent dendrimer (Figure 15b in [9]), confocal microscopy G5 TEA-coreprovided PAMAMimages showing covalent significant dendrimer co-localization (Figure 15b in of [9 ]),the confocal siRNA/AD microscopy complexes provided with the images showingmacropinocytosis significant co-localization biomarker dextran. of the Whereas, siRNA/AD negligiblecomplexes co-localization with the macropinocytosis was observed in biomarkercells dextran.treated Whereas, with the negligibletwo alternative co-localization biomarkers transf was observederrin (clathrin-mediated in cells treated endocytosis) with the twoand CTX-B alternative biomarkers(caveolae-mediated transferrin endocytosis). (clathrin-mediated Thus, these endocytosis) experiments and further CTX-B corroborate (caveolae-mediated the concept that endocytosis). these Thus,self-assembling these experiments PAMAM-based further corroborate amphiphilic thedendrons concept behave that very these similarly self-assembling to their covalent PAMAM-based high amphiphilicgeneration dendrons counterpart. behave very similarly to their covalent high generation counterpart. TheThe gene gene silencing silencing e ffeffectect inducedinduced by by Hsp-27 Hsp-27 targeting targeting siRNA siRNA delivered delivered by AD bynanomicellesAD nanomicelles was next investigated both at the mRNA and protein levels using prostate (PC-3) and breast (MCF-7 and was next investigated both at the mRNA and protein levels using prostate (PC-3) and breast (MCF-7 MDA-MB231) cancer cell lines. As seen in Figure 10, a remarkable attenuation of the Hsp27 and MDA-MB231) cancer cell lines. As seen in Figure 10, a remarkable attenuation of the Hsp27 expression was achieved in all cases. This effect was comparable, if not greater, than that achieved expressionwith Lipofectamine, was achieved the in allreference cases. Thisstandard effect reagent was comparable, for siRNA/DNA if not transfection greater, than (e.g., that AD achieved was 6 with Lipofectamine,times more theefficient reference than Lipofectamine standard reagent in downregu for siRNAlating/DNA Hsp27 transfection protein expression (e.g., AD wasin PC-3 6 times cells, more efficientFigure than 10b). Lipofectamine in downregulating Hsp27 protein expression in PC-3 cells, Figure 10b).
(a) (b)
Figure 10. Cont.
Pharmaceutics 2019, 11, 324 13 of 26 Pharmaceutics 2019, 11, x FOR PEER REVIEW 13 of 26
(c) (d)
FigureFigure 10. Downregulation10. Downregulation of of Hsp27 Hsp27 atat thethe mRNAmRNA ( (aa) )and and protein protein (b) (b) level level in prostate in prostate cancer cancer PC-3 PC-3 cellscells treated treated with with siRNA siRNA (50 (50 nM) nM) delivered delivered byby the self-assembled double-tail double-tail dendron dendron AD (N/PAD (N= 5)./P = 5). InhibitionInhibition of Hsp27 of Hsp27 expression expression by by siRNA siRNA//ADAD complexes (50 (50 nM nM siRNA, siRNA, N/P N =/P 5)= in5) breast in breast cancer cancer MCF-7 (c) and MDA-MB231 (d). Adapted from [6] with permission of John Wiley and Sons, 2014. MCF-7 (c) and MDA-MB231 (d). Adapted from [6] with permission of John Wiley and Sons, 2014. The knockdown of Hsp27 in these cancer cells by siRNA delivered by AD micelles was The knockdown of Hsp27 in these cancer cells by siRNA delivered by AD micelles was paralleled paralleled by significant anti-proliferative effects (~75% of cancer cell growth inhibition), in by significant anti-proliferative effects (~75% of cancer cell growth inhibition), in agreement with agreement with the evidence that Hsp27 silencing negatively affects PC-3 cell survival [9,17]. the evidenceSimilarly thatto what Hsp27 was silencing observed negatively for the high aff ectsgeneration PC-3 cellcovalent survival PAMAM [9,17 ].dendrimers Similarly to[9], what the was observedmechanism for the highleading generation to cell death covalent relied PAMAM on caspase-dependent dendrimers [9], theinduced mechanism apoptosis, leading that tois, cellthe death reliedprogrammed on caspase-dependent cell death was induced promoted apoptosis, by apoptotic that ca is,spases, the programmed a family of endoproteases cell death was that promotedprovide by apoptoticcritical caspases, links in cell a family regulatory of endoproteases networks controlling that provide cell death. critical Accordingly, links in cell a 3-fold regulatory increase networks in controllingcaspase-3/7 cell death.activation Accordingly, was observed a 3-fold on average, increase confirming in caspase-3 that siRNA/7 activation delivered was by observed the nanovectors on average, confirmingbased on that self-assembled siRNA delivered AD is byvery the effective nanovectors in silencing based Hsp27 on self-assembled and thereby inducingAD is verycaspase- effective in silencingdependent Hsp27 anticancer and thereby activity inducing in vitro. A caspase-dependent remarkable additional anticancer feature of activity this biologicalin vitro outcome. A remarkable is that the silencing effect was completely maintained for one week, and was also effective when additional feature of this biological outcome is that the silencing effect was completely maintained transfection was performed in the presence of serum, a prototypical Achilles’ heel of cationic for one week, and was also effective when transfection was performed in the presence of serum, nanovectors in siRNA delivery. In fact, the negatively charged serum proteins can not only compete a prototypicalwith siRNA Achilles’ binding heelto the of nanocarrier, cationic nanovectors but also may, in by siRNA virtue delivery. of strong Inelectrostatic fact, the negatively forces, lead charged to serumthe proteins the disintegration can not only of the compete nanoassemblies with siRNA with binding consequent to the premature nanocarrier, nucleic but acid also release may, byand virtue of strongdegradation electrostatic [9]. forces, lead to the the disintegration of the nanoassemblies with consequent prematureFrom nucleic another acid perspective, release and when degradation confronted [9 ].with its single-tail analogue 4, the efficiency of in Fromvitro silencing another induced perspective, by the double-tailed when confronted AD dendron/siRNA with its single-tail complexes analogue was practically4, the identical efficiency of in vitro(e.g.,silencing 97% for AD induced and 96% by thefor 4 double-tailed in PC-3 cells, FigureAD dendron 10b,c, respec/siRNAtively). complexes Most importantly, was practically however, identical (e.g.,remarkably 97% for AD goodand 96%results for were4 in PC-3obtained cells, with Figure AD 10 whenb,c, respectively). challenged against Most importantly,cancer stem cells however, (notoriously the cancer cell population most refractory to treatments), and also stem and primary remarkably good results were obtained with AD when challenged against cancer stem cells (notoriously cells that constitute the target of deadly viruses such as HIV-1. As shown in Figure 11, AD was indeed the cancerable to cell elicit population significant most RNAi refractory interference to treatments),(RNAi) in glioblastoma and also stem stem and cells primary (PBT003), cells and that in HIV-1 constitute the targetinfected of human deadly primary viruses peripheral such as blood HIV-1. mononuclear As shown cells in Figure(PBMC-CD4 11, AD+), andwas hematopoietic indeed able stem to elicit significantcells (HSC-CD34 RNAi interference+). Specifically, (RNAi) upon in treatment glioblastoma of the stem hardly cells tractable (PBT003), PBT003 and cells in HIV-1 with AD infected carrying human primarythe siRNA peripheral was directed blood mononuclear against the signal cells transducer (PBMC-CD4 and+ ),activator and hematopoietic of transcription stem 3 (STAT3), cells (HSC-CD34 which +). Specifically,is a key player upon protein treatment in glioma-initiating of the hardly cells, tractable thought PBT003 to be responsible cells with forAD glioblastomacarrying the induction, siRNA was directedprogression against and the recurrence. signal transducer A 35% reduction and activator in the corresponding of transcription mRNA 3 (STAT3), level was which observed, is a keywhile player proteinonly in 24% glioma-initiating mRNA reduction cells, was thought achieved to beupon responsible transfection for with glioblastoma Lipofectamine induction, (Figure progression 11a). The and + + recurrence.data relative A 35% to reductionthe two HIV-infected in the corresponding cell lines (PBMC-CD4 mRNA level and was HSC-CD34 observed,) transfected while only with 24% the mRNA siRNA targeting the HIV-1 Tat/Rev gene are more striking. The Tat and Rev HIV-1 proteins are reduction was achieved upon transfection with Lipofectamine (Figure 11a). The data relative to the two essential positive regulators of +gene expression through+ interaction with RNA target elements HIV-infectedpresent within cell lines the (PBMC-CD45′ translated leaderand sequence HSC-CD34 and) envelope transfected gene, with respectively. the siRNA Importantly, targeting the the HIV-1 Tat/Revgenes gene encode are morethe Rev striking. and Tat overlap, The Tat with and each Rev being HIV-1 produced proteins from are a essential different positivereading frame. regulators As of geneseen expression from Figure through 11b,c, interaction in both cases with a remarkab RNA targetle 50% elements reduction present in the withinTat/Rev the mRNA 50 translated expression leader sequencewas observed, and envelope while the gene, prototypical respectively. delivery Importantly, vector Lipofectamine the genes (aka encode RNAiMAX) the Rev failed and to Tatinduce overlap, with each being produced from a different reading frame. As seen from Figure 11b,c, in both cases a remarkable 50% reduction in the Tat/Rev mRNA expression was observed, while the prototypical delivery vector Lipofectamine (aka RNAiMAX) failed to induce any gene silencing in these cells Pharmaceutics 2019, 11, 324 14 of 26 Pharmaceutics 2019, 11, x FOR PEER REVIEW 14 of 26
any gene silencing in these cells infected by the acquired immunodeficiency syndrome (AIDS) virus. infectedFundamentally, by the acquired this translated immunodeficiency into a 50% reductio syndromen of (AIDS)the viral virus.infection Fundamentally, (Figure 11d,e) and, this to translated the into abest 50% of reductionthe authors’ of knowledge, the viral infection constitutes (Figure the first 11 d,e)report and, on toth thee successful best of theand authors’ safe delivery knowledge, of constitutessiRNA theinto firstprimary report and on stem the cells. successful and safe delivery of siRNA into primary and stem cells.
