For Liposomal Loading of Poorly Water-Soluble Compounds

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Review Development and Characterization of the Solvent-Assisted Active Loading Technology (SALT) for Liposomal Loading of Poorly Water-Soluble Compounds

Griﬃn Pauli, Wei-Lun Tang and Shyh-Dar Li *

Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada * Correspondence: [email protected]

Received: 22 March 2019; Accepted: 3 September 2019; Published: 9 September 2019

Abstract: A large proportion of pharmaceutical compounds exhibit poor water solubility,impacting their delivery. These compounds can be passively encapsulated in the lipid bilayer of liposomes to improve their water solubility, but the loading capacity and stability are poor, leading to burst drug leakage. The solvent-assisted active loading technology (SALT) was developed to promote active loading of poorly soluble drugs in the liposomal core to improve the encapsulation efficiency and formulation stability. By adding a small volume (~5 vol%) of a water miscible solvent to the liposomal loading mixture, we achieved complete, rapid loading of a range of poorly soluble compounds and attained a high drug-to-lipid ratio with stable drug retention. This led to improvements in the circulation half-life, tolerability, and efficacy profiles. In this mini-review, we summarize our results from three studies demonstrating that SALT is a robust and versatile platform to improve active loading of poorly water-soluble compounds. We have validated SALT as a tool for improving drug solubility, liposomal loading efficiency and retention, stability, palatability, and pharmacokinetics (PK), while retaining the ability of the compounds to exert pharmacological effects.

Keywords: liposome; water miscible solvents; remote loading; staurosporine; cancer; gambogic acid; loading gradients; meﬂoquine; child friendly formulation

1. Introduction

1.1. Challenges in Delivery of Poorly Water-Soluble Drugs Clinical translation of pharmaceutical compounds is often hindered by poor solubility. Over 40% of new chemical entities are not appreciably soluble in water, often resulting in limited therapeutic use, or abandonment as drug candidates [1,2]. Methods for improving apparent solubility include salt formulations, excipients, amorphous solid dispersions, pH adjustment, co-solvents, and nanocarriers [1,2]. There has been interest in the development of systematic approaches to overcoming low soluble compounds, as outlined in the developability classiﬁcation system which describes a categorization of compounds based on permeability and solubility [3]. There has also been growing interest in the use of nanocarriers as a vehicle to overcome issues of solubility, as they serve as a platform technology and provide cell targeting [4]. Lipid-based nanocarriers (LNCs) represent a class of lipid particles, including solid lipid nanoparticles, micro- or nano-emulsions, polymer-lipid hybrid nanoparticles, and liposomes.

Pharmaceutics 2019, 11, 465; doi:10.3390/pharmaceutics11090465 www.mdpi.com/journal/pharmaceutics PharmaceuticsPharmaceutics2019 2019, 11, 11, 465, x FOR PEER REVIEW 2 of 12

1.1.1. Liposomes and Drug Loading 1.1.1. Liposomes and Drug Loading Liposomes are among the most studied class of LNCs for improving drug delivery [5]. LiposomesLiposomes are nano-scale are among spheroid the most vesicles studied composed class of one LNCs or multiple for improving lipid bilayer(s) drug deliveryenclosing [an5]. Liposomesaqueous core are nano-scale[5]. Liposomes spheroid can vesiclesbe manufactured composed using of one several or multiple methods, lipid bilayer(s)including enclosingthin-film anhydration aqueous core[6], reverse [5]. Liposomes phase evaporation can be manufactured [7], and microfluidic using several mixing methods, [8]. The including process thin-filmused to hydrationgenerate [the6], reverseliposome phase will evaporationimpact its size [7 ],and and lamellarity microfluidic [9–11]. mixing Likewise, [8]. The various process lipids used can to generate be used theto liposomeformulate willliposomes impact with its differen size andt properties, lamellarity such [9–11 as]. size, Likewise, drug release various kinetics, lipids biocompatibility, can be used to formulatesurface charge, liposomes and withcell targeting different [12,13]. properties, To encapsulate such as size, drugs drug into release liposomes, kinetics, two biocompatibility, methods have surfacebeen implemented; charge, and cell passive targeting and active [12,13 loading,]. To encapsulate both of which drugs are into briefly liposomes, reviewed two below. methods have been implemented; passive and active loading, both of which are briefly reviewed below. 1.1.2. Passive Loading 1.1.2. Passive Loading Passive loading describes the procedure in which liposomes are formed concurrently with drug loadingPassive (Figure loading 1A). describes In general, the procedure hydrophilic in whichcompounds liposomes are aredistributed formed concurrentlyhomogenously with in drug the loadingaqueous (Figure phase1A). (both In general, inside and hydrophilic outside compoundsthe liposomes), are distributedwhereas hydrophobic homogenously drugs in theare aqueousretained phaseinside (both the insidelipid bilayer and outside of liposomes, the liposomes), respectively whereas. Specifically, hydrophobic whendrugs working are with retained poorly inside water- the lipidsoluble bilayer drugs, of liposomes, the drugs respectively.are first dissolved Specifically, with lip whenids in working an organic with solvent, poorly water-solublefollowed by solvent drugs, theevaporation drugs are firstto prepare dissolved a drug with containing lipids in thin an organic film, which solvent, is later followed hydrated by solventwith an evaporationaqueous phase to prepareto prepare a drug liposomes. containing When thin film, loading which water-soluble is later hydrated drugs, with the an aqueouslipid film phase is dispersed to prepare in liposomes. a drug- Whencontaining loading aqueous water-soluble phase. drugs, the lipid film is dispersed in a drug-containing aqueous phase.

