Drug Nanocrystallisation Within Liposomes

Drug Nanocrystallisation within Liposomes

Tang Li a, David Cipolla b, Thomas Radesc , Ben J Boyd a,*

* To whom correspondence should be addressed. Email: [email protected] a ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia. b Aradigm Corporation, Hayward, California 94545 c Faculty of Health and Medical Sciences, Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark

Abstract

Liposomes are phospholipid bilayer vesicles that have been explored in pharmaceutical research as drug delivery systems for more than 50 years. Despite being important to their morphology and drug release pattern, the physical state of the drug within liposomes (liquid, solid, crystalline form) is often overlooked. This review focuses on precipitation of drug within liposomes, which can result in the formation of confined nanocrystals, and consequent changes in liposome morphology and drug release patterns. The type of drugs that form nanocrystals within liposomes, preparation and characterisation of liposomal drug nanocrystals, and the in vitro drug release behaviour from these systems are communicated, with a discussion of their potential as drug delivery systems.

KEYWORDS

Liposomes; precipitation; nanocrystal; solid state; polymorph

1. Introduction

Liposomes are spherical colloidal systems consisting of a phospholipid bilayer shell with an aqueous internal core. These systems are widely explored in pharmaceutical research to reduce the toxicity of the drug candidates [1, 2] and enable effective drug delivery of lipophilic, hydrophilic and amphiphilic drugs [3, 4]. Since their discovery by Alec D Bangham 50 years ago [5], liposomes have found success as nano-sized delivery carriers in the pharmaceutical, food, agriculture and cosmetic industry [6-10]. With respect to utilizing liposomes as vehicles for drug delivery, the success of the liposomal doxorubicin formulation (Doxil®) [11, 12] has led to the development of many other approved liposomal products. Some of the examples include Ambisome® (amphotericin B) [13, 14], DaunoXome® (daunorubicin) [15, 16], Visudyne® (verteporphin) [17], Exparel® (bupivacaine) [18], Marqibo® (vincristine) [19] and the combination product Vyxeos® (daunorubicin-cytarabine) [20]. Most research into liposomes focuses on the modification of the composition of the liposome bilayer and surface chemistry [21]. This includes passive, long-circulating PEGylated liposomes [22-25], ligand targeted liposomes [26-28] and stimuli-responsive liposomes [29- 32]. On the other hand, despite studies that focus on the physical state of the doxorubicin sulfate crystals in Doxil® [33-35], the physical state of the encapsulated drug inside other liposomal formulations has often been overlooked. The physical state of encapsulated drug within the liposomes, whether it is in solution or forms an amorphous or crystalline precipitate, would affect the amount of free drug dissolved inside the liposome; hence affecting the rate of drug release from these drug delivery systems. The precipitation of confined nanocrystals also presents opportunities for alternative applications. For these reasons, this review explores the current status of liposomes in drug delivery from the perspective of drug precipitation and crystallisation inside liposomes, characterisation techniques for the physical state of drug within liposomes and the in vitro drug release behaviour from liposomal drug nanocrystals.

2. Liposomes as drug delivery systems Liposomal systems have been well exploited in drug delivery research especially in cancer treatment and the liposomal doxorubicin HCl injection; Doxil® is the first FDA approved nanomedicine [11, 12]. For liposomal products given via systemic administration, one of the major hurdles is opsonisation of the liposomes in plasmaThis can be addressed by functionalising the liposome surface with a hydrophilic polymer chain, most commonly

2 polyethylene glycol (PEG) (Figure 1), creating a hydrophilic surface that hinders the electrostatic and hydrophobic interactions with blood plasma opsonins, and therefore inhibits the uptake of the liposomes via the mononuclear phagocytic system. These “stealth” liposomes will thus extend the blood circulation time, which allows the liposomes to passively accumulate inside organs and tissues [24]. The physiochemical properties of the liposome including its size, lipid composition, surface charge, route of administration can also influence its immunological response [36, 37]. It has also been reported that shape is important in phagocytosis of nanoparticles and immune response [38-41]. The shape distortion of the liposome to non-spherical shape induced by in situ drug crystallisation can be seen as shape engineering of liposomes. The relationship between the shapes of the liposome and the immune system is still not yet understood, although one study reported an increased reactogenicity of Doxil® compares to placebo Doxil and SPI-77 (liposomal cisplatin with no intraliposomal precipitate and identical lipid composition to Doxil). The increased complement activation of Doxil could be due to the low surface curvature of the oval Doxil® vesicles [42]. Active targeting of liposomes that are functionalized with antibodies to enhance interaction with target antigens on cells at the site of interest, has also gained interest especially for cancer therapy [43]. These so-called ‘immunoliposomes’ bind to overexpressed receptors in the tumour cells, improving therapeutic efficacy and reducing systemic toxicity [28, 44]. Both anti-epidermal growth factor receptor (EGFR) and anti-HER2 tyrosine kinase receptor antibodies have been successfully conjugated to liposomes and are under clinical investigations. An anti-EGFR immunoliposome containing doxorubicin is in phase 2 clinical trials [45, 46].

Liposomes are also used as stimuli-responsive drug delivery systems. The lipid composition can be designed to respond to changes in pH, redox state and enzymes or to external stimuli such as heat, magnetic fields, ultrasound and light [30]. The stimuli will trigger the release of the encapsulated drugs allowing “on demand” drug release from the liposomes. ThermoDox® is a heat-sensitive liposomal doxorubicin that changes structure when heated to 40-45°C, releasing doxorubicin at the target tumour site. This formulation is in phase III clinical trials in combination with radiofrequency ablation (RFA) for the treatment of primary liver cancer [47-49].

Due to the amphiphilic nature of the lipid bilayers, drugs with various physicochemical properties can be encapsulated and delivered in liposomes. Hydrophilic drugs are typically encapsulated in the liposomal aqueous core or near the lipid-water interface. Poorly water- soluble lipophilic pharmaceutical actives can be solubilised within the acyl hydrocarbon chains of the lipid bilayers (Figure 1). The encapsulation efficiency of hydrophobic actives is dictated by the partition coefficient of the drug, the acyl chain length of the phospholipids, the packing density of the bilayer as well as the lipid to drug ratio [4, 50, 51]. Other advantages of drug encapsulation into liposomes include improved solubility for hydrophobic drugs [52] and reduction of non-specific organ toxicity compared to the free drug [2, 53, 54].

Whilst most studies have focussed on the pharmacokinetic and therapeutic efficacy of liposomal drug delivery systems [55-57], the physical characteristics of the encapsulated drugs are not well understood. A review of the literature shows only a few liposomal drug delivery systems for which the physical state of the drug has been reported. Lipophilic drugs that are solubilised within the bilayer (e.g. verteporphin, paclitaxel and Grb2 antisense oligodeoxynucleotide) as well as drugs that form ionic complexes with the oppositely charged lipid (e.g. amphotericin B) do not precipitate in a crystalline form within the liposome. In contrast, hydrophilic drugs that are encapsulated within the aqueous core of the liposome can exhibit different physical states. This includes supersaturated solutions (e.g. cisplatin, ciprofloxacin), amorphous precipitates (e.g. vinorelbine, vincristine) and nanocrystals (e.g. doxorubicin, topotecan, idarubicin) (Figure 1). The crystalline precipitate that forms within liposomes for some drugs is intriguing both from a lipid biophysics perspective as it can stretch or deform the bilayer, as well as by providing an additional means to control the drug release rate by the addition of a dissolution step, and as such is the main focus of this review.

Figure 1. Left: a spherical vesicle with an aqueous core formed by a phospholipid and cholesterol bilayer. The liposome can also be functionalised with polyethylene glycol coating to avoid opsonisation. The liposome aqueous core can encapsulate hydrophilic drugs whereas hydrophobic drugs reside in the lipid bilayer. Right: schematic of a crystalline precipitate formed within a liposome. Depending on the shape and length of the drug precipitate, the bilayer may be stretched to accommodate the growth of the crystalline drug precipitate within the liposome.

1 3. Drug loading methods

2 Different drug loading methods can be used to enable drug encapsulation into 3 liposomes but can also influence the physical state of the encapsulated drug and in some 4 instances lead to precipitation as nanocrystals within the liposomes. Additional sample 5 preparation methods (eg. freeze-thawing) can be also applied to modify the physical state of 6 the encapsulated drug. These methods are discussed in this section and the relevance to the 7 physical state is further discussed in section 5.

8 The simplest approach for drug encapsulation into liposomes is the passive loading 9 method, where drugs are dissolved in the aqueous buffer used to hydrate the lipid bilayer. 10 Thus, the concentration of drug in the aqueous core within the liposomes is similar to that in 11 the aqueous phase exterior to the vesicles. This method is relatively easy and convenient in 12 preparation and manufacturing. However it results in low encapsulation efficiency, high levels 13 of non-encapsulated drug (which can be removed by dialysis, gel filtration and ultrafiltration) 14 and high drug leakage for bilayer permeable drugs. The lipid bilayer is not permeable towards 15 ions and charged molecules. When the drug is unprotonated, it can move in and out of the 16 lipid bilayer of the liposome. The diffusion rate of the drug through the bilayer is dependent 17 upon its partition coefficient. Due to the permeability of the bilayer to uncharged drug 18 molecules, the passively loaded drug molecules of weak acid and weak bases typically exhibit 19 rapid leakage out of the liposomes. Liposomal ciprofloxacin passively loaded has exhibited a 20 50% loss of encapsulated drug after 2 days at 25 °C [58], whereas actively loaded liposomal 21 ciprofloxacin remained at around 99% encapsulation over 24 month period stored at 2-8 °C 22 [59] .

23 This active loading approach [60] addresses the low encapsulation efficiency of 24 passive loading by generating a transmembrane pH (or ion) gradient between the inside of 25 preformed liposomes and the outside continuous aqueous medium. When the uncharged 26 drug molecules diffuse through the lipid bilayer, they become protonated, which inhibits 27 diffusion out of the liposome, thus enhancing the drug loading efficiency and the retention of 28 drug inside the liposome. The loading efficiency is optimal when the drug is an amphipathic 29 weak acid (pKa >3) or weak base (pKa ≤ 11) with a log D of -2.5 to 2.0 at pH 7 [61]. Table 1 30 compares the active and passive loading methods for liposomal drug delivery systems.

31 For amphipathic weak bases, an ammonium sulfate transmembrane gradient [62] is 32 usually used. Firstly, the liposome is formed with an acidic ammonium sulfate buffer, then the 33 transmembrane gradient is generated by replacement of the external buffer with a neutral 34 pH buffer through dialysis or gel exclusion chromatography. When an uncharged ammonia

35 molecule (NH3) escapes the liposome, one proton is generated and retained in the liposome, 36 which results in a more acidic liposome core. The candidate drug (e.g. doxorubicin) is added 37 to the liposome dispersion and loading is performed at a temperature above the phase 38 transition temperature of the phospholipid used to make the liposome for a period of 30-60 39 minutes. During the drug loading process, the candidate drug diffuses into the liposome and 40 becomes protonated due to the acidic liposomal core, and is then trapped within the 41 liposome. The drug loaded into the liposome will transiently increase the internal pH, 42 increasing the level of ammonia and therefore creating more protons, enabling more drug 43 molecules to move into the liposome (Figure 2). After the majority of the drug is loaded into 44 the liposome, non-encapsulated drug is usually removed by dialysis, diafiltration or gel 45 chromatography.