(a) (b) (c)
(d) (e) (f)
FigureFigure 11. ( a11.) STAT3 (a) STAT3 knockdown knockdown in glioblastoma in glioblastoma stem cellsstem (PBT003)cells (PBT003) treated treated with siRNA with siRNA//AD complexesAD (50 nMcomplexes siRNA, (50 N/P nM= 5).siRNA, The N/P knockdown = 5). The ofknockdown Tat/Rev in of primary Tat/Rev in human primary peripheral human peripheral blood mononuclear blood + + + cells (PBMC-CD4mononuclear +cells)(b (PBMC-CD4) and hematopoietic) (b) and hematopoietic CD34+ stem cellsCD34 (HSC-CD4 stem cells +(HSC-CD4)(c) treated) (c as) treated in (a). as E ffinective + + inhibition(a). Effective of HIV inhibition replication of inHIV PBMC-CD4 replication+ in(d PBMC-CD4) and HSC-CD4 (d) and+ (e HSC-CD4) after treatment (e) after with treatment the anti-Tat with /Rev the anti-Tat/Rev siRNA delivered by AD (50 nM siRNA, N/P = 5). Viral loading was assessed using siRNA delivered by AD (50 nM siRNA, N/P = 5). Viral loading was assessed using HIV-1 p24 antigen HIV-1 p24 antigen ELISA 3 days post-treatment. PBMC-CD4+ cells and HSC-CD34+ cells were infected ELISA 3 days post-treatment. PBMC-CD4+ cells and HSC-CD34+ cells were infected by NL4-3 virus by NL4-3 virus at a multiplicity of infection (MOI) = 0.001 and by JR-FL virus (MOI = 0.005), at a multiplicityrespectively, offor infection 5 days before (MOI) transfection.= 0.001 and (f) by Proton-sponge JR-FL virus effect (MOI in= 0.005),PC-3 cells respectively, transfected forwith 5 days beforesiRNA/ transfection.AD in the ( fabsence) Proton-sponge (solid bars) e ffandect presence in PC-3 cells(patterned transfected bars) of with the proton siRNA pump/AD in inhibitor the absence (solidBafilomycin bars) and A1. presence Untreated (patterned cells were bars) used of as the the protoncontrol. pumpAdapted inhibitor from [6] Bafilomycinwith permission A1. of Untreated John cellsWiley were usedand Sons, as the 2014. control. Adapted from [6] with permission of John Wiley and Sons, 2014.
As forAs thefor the high high generation generation covalent covalent PAMAMPAMAM dendrimer analogues analogues presented presented in the in companion the companion paperpaper [9], it [9], was it was reasoned reasoned that that also also in thein the case case of of the theAD ADmicelles, micelles, the the so-called so-called protonproton sponge effect effect [18] could[18] be could invoked be invoked to explain to explain nucleic nucleic acid releaseacid rele fromase from the the self-assembled self-assembled double-tail double-tail nanovectorsnanovectors and, therefore,and, therefore, potent siRNA potent delivery siRNA delivery within within the cell th cytoplasm.e cell cytoplasm. According According to the to debatedthe debated proton proton sponge concept,sponge when concept, cationic when nanoparticles cationic nanoparticles such as such those as formedthose formed by AD byand AD and its siRNA its siRNA cargo cargo enter enter acidic acidic endosomal (and lysosomal) vesicles, unsaturated amino groups sequester protons supplied by endosomal (and lysosomal) vesicles, unsaturated amino groups sequester protons supplied by the the proton pump v-ATPase. These sequestered protons cause the pump to continue functioning, protonleading pump to v-ATPase. the retention These of chloride sequestered ions and protons water cause molecules. the pump Eventually, to continue osmotic functioning, swelling causes leading to the retentionthe rupture of chlorideof the vesicle, ions andallowing water the molecules. cationic nanoparticles Eventually, osmoticto enter swellingthe cytoplasm. causes As the the rupture AD of the vesicle,amphiphiles allowing feature the tertiary cationic amines nanoparticles in the interi toor enter of their the PAMAM cytoplasm. heads As which the AD becomeamphiphiles protonated feature tertiaryat the amines endosomal in the acidic interior pH of (5.5), their the PAMAM authors heads verified which whether become the protonatedproton sponge at theeffect endosomal was acidiceffectively pH (5.5), at the play authors also during verified their whether mediated the protonsiRNA spongedelivery. e ffIndeed,ect was as e ffilluectivelystrated atin play Figure also 11f, during theirHsp27 mediated silencing siRNA in PC-3 delivery. cells was Indeed, significantly as illustrated reduced in in the Figure presence 11f, of Hsp27 Bafilomycin silencing A1 (a in proton PC-3 cells was significantlypump inhibitor reduced that prevents in the presenceendosome ofacidification). Bafilomycin This A1 implies (a proton that pump the AD inhibitor-mediated that siRNA prevents endosome acidification). This implies that the AD-mediated siRNA delivery was indeed dependent on the endosomal acidification process and that the proton sponge effect played a role in the endosomal escape and cytoplasmic release of the nucleic acid. Finally, the same protocol adopted for investigating the cellular toxicity of the single-tail self-assembled dendrons described in Section 2.3 was applied to AD. As expected, no discernible PharmaceuticsPharmaceutics2019, 11 2019, 324, 11, x FOR PEER REVIEW 15 of 26 15 of 26
delivery was indeed dependent on the endosomal acidification process and that the proton sponge effect played a role in the endosomal escape and cytoplasmic release of the nucleic acid. toxicity was observed in all cell lines (PC-3, PBMC-CD4+, HSC-CD34+, and PBT003 transfected with Finally, the same protocol adopted for investigating the cellular toxicity of the single-tail self- 50 nM siRNAassembled and dendronsAD at described N/P = 5 andin Section 10) using2.3 was both applied the to MTT AD. As and expected, LDH assays,no discernible supporting toxicity further in vivowas gene observed silencing in all experiments cell lines (PC-3, with PBMC-CD4 this nanocarrier.+, HSC-CD34+, and PBT003 transfected with 50 nM siRNA and AD at N/P = 5 and 10) using both the MTT and LDH assays, supporting further in vivo 3.3. In Vivogene siRNAsilencing Delivery experiments Performance with this ofnanocarrier. the Double-Tail Self-Assembling Dendron AD
The3.3. final In Vivo steps siRNA to Delivery assess thePerformance therapeutic of the Double-Tail potential Self-Assembling of siRNA delivery Dendron basedAD on the double-tail self-assembled amphiphilic dendron AD were constituted by in vivo experiments using prostate cancer The final steps to assess the therapeutic potential of siRNA delivery based on the double-tail (PC-3) xenograftself-assembled mouse amphiphilic models anddendron Hsp27 AD as were the constituted target gene by for in knockdown.vivo experiments Figure using 12 prostatea,b reveal that, followingcancer a 5-week (PC-3) xenograft animal treatment mouse models by intraperitoneal and Hsp27 as the injections target gene (2 for/week) knockdown. of siRNA Figure/AD 12a,bcomplexes (3 mg/kgreveal Hsp27 that, siRNA,followingAD a 5-weekat N /animalP = 5), treatment there was by intraperitoneal a remarkable injections downregulation (2/week) of ofsiRNA/ Hsp27AD at both mRNAcomplexes (62%) and (3 protein mg/kg Hsp27 (73%) siRNA, levels. AD This at N/P was = paralleled5), there was by a remarkable an effective downregulation inhibition of of tumor Hsp27 growth, as shownat both in Figure mRNA 12(62%)c. Further, and protein as (73%) for the levels. case This of was the paralleled single-tail by amphiphilican effective inhibition analogue of tumor4, no major growth, as shown in Figure 12c. Further, as for the case of the single-tail amphiphilic analogue 4, no organ injury or histopathological changes were evidenced in the animal subjected to AD/siRNA major organ injury or histopathological changes were evidenced in the animal subjected to treatmentsAD/siRNA (Figures treatments 12d and (Figure A2). 12d and A2).