FigureFigure 1. 1.The The two two major major methods methods for for liposomal liposomal drug drug loading. loading. ((AA)) PassivePassive loadingloading involves co-current loadingloading and and liposomal liposomal formation. formation. ( B(B)) In In active active loading, loading, liposomes liposomes areare formedformed containingcontaining a gradientgradient usedused to to load load drugs. drugs.

1.1.3.1.1.3. Limitations Limitations of of Passive Passive Loading Loading TheThe trapping trapping effi ciencyefficiency of passiveof passive loading loading varies varies due to due several to factors,several includingfactors, including drug solubility, drug vesiclesolubility, size, lipidvesicle concentration, size, lipid concentration, and preparation and procedure.preparation In procedure. most cases, In the most typical cases, drug-to-lipid the typical ratiodrug-to-lipid (D/L) achieved ratio (D/L) by this achieved passive by loading this passive technique loading is technique less than is 0.05 less (w than/w)[ 0.0514,15 (w]./w In) [14,15]. addition, In theaddition, entrapped the drugs entrapped often cannotdrugs often be retained cannot stably be reta dueined to stably weak associationdue to weak between association the drugs between and the liposomes,drugs and resulting the liposomes, in poor resulting drug retention in poor and drug storage retention stability. and storage Passive stability. loading Passive often also loading results often in a “burstalso results release” in phenomenon, a “burst release” whereby phenomenon, a large percentage whereby a of large the entrappedpercentage drug of the is releasedentrapped quickly. drug is released quickly. 1.1.4. Active Loading 1.1.4.In Active active Loading loading, liposomes are first generated containing a transmembrane gradient, i.e., the aqueousIn active phases loading, inside liposomes and outside are the first liposomes generated are containing different. a Subsequently, transmembrane anamphipathic gradient, i.e., drug the aqueous phases inside and outside the liposomes are different. Subsequently, an amphipathic drug

Pharmaceutics 2019, 11, 465 3 of 12 dissolved in the exterior aqueous phase can permeate across the phospholipid bilayer(s), followed by interactions with a trapping agent in the core to lock-in the drug (Figure1). In 1976, Deamer and Nicols [16–18] demonstrated that a pH gradient could be utilized to load catecholamine into liposomes, leading to stable retention in vitro. They formed liposomes in a low pH (pH 5) solution and then + bathed the liposomes in an alkaline solution (pH 8) to create a 1000-fold difference in H3O ions across the bilayer. As the catecholamine molecules remained as the free form in the basic exterior, they freely permeated through the bilayer into the low pH core, where they were subsequently protonated. The charged catecholamine molecules were no longer membrane permeable and thus locked in. An accumulation of catecholamine molecules in the core of the liposomes was observed. Subsequent to the work of Dreamer et al. [18], Haran et al. [19] demonstrated that active loading can be achieved using an ammonium sulfate gradient. They produced liposomes with an interior containing ammonium sulfate. A concentrated solution of anthracycline was added to the liposomes and loading was then achieved after incubation at an elevated temperature. Anthracycline molecules formed aggregates in the core of the liposomes through precipitation with sulfate ions, and a high drug loading efficiency of >90% was achieved without burst drug leakage. Several other pH gradients have been established, including phosphate and calcium acetate [20]. Similar to the calcium acetate gradient, basified copper acetate gradients have also been employed for liposomal loading.

1.1.5. Limitations of Standard Active Loading Active loading remains a powerful tool that can be used to effectively and stably retain drugs in the core of liposomes. However, traditional active loading is predicated on compounds having high solubility and membrane permeability (i.e., amphipathic), so that they can be solubilized in the exterior aqueous phase and permeable through the lipid bilayer into the liposomal core. These selection criteria exclude a large number of hydrophobic drugs currently on the market or in the development pipeline. Excipients such as ß-cyclodextrin and super saturated solutions have been also used to increase the active loading efficiency of liposomes. These techniques have helped somewhat in overcome issues of poor solubility [21]. In addition, recent approaches have focused on examining the role of nanocrystillazaion inside liposomes to improve drug retention [22]. Although discussion of these techniques is beyond the scope of this paper, these methods have significant limitations, and consequently there is a need for an improved loading technology.

2. Solvent-Assisted Active Loading Technology (SALT)

2.1. Introduction Our laboratory has developed an innovative technology for liposomal loading of poorly water-soluble drugs. Solvent-assisted active loading technology (SALT) allows for stable and efficient loading of poorly water-soluble drugs into the aqueous core of liposomes. SALT has demonstrated efficacy across a range of poorly soluble compounds and is a versatile method for drug loading. We hypothesized that including a small volume of water-miscible solvent in the loading mixture of liposomes and a hydrophobic drug could help solubilize the drug in the aqueous phase, increase the drug penetration through the liposomal bilayer and boost active loading encapsulation eﬃciency. pH adjustment is among the most commonly-used methods to increase drug solubility. When compared to pH adjustment, the SALT/liposome technique enhanced the solubility of our model drugs Staurosporine, Gambogic Acid and Meﬂoquine from 120 µg/mL to 1 mg/mL, 5 µg/mL to 1 mg/mL, and 0.6 mg/mL to 8 mg/mL, respectively [13,23,24].