46 A phosphate gradient method has also been applied to load doxorubicin into 47 liposomes by Fritze et al. [63]. In their study, a higher drug encapsulation efficiency (EE) was 48 achieved using the phosphate gradient method compared to the ammonium sulfate or 49 acetate gradient methods. The phosphate gradient loaded doxorubicin liposomes also 50 showed a pH-dependent drug release profile that was not observed in the ammonium sulfate 51 gradient loaded liposomes.

52 An EDTA gradient method has also been used to encapsulate idarubicin [64], 53 topotecan [65] and doxorubicin [66]. The EDTA gradient method enabled efficient drug 54 accumulation and retention of idarubicin due to formation of crystals inside the liposomes 55 providing slower drug release in vivo compared with other gradient loading methods [64]. 56 Liposomes produced by this method have demonstrated decreased cytotoxic activities 57 compared with liposomes prepared by an ammonium sulfate gradient for doxorubicin and 58 topotecan [65, 66]. The calcium acetate gradient loading is applied to weakly acidic drugs 59 (Figure 2). In this case calcium ions remain inside the liposome and the neutral acetate moves 60 out of the liposome resulting in a high intraliposomal pH [67].

63 Figure 2. Left: Schematic of the active loading method of doxorubicin HCl into preformed liposomes using an 64 ammonium sulfate gradient. Doxorubicin forms a crystalline precipitate due to the presence of sulfate anions 65 inside the liposome. Right: Schematic of the active loading method of an amphipathic weak acid into preformed 66 liposomes using a calcium acetate method (adapted with permission from [61]).

68 The ionophore loading method is another method that has been applied to many 69 drugs. Nigericin or A23187 are common ionophores used for unilamellar liposomes. These

70 ionophores facilitate outward transport of K+, Mn2+ and Ca2+ ions and shift protons into the 71 liposome generating an acidic intraliposomal environment. This method has been used in 72 conjunction with a metal ion gradient for encapsulating e.g. ciprofloxacin, vincristine [68], 73 doxorubicin [69], topotecan [70, 71], irinotecan [72] and mitoxantrone [73, 74]. This drug 74 loading method can generate high pH gradient, high drug loading and encapsulation efficiency 75 and allows the encapsulation of different drugs by varying the selection of the ion gradient 76 and ionophore. Depending on the ion used, the encapsulated drug molecules can also form 77 ion-drug complexes within the liposome. The newly formed complex could alter the drug 78 solubility and hence the drug release profile [60].

79 Although the active loading methods are very effective in encapsulating amenable 80 drugs to achieve high and stable drug loading, the preparation methods are more complicated 81 and time consuming compared to the passive loading method. Recently, microfluidic systems 82 have been utilised to enable rapid and efficient remote loading of amphipathic drugs into 83 liposomes. This method incorporates the liposome production, buffer exchange, and remote 84 drug loading and mixing into a continuous process on a single microfluidic chip. This allows a 85 multi-day sample preparation method to be performed in a few minutes [75].

86 Freeze-thawing of MLVs is commonly applied before extrusion (Table 1) to break apart 87 the closely spaced lamellae of the liposomes; this increases the ratio of aqueous solute to 88 lipid and improves the encapsulation efficiency [76-78]. The extrusion of freeze-thawed MLVs 89 results in more monodispersed LUVs with a higher intraliposomal aqueous volume than LUVs 90 prepared without freeze-thawing [79]. Liposomes (both MLVs and LUVs) have also been used 91 as a model membrane to study the effect of freezing and thawing on the biological cells. 92 During the freeze-thawing process, a faster freezing rate (>10 °C) results in intraliposomal ice 93 formation whereas a slow freezing rate (<2 °C) showed osmotic shrinkage of the liposome 94 and no intraliposomal ice formation [80, 81]. Recently, the use of a single freeze-thaw cycle 95 was performed on the ammonium sulfate loaded ciprofloxacin liposomes [82]. Freeze-thaw 96 treatment resulted in ciprofloxacin precipitation inside the liposome. The precipitate was 97 observed as a single rod-shaped ciprofloxacin nanocrystal within each liposome. The 98 formations of intraliposomal ciprofloxacin nanocrystals have also stretched the liposome 99 from a spherical vesicle to a rugby ball-shaped vesicle.

100 Table 1. Comparison of active and passive drug loading methods used for liposomal drug delivery systems.

Drug loading type Method of Liposome Production Advantages Disadvantages References

Passive loading method  Formation of dried lipid film  Easy and convenient  Only for water soluble drugs [3, 83, 84]  Thin layer hydration (TLH)  Drug is solublised in the aqueous method  Low drug encapsulation  Reverse phase evaporation (REV) buffer used to hydrate the dried  Can be used to efficiency  Dehydration-rehydration vesicles lipid film to generate MLVs encapsulate any water  High amount of non- (DRV)  Sonication/extrusion of the MLVs soluble drug encapsulated drug (can be to make the LUVs removed by dialysis or diafiltration)  Rapid drug leakage of bilayer permeable drugs Active loading method  Formation of dried lipid film  High encapsulation  Drug needs to be water [60, 62, Examples  Active loading buffer used to efficiency soluble and have a weakly 67, 85,  Ammonium sulfate gradient, citric hydrate the dried lipid film to  Minimal drug wastage basic or weakly acidic 86] acid gradient (amphipathic weakly generate MLVs (less non-encapsulated functional group basic drugs)  Sonication/extrusion of the MLVs drug loss)  Time consuming and multi  Calcium acetate gradient to make LUVs  Better drug retention day preparations are (amphipathic weakly acidic drugs)  Generation of transmembrane pH required  Ionophore loading (Nigericin/ gradient across the liposomal A23187) with metal ion gradient bilayer through buffer exchange (Mg2+, Mn2+, Cu2+)  Drug solution is added, uncharged  Phosphate gradient drug molecules diffuse through the  EDTA gradient lipid bilayer, become protonated and trapped inside the liposome 101 Abbreviations: MLVs, multilamellar vesicles; LUVs, unilamellar vesicles

102 4. Structural characterisation techniques for the state of drugs inside liposomes

103 The state of the drug loaded by the above methods is important in understanding 104 behaviour, and there is a relative paucity of methods for studying this. Table 2 summarises 105 the advantages and disadvantages associated with the different techniques described in the 106 following section. The physical state of the drug inside the liposome that each methods are 107 capable of detecting are also incorporated within the table.

108 4.1 Cryogenic transmission electron microscopy (Cryo-TEM)/Cryogenic electron 109 tomography

110 The most direct method to visualise the state of a drug inside a liposomal dispersion is 111 using cryo-electron microscopy imaging techniques. In cryo-TEM investigations, the liposomal 112 dispersion is loaded onto the microscopy grid as a thin aqueous film, which is then vitrified by 113 plunging the sample-loaded grid into liquid ethane. The transmission micrograph is then 114 taken at liquid nitrogen temperature to minimize the formation of ice crystals, which can 115 compromise sample imaging. The transfer of the sample grid to the microscope is also 116 performed under liquid nitrogen to prevent formation of crystalline ice [87, 88]. The vitrified 117 sample retains the structures present in their native form in contrast to conventional TEM 118 where drying and staining of the samples disrupts the structures that exist in solution and 119 produces artefacts. The cryo-TEM technique can analyse samples in the nanometer scale; it 120 is best suited for nanoparticles in the size range from 4 to about 500 nm [89]. Since the 121 resolution of the electron microscope correlates to the thickness of the object, smaller 122 liposomes which form a thin film of sample (see above) gives a better signal to noise ratio in 123 the image [90]. This technique is ideal to determine the overall morphology of 124 nanoprecipitates inside liposomes (Figure 3A and Figures 4-6) where such small sized particles 125 (<500 nm) are not visible using light microscopic techniques such as polarised light microscopy 126 [91]. Cryogenic electron tomography (cryo-ET) acquires multiple cryo-TEM images (method 127 as described above) at different tilt positions. These collected images are then used to 128 generate a three dimensional field of view (a tomogram) of the sample. For liposome with 129 encapsulated drug nanocrystals, this technique provides information about the size and 130 shape variations of the nanocrystals within the liposomes in three-dimensional space and has 131 been used to characterize Doxil® [92] and liposomal ciprofloxacin nanocrystals [82]. The

132 advantage and application of the cryo-ET technique is similar to that of the cryo-TEM 133 technique. Moreover it can provide additional details to the drug crystal morphology in three 134 dimensional space that cannot be observed through 2D cryo-TEM images. Both cryo-TEM 135 and cryo-ET can provide a direct visualization of the drug’s solid state inside the liposome. 136 Crystalline drug are displayed as electron dense band, however if the drug formed an 137 amorphous gel or is dissolved inside the liposome, this cannot be easily distinguished in cryo- 138 TEM or cryo-ET.

139 4.2 Small/Wide angle X-ray scattering (SAXS/WAXS)

140 Small angle X-ray scattering, in the range of scattering angles 0.001°<2Ѳ<3° is often 141 used to assess the colloidal structure of dispersed formulations, and the structural attributes 142 of liposomes, including their shape, size, bilayer thickness and lamellarity [93, 94].

143 Of specific relevance to this review, the same technique can be used to determine the 144 structure of the drug precipitates from diffraction in the wider-angle scattering range 145 (5°<2Ѳ<90°), where diffraction from well-defined planes of symmetry within crystalline 146 material enables determination of the solid state form against known crystalline forms (Figure 147 3B), or indeed detection of new polymorphic forms. Alternatively, an amorphous precipitate 148 will present a halo in the diffractogram without defined peaks due to the disordered 149 arrangement of drug molecules in an amorphous form. This scattering technique coupled to 150 a synchrotron source provides high sensitivity and fast acquisition times and has been used 151 to determine the solid state properties for doxorubicin sulfate [35, 95] and doxorubicin citrate 152 nanocrystals inside liposomes [33].

153 4.3 Differential scanning calorimetry (DSC)

154 Differential scanning calorimetry (DSC) is a thermal analytical technique that measures 155 the specific heat capacity of phase transitions as a function of temperature [96]. However due 156 to its non-selective nature, other techniques are required to distinguish whether the sample 157 is undergoing melting, polymorphic transitions or decomposition [97]. DSC has been used to 158 determine the thermal behaviour of drug nanocrystals [98], the thermal behaviour and 159 stability of lipid bilayers and liposomes [99, 100], and to characterise amorphous 160 pharmaceutical solids [101] and drug polymorphism [102]. Consequently, DSC can be applied

161 to qualitatively indicate the thermotropic behaviour of the drug within the liposome and thus 162 its physical state (i.e., amorphous or crystalline nature). For example, recently, studies using 163 “high-sensitivity” DSC observed a two-phase transition for the Doxil® formulation (Figure 3C). 164 The first transition overlapped with that for empty liposomes indicating the “melting” of the 165 membrane lipid while the second transition indicated the melting of the intraliposomal 166 doxorubicin sulfate nanocrystals [34, 103].

167

168 Figure 3. Solid state characterisation of intraliposomal doxorubicin precipitates. (A) Cryo-TEM image of Doxil®, 169 which is ammonium sulfate loaded doxorubicin pegylated liposomes; a linear rod-shaped doxorubicin sulfate 170 nanocrystal is observed inside each liposome (adapted with permission from [11]). (B) Background-subtracted 171 radially-integrated scattering intensity from the Doxil® formulation. The peak identified at q= 2.25 nm-1 for the 172 fitted data (red curve) aligns with the (1,0,0) peak of the doxorubicin sulfate crystal, indicating that the 173 doxorubicin inside the liposome was in a crystalline state (adapted with permission from [35]). (C) Thermotropic 174 behaviour of Lipodox (FDA approved generic liposomal form of Doxil®) compared to empty liposomes using high 175 sensitivity DSC. The first endotherm for both empty and doxorubicin loaded liposomes represents the 176 membrane lipid phase transition. The second endotherm observed with Lipodox at 70 °C represents melting of 177 the intraliposomal doxorubicin-sulfate nanocrystals (adapted with permission from [34]).