(a) (b)
(c) (d)
Figure 12.FigureIn vivo12. In evivoffective effective gene gene silencing silencing of of Hsp27 Hsp27 at at the the mRNA mRNA (a) (anda) and protein protein (b) levels (b) levelsobserved observed in prostatein prostate cancer cancer (PC-3) (PC-3) xenograft xenograft mice mice after after treatment treatment with with Hsp27 Hsp27 siRNA siRNA delivered delivered by AD by (3 mg/kgAD (3 mg/kg siRNA and AD at N/P = 5, injection twice a week for 5 weeks). Hsp27 mRNA and protein levels were siRNA and AD at N/P = 5, injection twice a week for 5 weeks). Hsp27 mRNA and protein levels quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western were quantifiedblotting, respectively. by quantitative Non-treated reverse mice transcriptionwere used for the polymerase control. AD chain alone, reactionsiRNA alone (qRT-PCR) and a and westernscrambled blotting, (non-silencing) respectively. siRNA Non-treated sequence mice delivered were by used AD for were the used control. as negativeAD alone, controls. siRNA (c) alone and a scrambledTumor volume (non-silencing) during treatment siRNA of PC-3 sequence mice xenografts delivered with siRNA/ by ADADwere complexes usedas (red negative symbols), controls. (c) Tumorcompared volume with during non-treated treatment animal ofs PC-3 (green), mice and xenografts animals treated with siRNAby AD /aloneAD complexes(light blue), (redsiRNA symbols), comparedalone with (dark non-treated blue), and a scrambled animals (green),(non-silencing) and animalssiRNA sequence treated delivered by AD by alone AD (light(gray symbol) blue), siRNA used as negative controls. (d) Hepatic enzyme levels (alanine transferase ALT (light green bars), alone (dark blue), and a scrambled (non-silencing) siRNA sequence delivered by AD (gray symbol)
used as negative controls. (d) Hepatic enzyme levels (alanine transferase ALT (light green bars), gamma-glutamine transferase γ-GLT (teal bars), (kidney functions (creatinine CRE (mmol/L, sky blue bars), blood urea nitrogen BUN (µmol/L, dark blue bars), and cholesterol level in the blood (gray bars, values on the right axis) measured in male C57BL/6 mice treated with intravenous administration of siRNA/AD (3 mg/kg siRNA and AD at N/P = 5). Untreated mice were used for control. Adapted from [6] with permission of John Wiley and Sons, 2014. Pharmaceutics 2019, 11, 324 16 of 26
In summary, the nanosized micelles generated upon self-assembly of the double-tail amphiphilic PAMAM-based dendron AD were not able to deliver siRNA to a different panel of cancer cell lines with efficiency in vitro and in vivo comparable to those formed by its single-tail precursor 4. However, they were also capable of eliciting gene silencing effects in the highly challenging and treatment-refectory human primary cells and stem cells. Accordingly, these versatile and safe delivery properties of AD, coupled with easy formulation, render AD a promising nanocarrier for highly efficient, cell-type-independent RNAi therapeutics.
4. siRNA Delivery by the Double-Tail, Dual Targeting Self-Assembling Amphiphilic Dendron AD/E16G6RGDK
4.1. Design, Optimization and Chemico-Physical Charactrization of the Double-Tail, Dual Targeting Self-Assembling Amphiphilic Dendron AD/E16G6RGDK In the general design strategy of nanocarriers to achieve effective siRNA (and in general drug) delivery, two well-known criteria must be satisfied: (i) The delivered therapeutic is be able to reach the desired disease (e.g., tumor) site(s) after administration with minimal loss in their quantity and activity during their journey in the blood stream; and (ii) they only affect diseased cells without exerting harmful effects to healthy organs and tissues. These requirements may be enabled using two strategies: Passive and active drug targeting of drugs. The former approach relies on the (still controversial) intrinsic enhanced permeability and retention effect (EPR) [19,20], according to which molecules with size in the nanometer range tend to accumulate in diseased (especially tumor) tissues much more than they do in normal tissues, thereby leaving little space for specific molecular design and optimization. Active targeting employs directed intermolecular interactions (e.g., ligand binding to disease-overexpressed receptors or other molecular recognition processes) to confer more specificity to the delivery system. In the end, besides enhanced therapeutic action, active targeting can result in reduced non-specific interactions and localization of the drug in peripheral tissues, thus minimizing unwanted side-effects. One of the biggest challenges in active targeting consists in designing new nanovectors (or modifying existing performing ones) with targeting moieties that do not interfere with its cargo loading capacity and its final mission purposes. Obviously, these chemical entities must be fully available to interact with their cellular counterparts and be endowed with strong affinity towards their targets (e.g., surface receptors) in order to trigger endocytosis. With the goal of empowering the high-performance double-tail self-assembling amphiphilic dendron AD with active targeting ability for siRNA specific delivery to cancer cells, the authors considered the RGDK peptide as the starting point for molecular design and optimization. The RGDK peptide is an ideal moiety for cancer targeted nanomedicine in that it features a dual targeting capacity within a single, small molecular scaffold. On the one side, the RGD sequence is known to be able to target tumor endothelium via interaction with ανβ3-integrin (a receptor overexpressed in tumor vasculature [21]); while on the other side, the RGDK sequence has been shown to bind the neutropilin-1 (Nrp-1, a transmembrane hub receptor with multiple ligands that is abundant on the surface of cancer cells [22]), and in so doing, promoted cell uptake and penetration. Several sequence rounds of computer optimization led to the final design of the E16G6RGDK peptide shown in Figure 13a [8]. This new chemical entity features three distinct segments serving different functions, as follows: (1) A highly negatively charged tail composed of 16 glutamic acid residues (E16), for anchoring the peptide to the positively charged surface of the self-assembled AD micelles via strong, favorable electrostatic interactions; (2) the positively charged RGDK warhead for fostering cancer cell targeting and homing by virtue of the dual interaction with ανβ3-integrin and Nrp-1 receptors; and (3) a neutral spacer of optimal length composed of 6 glycine residues (G6) separating the oppositely charged segments, (1) and (2). Pharmaceutics 2019, 11, x FOR PEER REVIEW 17 of 26
PharmaceuticsNrp-1 receptors;2019, 11 ,and 324 (3) a neutral spacer of optimal length composed of 6 glycine residues 17(G of6)26 separating the oppositely charged segments, (1) and (2).
(a) (b) (c)
FigureFigure 13.13. ((aa)) ChemicalChemical structurestructure ofof the the E E1616GG66RGDKRGDK peptidepeptide designeddesigned asas a a double double targeting targeting moiety moiety for enhancingfor enhancing the siRNAthe siRNA delivery delivery to cancer to cancer cells mediatedcells mediated by the by double-tail the double-tail self-assembling self-assemblingAD dendron. AD
Thedendron. light redThe boxlight encases red box the encases E16 fragment, the E16 fragment, the light the green light box green marks box the marks G6 spacer the G6 whilespacer the while light bluethe light box highlightsblue box highlights the RGDK the warhead. RGDK warhead. (b) TEM imaging(b) TEM of imaging the siRNA of the/AD siRNA// E16G6ADRGDK/ E16G nanosized6RGDK micellesnanosized formed micelles upon formed simple upon addition simple of addition the targeting of the peptidetargeting to peptide pre-formed to pre-formed siRNA/AD siRNA/complexes.AD
(complexes.c) Integrated (c) ITCIntegrated profile ITC for profile the titration for the of titration siRNA /ofAD siRNA/complexesAD complexes with the with E16G the6RGDK E16G6RGDK peptide. Thepeptide. solid The red linesolid represents red line repres data fittingents data with fitting a sigmoidal with a function. sigmoida Thel function. insert shows The insert the corresponding shows the ITCcorresponding raw data. AdaptedITC raw fromdata. [Adapted8] with permission from [8] with of the permission American of Chemical the American Society, Chemical 2018. Society, 2018. With the optimized new peptide structure at hand, and the previous knowledge about the self-assemblingWith the optimized process of newAD peptideand its related structure performance at hand, and as siRNA the previous nanocarrier knowledge (Section about3), the the authors self- initiallyassembling verified process the of interaction AD and its of related the E16 Gperformance6RGDK molecules as siRNA with nanocarrier pre-formed (Section siRNA 3),/AD thecomplexes authors ininitially solution verified by means the interaction of different of experimental the E16G6RGDK techniques. molecules Both with DLS pre-formed and TEM confirmedsiRNA/AD the complexes formation ofin nanosizedsolution by (30–45 means nm of indifferent diameter) experiment sphericalal micellestechniques. (shown Both inDLS Figure and 13TEMb), characterizedconfirmed the by aformationζ-potential of value nanosized of +15 mV,(30–45 approximately nm in diameter equal)to spherical half of the micelles value measured (shown forin theFigure siRNA 13b),/AD assembliescharacterized (+ 32by mV).a ζ-potential This substantial value of decrease+15 mV, approximately in positive surface equal charges to half of further the value supported measured the interactionfor the siRNA/ of theAD negatively assemblies charged (+32 mV). ( 15) This peptide substantial with decrease the siRNA in/ ADpositivecomplexes. surface charges further − supportedHowever, the interaction the ultimate of the confirmation negatively charged of the e(−ff15)ective peptide binding with the of thesiRNA/ E16GAD6RGDK complexes. targeting peptidesHowever, to the the siRNA ultimate/AD nanomicellesconfirmation wasof the obtained effective by binding isothermal of the titration E16G6RGDK calorimetry targeting (ITC). ITCpeptides is a straightforwardto the siRNA/AD and nanomicelles noninvasive was titration-based obtained by isothermal method fortitration binding calorimetry interaction (ITC). analysis ITC performedis a straightforward in solution and at constant noninvasive temperature titration-based [23]. The method analysis for of binding ITC curves interaction directly yieldsanalysis the bindingperformed enthalpy in solution (∆H at )constant and, upon temperature data fitting [23] with. The a suitable analysis thermodynamic of ITC curves directly model, theyields binding the Δ bind constantbinding enthalpy K can be ( obtained.Hbind) and, Once upon K datais known, fitting with the free a suit energyable thermodynamic of binding ∆G model,is calculated the binding using b b Δ bind theconstant relationship Kb can be∆G obtained.