2.2. Applications and Mechanism A drug needs to be solubilized in the free form in the exterior phase of liposomes in order to eﬀectively penetrate the lipid bilayer. Therefore, a small amount of solvent is included in the liposomal suspension for complete solubilisation of the compound. After penetration into the liposomal core, Pharmaceutics 2019, 11, 465 4 of 12

thePharmaceutics drug can 2019 then, 11, interactx FOR PEER with REVIEW a trapping agent inside the liposomes to form insoluble precipitates 4 of 12 for stable a “lock-in” eﬀect. Finally, the solvent can be removed by dialysis or gel ﬁltration (Figure2). Togel test filtration the feasibility (Figure of2). thisTo idea,test the we feasibility performed of proof-of-principle this idea, we performed studies proof-of-principle [13,23,24] with a range studies of poorly[13,23,24] soluble with drugs.a range We of furtherpoorly characterizedsoluble drugs. SALT We further through characterized the use of various SALT solvents,through liposomalthe use of formulationsvarious solvents, and liposomal trapping agents formulations to demonstrate and trapping its versatility. agents to demonstrate its versatility.

FigureFigure 2. 2.Solvent-assisted Solvent-assisted active active loading loading technology technology (SALT) (SALT) mechanism mechanism overview overview for liposomal for liposomal loading ofloading a poorly of a water-soluble poorly water-soluble drug. drug.

2.3.2.3. Proof-of-Principle with aa Weak BaseBase DrugDrug Staurosporine and Liposomal Loading Staurosporine and Liposomal Loading Staurosporine (STS) is a broadly acting alkaloid protein kinase inhibitor with demonstrated Staurosporine (STS) is a broadly acting alkaloid protein kinase inhibitor with demonstrated efficacy as an antitumor agent against several cancer types [25]. STS contains a secondary amine efficacy as an antitumor agent against several cancer types [25]. STS contains a secondary amine group, but its solubility remains negligible even at a low pH [26]. Despite promising in vitro results, group, but its solubility remains negligible even at a low pH [26]. Despite promising in vitro results, STS development was hindered due to poor solubility and non-specificity [26]. We hypothesized that STS development was hindered due to poor solubility and non-specificity [26]. We hypothesized that liposomal delivery of STS would minimize its systemic toxicity through enhanced tumor targeting liposomal delivery of STS would minimize its systemic toxicity through enhanced tumor targeting while also overcoming issues surrounding poor solubility. Liposomal loading of STS was previously while also overcoming issues surrounding poor solubility. Liposomal loading of STS was previously reported by Mukthavaram et al. [27]. They utilized an active loading strategy employing a reverse pH reported by Mukthavaram et al. [27]. They utilized an active loading strategy employing a reverse gradient [27]. However, they were only able to achieve 70% drug encapsulation and a low drug-to-lipid pH gradient [27]. However, they were only able to achieve 70% drug encapsulation and a low drug- ratio (D/L) of 0.09 mol/mol [27]. We hypothesized that introducing a limited amount of dimethyl to-lipid ratio (D/L) of 0.09 mol/mol [27]. We hypothesized that introducing a limited amount of sulfoxide (DMSO) to the liposomal suspension could keep STS soluble during the active loading process dimethyl sulfoxide (DMSO) to the liposomal suspension could keep STS soluble during the active and would also facilitate the permeation of the drug into the inner core of liposomes. We employed loading process and would also facilitate the permeation of the drug into the inner core of liposomes. an ammonium sulfate gradient to effectively trap STS via sulfate-STS nano-aggregates inside the We employed an ammonium sulfate gradient to effectively trap STS via sulfate-STS nano-aggregates liposomal core and achieve 100% drug encapsulation at a high D/L of 0.31/1 (mol/mol). Subsequently, inside the liposomal core and achieve 100% drug encapsulation at a high D/L of 0.31/1 (mol/mol). gel filtration facilitated the removal of DMSO generating a final liposomal product. In the following Subsequently, gel filtration facilitated the removal of DMSO generating a final liposomal product. In section, we report the SALT method optimization and results of PK and efficacy studies. the following section, we report the SALT method optimization and results of PK and efficacy studies. Liposomal Formulation: A thin lipid film composed of 1,2-distearoyl-sn-glycero-3-phosphocholine Liposomal Formulation: A thin lipid film composed of 1,2-distearoyl-sn-glycero-3- (DSPC), cholesterol and PEGylated 1,2-distearoyl-sn-glycero-3-phosphoetholamine (DSPE-PEG2000) phosphocholine (DSPC), cholesterol and PEGylated 1,2-distearoyl-sn-glycero-3-phosphoetholamine (55/40/5 mole ratio) (100 mg total lipids) was hydrated with 1 mL of 350 mM ammonium sulfate at (DSPE-PEG2000) (55/40/5 mole ratio) (100 mg total lipids) was hydrated with 1 mL of 350 mM 60 C, followed by membrane extrusion to control the size ~100 nm with a polydispersity index (PDI) ammonium◦ sulfate at 60°C, followed by membrane extrusion to control the size ~100 nm with a of <0.06. The liposomes were then dialyzed against an acetate buffer (100 mM, pH 5) to create a polydispersity index (PDI) of <0.06. The liposomes were then dialyzed against an acetate buffer (100 transmembrane gradient of ammonium sulfate. mM, pH 5) to create a transmembrane gradient of ammonium sulfate. Liposomal STS Loading Using SALT: We dissolved STS in DMSO and added into the liposomal Liposomal STS Loading Using SALT: We dissolved STS in DMSO and added into the liposomal suspension at a range of D/L with a final DMSO content of 5–60% and incubated the mixture at room suspension at a range of D/L with a final DMSO content of 5–60% and incubated the mixture at room temperature or 60 C. We found that at least 5% DMSO was required to achieve complete drug loading, temperature or 60◦ °C. We found that at least 5% DMSO was required to achieve complete drug and that complete drug loading was maintained over a range of DMSO concentrations between loading, and that complete drug loading was maintained over a range of DMSO concentrations 5% and 60%. Loading kinetics was impacted by the temperature. In the presence of 5% DMSO, between 5% and 60%. Loading kinetics was impacted by the temperature. In the presence of 5% complete loading was achieved more rapidly (5 min) with incubation at 60 C compared to room DMSO, complete loading was achieved more rapidly (5 min) with incubation◦ at 60 °C compared to temperature (15 min). The highest D/L achieved for complete drug loading was 0.31 w/w. The size room temperature (15 min). The highest D/L achieved for complete drug loading was 0.31 w/w. The (~100 nm) and the PDI (<0.06) of the final STS-Lipo produced with various amounts of DMSO were size (~100 nm) and the PDI (<0.06) of the final STS-Lipo produced with various amounts of DMSO comparable and remained unchanged compared to the empty liposomes. We also determined that STS were comparable and remained unchanged compared to the empty liposomes. We also determined that STS leakage from the liposomes was minimal (<5%) after seven days incubation in 50% fetal bovine serum (FBS) at 37 °C. Finally, the cryo-transmission electron microscopy revealed that STS