178 4.4 Other techniques

179 Other methods have also been used to study the encapsulated drug inside liposomes. 180 Nuclear magnetic resonance (NMR) is a powerful analytical tool to evaluate both the lipid 181 bilayer physical state and rate of motion as well as the physical and chemical nature of the 182 drug inside the liposome. It can also identify any drug to lipid interactions. The advantage of 183 NMR compared to other fluorescent techniques is that the method is based on the use of 184 intrinsic reporter moieties [104]. A study used 195Pt NMR measurement to quantify the 185 soluble cisplatin where any platinum in crystalline state would be undetected by 195Pt NMR

186 due to line broadening. The study confirmed that the cisplatin in the liposome are in the 187 soluble aqueous phase [104]. 1H-NMR was used to study the state of ciprofloxacin located in 188 the aqueous core of the ammonium sulfate gradient loaded liposome. Results demonstrated 189 that ciprofloxacin was located in the aqueous core in a supersaturated state and did not 190 precipitate inside the liposomes [105]. Li et al. used 13C-NMR to assess the interaction of 191 doxorubicin with the intraliposomal citrate ion. In citrate gradient loaded liposomes, the 192 citrate resonance was shifted and broadened by loading of DOX indicating an interaction of 193 DOX with citrate to form a 3:1 charge ratio precipitate [33]. In another study, Arcon et al. used 194 an extended X-ray absorption fine structure method and confirmed that the cisplatin within 195 liposomes did not crystallize but rather formed a supersaturated solution in the liposome core 196 containing 2000-3000 cisplatin molecules per liposome [106]. Circular dichroism 197 spectroscopy was also used to confirm the complexation changes of doxorubicin inside 198 liposomes and monitor the electronic state of the drug for different gradient loaded 199 liposomes [33, 107] (Table 3). Furthermore, electronic paramagnetic resonance spectroscopy 200 (EPR) and UV-vis electronic absorption spectroscopy (UV-vis) were used to characterise the 201 liposomal formulation VyxeosTM (daunorubicin and cytarabine liposomes). EPR is a selective, 202 sensitive spectroscopic technique based on magnetic resonance signals from unpaired 203 electrons in an external magnetic field. Signal intensity is directly proportional to the 204 concentration of paramagnetic species. The presence of the paramagnetic copper (II) ion in 205 the copper gluconate/TEA buffer system used in liposomal cytarabine and daunorubicin 206 formulation allow the use of EPR to investigate the coordination state of the buffer. With the 207 use of different techniques, the interaction of copper gluconate/TEA with daunorubicin and 208 cytarabine showed a 1:1 or 2:1 daunorubicin:copper complexation within the liposomes [108].

209

210

211 Table 2. The advantages and disadvantages associated with different characterisation techniques for the physical state analysis of encapsulated drugs inside liposome.

212

Characterisation Advantages Disadvantages References techniques Cryogenic transmission  Non-destructive (preserve liposome in its suspension form  Only liposome of certain sizes can be viewed [82, 87, 90, electron microscopy while imaging)  Cannot provide a clear picture of the whole sample 92] (Cryo-TEM)  Direct visualisation of both the lipid membrane morphology distribution Cryogenic electron and the structure of encapsulated drug  Cannot be used for physical state/phase identification of tomography drug in the liposome (Cryo-ET)  Expensive and time consuming  Susceptible to electron beam radiation damage Small/Wide angle X-ray  Direct physical state identification  Weak signal for liposomal suspension samples [35, 95, 109] scattering (SAXS/WAXS)  Phase identification (crystal structure)  The liposome signal and drug signal are combined  Rapid data acquisition  Limited access to synchrotron sourced SAXS/WAXS  Sample is susceptible to radiation damage Differential scanning  Indirect physical state identification  Cannot be used for phase identification [34, 96, 100, calorimetry  Provide thermotropic behaviour for crystalline drug in the  Low sensitivity for liquid samples 102, 103] (DSC) liposome  Only crystals with highly ordered structures (high  Identify any drug interaction with phospholipid membrane crystallinity) are detectable Circular dichroism  Rapid and non-destructive  Only drugs with several chromophores or chiral centres [33, 107, 110, (CD)  Identify intermolecular interaction of the drug in the can be studied 111] liposome (changes in physical state)  Background solution can contribute to the CD spectrum  Monitor both the conformation and stability of drug in the (350-200 nm) liposome  Cannot be used for phase identification 15

NMR  Versatile technique based on the use of intrinsic reporter  Cannot be used for phase identification [104, 112] moieties (e.g. 13C, 31P, 1H, 15N, 195Pt)  Low sensitivity (Drug needs to be above a certain  Identify physical and chemical nature of the drug in the concentration for detection) liposome  Time consuming  Identify any drug interaction with the phospholipid membrane Extended X-ray  Study arrangement of atoms in materials without long range  Cannot be used for phase identification [106, 113] absorption fine structure order  Inability to distinguish between scattering atoms with little (EXAFS)  Can apply to both crystalline or amorphous materials, liquid difference in atomic number (C, N, O or S, Cl or Mn, Fe) or molecular gases 213

214 Table 3. List of liposomal drug delivery systems that have been FDA approved, are currently under clinical investigation or were reported in literature with a focus on the 215 physical state of the drug within the aqueous core of the liposome.

Liposome type Drug Drug loading Physical state of the Characterisation techniques Comments References (Formulation) method encapsulated drug

FDA approved formulations Stealth Doxorubicin Ammonium Doxorubicin sulfate drug  Cryo-TEM The doxorubicin sulfate [11, 35, 85, 95, liposome (Doxil®/Caelyx®) sulfate gradient nanocrystals. Observed as  SAXS/XRD nanocrystals showed a 114] rod shaped fibrous  DSC diffraction peak at q=2.30 nm-1 bundles. and melting point of 70°C.

Stealth Irinotecan TEA-sucrose Irinotecan form a gelated NA [115, 116] liposome (Onivyde®) octasulfate (TEA- or precipitated state with SOS) gradient sucrose octasulfate salt Conventional Daunorubicin and Copper Spherical internal  Cryo-TEM Copper interacts with both [108, 117] liposome cytarabine gluconate, TEA structure inside the  Electron paramagnetic cytarabine and daunorubicin (VyxeosTM) buffer liposome identified as a resonance and assists in retaining both second lamella spectroscopy drugs inside the liposome.  1H-NMR  UV-vis electronic absorption spectroscopy Formulations under clinical investigation

Stealth Doxorubicin Citrate gradient Doxorubicin precipitated  Cryo-TEM Precipitate shape similar to [47, 118-120] liposome (ThermoDox®) as fibrous bundles Doxil® (Stimuli (Phase III) responsive) Conventional Ciprofloxacin Ammonium No precipitation  Cryo-TEM/ET Ciprofloxacin drug nanocrystals [82, 121, 122] liposome (Pulmaquin®/Lipoquin®) sulfate gradient  1H-NMR can be induced inside the (Phase III/Phase II) liposome after freeze-thaw in the presence of a cryopreservative (e.g., sucrose) Stealth Doxorubicin Ammonium Doxorubicin precipitated  Cryo-TEM Precipitate shape similar to [123, 124] liposome (2B3-101 or 2X-111) sulfate gradient as fibrous bundles Doxil® (Glutathione) (Phase II) Stealth Cisplatin Passive loading No precipitation  EXAFS Cisplatin form a supersaturated [57, 104, 106] liposome (SPI-077)  Cryo-TEM solution inside the liposome (Phase II)  31P-NMR  195Pt-NMR Conventional Topotecan Manganese Topotecan precipitated as  Cryo-TEM NA [70] liposome (INX-0076) sulfate gradient + thin linear structures (Phase II) ionophore A23187 Conventional Vinorelbine Magnesium Amorphous drug  Cryo-TEM Increased electron density [125, 126] liposome (INX-0125) sulfate gradient + precipitate observed for vinorelbine loaded (Phase I) ionophore liposomes A23187

Experimental formulations Conventional Vincristine/ Manganese Amorphous drug  Cryo-TEM Vincristine can form granular [126, 127] liposome Vinblastine/ sulfate gradient + precipitate structures inside the liposome Vinorelbine ionophore at higher drug to lipid ratio A23187 Conventional Doxorubicin Citrate gradient Doxorubicin precipitated  Cryo-TEM Linear, circular fiber bundles [33] liposome as fibrous bundles  Circular dichroism aligned longitudinally in  13C-NMR hexagonal array  SAXS Manganese No precipitation  Cryo-TEM Doxorubicin manganese [107] sulfate gradient  Circular dichroism complex Manganese Doxorubicin precipitated Precipitate consisted of linear

sulfate gradient as fibrous bundles fibrous bundles + ionophore A23187 Manganese No precipitation NA chloride gradient Manganese No precipitation NA chloride gradient + ionophore A23187 Phosphate Doxorubicin precipitated  Cryo-TEM NA [63] gradient as linear bundles

Magnesium Doxorubicin precipitated  Cryo-TEM Doxorubicin can form triangular [128] sulfate gradient + as linear bundles or even rectangular precipitate ionophore structures at higher drug to A23187 lipid ratios Conventional Topotecan Copper sulfate Topotecan precipitated as  Cryo-TEM NA [71, 129] liposome gradient + needle-like crystals ionophore A23187 Ammonium sulfate gradient Manganese sulfate gradient + Topotecan precipitated as  Cryo-TEM Visually all topotecan [70] ionophore thin needle- like crystals precipitates are similar in A23187 morphology and size regardless Citrate gradient of the D/L ratio and loading Manganese method chloride gradient + ionophore A23187 Stealth Idarubicin EDTA gradient Idarubicin precipitated as  Cryo-TEM Precipitate bundles were mostly [64] liposome elongated bundles bent or circular-shaped structures Stealth Epirubicin EDTA gradient Epirubicin precipitated  Cryo-TEM Precipitate structures were [130] liposome either circular or button-shaped

Stealth Ciprofloxacin Magnesium No precipitation  Cryo-TEM NA [131] liposome sulfate gradient + ionophore A23187 Calcium Ciprofloxacin precipitated Ciprofloxacin precipitate hydrooxybenzens formed between the lamellae ulfonate gradient of bilamellar liposomes. + ionophore A23187 Stealth Mitoxantrone Ammonium Mitoxantrone precipitated  Cryo-TEM NA [132] liposome sulfate loading as linear nanocrystals 216

217 5. Precipitation of drug within liposomes

218 Depending on the physiochemical properties and chemical structures of the drug, the 219 active loading method used, and the process parameters applied, the encapsulated drug may 220 exist as a crystalline precipitate, amorphous precipitate, or in a supersaturated or sub- 221 saturated solution. The specific physical states of the drug within the liposome may impact 222 on both, the stability of the encapsulated drug and the apparent drug release rate from the 223 liposomes. Table 3 list the liposomal formulation that has been FDA approved, under clinical 224 investigation or experimental formulations with a focus on the physical state of the drug 225 inside the liposome. In the following section, different classes of drugs that have been 226 reported to form precipitates within liposomes will be discussed.

227 5.1 Doxorubicin

228 Lasic et al. first reported the physical state of doxorubicin (DOX) in the core of PEGylated 229 liposomes loaded by the ammonium sulfate gradient method. Drug was observed as an 230 electron opaque band within the liposomes and the authors concluded that the DOX sulfate 231 precipitated as crystalline one-dimensional rods, which did not interact with the bilayer (refer 232 to Figure 4B). The material within the liposome displayed a single sharp X-ray reflection at 27 233 Å which correlated with that of doxorubicin precipitated from ammonium sulfate solution, 234 confirming the precipitate to be DOX-sulfate nanocrystals [114].