= RT Once lnK Kb is, known, where Rthe is free the universalenergy of gasbinding constant Gbind and is calculated T is the absolute using Δ bind − bind temperature.the relationship Finally, Gbind the = variation−RT lnKbind in, entropywhere R upon is the binding universal is derivedgas constant from theand fundamental T is the absolute Gibbs temperature. Finally, the variation in entropy upon binding is derived from the fundamental Gibbs equation ∆Gbind = ∆Hbind T∆Sbind. The binding thermodynamics between the targeting peptides and equation ΔGbind = ΔHbind − −TΔSbind. The binding thermodynamics between the targeting peptides and the siRNA/AD micelles was found to be characterized by a favorable (i.e., negative) enthalpic variation the siRNA/AD micelles was found to be characterized by a favorable (i.e., negative) enthalpic (∆Hbind = 5.0 0.2 kcal/mol), as evidenced by the exothermic peaks in the corresponding thermogram variation (−ΔHbind± = −5.0 ± 0.2 kcal/mol), as evidenced by the exothermic peaks in the corresponding (Figure 13c). Also, a small, favorable (i.e., positive) entropic change (T∆S = 1.9 kcal/mol) was estimated, thermogram (Figure 13c). Also, a small, favorable (i.e., positive) entropic change (TΔS = 1.9 kcal/mol) leading to an overall favorable (i.e., negative) ∆Gbind value of 6.8 kcal/mol. The substantial enthalpic was estimated, leading to an overall favorable (i.e., negative)− ΔGbind value of −6.8 kcal/mol. The nature of the binding between the RGKD peptide and the siRNA/AD micelles is the result of electrostatic substantial enthalpic nature of the binding between the RGKD peptide and the siRNA/AD micelles forces as the main supramolecular interaction drivers. However, the favorable entropic contribution is the result of electrostatic forces as the main supramolecular interaction drivers. However, the was ascribed to the stabilizing peptide/micelle hydrophobic interactions and the concomitant release favorable entropic contribution was ascribed to the stabilizing peptide/micelle hydrophobic of ions and water molecules into the bulk solvent [24–26]. interactions and the concomitant release of ions and water molecules into the bulk solvent [24–26]. The structure of the siRNA/AD/E16G6RGDK was next investigated at the molecular level The structure of the siRNA/AD/E16G6RGDK was next investigated at the molecular level via via computer simulations. Extensive (1 µs) atomistic molecular dynamics (MD) simulations revealed computer simulations. Extensive (1 μs) atomistic molecular dynamics (MD) simulations revealed the the formation of stable, Janus-like nanoparticles featuring a siRNA molecule bound on one face of formation of stable, Janus-like nanoparticles featuring a siRNA molecule bound on one face of each each AD micelle and 4 peptides adsorbed on the opposite face (Figure 14a). Further, MD simulations AD micelle and 4 peptides adsorbed on the opposite face (Figure 14a). Further, MD simulations showed that the negatively charged peptide tails (E G ) were adsorbed onto the positively charged showed that the negatively charged peptide tails (E1616G6)6 were adsorbed onto the positively charged surface micelle while, most importantly, the 4 RGDK warhead was free from sterical hindrance and protruded deeply into the solvent (Figure 14b). Pharmaceutics 2019, 11, x FOR PEER REVIEW 18 of 26 surfacePharmaceutics micelle2019 ,while,11, 324 most importantly, the 4 RGDK warhead was free from sterical hindrance18 and of 26 protruded deeply into the solvent (Figure 14b).
(a) (b)
(c) (d)
FigureFigure 14. (a) ImageImage extracted extracted from from the the equilibrated equilibrated portion portion of the of MDthe simulationMD simulation of a siRNA of a /siRNA/AD micelleAD
micellein complex in complex with 4 E 16withG6RGDK 4 E16G peptides6RGDK peptides performed performed in 150 nM in NaCl 150 waternM NaCl solution. water The solution. 4 peptides The are 4 peptidesnumbered are and numbered colored and as follows: colored Eas16 follows:G6RGDK1, E16G tail6RGDK1, (E16G6 tail) sandy (E16G brown,6) sandy head brown, (RDGK) head firebrick;(RDGK) firebrick;E16G6RGDK2, E16G6 tailRGDK2, light green,tail light head green, olive drab;head Eolive16G6 RGDK3,drab; E16 tailG6RGDK3, khaki, head tail goldenkhaki, rod;head E 16goldenG6RGDK4, rod; Etail16G plum,6RGDK4, head tail magenta. plum, TheheadAD magenta.micelle is The in a darkAD slatemicelle blue is sticks-and-balls in a dark slate representation, blue sticks-and-balls the siRNA representation,molecule is highlighted the siRNA by molecule its gray vanis highlighted der Waals surface,by its gray the van peptides der Waals are shown surface, as the colored peptides spheres, are + shownNa and as colored Cl− ions spheres, are depicted Na+ and as purpleCl− ions and are greendepicted hollow as purple spheres, and whilegreen ahollow lightcyan spheres, transparent while a lightsurface cyan is usedtransparent to represent surface the is waterused to solvent. represent (b) the Zoomed water viewsolvent. of the (b) imageZoomed in panelview of (a ),the showing image
inthe panel E16G (6aRGDK4), showing peptide the E16 withG6RGDK4 its tail peptide (plum) attachedwith its tail to the(plum) micelle attached surface to andthe micelle its RGDK surface warhead and its(magenta) RGDK warhead protruding (magenta) into the protrudi solvent.ng In thisinto panel,the solvent. water In and this ions pa arenel, omitted water and for clarity.ions are ( comitted) Radial fordistribution clarity. (c function) Radial (RDF) distribution from the functionAD center (RDF) of mass from for the the AD positively center chargedof massAD forterminal the positively groups charged(dark slate AD blue), terminal and groups for each (dark of theslate four blue), RGDK and for warhead each of groups the four (colors RGDK as warhead in panel groups (a)). ( d(colors) Free
asenergy in panel of binding (a)). (d ()∆ FreeGbind energy) of each of E binding16G6RGDK (ΔG peptidebind) of each on the E16 siRNAG6RGDK/AD peptidemicelle. on The the patterned siRNA/ barsAD micelle.represent The the patterned∆Gbind values bars represent for the entire the Δ peptideGbind values molecule, for the while entire the peptide solid bars molecule, show the while contribution the solid
barsto ∆ Gshowbind athefforded contribution by the negatively to ΔGbind chargedafforded (Eby16 Gthe6) negatively tails only. Adaptedcharged from(E16G6 [)8 ]tails with only. permission Adapted of fromthe American [8] with permission Chemical Society, of the American 2018. Chemical Society, 2018. These pictorial views were quantified first by calculating the radial distribution function (RDF) These pictorial views were quantified first by calculating the radial distribution function (RDF) from the AD micelle center of mass for the positively charged AD terminal groups and the 4 targeting from the AD micelle center of mass for the positively charged AD terminal groups and the 4 targeting peptide RGDK warheads, as shown in Figure 14c. As seen from this Figure, all 4 RGDK moieties peptide RGDK warheads, as shown in Figure 14c. As seen from this Figure, all 4 RGDK moieties are are positioned far away from the nanomicelle periphery. Accordingly, they are potentially available positioned far away from the nanomicelle periphery. Accordingly, they are potentially available for for interaction with their cellular target receptors. Next, the free energy of binding ∆Gbind of each interaction with their cellular target receptors. Next, the free energy of binding ΔGbind of each single single E16G6RGDK peptide with the siRNA/AD nanomicelle was estimated by processing the MD E16G6RGDK peptide with the siRNA/AD nanomicelle was estimated by processing the MD data data according to the MM/PBSA methodology [9–15]. Figure 14d shows not only that all peptides according to the MM/PBSA methodology [9–15]. Figure 14d shows not only that all peptides are are characterized by favorableΔ ∆Gbind values (from 7.0 to 6.2 kcal/mol), but also that the major characterized by favorable Gbind values (from −7.0− to −6.2− kcal/mol), but also that the major contribution to binding is afforded by the strong electrostatic interaction between the negatively contribution to binding is afforded by the strong electrostatic interaction between the negatively charged peptide tails (E16G6) and the positively charged micellar surface, as expected by the original molecular design.
Pharmaceutics 2019, 11, x FOR PEER REVIEW 19 of 26 charged peptide tails (E16G6) and the positively charged micellar surface, as expected by the original Pharmaceutics 2019, 11, 324 19 of 26 molecular design.