Pharmaceutics 2019, 11, 465 5 of 12 leakage from the liposomes was minimal (<5%) after seven days incubation in 50% fetal bovine serum Pharmaceutics 2019, 11, x FOR PEER REVIEW 5 of 12 (FBS) at 37 ◦C. Finally, the cryo-transmission electron microscopy revealed that STS molecules form spherical precipitates inside the liposomal core (Figure3). The data support the contention that STS molecules form spherical precipitates inside the liposomal core (Figure 3). The data support the was actively and stably loaded into the liposomal core by the SALT. contention that STS was actively and stably loaded into the liposomal core by the SALT.

FigureFigure 3. 3.Cryo-TEM Cryo-TEM imagesimages ofof thethe drugdrug freefree liposomesliposomes ((aa)) andand thethe staurosporinestaurosporine (STS)-Lipo loaded loaded usingusing 5% 5% dimethyl dimethyl sulfoxide sulfoxide (DMSO) (DMSO) ( b(b)) 60% 60% DMSODMSO ((cc).). ScaleScale bar represents 100 nm. nm. Reprinted Reprinted with with permissionpermission from from Tang Tang et et al., al., Pharmaceutical Pharmaceutical Research, Research, published published byby SpringerSpringer Nature,Nature, 20162016 [[24].24].

SafetySafety and and E Efficacyfficacy Studies: Studies:It It waswas foundfound that the accumulated accumulated maximum maximum tolerated tolerated dose dose (MTD) (MTD) ofof STS-Lipo STS-Lipo was was 9 9 mg mg/kg/kg relativerelative toto 33 mgmg/kg/kg for free STS (dissolved in in acetate acetate buffer buffer and and 1% 1% DMSO). DMSO). STS-LipoSTS-Lipo exhibited exhibited significantlysignificantly improvedimproved eefficacyfficacy againstagainst multidrug resistant EMT6-AR1 murine murine breastbreast tumor tumor in in mice mice compared compared withwith freefree STSSTS andand docetaxel (DTX). The tumor tumor growth growth was was effectively effectively 3 impededimpeded by by STS-Lipo STS-Lipo therapy therapy and theand average the average tumor tumorvolume volume was controlled was controlled by 150 mm by in 150 comparison mm3 in 3 tocomparison ~800 mm into the~800 free mm STS3 in group the free by STS day group 18, while by allday tumors 18, while in the all DTXtumors and in bu theffer DTX treated and mice buffer all exceededtreated mice the endpoint all exceeded size (1000the endpoint mm3) before size (1000 day 14. mm The3) before STS-Lipo day treatment 14. The STS-Lipo did not cause treatment significant did bodynot cause weight significant loss. On thebody other weight hand, loss. 3 out On of the 3 mice other in thehand, free 3 STSout groupof 3 mice reached in the humane free STS endpoints group onreached day 9 duehumane to the endpoints drug toxicity, on day and 9 due there to wasthe drug ~5% toxicity, body weight and there loss inwas the ~5% mice body treated weight with loss DTX. in Thethedata mice indicate treated thatwith SALT DTX. enabled The data liposomal indicate deliverythat SALT of enabled STS, leading liposomal to enhanced delivery safety of STS, and leading efficacy comparedto enhanced to freesafety STS and and efficacy the standard compared taxane to free chemotherapy. STS and the standard taxane chemotherapy.

2.4.2.4. Proof-of-Principle Proof-of-Principle withwith aa WeakWeak AcidAcid DrugDrug

2.4.1.2.4.1. Introduction Introduction AfterAfter our our initial initial discovery discovery that that SALT SALT could could promote promote loading loading of an insoluble of an insoluble weak base weak compound base intocompound the liposomal into the aqueous liposomal core to aqueous improve core the drug to improve delivery, the we drug sought delivery, to further we explore sought applications to further forexplore SALT. applications We investigated for SALT. whether We SALTinvestigated could whether efficiently SALT load could other efficiently drug classes. load In other addition, drug weclasses. examined In addition, whether solvents we examined other than whether DMSO solvents could be other used than in this DMSO technology could and be usedthe role in these this solventstechnology played and inthe drug role loading.these solvents To achieve played this,in drug we loading. investigated To achieve the ability this, ofwe SALT investigated to improve the loadingability of gambogicSALT to improve acid (GA). loading GA was of gambogic selected as acid a model (GA). drug GA aswas it isselected water insolubleas a model yet drug dissolvable as it is inwater a range insoluble of water yet miscible dissolvable solvents in a range (>20 mg of /watermL). Asmiscible such, wesolvents could (>20 investigate mg/mL). both As thesuch, use we of could SALT oninvestigate a different both drug the class use (weakof SALT acid) on anda different how SALT drugperforms class (weak with acid) solvents and how other SALT than performs DMSO. with solvents other than DMSO. 2.4.2. Gambogic Acid (GA) 2.4.2.GA Gambogic is a naturally-derived Acid (GA) compound found in traditional Asian medicines. GA has been reported to have anti-cancerGA is a naturally-derived and anti-inflammatory compound properties found by acting in traditional through aAsian number medicines. of cell processes GA has [28 been–30]. reported to have anti-cancer and anti-inflammatory properties by acting through a number of cell processes [28–30]. Despite its promising results as an anti-cancer compound, its clinical development has been hindered by its poor water solubility (<5 μg/mL). Several methods have been attempted to