235 In another study by Li et al., the physical state of DOX in citrate containing liposomes 236 exhibited more curved and circular fibrous bundles (refer to Figure 4C) compared to the DOX- 237 sulfate nanocrystals where only straight rods were observed. This indicated that DOX-citrate 238 nanocrystals are more flexible. X-ray diffraction results showed that the DOX-citrate 239 nanocrystals exhibited an interfiber spacing of 30-35 Å [33]. In a more recent study by Schilt 240 et al. [35], solution X-ray scattering was performed in situ on Doxil® and other DOX-containing 241 liposomal generic formulations. The authors reaffirmed the crystalline state of the DOX- 242 sulfate precipitate within the liposomes identifying a strong diffraction peak at q=2.30 nm-1 243 (Figure 3A). The aggregation and ultimate crystallisation of DOX inside sulfate and citrate 244 containing liposomes is also related to the self-aggregation of DOX through π-π interactions 245 between the planar anthracycline rings in DOX (Figure 4C). Drug complexation with anions 246 present inside the liposome in the case of DOX-sulfate nanocrystals showed tighter packing

247 than DOX-citrate nanocrystals attributed to the sulfate ion being a smaller anion compared 248 to the citrate ion (refer to Figure 4) [33].

249 Manganese chloride and manganese sulfate-loaded DOX liposomes with and without 250 ionophore were also characterised with cryo-TEM to study the state of DOX inside the 251 liposome. Manganese chloride gradient-loaded DOX liposomes did not show any drug 252 precipitation regardless of ionophore addition. However, the manganese sulfate gradient 253 loaded DOX liposomes with ionophore exhibited linear bundle-like nanocrystal structures 254 similar to those of the citrate loaded DOX liposomes. The presence of the ionophore A23187 255 generated a >2.5 pH units gradient with an acidic core compared to little or no 256 transmembrane pH gradient across the liposome in the absence of the ionophore. This

257

258

259 Figure 4. Dependence of the form of doxorubicin precipitates under confinement in liposomes on the counterion 260 (A) Chemical structure of doxorubicin. (B) Cryo-TEM image of doxorubicin HCl loaded into liposomes using an 261 ammonium sulfate gradient (adapted with permission from [114]) showing linear rod like nanocrystals (C) Cryo- 262 TEM image of doxorubicin HCl loaded into liposomes using a citrate gradient showing circular and U-shaped 263 nanocrystals (adapted with permission from [33]).

264

265 DOX to ionise and interact with the sulfate anion and precipitate within the liposome [107]. 266 Other gradient loaded DOX liposomes such as the phosphate gradient [63] and magnesium 267 sulfate gradient (with ionophore) liposomes [128] also enabled DOX precipitation inside the 268 liposome (refer to Table 3).

269 Overall, it is clear that the counter ions used to actively load the DOX into the 270 liposomes result in differences in the structure of the precipitates, most likely due to the 271 formation of salts with different crystal habits and rigidity.

272

273 5.2 Vincristine, vinorelbine and vinblastine

274 Liposomal vincristine sulfate (trade name: Marqibo®) has been approved for the 275 treatment of lymphoblastic leukemia. Vincristine (VCR) is a vinca alkaloid with low aqueous 276 solubility at physiological pH but with high permeability, making it ideal to be encapsulated 277 into liposomes to reduce the acute toxicity of the free drug [133]. Johnston et al. compared 278 the physical states of magnesium sulfate gradient (with ionophore) loaded vincristine 279 liposomes at different drug to lipid (D/L) ratios (Figure 5). At the highest D/L ratio of 1.03, the 280 cryo-TEM image showed the liposomes with dense granular structures within their aqueous 281 core (Figure 5F). These structures are very different to the linear crystal rods formed in DOX 282 sulfate liposomes [127]. In this study, the higher the D/L ratio, the better the drug retention 283 (i.e., the lower the vincristine release rate). However a higher drug retention does not always 284 correlate to the most optimal therapeutic efficacy; in the above study, the optimal 285 therapeutic efficacy was achieved with liposomes formed at a D/L ratio of 0.1.

286

287 Figure 5. Cryo-TEM images of liposomal formulations of vincristine. The drugs were loaded into the liposomes

288 using the ionophore A23187/MgSO4 loading method at different D/L ratios. (A) Liposome in the absence of

289 drug, and at a D/L ratio of 0.06 (B), 0.27 (C), 0.6 (D) and 1.03 (E). (F) Enlarged section of (E) showing granulated 290 structures within the liposomes. The size bar represents 200 nm in each image (adapted with permission from 291 [127]).

292 In another study, other vinca alkaloids, including vinorelbine and vinblastine were also 293 investigated as liposomal formulations in comparison to vincristine [126]. None of the vinca 294 alkaloids, i.e., vinorelbine, vinblastine or vincristine showed crystalline precipitates within 295 magnesium sulfate (with ionophore) loaded liposomes (Figure 6). The more electron-dense 296 appearance in the cryo-EM micrographs compared with drug-free liposomes indicated that 297 the drug had precipitated in an amorphous rather than crystalline form within the liposome.

298

299

300 Figure 6. Cryo-TEM images of liposomal formulations of different vinca alkaloids. The drugs were loaded into the

301 liposomes using the ionophore A23187/MgSO4 loading method at a D/L ratio of 0.3. (A) Liposomes in the 302 absence of drug, (B) liposomal vinblastine, (C) liposomal vinorelbine and (D) liposomal vincristine. The size bar 303 represents 100 nm for all of the images (adapted with permission from [126]).

304 5.3 Topotecan

305 Topotecan (TPT), a camptothecin analogue, is a topoisomerase I inhibitor for the 306 management of ovarian cancer. Its closed α-lactone ring is therapeutically important for 307 tumour cell targeting; however the stability of this molecule presents a challenge as it has a 308 tendency to undergo hydrolysis at neutral physiological pH [134, 135]. Encapsulation of TPT 309 into liposomes can increase drug stability and enhance its therapeutic index [136, 137]. Four 310 different active loading methods were employed; namely ammonium sulfate, citrate, 311 manganese sulfate (with ionophore), and manganese chloride (with ionophore). All methods 312 induced the formation of intraliposomal linear nanocrystals at a low D/L ratio of 0.1 [70] (refer 313 to Figure 7). The crystalline TPT precipitates also altered the vesicle morphology to a more 314 stretched elliptical shape compared to the empty spherical liposomes.

315 Co-encapsulation of anticancer drugs, such as VCR and TPT, increases their 316 therapeutic efficacy because the combination acts on different targets in the cancer cell and 317 affects different phases in the cell cycle. TPT converts DNA topoisomerase I into a cellular

318 toxin, leading to arrest in the S phase or G2-M phase, whereas, VCR causes depolymerisation 319 of microtubules, leading to mitotic arrest [138]. In the case of VCR and TPT, both drugs are 320 amphiphilic weak bases and apparently do not interact with each other after co-encapsulation 321 into ammonium sulfate gradient loaded liposomes [109]. Analyses by SAXS showed that TPT 322 crystallizes in the shape of longitudinal rods while VCR did not precipitate. The author 323 suggested since the intraliposomal VCR concentration significantly exceeds its maximal 324 aqueous solubility, and that it probably formed a supersaturated amorphous gel-like phase 325 inside the liposome [109].

326

327

328 Figure 7. Cryo-TEM images of liposomes before and after active drug loading with topotecan (TPT). Top row: 329 Empty liposomes before TPT loading. Bottom row: loaded topotecan at 0.1 w/w drug to lipid ratio. The type of 330 active loading method is annotated above the images (adapted with permission from [70]). All loading methods 331 resulted in formation of linear crystalline precipitates for TPT liposomes. Scale bar is equivalent to 100 nm for 332 all the images.

333 5.4 Other drugs

334 Ciprofloxacin is a fluoroquinolone broad-spectrum antibiotic and is a weakly basic 335 drug with poor aqueous solubility at neutral pH [105]. Actively loading liposomes with

336 ciprofloxacin using an ammonium sulfate gradient can provide a high intraliposomal drug 337 concentration; however the drug does not precipitate as nanocrystals [85]. This led to 338 acceptable long-term stability of 6 months in an early research liposomal ciprofloxacin batch 339 [85] and 2 years in the GMP batches (e.g., with Lipoquin®) with no decrease in encapsulated 340 drug [121]. However, the drug leakage rate after dilution in plasma was faster for liposomal 341 ciprofloxacin than for the liposomal formulation containing doxorubicin in a nanocrystalline 342 state [85]. Thus, attenuation of the ciprofloxacin release rate may be possible if it could be 343 converted into a nanocrystalline state. Recently, an additional freeze-thawing step was 344 introduced to the ammonium sulfate loaded ciprofloxacin liposomes that generated a linear 345 rod-like ciprofloxacin precipitate inside the liposomes. The precipitate was visually similar to 346 DOX sulfate crystals as observed in Doxil® [82]. In another study with ciprofloxacin, 347 hydroxybenzenesulfonate (HBS) was used in the loading of ciprofloxacin into liposomes, and 348 resulted in the formation of intravesicular ciprofloxacin-HBS non-soluble ion pair complexes 349 [131]. The negatively charged sulfonic group of HBS and the positive charge on the protonated 350 amine in ciprofloxacin lead to the formation of insoluble ion pairs. Other drugs that have also 351 precipitated inside liposomes include EDTA loaded idarubicin [64] and epirubicin [130]. Both 352 idarubicin and epirubicin formed circular button shaped bundles inside the liposome. For 353 idarubicin, the EDTA loading method enabled efficient drug accumulation inside the liposome. 354 The formation of a low solubility idarubicin EDTA precipitate inside the liposome leads to 355 increased in vivo stability for the otherwise fast-leaking hydrophobic drug [64].

356 Although as discussed previously the type of active loading buffer can influence the 357 structure of the nanocrystal formation such as for doxorubicin. There are also slight 358 differences for the nanocrystal precipitate observed among different drugs (Table 3). For 359 example topotecan is reported to precipitate in thin needle like precipitate, whereas 360 doxorubicin is precipitated in a more linear bundle like structure and epirubicin having some 361 linear structures but mostly circular or button like structures. The difference in the 362 doxorubicin nanocrystals to topotecan nanocrystals could be due to the chemical structure of 363 the drug molecule itself. Topotecan is comprised of a planar pentacyclic ring structure, 364 whereas doxorubicin contains a tetracenequinone ring structure with a sugar attached by 365 glycosidic linkage. The difference in the molecular structure would affect the crystal packing 366 which changes the nanocrystal structure formed. In the case of epirubicin (EDTA loaded

367 liposomes), where the molecular structure is identical to doxorubicin, the difference in the 368 nanocrystal structure formed inside the liposome could be due to the difference in the active 369 loading buffer used.

370

371 6 Drug release from nanocrystallised drug within liposomes

372 The fundamental mechanism of drug release in liposomal drug delivery systems is through 373 passive drug permeation and diffusion. Drug release from liposomal carriers is a complex 374 process affected by the physiochemical properties of the liposome and the physical state of 375 the encapsulated drug, as well as external factors such as the release medium selection, 376 temperature and pH. The physiochemical properties that dictate drug release from liposomes 377 include the bilayer permeability of the drug, the drug ionisation constant, drug binding 378 behaviour with the lipid bilayer, self-association of the drug and the presence of 379 intraliposomal precipitate. The size and the cross sectional area of the molecule are also 380 important in dictating the permeability and diffusion of the drug across the lipid bilayer [139, 381 140]. Other parameters to be considered in the case of actively loaded liposomes are the 382 concentration and type of the loading buffer, the D/L ratio, intraliposomal pH, volume, as well 383 as external factors such as the presence of other permeable ions in the external buffer.