4.2. In Vitro Targeted siRNAsiRNA DeliveryDelivery PerformancePerformance ofof Double-Tail,Double-Tail, Dual Dual Targeting Targeting Self-Assembling Self-Assembling AmphiphilicAmphiphilic DendronDendron ADAD/E/E1616G66RGDK
The authorsauthors verified verified that that the presencethe presence of the of E16 theG6RGDK E16G6RGDK peptides peptides on the surface on the of surface the siRNA of/ ADthe micellessiRNA/AD was micelles indeed ewasffective indeed in specifically effective in targeting specifically cancer targeting cells via cancer dualbinding cells via with dual the binding designated with ανβ targetthe designated receptors ανβ target3integrin receptors and Nrp-1. The3integrin cellular uptakeand Nrp-1. of the siRNA The /ADcellular/E16G 6RGDKuptake complexes of the wassiRNA/ investigatedAD/E16G6 againRGDK with complexes PC-3 cells, was in investigated which both receptorsagain with were PC-3 highly cells, expressed in which [ 27both,28 ].receptors As seen inwere Figure highly 15a, theexpressed amount [27,28]. of siRNA As/AD seen/E16 Gin6 RGDKFigure complexes15a, the amount internalized of siRNA/ by PC-3AD cells/E16 wasG6RGDK three timescomplexes higher internalized than that by measured PC-3 cells for was the three siRNA times/AD highermicelle. than Further, that measured the cellular for the uptake siRNA/ ofAD the nanocarriermicelle. Further, modified the withcellular the targetinguptake of moieties the nanocarrier was inhibited modified in the with presence the oftargeting a cyclic moieties RGD peptide. was Thisinhibited clearly in indicates the presence that this of lasta cyclic molecule RGD competed peptide. withThis theclearly peptide-coated indicates that nanovector this last for molecule integrin bindingcompeted via with the RDGthe peptide-coated motif. nanovector for integrin binding via the RDG motif.
(a)
(A) (B) (C) (D) (E) (b)
Figure 15.15. ((aa)) FlowFlow cytometrycytometry analysisanalysis ofof cellularcellular uptakeuptake ofof siRNAsiRNA//ADADand and siRNA siRNA//ADAD/E/E1616GG66RGDK nanoparticles in in PC-3 PC-3 cells cells using using a siRNA a siRNA labeled labeled with with the thefluorescent fluorescent Cy5 Cy5die. The die. last The column last column shows
showsthe cell theuptake cell results uptake for results the siRNA/ for theAD/E siRNA16G6RGDK/AD/E obtained16G6RGDK in the obtained presence in of the the presencecyclic peptide of the c- cyclicRGD, peptidean ανβ3-integrin c-RGD, anintegrinανβ3 -integrinbinder. (b integrin) Cellular binder. uptake (b visualized) Cellular uptakeby confocal visualized microscopy by confocal of the microscopysiRNA/AD/E of16 theG6RGDK siRNA complexes/AD/E16G 6inRGDK the absence complexes (top inpanel) the absenceand in the (top presence panel) and(bottom in the panel) presence of a (bottomNrp-1 antibody. panel) of Images a Nrp-1 from antibody. left to Imagesright: ( fromA) cy5-labelled left to right: siRNA; (A) cy5-labelled (B) nucleus siRNA; (4′,6-diamidino-2- (B) nucleus (4phenylindole0,6-diamidino-2-phenylindole (DAPI) staining); (DAPI)(C) merged staining); (A,B (C). )The merged last (twoA,B ).images The last (D two,E) imagesare the ( Dbright,E) are field the brightversion field of ( versionB,C). In ofall ( Bthese,C). Inexperiments, all these experiments, the siRNA/ theAD siRNA/E16G6/RGDKAD/E16 nanoassembliesG6RGDK nanoassemblies were prepared were preparedusing 50 nM using siRNA, 50 nM siRNA/ siRNA,AD siRNA N/P =/ AD10, andN/P AD= 10,/E16 andG6RGDKAD/E molar16G6RGDK ratio = molar 5. Adapted ratio = from5. Adapted [8] with frompermission [8] with of permissionthe American of theChemical American Society, Chemical 2018. Society, 2018.
Next, PC-3 PC-3 cells cells pretreated pretreated with with a monoclonal a monoclonal antibody antibody (mAb) (mAb) directed directed against against Nrp-1 Nrp-1 were wereused used to assay the internalization of the siRNA/AD/E16G6RGDK nanoassemblies in comparison with to assay the internalization of the siRNA/AD/E16G6RGDK nanoassemblies in comparison with untreated cells. As can be easily seen in the corre correspondingsponding confocal microsco microscopypy images (Figure 15b),15b), in the presence of the Nrp-1 mAb (bottom panel), the uptake of the siRNA-loaded peptide-modified AD micelles was drastically reduced when the mAb was absent (upper panel). This indicates that Pharmaceutics 2019, 11, x FOR PEER REVIEW 20 of 26 in the presence of the Nrp-1 mAb (bottom panel), the uptake of the siRNA-loaded peptide-modified AD micelles was drastically reduced when the mAb was absent (upper panel). This indicates that the targetingPharmaceutics peptide-decorated2019, 11, 324 siRNA delivery system was effectively cell internalized also via20 of its 26 interaction of its RGDK warhead with the Nrp-1 receptor. Following verification of the successful attribution of the desired targeting properties to our double-tailthe targeting self-assembling peptide-decorated AD nanovector, siRNA delivery the systemauthors waschecked effectively whether cell its internalized excellent endosomal also via its escapeinteraction features, of its RGDKrequired warhead for efficient with the siRNA Nrp-1 deli receptor.very and release, was preserved. By confocal microscopy,Following it was verification observed of that the successful2 h after transf attributionection, ofalmost the desired all of the targeting siRNA properties molecules to were our localizeddouble-tail inself-assembling the cell cytoplasmAD (Figurenanovector, A3). theThis authors confirms checked that the whether peptide-decorated its excellent endosomal AD nanovectors escape werefeatures, also requiredable to effectively for efficient circumvent siRNA delivery endosoma andl release,trapping was and preserved. release their By nucleic confocal acid microscopy, cargo for subsequentit was observed RNAi. that 2 h after transfection, almost all of the siRNA molecules were localized in the cell cytoplasmThe authors (Figure furtherA3). This assessed confirms whether that the the peptide-decorated endowment ofAD thenanovectors double-tail wereself-assembling also able to dendroneffectively AD circumvent with targeting endosomal properties trapping eventually and release conferred their nucleicby the RGDK-base acid cargo ford peptides subsequent rendered RNAi. this nanovectorThe authors more further effective assessed in siRNA whether delivery the endowment to cancer ofcells the with double-tail respect self-assembling to the undecorated dendron AD micelles.AD with Their targeting RNAi properties performance eventually in silencing conferred Hsp27 by in the PC-3 RGDK-based cancer cells peptides was compared. rendered The this relevantnanovector results more showed effective that in siRNA a remarkable delivery Hsp27 to cancer knockdown cells with was respect achieved to the undecoratedboth at the mRNAAD micelles. (56%, FigureTheir RNAi 16a) performanceand the protein in silencing (90%) Hsp27levels inwith PC-3 a cancervery low cells (20 was nM) compared. siRNA Theconcentration relevant results (for comparison,showed that a remarkable50 nM siRNA Hsp27 concentration knockdown was was requir achieveded to both produce at the similar mRNA results (56%, Figureeither with16a) andthe covalentthe protein G5 (90%) PAMAM levels dendrimer with a very [9] low or (20with nM) the siRNA AD micelles concentration without (for targeting comparison, peptide a 50 decoration nM siRNA (Figureconcentration 10)). Moreover, was required the tosame produce experiments similar results perfor eithermed in with the thepresence covalent of Geither5 PAMAM the c-RGD dendrimer peptide [9] or the with Nrp-1 the AD directedmicelles mAb without resulted targeting in a decreased peptide gene decoration silencing (Figure effect 10(as)). shown Moreover, by the the last same two columnsexperiments in Figure performed 16a). inThis the confirms presence that of either the siRNA/AD/E the c-RGD peptide16G6RGDK or the nanoparticles Nrp-1 directed indeed mAb resultedinteract within a decreasedthe two target gene receptors. silencing effect (as shown by the last two columns in Figure 16a). This confirms that the siRNA/AD/E16G6RGDK nanoparticles indeed interact with the two target receptors.
(a) (b)
Figure 16. (a(a) )mRNA mRNA levels levels following following knockdown knockdown of of Hsp27 Hsp27 in in PC-3 PC-3 cells cells with with siRNA/ siRNA/ADAD and
siRNA/siRNA/AD//EE1616GG66RGDKRGDK nanoparticles. nanoparticles. The The last last two two columns columns show show the the results results of of the the same same experiments experiments performed in the presence of the integrin binder c-RGD and of the mAb directed toward the Nrp-1 receptor,receptor, respectively. (b (b) )Comparison Comparison of of the the antiproliferative antiproliferative activity activity exerted exerted on on PC-3 PC-3 cells cells by
siRNA/siRNA/ADAD andand siRNA/ siRNA/ADAD/E/E1616GG6RGDK6RGDK nanoparticles. nanoparticles. In Inboth both panels, panels, non-treated non-treated cells cells were were used used for control,for control, siRNA siRNA concentration concentration = 20= 20nM, nM, siRNA/ siRNAAD/AD N/PN/ P= =10,10, and and ADAD/E/E1616GG6RGDK6RGDK molar molar ratio ratio == 5.5. Adapted from [8] [8] with permission of the American ChemicalChemical Society, 2018.2018.