Pharmaceutics 2019, 11, 465 6 of 12

Despite its promising results as an anti-cancer compound, its clinical development has been hindered by its poor water solubility (<5 µg/mL). Several methods have been attempted to overcome its issues of poor solubility, yet parameters relating to PK were only marginally improved [31–35]. We hypothesized that SALT could facilitate stable and efficient loading of GA into liposomes, leading to prolonged PK and tumor-targetedPharmaceutics 2019 delivery., 11, x FOR PEER REVIEW 6 of 12 SALT Promotes GA Loading into Liposomes: We began the GA study by seeking to find answers overcome its issues of poor solubility, yet parameters relating to PK were only marginally improved to three[31–35]. questions. We hypothesized Primarily, that we SALT were could interested facilitate in stable determining and efficient whether loading SALTof GA into could liposomes, be applied to load otherleading drugs to prolonged besides weak PK and bases. tumor-targeted Second, we delivery. sought to determine if other water miscible solvents were compatibleSALT with Promotes the SALT GA Loading system. into Third, Liposomes: we wanted We to began elucidate the GA the study mechanism by seeking behind to find the SALT system,answers specifically, to three to questions. investigate Primarily, the functions we were of interested solvents in in determining promoting whether drug loading.SALT could GA be is freely solubleapplied in eight to di loadfferent other water-miscible drugs besides weak solvents; bases. DMSO, Second, DMF, we sought EtOH, to determine MeOH, acetonitrile, if other water acetone, miscible solvents were compatible with the SALT system. Third, we wanted to elucidate the 1,4-dioxane, and NMP. We examined whether these solvents could be used to promote GA loading into mechanism behind the SALT system, specifically, to investigate the functions of solvents in 2+ liposomes.promoting We prepared drug loading. liposomes GA is freely containing soluble ain gradient eight different of basified water-miscible copper solvents; acetate DMSO, where [CuDMF, ]interior 2+ > [Cu EtOH,]exterior MeOH,. We rationalized acetonitrile, acetone, that after 1,4-dioxane, drug solubilization and NMP. We and examined loading whether into these the liposomal solvents core, GA wouldcould bind be used cooper to promote via coordination GA loading complex.into liposomes. This We drug-copper prepared liposomes conjugate containing forms stablea gradient complexes and effectivelyof basified traps copper GA in acetate the liposome. where [Cu This2+]interior mechanism, > [Cu2+]exterior which. We parallels rationalized other that trapping after drug strategies using compoundssolubilization such and loading as calcium into the acetate, liposomal helps core, boost GA would drug bind retention cooper andvia coordination minimize leakagecomplex. during This drug-copper conjugate forms stable complexes and effectively traps GA in the liposome. This storage. Our results showed that all 8 solvents could facilitate GA loading and the optimal solvent mechanism, which parallels other trapping strategies using compounds such as calcium acetate, contenthelps was boostunique drug to retention the solvent. and minimize The data leakage suggests during the storage. solvent Our function results showed is twofold. that all Primarily, 8 the solventsolvents is used could tofacilitate completely GA loading dissolve and the the optimal drug solvent in the content liposomal was unique exterior to waterthe solvent. phase. The This is requireddata as largesuggests drug the precipitatessolvent function are is impermeable twofold. Primarily, to the the lipid solvent bilayer. is used However, to completely we discovered dissolve that just completethe drug solubilisation in the liposomal did exterior not promote water completephase. This drug is required loading. as largeAfter drug the solvent precipitates has are dissolved the drug,impermeable additional to solventthe lipid addedbilayer. servesHowever, to we increase discovered the permeabilitythat just complete of solubilisation the liposomal did membrane not promote complete drug loading. After the solvent has dissolved the drug, additional solvent added (Figure4serves). However, to increase additional the permeability solvent of must the liposomal not exceed membrane the limit (Figure that 4). induces However, lipid additional membrane instability.solvent In these must studies,not exceed we the have limit demonstrated that induces lipid that membrane SALT is instability. capable of In loadingthese studies, both we poorly have soluble weak aciddemonstrated and base modelthat SALT drugs is capable with high of loading efficiency both poorly and retention. soluble weak SALT acid is and a versatile base model platform drugs which could bewith used high to efficiency load an and array retention. of compounds SALT is a versatile that may platform have which been could previously be used non-deliverableto load an array with liposomes.of compounds In addition that to may STS have and GA,been wepreviously further non-deliverable demonstrated with that liposomes. this method In addition could beto appliedSTS for and GA, we further demonstrated that this method could be applied for loading other drugs, loading other drugs, including artesunate, prednisolone hemisuccinate, and quercetin [13]. including artesunate, prednisolone hemisuccinate, and quercetin [13].