384 Dialysis is commonly used to study the drug release kinetics from liposomal formulations. 385 The liposomes dispersion is placed inside a dialysis bag, which is then sealed and placed into 386 the release medium reservoir to generate sink conditions and drive drug release. However 387 this method has serious, often unrecognised limitations in the ability to measure the release 388 rate and kinetics from liposomal formulations. The barrier nature of the dialysis membrane 389 itself can retard the apparent drug release rate, meaning that although drug is released from 390 the liposome, it is not recognised as such until it reaches the receptor medium [141, 142] The 391 drug could also reversibly or irreversibly bind to the dialysis membrane or the liposome after 392 release from the carrier, which reduces the apparent release rate and concentration gradient 393 across the membrane [143, 144]. Another conventional method to evaluate drug release from 394 liposomal formulations is the “sample and separate” method. The liposomal formulations are 395 diluted into the release medium under sink condition and aliquots are removed from the 396 release medium at different time intervals. The amount of drug release is determined after

397 separation of the dispersed liposomal formulation (e.g. by ultracentrifugation or 398 ultrafiltration) from the release medium [145-147]. The limiting factor for this method is the 399 efficiency and accuracy of the sample separation method that is used. The force and time 400 applied for centrifugation to separate the liposomes from the free drug can potentially induce 401 unwanted drug release and affect the apparent drug release [141, 148]. It is important to note 402 that despite Doxil® having been on the market for two decades now, there is still no 403 pharmacopoeial method for the measurement of drug release from liposomes, although 404 recent FDA sponsored work towards a continuous flow USP-4 method has been reported 405 [149].

406 Several mechanistic models have been developed to predict drug release for Doxil® [143, 407 150] and liposomal topotecan [151, 152]. These models explain the physiochemical 408 properties important in dictating the drug release behaviour as well as gaining insight into the 409 in vivo - in vitro correlation to better predict the drug release behaviour in vivo. Csuhai et al. 410 determined the key parameters for mechanism-based models to help predict doxorubicin 411 release from actively loaded liposomes [150]. These parameters included drug pKa, 412 permeability coefficients, drug self-association and relevant equilibrium constants, solubility 413 product of the DOX-sulfate nanocrystals, and partition coefficients of DOX. The amount of 414 drug loaded, and the concentration of the sulfate ion dictate how much drug is precipitated

415 within the liposome. The solubility product constant (Ksp) for DOX-sulfate nanocrystals of 416 Doxil® was reported to be 2.9×10-7 M3 at 37°C. The solubility of DOX decreases with increasing 417 ammonium sulfate concentration, and the solubility product of the DOX-sulfate nanocrystals 418 is temperature dependent. The authors assumed that only the unionised monomeric form of 419 DOX can cross the liposomal membrane. Therefore the concentration of DOX in the 420 monomeric form rather than the self-associated dimeric and oligomeric form will be the 421 driving force for drug release.

422 It has also been reported that the precipitation of DOX-sulfate salt as nanocrystals inside 423 the liposomes lowers the fraction of drug in solution [143]. This reduces the driving force for 424 release and retards DOX release from Doxil®. The solubility product of the precipitate is the 425 rate limiting factor to control drug release rather than the dissolution rate. Since more than 426 90% of the total entrapped drug is precipitated as drug nanocrystals, the driving force for drug 427 release remains constant, resulting in zero order release kinetics. Also the study observed

428 that when there is no external ammonia in the release media, the internal pH is acidic, which 429 protonates the DOX and the ionised drug is then membrane impermeable, resulting in an 430 extremely slow release profile. However when ammonia is added externally, the reservoir pH 431 is elevated and the influx of ammonia will raise the intraliposomal pH and accelerate drug 432 release. This zero order release kinetics model is also consistent with the data reported for 433 liposomes containing precipitated vincristine with the drug efflux rate being proportional to 434 the soluble drug concentration [127].

435 A more recent study by Russell et al. assessed the kinetics of doxorubicin leakage from 436 the Doxil® formulation [153], namely that the release of doxorubicin from Doxil® undergoes 437 three processes, first the dissolution of the solid DOX-sulfate nanocrystals, then the 438 protonation/deprotonation of the soluble DOX and finally passive transport of neutral DOX 439 across the lipid bilayer to the external medium. They proposed that the dissolution of the 440 DOX-sulfate nanocrystals followed first order kinetics with a rate constant of 1.0 x 10-9 cm s- 441 1. The DOX leakage for transport across the lipid bilayer was also modelled by first order 442 kinetics with a rate constant of 1-3 x 10-12 cm s-1 depending on the pH, with a more acidic pH 443 resulting in a higher rate constant. The dissolution rate of the DOX-sulfate nanocrystal is three 444 orders of magnitude faster than the leakage rate constant of DOX across the lipid bilayer. 445 Hence, the kinetics of DOX leakage from the liposome are dominated by the rate constant for 446 transport across the liposome bilayer and the pKa [153].

447

448

449 Figure 8 Schematics of different drug release kinetics. A Drug is present inside the liposome only in a solubilised 450 form. The drug release rate follows first order kinetics. B Drug precipitation/crystallisation has occurred within 451 the liposome core. The drug release rate follows zero order kinetics with (adapted with permission from [131]).

452

453 As described earlier, Zhigaltsev et al. prepared HBS loaded liposomal vinorelbine and 454 liposomal ciprofloxacin. The drug and HBS formed non-soluble ion pair complexes that 455 precipitated inside the liposomes leading to improved drug retention [131]. The in vitro 456 release studies showed that when the precipitates had formed, release was governed by zero 457 order kinetics, but in the absence of intraliposomal precipitation the release followed first 458 order kinetics (Figure 8). Also they suggested that if the dissolution rate of the drug is slower 459 than the membrane diffusion rate of the unionised species, a biphasic release with a rapid 460 initial loss of drug followed by slower linear zero order release will be observed. However if 461 the membrane diffusion rate is slower than the dissolution of the precipitate, only a zero 462 order release rate is observed.

463 The formation of ciprofloxacin nanocrystals inside the liposomes after using the freeze- 464 thaw method was briefly discussed in section 5.4. In another study, addition of polysorbate

465 20 or Brij 30 during the preparation of liposomal ciprofloxacin before the freeze-thaw method 466 was applied reduced the extent of ciprofloxacin nanocrystal growth inside the liposomes and 467 hence allowed modification of the drug release profile [122]. It was also noted that the 468 samples that had nanocrystals formed inside the liposomes post the freeze-thaw process 469 showed slower drug release compared to liposomal ciprofloxacin in the absence of 470 nanocrystals, regardless whether surfactant was added or not. This study suggests the 471 potential possibilities of controlling the size of nanocrystal formation within the liposomes, 472 which may be useful in developing future liposomal drugs with tailored drug release profiles.

473

474 7 Future applications of nanocrystallised liposomes and conclusions

475 Nanocrystalline precipitates formed within liposomes provide many potential benefits for 476 liposomal drug formulation including higher drug retention, better formulation stability, and 477 more efficient drug loading. However, the solid state properties of the precipitates have not 478 been studied in a detailed and systemic way (as shown in Table 3). To date, only DOX-sulfate 479 and DOX-citrate precipitates in liposomal DOX have been fully characterised and confirmed 480 to show the precipitates are in a crystalline form. This greatly limits the investigation into the 481 mechanism of drug crystallisation within liposomes as well as the assessment of additional 482 benefits in drug delivery applications. From Table 3, some observations can be drawn from 483 the drugs that form “nanocrystal like” fibrous bundles/precipitate inside the liposomes. 484 Firstly, these liposomal formulations are prepared using an active loading method (as 485 described in section 3) so the drug needs to be an amphiphilic base or acid. Also the selection 486 of ion for the active loading method is crucial in generating insoluble drug-ion complexes, 487 which promote precipitation. Secondly, these actively loaded liposomal formulations have 488 achieved high drug loading efficiency and high drug retention properties. The high 489 intraliposomal drug concentration means that more ionized drug molecules are able to form 490 insoluble complexes with the ions inside the liposome. Lastly, these drugs have many 491 aromatic rings in their structures that can form stacks through aromatic pi-pi interactions.

492 For drugs that did not precipitate inside the liposome after active loading, the freeze- 493 thawing method which induced crystallisation of ciprofloxacin within liposomes [82, 122] 494 could potentially be applied to enable crystallisation inside the liposome. This method may

495 be easily incorporated in the process to form drug nanocrystals inside the liposome. Although 496 there is no definitive mechanistic understanding of the formation of drug precipitate within 497 the liposome, the expansion and study of a wider range of compounds exhibiting this 498 phenomenon would generate increased interest and experimental studies to support 499 theoretical modeling of possible mechanisms.

500 One possible future application for the liposomal drug nanocrystals may be in oral drug 501 delivery. Traditionally, nanocrystals range from 10-1000 nm in particle size [154]. The smaller 502 particle size of these nanocrystals would increase the apparent surface area, which, together 503 with the enhanced solubility by virtue of the lipids being present, will increase the dissolution 504 rate of the poorly water soluble drugs predicted from the Noyes-Whitney equation [155]. 505 Hence, the nanocrystals present inside the liposome would provide a source for enhanced 506 dissolution rate due to their small size. Liposomes, at least in theory, are inherently a lipid- 507 based formulation (LBF) [156]. During the digestion of LBF, bile salts can interact with 508 liposomes forming mixed micelles [157, 158] and lipases and phospholipases have the ability 509 to hydrolyse the phospholipid in the liposomes [159, 160]. In the case of liposomes with 510 encapsulated nanocrystals, if the crystallised drug is weakly basic in nature, the hydrolysis of 511 phospholipid into free fatty acid by the nature of the digestion process would interact with 512 the oppositely charged weakly basic drug to form an amorphous drug salt [161]. This 513 formation of the amorphous drug salt during digestion of liposomal drug nanocrystals could 514 increase the drug dissolution rate in the GI tract, and enable enhanced absorption by 515 providing an increased pool of non-crystalline drug.

516 Another possible future application for liposomal drug nanocrystals is their use as a new 517 formulation approach for current drugs formulated as nanosuspensions. Nanosuspensions 518 are being developed for drugs whose low solubility in lipid or water precludes their 519 solubilisation into conventional carrier systems for IV administration. Preparing intravenous 520 nanosuspensions of drugs, such as Abraxane® for paclitaxel [162], requires a complex 521 manufacturing process with dedicated facilities and onerous quality control. In the 522 nanocrystal examples provided above, the confinement of the liposome appears to exclude 523 external crystallization or growth through Ostwald ripening, leading to potentially greater size 524 stability and thus increased safety for such products, while enabling the administration of a 525 meaningful dose.

526 Liposomes, including conventional liposomes [163], highly deformable liposomes 527 (transfersomes) [164, 165] and niosomes [166] have been explored to improve drug delivery 528 through the skin [167, 168]. The phospholipid of the liposomal bilayer acts as a penetration 529 enhancer by loosening the lipid structure of the stratum corneum and this impaired barrier 530 function of the skin subsequently increases skin partitioning of the drug [169, 170]. There are 531 currently three nanocrystalline-based products for topical drug delivery on the market and a 532 few under clinical trials [171]. Drug nanocrystals with their larger surface area compared to 533 for example micronized drug particles, increase the drugs’s dissolution rate and even 534 saturation solubility [172, 173]. The increased saturation solubility helps to maintain the 535 concentration gradient between the suspension and the target cell which improves drug 536 absorption by diffusion [171]. Currently, drug nanocrystal suspensions are formulated in gels 537 or creams for ease of application [174, 175]. Liposomal drug nanocrystals however, can be 538 beneficial in these cases as the liposomal bilayer acts as the stabiliser for the drug 539 nanocrystals as noted above. This may reduce the requirement for additional formulation 540 development. The liposomal bilayer may also act as a penetration enhancer for the 541 encapsulated drug nanocrystals.