Following siRNA delivery to PC-3 cells with the peptide-decorated AD nanomicelles, a drastic anticancer activity activity was was observed observed again again using using only only a 20 a nM 20 nMsiRNA siRNA concentration concentration (Figure (Figure 16b). 16Thisb). correspondsThis corresponds to an to effect an eff ofect 30% of 30% higher higher than than that that achieved achieved with with the the pristine pristine ADAD nanovectors.nanovectors. Concomitantly, the AD//EE16GG66RGDKRGDK nanoassemblies nanoassemblies exhibited exhibited a a safe safe to toxicityxicity profile, profile, similar similar to to that presented byby thetheAD ADmicelles micelles (Figure (Figure A4 A4),), and and in addition in addition to its to notable its notable improvement improvement in hemolytic in hemolytic effects (Figure A4c). All these data led the authors to perform the final testing of the siRNA/AD/E16G6RGDK nanoparticles in vivo.
Pharmaceutics 2019, 11, x FOR PEER REVIEW 21 of 26
effects (Figure A4c). All these data led the authors to perform the final testing of the Pharmaceutics 2019 11 siRNA/AD/E16G6RGDK, , 324 nanoparticles in vivo. 21 of 26
4.3.4.3. In In Vivo Vivo Targeted Targeted siRNA siRNA Delivery Delivery Performance Performance of of Double-Tail, Double-Tail, Dual Dual Targeting Targeting Self-Assembling Self-Assembling AmphiphilicAmphiphilic Dendron Dendron AD AD/E/E16G16G6RGDK6RGDK TheThein in vivo vivogene gene silencingsilencing and antica anticancerncer effects effects of of the the peptide-decorated peptide-decorated ADAD nanomicellesnanomicelles was wasinvestigated investigated again again using using PC-3 PC-3xenograft xenograft nude mice. nude Th mice.e first The analysis first was analysis conducted was conducted to verify their to verifyenhanced their tumor enhanced targeting tumor ability targeting with ability respect with to respect the undecorated to the undecorated AD nanovectorsAD nanovectors using a usingfluorescent-labeled a fluorescent-labeled Hsp27 Hsp27 siRNA. siRNA. Accordingly, Accordingly, ex exvivo vivo imaging imaging of of isolated isolated tumors andand thethe quantificationquantification of of the the relevant relevant mean mean fluorescence fluorescence intensity intensity were were performed performed (Figure (Figure 17 17a,b),a,b), from from which which thethe substantially substantially higher higher tumor tumor accumulation accumulation of of the the targeted targeted nanoparticles nanoparticles was was substantiated. substantiated.
(a) (b)
(c) (d)
FigureFigure 17. 17.(a ()a Ex) Ex vivo vivo fluorescence fluorescence images images obtained obtained from tumorfrom tumor isolated isolated from PC-3 from xenograft PC-3 xenograft mice treated mice
withtreated PBS bufferwith (deliveryPBS buffer medium (delivery used as medium control, left),used siRNA as /control,AD (center), left), and siRNA/ siRNAAD/AD /E(center),16G6RGDK and nanoparticlessiRNA/AD/E (right).16G6RGDK (b) nanoparticles Quantification (right). of the mean(b) Quantification fluorescence of intensity the mean for fluorescence the images in intensity panel a.for PBS the buffer images (c )in Tumor panel growtha. PBS buffer in untreated (c) Tumor animals growth (green in untreated filled symbols), animals and (green in animals filled symbols), treated withand siRNAin animals/AD (redtreated filled with symbols). siRNA/AD (d )(redIn vivo filledknockdown symbols). (d of) In Hsp27 vivo withknockdown siRNA/ ADof Hsp27(-RGKD) with andsiRNA/ siRNAAD/AD (-RGKD)/E16G6RGDK and siRNA/ (+ RGDK)AD/E nanoparticles.16G6RGDK (+ RGDK) Untreated nanoparticles. animals were Untreated used as animals the control. were Theused treatments as the control. (0.25 mg The/kg treatm siRNA,ents siRNA (0.25/ ADmg/kgN/P siRNA,= 10, and siRNA/AD/EAD16G 6N/PRGDK = 10, molar and ratioAD/E=16G5)6RGDK were administeredmolar ratio by= 5) intravenous were administered injection twice by intravenou per week.s Adapted injection from twice [8 ]per with week. permission Adapted of the from American [8] with Chemicalpermission Society, of the 2018. American Chemical Society, 2018.
AfterAfter 4 4 weeks weeks of of mice mice treatment treatment with with a a very very low low siRNA siRNA concentration concentration (0.25 (0.25 mg mg/kg)—12-fold/kg)—12-fold less less thanthan the the conventional conventional concentration concentration used used for for siRNA siRNA delivery delivery in mice—both in mice—both the tumorthe tumor volume volume and theand correspondingthe corresponding levels levels of Hsp27 of Hsp27 protein protein were drastically were drastically reduced reduced when siRNA when wassiRNA administered was administered via the peptide-decoratevia the peptide-decorateAD nanovectors, AD nanovectors, as shown as in Figureshown 17inc,d. Figure In particular, 17c,d. In particular, at day 31 after at day treatment 31 after withtreatment the siRNA with/AD the/ EsiRNA/16G6RGDKAD/E nanoparticles,16G6RGDK nanoparticles, tumor growth tumor was growth reduced was by reduced 73% with by respect 73% with to therespect control to andthe control by 50% and relative by 50% to siRNA relative/AD to treatmentsiRNA/AD (Figure treatment 17c). (Figure Consistently, 17c). Consistently, Hsp27 expression Hsp27 inexpression tumors was in reduced tumors bywas 65% reduced and 41% by upon 65% siRNAand 41% treatment upon siRNA with the treatment targeted nanovectorswith the targeted with respectnanovectors to the controlwith respect and AD to -deliveredthe control siRNA,and AD respectively-delivered siRNA, (Figure respectively 17d). (Figure 17d).
Pharmaceutics 2019, 11, 324 22 of 26
These highly promising results, coupled with the excellent in vivo toxicity profile exhibited by the AD/E16G6RGDK nanocarriers confirmed the high potential for these targeting nanosystems as safe and efficient siRNA delivery, gene silencing and consequent anticancer nanotherapeutics.
5. Conclusions As defined by Nature.com [29], “self-assembly is the process by which an organized structure spontaneously forms from individual components, as a result of specific, local interactions among the components. When the constitutive components are molecules, the process is termed molecular self-assembly”. A key feature of molecular self-assembly is the multivalent, cooperative and synergistic nature of the intermolecular interactions leading to the organization of individual molecular entities into well-defined nanosized structures. This approach presents at least two major conceptual advantages. The first relies on the evidence that the driving forces governing self-assembly lead to the formation of the nano-objects virtually with no flaws, as interactions between the nanomicelle building blocks are mediated by specific molecular recognition ultimately resulting in complex and ordered nanoscale structures. The second, by no means of less important benefit to self-assembly is that very small amounts of material are required to accomplish the process. In the companion paper [9], the authors presented the design, synthesis and gene silencing activity of modified PAMAM-based covalent nanovectors for siRNA delivery in cancer therapeutics. However, high generation dendrimer synthesis is extremely laborious and time-consuming, since the final product purification is difficult and hampered by the presence of highly similar side products. Thus, notwithstanding the highly promising results achieved with these molecules, the difficulties inherent in large-scale good manufacturing practice (GMP) production of high generation dendrimers led the authors to explore the potential of self-assembly in the design of new efficient siRNA nanocarriers. Thus, as presented in this second short review, the small PAMAM-based amphiphilic dendron 4, composed by a PAMAM head and a C18-long hydrocarbon tail, was initially developed. This was safe and effective in delivering siRNA both in vitro and in vivo. The natural evolution of 4 was its double-tail counterpart AD. The in vivo gene silencing effect obtained with siRNA delivered by AD nanomicelles in cancer treatment was comparable to that obtained with its precursor dendron 4. However, AD nanovectors were also capable of eliciting gene silencing effects in the highly challenging and treatment-refectory human primary cells and stem cells. The RNAi results of both the amphiphilic dendrons 4 and AD could be mainly attributed to passive targeting via the EPR effect in addition to their excellent siRNA delivery and endosomal escape capabilities. The authors focused on equipping AD with the dual E16G6RGDK targeting peptide, with the final goal of endowing the resulting nanovectors with cancer cell-directing specificity and, accordingly, higher anticancer activity. Compared to AD, the peptide-decorated AD nanomicelles exhibited more than 10-fold greater in vivo RNAi at significantly lower siRNA doses with respect to both covalent PAMAM dendrimers [9] and the non-targeted AD nanovectors. Our current activity in the field is progressing along this line, with the computer-assisted design of new targeting moieties for decorating self-assembling nanovectors in the development of gene silencing-based personalized medicine against different highly challenging and deadly (e.g., glioblastoma and pancreatic) human cancers.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/11/7/324/s1, Figure S1. Schematic representation of the coarse-grained DPD model of the AD dendron. The different bead types are colored as follows: RC, dark magenta; R, plum; L, dark turquoise; G, chartreuse; C, light gray. Table S1. Example of DPD interaction parameters used to simulate the self-assembling of the amphiphilic dendron AD per se and in the presence of siRNA molecules. Funding: This research was funded by the Italian Association for Cancer Research (AIRC), grant IG17413 to SP. The assistant position (RTDa) of SA is fully supported by the University of Trieste, in agreement with the actuation of the strategic planning financed by the Italian Ministry for University and Research (MIUR, triennial program 2016–2018) and the Regione Friuli Venezia Giulia (REFVG, strategic planning 2016–18), assigned to SP. This award is deeply acknowledged. Pharmaceutics 2019,, 11,, x 324 FOR PEER REVIEW 2323 of of 26 26 Pharmaceutics 2019, 11, x FOR PEER REVIEW 23 of 26 Acknowledgments: Authors wish to thank Ling Peng and her group for the longstanding, fruitful collaboration, Acknowledgments: Authors wish to thank Ling Peng and her group for the longstanding, fruitful collaboration, theAcknowledgments: challenges in siRNAAuthors delivery wish nanovector to thank Ling design Peng and and optimization, her group for the the inspiring longstanding, discussions fruitful and, collaboration, above all, the challenges in siRNA delivery nanovector design and optimization, the inspiring discussions and, above all, thethe personal challenges friendship. in siRNA delivery nanovector design and optimization, the inspiring discussions and, above all, the personal friendship.friendship. Conflicts of Interest: The authors declare no conflict of interest. ConflictsConflicts ofof Interest:Interest: The authors declaredeclare nono conflictconflict ofof interest.interest. Appendix Appendix A
Figure A1. Experimental determination of siRNA binding to self-assembling dendrons 1 (gray), 2 Figure A1.A1. ExperimentalExperimental determination determination of of siRNA siRNA binding binding to self-assemblingto self-assembling dendrons dendrons1 (gray), 1 (gray),2 (light 2 (light green), 3 (purple), 4 (red), 5 (black), and 6 (yellow) by EB assay. Non-self-assembling dendrons green),(light green),3 (purple), 3 (purple),4 (red), 45 (red),(black), 5 (black), and 6 (yellow) and 6 (yellow) by EB assay. by EB Non-self-assembling assay. Non-self-assembling dendrons dendrons7 (cyan) 7 (cyan) and 8 (orange) were used as negative controls. Adapted from [7] with the permission of John and7 (cyan)8 (orange) and 8 (orange) were used were as used negative as negative controls. controls. Adapted Adapted from [7 from] with [7] the with permission the permission of John of Wiley John Wiley and Sons, 2016. andWiley Sons, and 2016.Sons, 2016.