Figure 4.FigureThe solvent 4. The solvent eﬀect on effect drug on loading drug loading in the SALTin the system. SALT system The. ﬁgure The figure is reprinted is reprinted with with permission permission from Tang et al., Biomaterials; published by Elsevier, 2018 [13]. from Tang et al., Biomaterials; published by Elsevier, 2018 [13].

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Formulation Optimization: After confirming that SALT could facilitate active loading of GA, we nextFormulation sought to optimize Optimization: the formulationAfter confirming by modifying that the SALT loading could gradient facilitate and activelipid composition loading of toGA, improve we next the sought D/L and to optimize drug retention. the formulation We studied by GA modifying loading the efficiency loading in gradient 1,2-distearoyl-sn- and lipid glycero-3-phosphocholine/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-composition to improve the D/L and drug retention. We studied GA loading efficiency in 1,2-distearoyl- [amino(polyethyleneglycol)-2000sn-glycero-3-phosphocholine/cholesterol (DSPC/Chol/DSPE-PEG2K)/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino liposomes with a range of (polyethyleneglycol)-2000transmembrane gradients, including (DSPC/Chol magnesium/DSPE-PEG2K) gluconate, liposomes calcium with formate, a range and ofcopper transmembrane acetate. All gradientsgradients, achieved including complete magnesium drug gluconate, loading in calciumthe presence formate, of 5 and vol% copper DMSO acetate. at a D/L All of gradients 1/5 w/w. However,achieved completethe basified drug copper loading acetate in thegradient presence (pH 9) of demonstrated 5 vol% DMSO the at highest a D/L ofdrug 1/5 retentionw/w. However, in 50% serum:the basified ~45% copper GA retained acetate gradient in the liposomes (pH 9) demonstrated after 4 h incubation. the highest We drug then retention modified in 50% the serum: lipid ~45%composition GA retained to further in the enhance liposomes GA after retention. 4 h incubation. Our results We then show modified that decreasing the lipid compositionthe cholesterol to contentfurther enhancefrom 45% GA to 0% retention. increased Our GA results retention show from that 40% decreasing to 68% after the 2.5 cholesterol h incubation content in serum. from 45%We thento 0% compared increased GA formulations retention from prepared 40% to 68% with after an 2.5 unsaturated h incubation lipid in serum. 1,2-dioleoyl-sn-glycero-3- We then compared phosphocholineformulations prepared (DOPC) with or a ansaturated unsaturated lipid (DSPC): lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine the DOPC-liposomes retained >95% of the (DOPC) drug afteror a saturated24 h incubation lipid (DSPC): in serum the without DOPC-liposomes significant retainedsize change.>95% This of thefinding drug was after unanticipated 24 h incubation as saturatedin serum without liposomes significant containing size lipids change. with This a high finding transition was unanticipated temperature as have saturated been liposomes shown to increasecontaining doxorubicin lipids with retention a high transition relative temperature to unsaturated have lipids been shown[36]. However, to increase it doxorubicinhas been discovered retention thatrelative unsaturated to unsaturated lipids, lipids such [ 36 as]. DOPC, However, may it has form been flexible discovered liposomes that unsaturated which trap lipids, hydrophobic such as moleculesDOPC, may more form effectively flexible liposomes [37]. which trap hydrophobic molecules more effectively [37]. Characterization of optimized Lipo-GA: Transmission electron cryomicroscopy (CryoTEM(CryoTEM)) imaging revealed thatthat liposomesliposomes displayed displayed bi-lamellar bi-lamellar structure structure with with an an electron electron dense dense core core (Figure (Figure5). 5).Both Both these these features features have have been been previously previously reported reported with with liposomes liposomes containing containing a copper a copper gradient gradient for fordrug drug loading loading [38 ,[38,39],39], indicative indicative of bilayer of bilayer rearrangement rearrangement and and copper-GA copper-GA complex complex formation formation in the in thecore. core. The The formation formation of copper-GA of copper-GA complexes, complexes, in addition in addition to the optimizedto the optimized lipid composition lipid composition resulted resultedin stable in retention stable retention of GA. of GA.

Figure 5. Cryo-TEM images images of of empty liposomes (DOPC/Chol/DSPE-PEG2K, (DOPC/Chol/DSPE-PEG2K, 85/10/5 85/10/5 by by mol%) (A)) and Lipo-GA ((BB).). TheThe ﬁgurefigure is is reprinted reprinted with with permission permission from from Tang Tang et et al., al., Biomaterials; Biomaterials; published published by byElsevier, Elsevier, 2018 2018 [13 ].[13].

Safety, PK and Efficac Efficacy:y: GA is known to induce apoptosis of red blood cells, leading to potential potential toxicity in vivo [[40].40]. While free GA waswas highlyhighly hemolytic, the equivalent concentration of Lipo-GA showed no activityactivity inin inducinginducing hemolysis.hemolysis. Compared Compared to to free free GA, GA, the the Lipo-GA Lipo-GA formulation formulation resulted resulted in inan an 18-fold 18-fold increase increase in in plasma plasma half-life, half-life, a a 20-fold 20-fold higher higher mean mean residence residence time,time, aa 7.5-fold7.5-fold higher area under the curve curve (AUC (AUC0–0-∞),), and and 10-fold 10-fold decreased decreased clearance, clearance, confirming confirming its its prolonged circulation ∞ relative to to free free GA. GA. In two In two murine murine tumor tumor models, models, we observed we observed a significant a significant dose-dependent dose-dependent reduction reductionin tumour involume tumour after volume Lipo-GA after therapy. Lipo-GA In particular, therapy. In in the particular, EMT6-AR1 in the multidrug EMT6-AR1 resistant multidrug breast resistanttumor model, breast one tumor dose model, of Lipo-GA one dose completely of Lipo-GA suppressed completely the suppressed tumor growth, the tumor while growth, free GA while only freemoderately GA only inhibited moderately 65% inhibited tumor growth. 65% tumor Mice growth. treated Mice with treated Lipo-GA with showed Lipo-GA nobody showed weight no body loss, weightsuggesting loss, good suggesting safety. good safety.