542 In conclusion, this review has explored the importance of the physical state of the drug 543 encapsulated within liposomes and the role this state plays with respect to drug dissolution 544 and release. The ability to modify the drug’s physical state inside a liposome may provide an 545 opportunity to modify the pharmacokinetics of the drug to optimise the safety and efficacy 546 of treatment with liposomal products.

547 Conflict of Interest

548 At the time of preparation of this manuscript, David Cipolla was an employee of Aradigm 549 Corporation, developing liposomal ciprofloxacin.

550

551 References

552 [1] G. Batist, G. Ramakrishnan, C.S. Rao, A. Chandrasekharan, J. Gutheil, T. Guthrie, P. Shah, A. 553 Khojasteh, M.K. Nair, K. Hoelzer, K. Tkaczuk, Y.C. Park, L.W. Lee, Reduced Cardiotoxicity and Preserved 554 Antitumor Efficacy of Liposome-Encapsulated Doxorubicin and Cyclophosphamide Compared With

555 Conventional Doxorubicin and Cyclophosphamide in a Randomized, Multicenter Trial of Metastatic 556 Breast Cancer, Journal of Clinical Oncology, 19 (2001) 1444-1454. 557 [2] M.E.R. O’Brien, N. Wigler, M. Inbar, R. Rosso, E. Grischke, A. Santoro, R. Catane, D.G. Kieback, P. 558 Tomczak, S.P. Ackland, F. Orlandi, L. Mellars, L. Alland, C. Tendler, Reduced cardiotoxicity and 559 comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX™/Doxil®) 560 versus conventional doxorubicin for first-line treatment of metastatic breast cancer, Annals of 561 Oncology, 15 (2004) 440-449. 562 [3] J.O. Eloy, M. Claro de Souza, R. Petrilli, J.P.A. Barcellos, R.J. Lee, J.M. Marchetti, Liposomes as 563 carriers of hydrophilic small molecule drugs: Strategies to enhance encapsulation and delivery, 564 Colloids and Surfaces B: Biointerfaces, 123 (2014) 345-363. 565 [4] A. Fahr, P.v. Hoogevest, S. May, N. Bergstrand, M.L. S. Leigh, Transfer of lipophilic drugs between 566 liposomal membranes and biological interfaces: Consequences for drug delivery, European Journal of 567 Pharmaceutical Sciences, 26 (2005) 251-265. 568 [5] A.D. Bangham, Physical Structure and Behavior of Lipids and Lipid Enzymes, in: R. Paoletti, D. 569 Kritchevsky (Eds.) Adv Lipid Res, Elsevier, 1963, pp. 65-104. 570 [6] T.M. Allen, P.R. Cullis, Liposomal drug delivery systems: From concept to clinical applications, 571 Advanced Drug Delivery Reviews, 65 (2013) 36-48. 572 [7] C. Zylberberg, S. Matosevic, Pharmaceutical liposomal drug delivery: a review of new delivery 573 systems and a look at the regulatory landscape, Drug Delivery, 23 (2016) 3319-3329. 574 [8] T.M. Taylor, J. Weiss, P.M. Davidson, B.D. Bruce, Liposomal Nanocapsules in Food Science and 575 Agriculture, Critical Reviews in Food Science and Nutrition, 45 (2005) 587-605. 576 [9] M. Reza Mozafari, C. Johnson, S. Hatziantoniou, C. Demetzos, Nanoliposomes and Their 577 Applications in Food Nanotechnology, Journal of Liposome Research, 18 (2008) 309-327. 578 [10] Y. Rahimpour, H. Hamishehkar, Liposomes in cosmeceutics, Expert Opinion on Drug Delivery, 9 579 (2012) 443-455. 580 [11] Y. Barenholz, Doxil® — The first FDA-approved nano-drug: Lessons learned, Journal of Controlled 581 Release, 160 (2012) 117-134. 582 [12] N.D. James, R.J. Coker, D. Tomlinson, J.R.W. Harris, M. Gompels, A.J. Pinching, J.S.W. Stewart, 583 Liposomal doxorubicin (Doxil): An effective new treatment for Kaposi's sarcoma in AIDS, Clinical 584 Oncology, 6 (1994) 294-296. 585 [13] J. Adler-Moore, R.T. Proffitt, AmBisome: liposomal formulation, structure, mechanism of action 586 and pre-clinical experience, Journal of Antimicrobial Chemotherapy, 49 (2002) 21-30. 587 [14] G.W. Boswell, D. Buell, I. Bekersky, AmBisome (Liposomal Amphotericin B): A Comparative Review, 588 The Journal of Clinical Pharmacology, 38 (1998) 583-592. 589 [15] E.A. Forssen, The design and development of DaunoXome® for solid tumor targeting in vivo, 590 Advanced Drug Delivery Reviews, 24 (1997) 133-150. 591 [16] E. Rosenthal, I. Poizot-Martin, T. Saint-Marc, J.P. Spano, P. Cacoub, D.N.X.S. Group, Phase IV study 592 of liposomal daunorubicin (DaunoXome) in AIDS-related Kaposi sarcoma, Am J Clin Oncol, 25 (2002) 593 57-59. 594 [17] G. Treatment of Age-related Macular Degeneration With Photodynamic Therapy Study, 595 Photodynamic therapy of subfoveal choroidal neovascularization in age-related macular degeneration 596 with verteporfin: One-year results of 2 randomized clinical trials—tap report 1, Archives of 597 Ophthalmology, 117 (1999) 1329-1345. 598 [18] J.B. McAlvin, R.F. Padera, S.A. Shankarappa, G. Reznor, A.H. Kwon, H.H. Chiang, J. Yang, D.S. 599 Kohane, Multivesicular liposomal bupivacaine at the sciatic nerve, Biomaterials, 35 (2014) 4557-4564. 600 [19] J.A. Silverman, S.R. Deitcher, Marqibo(®) (vincristine sulfate liposome injection) improves the 601 pharmacokinetics and pharmacodynamics of vincristine, Cancer Chemotherapy and Pharmacology, 71 602 (2013) 555-564. 603 [20] M.S. Franco, M.C. Oliveira, Liposomes co-encapsulating anticancer drugs in synergistic ratios as 604 an approach to promote increased efficacy and greater safety, Anticancer Agents Med Chem, (2018).

605 [21] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nature Reviews Drug 606 Discovery, 4 (2005) 145. 607 [22] O.K. Nag, V. Awasthi, Surface Engineering of Liposomes for Stealth Behavior, Pharmaceutics, 5 608 (2013) 542-569. 609 [23] B. Cˇeh, M. Winterhalter, P.M. Frederik, J.J. Vallner, D.D. Lasic, Stealth® liposomes: from theory 610 to product, Advanced Drug Delivery Reviews, 24 (1997) 165-177. 611 [24] M.L. Immordino, F. Dosio, L. Cattel, Stealth liposomes: review of the basic science, rationale, and 612 clinical applications, existing and potential, International Journal of Nanomedicine, 1 (2006) 297-315. 613 [25] S.M. Moghimi, J. Szebeni, Stealth liposomes and long circulating nanoparticles: critical issues in 614 pharmacokinetics, opsonization and protein-binding properties, Progress in lipid research, 42 (2003) 615 463-478. 616 [26] E. Forssen, M. Willis, Ligand-targeted liposomes, Advanced Drug Delivery Reviews, 29 (1998) 249- 617 271. 618 [27] C.O. Noble, D.B. Kirpotin, M.E. Hayes, C. Mamot, K. Hong, J.W. Park, C.C. Benz, J.D. Marks, D.C. 619 Drummond, Development of ligand-targeted liposomes for cancer therapy, Expert Opinion on 620 Therapeutic Targets, 8 (2004) 335-353. 621 [28] G.T. Noble, J.F. Stefanick, J.D. Ashley, T. Kiziltepe, B. Bilgicer, Ligand-targeted liposome design: 622 challenges and fundamental considerations, Trends in Biotechnology, 32 (2014) 32-45. 623 [29] B. Kneidl, M. Peller, G. Winter, L.H. Lindner, M. Hossann, Thermosensitive liposomal drug delivery 624 systems: state of the art review, International Journal of Nanomedicine, 9 (2014) 4387-4398. 625 [30] B.J. Boyd, W.-K. Fong, Stimuli-Responsive Lipid-Based Self-Assembled Systems, in: Self- 626 Assembled Supramolecular Architectures, John Wiley & Sons, Inc., 2012, pp. 257-288. 627 [31] A. Jain, S.K. Jain, Stimuli-responsive Smart Liposomes in Cancer Targeting, Current drug targets, 628 19 (2018) 259-270. 629 [32] F. Movahedi, R.G. Hu, D.L. Becker, C. Xu, Stimuli-responsive liposomes for the delivery of nucleic 630 acid therapeutics, Nanomedicine: Nanotechnology, Biology and Medicine, 11 (2015) 1575-1584. 631 [33] X. Li, D.J. Hirsh, D. Cabral-Lilly, A. Zirkel, S.M. Gruner, A.S. Janoff, W.R. Perkins, Doxorubicin 632 physical state in solution and inside liposomes loaded via a pH gradient, Biochimica et Biophysica Acta 633 (BBA) - Biomembranes, 1415 (1998) 23-40. 634 [34] X. Wei, R. Cohen, Y. Barenholz, Insights into composition/structure/function relationships of 635 Doxil® gained from “high-sensitivity” differential scanning calorimetry, European Journal of 636 Pharmaceutics and Biopharmaceutics, 104 (2016) 260-270. 637 [35] Y. Schilt, T. Berman, X. Wei, Y. Barenholz, U. Raviv, Using solution X-ray scattering to determine 638 the high-resolution structure and morphology of PEGylated liposomal doxorubicin nanodrugs, 639 Biochimica et Biophysica Acta (BBA) - General Subjects, 1860 (2016) 108-119. 640 [36] D. Landesman-Milo, D. Peer, Altering the immune response with lipid-based nanoparticles, 641 Journal of controlled release : official journal of the Controlled Release Society, 161 (2012) 600-608. 642 [37] D.S. Watson, A.N. Endsley, L. Huang, Design considerations for liposomal vaccines: Influence of 643 formulation parameters on antibody and cell-mediated immune responses to liposome associated 644 antigens, Vaccine, 30 (2012) 2256-2272. 645 [38] N. Doshi, S. Mitragotri, Macrophages Recognize Size and Shape of Their Targets, PLOS ONE, 5 646 (2010) e10051. 647 [39] J.A. Champion, S. Mitragotri, Shape Induced Inhibition of Phagocytosis of Polymer Particles, 648 Pharmaceutical Research, 26 (2009) 244-249. 649 [40] J.A. Champion, S. Mitragotri, Role of target geometry in phagocytosis, P Natl Acad Sci USA, 103 650 (2006) 4930. 651 [41] S.E.A. Gratton, P.A. Ropp, P.D. Pohlhaus, J.C. Luft, V.J. Madden, M.E. Napier, J.M. DeSimone, The 652 effect of particle design on cellular internalization pathways, Proceedings of the National Academy of 653 Sciences, 105 (2008) 11613. 654 [42] J. Szebeni, P. Bedőcs, Z. Rozsnyay, Z. Weiszhár, R. Urbanics, L. Rosivall, R. Cohen, O. Garbuzenko, 655 G. Báthori, M. Tóth, R. Bünger, Y. Barenholz, Liposome-induced complement activation and related