FigureFigure A2. PathologyPathology of of major major organs organs from from treated treated mice mice (H (HESES staining). staining). Columns Columns (from (from left left to to right): right): Figure A2. Pathology of major organs from treated mice (HES staining). Columns (from left to right): control,control, siRNA, siRNA, ADAD,, AD/siRNA./siRNA. Rows (from to bottom): heart, heart, kidney, kidney, liver, lung, spleen. Adapted control, siRNA, AD, AD/siRNA. Rows (from to bottom): heart, kidney, liver, lung, spleen. Adapted fromfrom [6] [6] with with the the permission permission of John Wiley and Sons, 2014. from [6] with the permission of John Wiley and Sons, 2014.
Pharmaceutics 2019, 11, x FOR PEER REVIEW 24 of 26 Pharmaceutics 2019, 11, 324 24 of 26 Pharmaceutics 2019, 11, x FOR PEER REVIEW 24 of 26
Figure A3. Confocal microscopy images of the successful endosomal escape of the siRNA/AD/E16G6RGDK nanoparticles in PC-3 cells after incubation times 0 (control), 0.5, 1, and 2 h Figure A3.A3. ConfocalConfocal microscopy microscopy images images of theof successfulthe successful endosomal endosomal escape ofescape the siRNA of /ADthe/ Red channel images show the cy5-labeled siRNA/AD/E16G6RGDK nanoparticles, the green channel siRNA/E16G6RGDKAD/E16 nanoparticlesG6RGDK nanoparticles in PC-3 cells in after PC-3 incubation cells after timesincubation 0 (control), times 0.5,0 (control), 1, and 2 0.5, h Red 1, channeland 2 h images reveal the Lyso Tracker red-marked endosomes, and the blue channel images show the cell Redimages channel show images the cy5-labeled show the siRNA cy5-labeled/AD/E16 siRNA/AD/EG6RGDK nanoparticles,16G6RGDK nanoparticles, the green channel the green images channel reveal nuclei stained with Hoechst 33342. In all these experiments, the siRNA/AD/E16G6RGDK imagesthe Lyso reveal Tracker the red-marked Lyso Tracker endosomes, red-marked and endosomes, the blue channel and imagesthe blue show channel the cell images nuclei show stained the with cell nanoassemblies were prepared using 50 nM siRNA, siRNA/AD N/P = 10, and AD/E16G6RGDK molar nucleiHoechst stained 33342. Inwith all theseHoechst experiments, 33342. theIn siRNAall these/AD /Eexperiments,16G6RGDK nanoassemblies the siRNA/AD were/E16G prepared6RGDK ratio = 5. Adapted from [8] with the permission of the American Chemical Society, 2018. nanoassembliesusing 50 nM siRNA, were siRNAprepared/AD usingN/P 50= 10,nM andsiRNA,AD /siRNA/E16G6RGDKAD N/P molar = 10, ratio and AD= 5./E Adapted16G6RGDK from molar [8] ratiowith the= 5. permissionAdapted from of the [8] Americanwith the permissi Chemicalon Society,of the American 2018. Chemical Society, 2018.
(a) (b)
(a) Figure A4. Cont. (b)
PharmaceuticsPharmaceutics 20192019,, 1111,, x 324 FOR PEER REVIEW 2525 of of 26 26
(c)
FigureFigure A4. A4. ((aa)) Toxicity Toxicity assessment assessment using using the the MTT MTT assay assay in in pr prostateostate cancer cancer PC-3 PC-3 cells cells (solid (solid bars) bars) and and inin human human embryonic embryonic kidney kidney (HEK293) (HEK293) cells cells (patterned (patterned bars) bars) of of the the ADAD micellesmicelles alone, alone, ADAD inin complex complex withwith a a non-silencing non-silencing siRNA siRNA (nssiRNA/ (nssiRNA/ADAD)) and and the the targeting targeting peptide-decorated peptide-decorated ADAD inin complex complex with with
aa non-silencing non-silencing siRNA siRNA (nssiRNA/ (nssiRNAAD/AD/E/E1616GG6RGDK).6RGDK). (b) ( bToxicity) Toxicity assessment assessment using using the the LDH LDH assay assay in prostatein prostate cancer cancer PC-3 PC-3 cells cells (sol (solidid bars) bars) and and in in human human embryonic embryonic kidney kidney (HEK293) (HEK293) cells cells (patterned (patterned bars)bars) of of the the ADAD micelles alone, alone, ADAD inin complex complex with with a a non-silencing non-silencing siRNA siRNA (nssiRNA/ (nssiRNA/ADAD)) and and the the
targetingtargeting peptide-decorated peptide-decorated ADAD inin complex complex with with a a non-silencing non-silencing siRNA siRNA (nssiRNA/ (nssiRNA/AD/E/E16GG66RGDK).RGDK). ((cc)) Hemolysis Hemolysis effect effect measured measured via UV absorption absorption at 540 540 nm nm for for the the ADAD micellesmicelles alone, alone, ADAD inin complex complex withwith a a non-silencing non-silencing siRNA siRNA (nssiRNA/ (nssiRNA/ADAD)) and and the the targeting targeting peptide-decorated peptide-decorated ADAD inin complex complex with with aa non-silencing non-silencing siRNA siRNA (nssiRNA/ (nssiRNAAD/AD/E/E1616GG6RGDK)6RGDK) at atdifferent different ADAD concentrationsconcentrations (corresponding (corresponding to nssiRNAto nssiRNA concentrations concentrations of 10, of 10,20, 20,50, 50,100, 100, and and200 200nM, nM, respectively). respectively). In al Inl these all these experiments, experiments, the siRNA/the siRNAAD//EAD16G/E6RGDK16G6RGDK nanoassemblies nanoassemblies were were prepared prepared using using 50 nM 50 siRNA, nM siRNA, siRNA/ siRNAAD /N/PAD =N 10,/P =and10, ADand/EAD16G/6ERGDK16G6RGDK molar molar ratio ratio= 5. =Adapted5. Adapted from from [8] [with8] with the the permission permission of of the the American American Chemical Chemical Society,Society, 2018.