3. Pediatric Formulation

Pharmaceutics 2019, 11, 465 8 of 12

3. Pediatric Formulation We next explored a unique application for SALT. Malaria is the world’s leading parasitic disease [41], with over 200 million cases reported in 2015. Children under 5 years old are the most vulnerable population and account for over 70% of malaria associated deaths [42]. Treatment or prophylaxis of malaria often relies on the first line drug mefloquine (Mef) [43]. Despite its effectiveness as an antimalarial agent, Mef is difficult to accurately dose in children and neonates [44]. This is due to the lack of a pediatric formulation and Mef is typically administered by crushing up a portion of an adult tablet and administering with milk or food, in an effort to mask the extremely bitter taste. However, children often spit out the medicine, leading to subtherapeutic levels. Microemulsions of Mef have been developed to overcome poor solubility and increase bioavailability [45,46], yet their use in newborns is contradicted by risk of renal and liver damage caused by high concentrations of surfactants [47]. A pediatric pill of Mef is available (Artequin pediatric), yet this pill remains too large for neonatal dosing. We hypothesized that liposomal delivery of Mef could overcome many of these issues. Liposomal suspension of Mef could increase the solubility and subsequent absorption leading to higher bioavailability [48]. In addition, dosing the liquid suspension to children is easy and accurate. We hypothesized liposomes could also mask the taste of the bitter drug by shielding it from the taste buds. Liposomes can be lyophilized to prepare a powder formulation that is stable upon room temperature storage and orally dispersible for pediatric use. Lastly, liposomal formulations using only the neutral lipid DSPC and cholesterol can be considered very safe for oral administration in young children. The remaining challenge was how to prepare a stable Mef-Lipo formulation. Liposomal Loading of Mef Using SALT: We first prepared liposomes (DSPC and cholesterol) containing an ammonium gradient, and then incubated the liposomes with Mef in the presence of 10% DMSO at a D/L concentration of 0.1 w/w at room temperature for 30 min to achieve complete loading. The liposomes were then purified by dialysis to remove DMSO and a formulation containing 8 mg/mL of Mef was obtained. Lyophilizaition: The Mef-Lipo suspension was subjected to freeze drying, aiming to prepare a solid powder formulation to facilitate longer storage time and produce a rapidly dissolvable formulation. To protect the integrity of the liposomes during lyophilization we buffered the exterior with 300 mM sucrose and 20 mM phosphate. Sucrose is a known lyoprotectant and phosphate was shown to increase the stability of liposomes [49]. The lyophilization process only increased the liposome size from 110 nm to 130 nm and the PDI remained <0.1. Importantly, we observed no significant drug leakage. The lyophilized liposomes containing Mef remained stable under the storage at room temperature for >3 months. In addition, they were rapidly dissolvable in water within 10 s, indicating an orally dispersible formulation. Drug Release: Both the liquid suspension and the lyophilized Mef-Lipo exhibited similar drug release profiles in simulated saliva, gastric, and intestinal fluids. In simulated saliva, both liquid and lyophilized Mef-Lipo demonstrated no observable drug release. Drug release was highest in the simulated stomach fluid for both formulations. There was no drug release in the simulated intestinal fluid in the absence of a bile salt for both preparations, while Mef was effectively released from the liposomes in simulated intestinal fluid supplemented with a bile salt. Our data indicate that Mef release from the liposomes was triggered by acid- or surfactant-induced destabilization of liposomes. The results also suggest that the liposomal formulation would effectively mask the drug taste in the oral cavity but rapidly release the drug in the gastrointestinal fluids. Bitterness Masking: To determine the bitterness masking effect of the liposomal technology, we employed the Astree e-tongue technology and compared the bitterness of Mef-Lipo to 10% sucrose, Mef suspension and Infant Tylenol®. As shown in Figure6, the bitterness of the Mef-Lipo was similar to 10% sucrose indicating a palatable formulation, while the standard Mef suspension displayed high bitterness. Pharmaceutics 2019, 11, 465 9 of 12 Pharmaceutics 2019, 11, x FOR PEER REVIEW 9 of 12

FigureFigure 6.6. QuantiﬁcationQuantification ofof bitternessbitterness ofof variousvarious drugdrug formulationsformulations using using the the e-tongue. e-tongue. Data Data= =mean mean ± ± StandardStandard DeviationDeviation (SD)(SD) ((nn == 3). The figure ﬁgure is adapted with permission from Tang etet al.,al., MolecularMolecular Pharmaceutics;Pharmaceutics; publishedpublished byby AmericanAmerican ChemicalChemical Society,Society,2017 2017[ 23[23].].

PharmacokineticsPharmacokinetics andand Bioavailability:Bioavailability: InIn PKPK andand bioavailabilitybioavailability (BA)(BA) studiesstudies inin mice,mice, ourour datadata confirmedconfirmed thatthat thethe liquidliquid andand lyophilizedlyophilized Mef-LipoMef-Lipo werewere comparablecomparable formulationsformulations with with similar similar C Cmaxmax, TTmaxmax,, area area under the curvecurve (AUC),(AUC), andand BABA (81(81−86%).86%). Mef suspension, however, displayeddisplayed ~20%~20% − decreaseddecreased CCmaxmax, AUC,AUC, andand BA BA compared compared to to the the liposomal liposomal formulation. formulation. There There was was no di nofference difference in Tmax in andTmax t1and/2 among t1/2 among these threethese formulations, three formulations, suggesting suggesting that the formulationsthat the formulations did not alter did the not metabolism alter the ormetabolism elimination or elimination of the drug, of and the thedrug, di ffanderence the diff in BAerence was in due BA was to the due absorption. to the absorption. Liposomes Liposomes might improvemight improve the absorption the absorption by preventing by preventing aggregation aggregation of Mef in the of gastrointestinal Mef in the gastrointestinal tract, protectiing tract, from degradation,protectiing from and degradation promoting direct, and promoting uptake by entericdirect uptake cells. by enteric cells.