656 cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome, 657 Nanomedicine: Nanotechnology, Biology and Medicine, 8 (2012) 176-184. 658 [43] K. Maruyama, O. Ishida, T. Takizawa, K. Moribe, Possibility of active targeting to tumor tissues 659 with liposomes, Advanced Drug Delivery Reviews, 40 (1999) 89-102. 660 [44] J.O. Eloy, R. Petrilli, L.N.F. Trevizan, M. Chorilli, Immunoliposomes: A review on functionalization 661 strategies and targets for drug delivery, Colloids and Surfaces B: Biointerfaces, 159 (2017) 454-467. 662 [45] C. Mamot, D.C. Drummond, U. Greiser, K. Hong, D.B. Kirpotin, J.D. Marks, J.W. Park, Epidermal 663 Growth Factor Receptor (EGFR)-targeted Immunoliposomes Mediate Specific and Efficient Drug 664 Delivery to EGFR- and EGFRvIII-overexpressing Tumor Cells, Cancer Research, 63 (2003) 3154. 665 [46] C. Mamot, R. Ritschard, A. Wicki, G. Stehle, T. Dieterle, L. Bubendorf, C. Hilker, S. Deuster, R. 666 Herrmann, C. Rochlitz, Tolerability, safety, pharmacokinetics, and efficacy of doxorubicin-loaded anti- 667 EGFR immunoliposomes in advanced solid tumours: a phase 1 dose-escalation study, The Lancet 668 Oncology, 13 (2012) 1234-1241. 669 [47] D. Needham, J.-Y. Park, A.M. Wright, J. Tong, Materials characterization of the low temperature 670 sensitive liposome (LTSL): effects of the lipid composition (lysolipid and DSPE-PEG2000) on the thermal 671 transition and release of doxorubicin, Faraday Discussions, 161 (2013) 515-534. 672 [48] R. Lencioni, D. Cioni, RFA plus lyso-thermosensitive liposomal doxorubicin: in search of the 673 optimal approach to cure intermediate-size hepatocellular carcinoma, Hepatic Oncology, 3 (2016) 674 193-200. 675 [49] R.T. Poon, N. Borys, Lyso-thermosensitive liposomal doxorubicin: an adjuvant to increase the cure 676 rate of radiofrequency ablation in liver cancer, Future Oncology, 7 (2011) 937-945. 677 [50] A.R. Mohammed, N. Weston, A.G.A. Coombes, M. Fitzgerald, Y. Perrie, Liposome formulation of 678 poorly water soluble drugs: optimisation of drug loading and ESEM analysis of stability, International 679 Journal of Pharmaceutics, 285 (2004) 23-34. 680 [51] M.H. Ali, B. Moghaddam, D.J. Kirby, A.R. Mohammed, Y. Perrie, The role of lipid geometry in 681 designing liposomes for the solubilisation of poorly water soluble drugs, International Journal of 682 Pharmaceutics, 453 (2013) 225-232. 683 [52] T. Yang, F.D. Cui, M.K. Choi, H. Lin, S.J. Chung, C.K. Shim, D.D. Kim, Liposome formulation of 684 paclitaxel with enhanced solubility and stability, Drug Deliv, 14 (2007) 301-308. 685 [53] C. Tremblay, M. Barza, F. Szoka, M. Lahav, J. Baum, Reduced toxicity of liposome-associated 686 amphotericin B injected intravitreally in rabbits, Invest Ophthalmol Vis Sci, 26 (1985) 711-718. 687 [54] R.T. Mehta, Liposome encapsulation of clofazimine reduces toxicity in vitro and in vivo and 688 improves therapeutic efficacy in the beige mouse model of disseminated Mycobacterium avium-M. 689 intracellulare complex infection, Antimicrobial agents and chemotherapy, 40 (1996) 1893-1902. 690 [55] A. Gabizon, D. Tzemach, L. Mak, M. Bronstein, A.T. Horowitz, Dose dependency of 691 pharmacokinetics and therapeutic efficacy of pegylated liposomal doxorubicin (DOXIL) in murine 692 models, J Drug Target, 10 (2002) 539-548. 693 [56] D. Papahadjopoulos, T.M. Allen, A. Gabizon, E. Mayhew, K. Matthay, S.K. Huang, K.D. Lee, M.C. 694 Woodle, D.D. Lasic, C. Redemann, Sterically stabilized liposomes: improvements in pharmacokinetics 695 and antitumor therapeutic efficacy, Proceedings of the National Academy of Sciences, 88 (1991) 696 11460-11464. 697 [57] M.S. Newman, G.T. Colbern, P.K. Working, C. Engbers, M.A. Amantea, Comparative 698 pharmacokinetics, tissue distribution, and therapeutic effectiveness of cisplatin encapsulated in long- 699 circulating, pegylated liposomes (SPI-077) in tumor-bearing mice, Cancer Chemotherapy and 700 Pharmacology, 43 (1999) 1-7. 701 [58] K.F.W. M.J. Hope, Liposomal formulation of ciprofloxacin, in: P.N. Shek (Ed.) Liposomes in 702 Biomedical Applications, Harwood Academic Publishers, New York, 1995, pp. 121-134. 703 [59] D. Cipolla, H. Wu, I. Gonda, H.-K. Chan, Aerosol Performance and Long-Term Stability of 704 Surfactant-Associated Liposomal Ciprofloxacin Formulations with Modified Encapsulation and Release 705 Properties, AAPS PharmSciTech, 15 (2014) 1218-1227.

706 [60] J. Gubernator, Active methods of drug loading into liposomes: recent strategies for stable drug 707 entrapment and increased in vivo activity, Expert Opin Drug Deliv, 8 (2011) 565-580. 708 [61] D. Zucker, D. Marcus, Y. Barenholz, A. Goldblum, Liposome drugs' loading efficiency: A working 709 model based on loading conditions and drug's physicochemical properties, Journal of Controlled 710 Release, 139 (2009) 73-80. 711 [62] G. Haran, R. Cohen, L.K. Bar, Y. Barenholz, Transmembrane ammonium sulfate gradients in 712 liposomes produce efficient and stable entrapment of amphipathic weak bases, Biochim Biophys Acta, 713 1151 (1993) 201-215. 714 [63] A. Fritze, F. Hens, A. Kimpfler, R. Schubert, R. Peschka-Süss, Remote loading of doxorubicin into 715 liposomes driven by a transmembrane phosphate gradient, Biochimica et Biophysica Acta (BBA) - 716 Biomembranes, 1758 (2006) 1633-1640. 717 [64] J. Gubernator, G. Chwastek, M. Korycińska, M. Stasiuk, G. Grynkiewicz, F. Lewrick, R. Süss, A. 718 Kozubek, The encapsulation of idarubicin within liposomes using the novel EDTA ion gradient method 719 ensures improved drug retention in vitro and in vivo, Journal of Controlled Release, 146 (2010) 68-75. 720 [65] Y. Yang, Y. Ma, S. Wang, A novel method to load topotecan into liposomes driven by a 721 transmembrane NH4EDTA gradient, European Journal of Pharmaceutics and Biopharmaceutics, 80 722 (2012) 332-339. 723 [66] Y. Song, Z. Huang, Y. Song, Q. Tian, X. Liu, Z. She, J. Jiao, E. Lu, Y. Deng, The application of EDTA in 724 drug delivery systems: doxorubicin liposomes loaded via NH(4)EDTA gradient, International Journal of 725 Nanomedicine, 9 (2014) 3611-3621. 726 [67] S. Clerc, Y. Barenholz, Loading of amphipathic weak acids into liposomes in response to 727 transmembrane calcium acetate gradients, Biochimica et Biophysica Acta (BBA) - Biomembranes, 1240 728 (1995) 257-265. 729 [68] D.B. Fenske, K.F. Wong, E. Maurer, N. Maurer, J.M. Leenhouts, N. Boman, L. Amankwa, P.R. Cullis, 730 Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting 731 transmembrane ion gradients, Biochimica et Biophysica Acta (BBA) - Biomembranes, 1414 (1998) 188- 732 204. 733 [69] B.C.L. Cheung, T.H.T. Sun, J.M. Leenhouts, P.R. Cullis, Loading of doxorubicin into liposomes by 734 forming Mn2+-drug complexes, Biochimica et Biophysica Acta (BBA) - Biomembranes, 1414 (1998) 735 205-216. 736 [70] S.A. Abraham, K. Edwards, G. Karlsson, N. Hudon, L.D. Mayer, M.B. Bally, An evaluation of 737 transmembrane ion gradient-mediated encapsulation of topotecan within liposomes, Journal of 738 Controlled Release, 96 (2004) 449-461. 739 [71] A.S. Taggar, J. Alnajim, M. Anantha, A. Thomas, M. Webb, E. Ramsay, M.B. Bally, Copper- 740 topotecan complexation mediates drug accumulation into liposomes, Journal of controlled release : 741 official journal of the Controlled Release Society, 114 (2006) 78-88. 742 [72] E. Ramsay, J. Alnajim, M. Anantha, J. Zastre, H. Yan, M. Webb, D. Waterhouse, M. Bally, A novel 743 liposomal irinotecan formulation with significant anti-tumour activity: Use of the divalent cation 744 ionophore A23187 and copper-containing liposomes to improve drug retention, European Journal of 745 Pharmaceutics and Biopharmaceutics, 68 (2008) 607-617. 746 [73] C. Li, J. Cui, Y. Li, C. Wang, Y. Li, L. Zhang, L. Zhang, W. Guo, J. Wang, H. Zhang, Y. Hao, Y. Wang, 747 Copper ion-mediated liposomal encapsulation of mitoxantrone: The role of anions in drug loading, 748 retention and release, European Journal of Pharmaceutical Sciences, 34 (2008) 333-344. 749 [74] J. Cui, C. Li, L. Wang, C. Wang, H. Yang, Y. Li, L. Zhang, L. Zhang, W. Guo, M. Liang, Ni2+-mediated 750 mitoxantrone encapsulation: Improved efficacy of fast release formulation, International Journal of 751 Pharmaceutics, 368 (2009) 24-30. 752 [75] R.R. Hood, W.N. Vreeland, D.L. DeVoe, Microfluidic remote loading for rapid single-step liposomal 753 drug preparation, Lab on a Chip, 14 (2014) 3359-3367. 754 [76] J.D. Castile, K.M.G. Taylor, Factors affecting the size distribution of liposomes produced by freeze– 755 thaw extrusion, International Journal of Pharmaceutics, 188 (1999) 87-95.