ReferencesReferences
1.1. Lehn,Lehn, J.-M. J.-M. Toward self-organizationself-organization and and complex complex matter. matter.Science Science2002 2002, 295, 295, 2400–2403., 2400–2403. [CrossRef ][PubMed] 2.2. Whitesides,Whitesides, G.M.; G.M.; Grzybowski, Grzybowski, B. B. Self-Assembly Self-Assembly at atall allscales. scales. ScienceScience 20022002, 295,, 2952418–2421., 2418–2421. [CrossRef] 3. Mattia,[PubMed E.;] Otto, S. Supramolecular systems chemistry. Nat. Nanotechnol. 2015, 10, 111–119. 4.3. Ariga,Mattia, K.; E.; Nishikawa, Otto, S. Supramolecular M.; Mori, T.; Takeya, systems J.; chemistry. Shrestha, Nat.L.K.; Nanotechnol.Hill, J.P. Self-assembly2015, 10, 111–119.as a key [playerCrossRef for] materials[PubMed ]nanoarchitectonics. Sci. Technol. Adv. Mater. 2019, 20, 51–95. 5.4. Whitty,Ariga, K.;A. Cooperativity Nishikawa, M.; and Mori, biological T.; Takeya, complexity. J.; Shrestha, Nat. Chem. L.K.; Biol Hill,. 2008 J.P. Self-assembly, 4, 435–439. as a key player for 6. Liu,materials X.; Zhou, nanoarchitectonics. J.; Yu, T.; Chen, C.;Sci. Cheng, Technol. Q.; Adv. Sengupt, Mater. K.;2019 Huang,, 20, 51–95.Y.; Li, H.; [CrossRef Liu, C.;][ Wang,PubMed Y.; ]et al. Adaptive 5. amphiphilicWhitty, A. Cooperativity dendrimer-based and biological nanoassemblies complexity. as robustNat. Chem.and versatile Biol. 2008 siRNA, 4, 435–439. delivery [CrossRef systems.] Angew. 6. Chem.Liu, X.; Int. Zhou, Ed. Engl J.;. Yu,2014 T.;, 53 Chen,, 11822–11827. C.; Cheng, Q.; Sengupt, K.; Huang, Y.; Li, H.; Liu, C.; Wang, Y.; et al. 7. Chen,Adaptive C.; Posocco, amphiphilic P.; Liu, dendrimer-based X.; Cheng, Q.; Laurini, nanoassemblies E.; Zhou, as J.; robust Liu, C.; and Wang, versatile Y.; Tang, siRNA J.; Dal delivery Col, V.; systems. et al. MasteringAngew. Chem. dendrimer Int. Ed. Engl.self-assembly2014, 53, 11822–11827.for efficient [siRNACrossRef delivery:][PubMed From] conceptual design to in vivo 7. efficientChen, C.; gene Posocco, silencing. P.; Liu, Small X.; 2016 Cheng,, 12, Q.;3667–3676. Laurini, E.; Zhou, J.; Liu, C.; Wang, Y.; Tang, J.; Dal Col, V.; et al. 8. Dong,Mastering Y.; Yu, dendrimer T.; Ding, self-assembly L.; Laurini, E.; for Huang, efficient Y.; siRNA Zhang, delivery: M.; Weng, From Y.; conceptual Lin, S.; Chen, design P.; toMarson,in vivo D.;effi etcient al. Agene dual silencing. targetingSmall dendrimer-mediated2016, 12, 3667–3676. siRNA [CrossRef delivery] system for effective gene silencing in cancer therapy. 8. J.Dong, Am. Chem. Y.; Yu, Soc T.;. 2018 Ding,, 140 L.;, Laurini,16264–16274. E.; Huang, Y.; Zhang, M.; Weng, Y.; Lin, S.; Chen, P.; Marson, D.; et al. 9. Marson,A dual targeting D.; Laurini, dendrimer-mediated E.; Aulic, S.; Fermeglia, siRNA delivery M.; Pricl, system S. Evolution for effective from gene covalent silencing to in self-assembled cancer therapy. PAMAM-basedJ. Am. Chem. Soc. dendrimers2018, 140, 16264–16274.as nanovectors [CrossRef for siRNA] delivery in cancer by coupled in silico-experimental 9. studies.Marson, Part D.; I: Laurini, Covalent E.; siRNA Aulic, nanocarriers. S.; Fermeglia, Pharmaceutics M.; Pricl,, S.2019 Evolution, in press. from covalent to self-assembled 10. Karatasos,PAMAM-based K.; Posocco, dendrimers P.; Laurini, as nanovectors E.; Pricl, forS. Poly(amidoamine)-based siRNA delivery in cancer bydendrimer/siRNA coupled in silico-experimental complexation Pharmaceutics 2019 studiedstudies. by Part computer I: Covalent simulations: siRNA nanocarriers. Effects of pH and generation on, dendrimer in press. structure and siRNA binding. 10. Karatasos, K.; Posocco, P.; Laurini, E.; Pricl, S. Poly(amidoamine)-based dendrimer/siRNA complexation Macromol. Biosci. 2012, 12, 225–240. studied by computer simulations: Effects of pH and generation on dendrimer structure and siRNA binding. 11. Posocco, P.; Laurini, E.; Dal Col, V.; Marson, D.; Karatasos, K.; Fermeglia, M.; Pricl, S. Tell me something Macromol. Biosci. 2012, 12, 225–240. [CrossRef] that I do not know. Multiscale molecular modeling of dendrimer/dendron organization and self-assembly in gene therapy. Curr. Med. Chem. 2012, 19, 5062–5087.
Pharmaceutics 2019, 11, 324 26 of 26
11. Posocco, P.; Laurini, E.; Dal Col, V.; Marson, D.; Karatasos, K.; Fermeglia, M.; Pricl, S. Tell me something that I do not know. Multiscale molecular modeling of dendrimer/dendron organization and self-assembly in gene therapy. Curr. Med. Chem. 2012, 19, 5062–5087. [CrossRef][PubMed] 12. Posocco, P.; Laurini, E.; Dal Col, V.; Marson, D.; Peng, L.; Smith, D.K.; Klajnert, B.; Bryszewska, M.; Caminade, A.-M.; Majoral, J.P.; et al. Multiscale modeling of dendrimers and dendrons for drug and nucleic acid delivery. In Dendrimers in Biomedical Applications; Klajnert, B., Peng, L., Ceña, V., Eds.; RSC Publishing: Cambrige, UK, 2013; pp. 148–166. 13. Pavan, G.M.; Posocco, P.; Tagliabue, A.; Maly, M.; Malek, A.; Danani, A.; Ragg, E.; Catapano, C.V.; Pricl, S. PAMAM dendrimers for siRNA delivery: Computational and experimental insights. Chem. Eur. J. 2010, 16, 7781–7795. [CrossRef][PubMed] 14. Marson, D.; Laurini, E.; Posocco, P.; Fermeglia, M.; Pricl, S. Cationic carbosilane dendrimers and oligonucleotide binding: An energetic affair. Nanoscale 2015, 7, 3876–3887. [CrossRef][PubMed] 15. Mehrabadi, F.S.; Hirsch, O.; Zeisig, R.; Posocco, P.; Laurini, E.; Pricl, S.; Haag, R.; Kemmner, W.; Calderón, M. Structure–activity relationship study of dendritic polyglycerolamines for efficient siRNA transfection. RSC Adv. 2015, 5, 78760–78770. [CrossRef] 16. Percec, V.; Wilson, D.A.; Leowanawat, P.; Wilson, C.J.; Huges, A.D.; Kaucher, M.S.; Hammer, D.A.; Levine, D.H.; Kim, A.J.; Bates, F.S.; et al. Self-assembly of Janus dendrimers into uniform dendrimersomes and other complex architectures. Science 2010, 328, 1009–1014. [CrossRef][PubMed] 17. Rocchi, P.; So, A.; Kojima, S.; Signaevsky, M.; Beraldi, E.; Fazli, L.; Hurtado-Coll, A.; Yamanaka, K.; Gleave, M. Heat shock protein 27 increases after androgen ablation and plays a cytoprotective role in hormone-refractory prostate cancer. Cancer Res. 2004, 64, 6595–6602. [CrossRef][PubMed] 18. Behr, J.P. The proton sponge: A trick to enter cells viruses did not exploit. Chimia 1997, 51, 34–36. 19. Nichols, J.W.; Bae, Y.H. EPR: Evidence and fallacy. J. Control. Release 2014, 190, 451–464. [CrossRef] 20. Danhier, F. To exploit the tumor microenvironment: Since the EPR effect fails in the clinic, what is the future of nanomedicine? J. Control. Release 2016, 244, 108–121. [CrossRef] 21. Desgrosellier, J.; Cheresh, D. Integrins in cancer: Biological implications and therapeutic opportunities. Nat. Rev. Cancer 2010, 10, 9–22. [CrossRef] 22. Teesalu, T.; Sugahara, K.; Kotamraju, V.; Ruoslahti, E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration. Proc. Natl. Acad. Sci. USA 2009, 106, 16157–16162. [CrossRef][PubMed] 23. Velázquez Campoy, A.; Freire, E. ITC in the post-genomic era...? Priceless. Biophys. Chem. 2005, 115, 115–124. [CrossRef][PubMed] 24. Thornalley,K.A.; Laurini, E.; Pricl, S.; Smith, D.K. Enantiomeric and diastereomeric self-assembled multivalent nanostructures: Understanding the effects of chirality on binding to polyanionic heparin and DNA. Angew. Chem. Int. Ed. Engl. 2018, 57, 8530–8534. [CrossRef][PubMed] 25. Rodrigo, A.C.; Laurini, E.; Vieira, V.M.P.; Pricl, S.; Smith, D.K. Effect of buffer at nanoscale molecular recognition interfaces - electrostatic binding of biological polyanions. Chem. Commun. 2017, 53, 11580–11583. [CrossRef][PubMed] 26. Marson, D.; Laurini, E.; Fermeglia, M.; Smith, D.K.; Pricl, S. Mallard Blue binding to heparin, its SDS micelle-driven decomplexation, and interaction with human serum albumin: A combined experimental/modeling investigation. Fluid Phase Equilib. 2018, 470, 259–267. [CrossRef] 27. Goel, H.L.; Li, J.; Kogan, S.; Languino, L.R. Integrins in prostate cancer progression. Endocr. Relat. Cancer 2008, 15, 657–664. [CrossRef][PubMed] 28. Soker, S.; Takashima, S.; Miao, H.Q.; Neufeld, G.; Klagsbrun, M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998, 92, 735–745. [CrossRef] 29. Available online: https://www.nature.com/subjects/self-assembly (accessed on 4 July 2019).
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