4.4. Perspectives and Future Directions WeWe havehave demonstrateddemonstrated thatthat SALTSALT isis aa versatileversatile loadingloading technologytechnology forfor promotingpromoting thethe activeactive loadingloading ofof poorly poorly water-soluble water-soluble drugs drugs into into the liposomalthe liposomal core. Wecore. performed We performed proof-of-principle proof-of-principle studies withstudies multiple with multiple drugs to drugs characterize to characterize the versatility the versatility and robustness and robustness of SALT. Our of SALT. studies Our show studies that SALTshow couldthat SALT improve could drug improve solubility, drug liposomal solubility, loading liposomal effi ciency,loading liposomal efficiency, retention, liposomal stability, retention, palatability, stability, PK,palatability, and efficacy. PK, SALTand efficacy. has the potentialSALT has to the overcome potential barriers to overcome currently barriers impeding currently delivery impeding of many compounds.delivery of many We have compounds. also demonstrated We have also applications demonstrated of SALT applications in cancer therapyof SALT and in cancer child-friendly therapy oraland formulations.child-friendly oral The formulations application of. The SALT application could extend of SALT beyond could therapeutic extend beyond drug loadingtherapeutic and drug also beloading employed and also in the be imagingemployed and in theranosticthe imaging fields. and theranostic Specifically, fields SALT. Specifically, could be useful SALT in could loading be poorlyuseful solublein loading imaging poorly probes soluble into imaging the core ofprobes liposomes. into the A frequentcore of liposomes. barrier to e ffAective frequent image-guided barrier to drugeffective targeting image-guided is the weak drug association targeting betweenis the weak the probeassociation and the between delivery the vehicle probe [and50]. the SALT delivery could helpvehicle retain [50]. imaging SALT could probes help inside retain the imaging core of liposomesprobes inside to improve the core theirof liposomes delivery to improve target tissues. their Despitedelivery the to hightarget performance tissues. Despite of SALT the ashigh a loading perfor platform,mance of thereSALT remain as a loading parameters platform, that canthere be furtherremain optimized.parameters Asthat demonstrated can be further in optimized. these specific As studies, demonst a limitationrated in these of SALT specific when studies, implemented a limitation across of diSALTfferent when compounds implemented is the across requirement different for compound optimizations is of the the requirement loading gradient, for optimization trapping method, of the andloading lipid gradient, composition trapping for maximal method, loading and lipid efficiency composition and retention. for maximal However, loading trapping efficiency agents and can beretention. rationalized However, based trapping on the drug agents structure can be and rationalized lipid composition based on can the be drug empirically structure determined. and lipid Incomposition addition, a can limitation be empirically of SALT determined. is found in the In needadditi toon, find a limitation a water miscible of SALT solvent is found that in canthe beneed used to tofind load a water certain miscible drugs, whichsolvent may that not can always be used be possible.to load certain Moreover, drugs, some which drugs may may not require always large be volumespossible. ofMoreover, solvent that some may drugs result may in degradationrequire large on volumes the liposome of solvent itself that prior may to eresultfficient in drug degradation loading althoughon the liposome this was itself not prior found to in efficient our studies. drug Theloading drugs although themselves this was must not almost found possess in our anstudies. ionizable The functionaldrugs themselves group, must limiting almost potential possess candidates an ionizable for thisfunctional technology. group, Despitelimiting thesepotential limitations candidates we anticipatefor this technology. that future Despite innovations these of limitations SALT could we help an overcometicipate that loading future of innovations other species of of SALT drugs could such ashelp the overcome poorly membrane-permeable loading of other species yet water-solubleof drugs such biomoleculesas the poorly suchmembrane-permeable as proteins and nucleotides, yet water- bothsoluble of whichbiomolecules may benefit such fromas proteins liposomal and delivery. nucleotides, SALT, both however, of which is notmay limited benefit to from liposomes liposomal and willdelivery. be implemented SALT, however, for drug is not loading limited in otherto liposom bilayer-basedes and will delivery be implemented systems, further for drug expanding loading the in other bilayer-based delivery systems, further expanding the impact of this exciting technology. In addition, SALT can be used to improve current formulations that are prepared using passive loading.

Pharmaceutics 2019, 11, 465 10 of 12 impact of this exciting technology. In addition, SALT can be used to improve current formulations that are prepared using passive loading. There exist several drugs on the market that are prepared using passive loading due to their hydrophobicity. SALT could be employed to re-formulate these existing liposomal products to improve the loading eﬃciency, stability and drug retention. SALT is a promising platform which may propel the ﬁeld of lipid-based drug delivery to novel areas.

Funding: This research was supported by grants from Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada and Canada Foundation of Innovation. W.-L.T. was supported by the Frederick Banting and Charles Best Canada Graduate Scholarship from CIHR as well as Four Year Fellowship (4YF) Tuition Award from the UBC. S.-D.L. is a recipient of CIHR New Investigator Salary Awards and the Angiotech Professorship in Drug Delivery. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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