756 [77] M.J. Hope, M.B. Bally, G. Webb, P.R. Cullis, Production of large unilamellar vesicles by a rapid 757 extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a 758 membrane potential, Biochimica et Biophysica Acta (BBA) - Biomembranes, 812 (1985) 55-65. 759 [78] L.D. Mayer, M.J. Hope, P.R. Cullis, A.S. Janoff, Solute distributions and trapping efficiencies 760 observed in freeze-thawed multilamellar vesicles, Biochimica et Biophysica Acta (BBA) - 761 Biomembranes, 817 (1985) 193-196. 762 [79] L.D. Mayer, M.J. Hope, P.R. Cullis, Vesicles of variable sizes produced by a rapid extrusion 763 procedure, Biochimica et Biophysica Acta (BBA) - Biomembranes, 858 (1986) 161-168. 764 [80] Y. Ozer, H. Talsma, D.J.A. Crommelin, A.A. Hincal, Influence of freezing and freeze-drying on the 765 stability of liposomes dispersed in aqueous media, Acta Pharmaceutica Technologica, 34 (1988) 129- 766 139. 767 [81] R.A. Callow, J.J. McGrath, Thermodynamic modeling and cryomicroscopy of cell-size, unilamellar, 768 and paucilamellar liposomes, Cryobiology, 22 (1985) 251-267. 769 [82] D. Cipolla, H. Wu, S. Salentinig, B. Boyd, T. Rades, D. Vanhecke, A. Petri-Fink, B. Rothin-Rutishauser, 770 S. Eastman, T. Redelmeier, I. Gonda, H.K. Chan, Formation of drug nanocrystals under 771 nanoconfinement afforded by liposomes, RSC Advances, 6 (2016) 6223-6233. 772 [83] L.D. Mayer, M.B. Bally, M.J. Hope, P.R. Cullis, Techniques for encapsulating bioactive agents into 773 liposomes, Chem Phys Lipids, 40 (1986) 333-345. 774 [84] P.R. Cullis, L.D. Mayer, M.B. Bally, T.D. Madden, M.J. Hope, Generating and loading of liposomal 775 systems for drug-delivery applications, Advanced Drug Delivery Reviews, 3 (1989) 267-282. 776 [85] D.D. Lasic, B. Čeh, M.C.A. Stuart, L. Guo, P.M. Frederik, Y. Barenholz, Transmembrane gradient 777 driven phase transitions within vesicles: lessons for drug delivery, Biochimica et Biophysica Acta (BBA) 778 - Biomembranes, 1239 (1995) 145-156. 779 [86] Y. Barenholz, Amphipathic Weak Base Loading into Preformed Liposomes Having a 780 Transmembrane Ammonium Ion Gradient: From the Bench to Approved Doxil, 2006. 781 [87] J. Lepault, F. Pattus, N. Martin, Cryo-electron microscopy of artificial biological membranes, 782 Biochimica et Biophysica Acta (BBA) - Biomembranes, 820 (1985) 315-318. 783 [88] R.A. Grassucci, D.J. Taylor, J. Frank, Preparation of macromolecular complexes for cryo-electron 784 microscopy, Nature protocols, 2 (2007) 3239-3246. 785 [89] M. Almgren, K. Edwards, G. Karlsson, Cryo transmission electron microscopy of liposomes and 786 related structures, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 174 (2000) 3- 787 21. 788 [90] P.M. Frederik, D.H.W. Hubert, Cryoelectron Microscopy of Liposomes, in: Methods in Enzymology, 789 Academic Press, 2005, pp. 431-448. 790 [91] M. Placzek, M. Kosela, Microscopic methods in analysis of submicron phospholipid dispersions, 791 Acta pharmaceutica (Zagreb, Croatia), 66 (2016) 1-22. 792 [92] J. Lengyel, J.L.S. Milne, S. Subramaniam, Electron tomography in nanoparticle imaging and 793 analysis, Nanomedicine (London, England), 3 (2008) 125-131. 794 [93] G. Pabst, M. Rappolt, H. Amenitsch, P. Laggner, Structural information from multilamellar 795 liposomes at full hydration: full q-range fitting with high quality x-ray data, Physical review. E, 796 Statistical physics, plasmas, fluids, and related interdisciplinary topics, 62 (2000) 4000-4009. 797 [94] M. Rappolt, Bilayer thickness estimations with “poor” diffraction data, Journal of Applied Physics, 798 107 (2010) 084701. 799 [95] P.P. Wibroe, D. Ahmadvand, M.A. Oghabian, A. Yaghmur, S.M. Moghimi, An integrated 800 assessment of morphology, size, and complement activation of the PEGylated liposomal doxorubicin 801 products Doxil®, Caelyx®, DOXOrubicin, and SinaDoxosome, Journal of Controlled Release, 221 (2016) 802 1-8. 803 [96] M.H. Chiu, E.J. Prenner, Differential scanning calorimetry: An invaluable tool for a detailed 804 thermodynamic characterization of macromolecules and their interactions, Journal of Pharmacy and 805 Bioallied Sciences, 3 (2011) 39-59.

806 [97] H. Bunjes, T. Unruh, Characterization of lipid nanoparticles by differential scanning calorimetry, 807 X-ray and neutron scattering, Advanced Drug Delivery Reviews, 59 (2007) 379-402. 808 [98] M.M. Chogale, V.N. Ghodake, V.B. Patravale, Performance Parameters and Characterizations of 809 Nanocrystals: A Brief Review, Pharmaceutics, 8 (2016) 26. 810 [99] R.L. Biltonen, D. Lichtenberg, The use of differential scanning calorimetry as a tool to characterize 811 liposome preparations, Chem Phys Lipids, 64 (1993) 129-142. 812 [100] C. Demetzos, Differential Scanning Calorimetry (DSC): A Tool to Study the Thermal Behavior of 813 Lipid Bilayers and Liposomal Stability, Journal of Liposome Research, 18 (2008) 159-173. 814 [101] L. Yu, Amorphous pharmaceutical solids: preparation, characterization and stabilization, 815 Advanced Drug Delivery Reviews, 48 (2001) 27-42. 816 [102] D. Giron, Thermal analysis and calorimetric methods in the characterisation of polymorphs and 817 solvates, Thermochimica Acta, 248 (1995) 1-59. 818 [103] X. Wei, D. Shamrakov, S. Nudelman, S. Peretz-Damari, E. Nativ-Roth, O. Regev, Y. Barenholz, 819 Cardinal Role of Intraliposome Doxorubicin-Sulfate Nanorod Crystal in Doxil Properties and 820 Performance, ACS Omega, 3 (2018) 2508-2517. 821 [104] T. Peleg-Shulman, D. Gibson, R. Cohen, R. Abra, Y. Barenholz, Characterization of sterically 822 stabilized cisplatin liposomes by nuclear magnetic resonance, Biochimica et Biophysica Acta (BBA) - 823 Biomembranes, 1510 (2001) 278-291. 824 [105] N. Maurer, K.F. Wong, M.J. Hope, P.R. Cullis, Anomalous solubility behavior of the antibiotic 825 ciprofloxacin encapsulated in liposomes: a 1H-NMR study, Biochimica et Biophysica Acta (BBA) - 826 Biomembranes, 1374 (1998) 9-20. 827 [106] I. Arčon, A. Kodre, R.M. Abra, A. Huang, J.J. Vallner, D.D. Lasič, EXAFS study of liposome- 828 encapsulated cisplatin, Colloids and Surfaces B: Biointerfaces, 33 (2004) 199-204. 829 [107] S.A. Abraham, K. Edwards, G. Karlsson, S. MacIntosh, L.D. Mayer, C. McKenzie, M.B. Bally, 830 Formation of transition metal–doxorubicin complexes inside liposomes, Biochimica et Biophysica Acta 831 (BBA) - Biomembranes, 1565 (2002) 41-54. 832 [108] A. Dicko, S. Kwak, A.A. Frazier, L.D. Mayer, B.D. Liboiron, Biophysical characterization of a 833 liposomal formulation of cytarabine and daunorubicin, International Journal of Pharmaceutics, 391 834 (2010) 248-259. 835 [109] D. Zucker, A.V. Andriyanov, A. Steiner, U. Raviv, Y. Barenholz, Characterization of PEGylated 836 nanoliposomes co-remotely loaded with topotecan and vincristine: relating structure and 837 pharmacokinetics to therapeutic efficacy, Journal of Controlled Release, 160 (2012) 281-289. 838 [110] R.M. Straubinger, S.V. Balasubramanian, Preparation and Characterization of Taxane-Containing 839 Liposomes, in: Methods in Enzymology, Academic Press, 2005, pp. 97-117. 840 [111] G. Siligardi, R. Hussain, S.G. Patching, M.K. Phillips-Jones, Ligand- and drug-binding studies of 841 membrane proteins revealed through circular dichroism spectroscopy, Biochimica et Biophysica Acta 842 (BBA) - Biomembranes, 1838 (2014) 34-42. 843 [112] O. Rezhdo, L. Speciner, R.L. Carrier, Lipid-associated Oral Delivery: Mechanisms and Analysis of 844 Oral Absorption Enhancement, Journal of controlled release : official journal of the Controlled Release 845 Society, 240 (2016) 544-560. 846 [113] J. Yano, V.K. Yachandra, X-ray absorption spectroscopy, Photosynthesis Research, 102 (2009) 847 241-254. 848 [114] D.D. Lasic, P.M. Frederik, M.C.A. Stuart, Y. Barenholz, T.J. McIntosh, Gelation of liposome interior 849 A novel method for drug encapsulation, FEBS Letters, 312 (1992) 255-258. 850 [115] AusPAR attachement 1: Product information for Irinotecan (as sucrosofate), in: D.o. Health (Ed.), 851 Therapeutic Goods Administration, 2017. 852 [116] D.C. Drummond, C.O. Noble, Z. Guo, K. Hong, J.W. Park, D.B. Kirpotin, Development of a Highly 853 Active Nanoliposomal Irinotecan Using a Novel Intraliposomal Stabilization Strategy, Cancer Research, 854 66 (2006) 3271.

855 [117] P. Tardi, S. Johnstone, N. Harasym, S. Xie, T. Harasym, N. Zisman, P. Harvie, D. Bermudes, L. 856 Mayer, In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances 857 therapeutic efficacy, Leukemia Research, 33 (2009) 129-139. 858 [118] C.D. Landon, J.-Y. Park, D. Needham, M.W. Dewhirst, Nanoscale Drug Delivery and Hyperthermia: 859 The Materials Design and Preclinical and Clinical Testing of Low Temperature-Sensitive Liposomes 860 Used in Combination with Mild Hyperthermia in the Treatment of Local Cancer, The open 861 nanomedicine journal, 3 (2011) 38-64. 862 [119] G.N.C. Chiu, S.A. Abraham, L.M. Ickenstein, R. Ng, G. Karlsson, K. Edwards, E.K. Wasan, M.B. Bally, 863 Encapsulation of doxorubicin into thermosensitive liposomes via complexation with the transition 864 metal manganese, Journal of Controlled Release, 104 (2005) 271-288. 865 [120] A.V. Andriyanov, E. Koren, Y. Barenholz, S.N. Goldberg, Therapeutic Efficacy of Combining 866 PEGylated Liposomal Doxorubicin and Radiofrequency (RF) Ablation: Comparison between Slow-Drug- 867 Releasing, Non-Thermosensitive and Fast-Drug-Releasing, Thermosensitive Nano-Liposomes, PLOS 868 ONE, 9 (2014) e92555. 869 [121] D. Cipolla, J. Blanchard, I. Gonda, Development of Liposomal Ciprofloxacin to Treat Lung 870 Infections, Pharmaceutics, 8 (2016) 6. 871 [122] D. Cipolla, H. Wu, S. Eastman, T. Redelmeier, I. Gonda, H.-K. Chan, Tuning Ciprofloxacin Release 872 Profiles from Liposomally Encapsulated Nanocrystalline Drug, Pharmaceutical Research, 33 (2016) 873 2748-2762. 874 [123] P.J. Gaillard, C.C.M. Appeldoorn, R. Dorland, J. van Kregten, F. Manca, D.J. Vugts, B. Windhorst, 875 G.A.M.S. van Dongen, H.E. de Vries, D. Maussang, O. van Tellingen, Pharmacokinetics, Brain Delivery, 876 and Efficacy in Brain Tumor-Bearing Mice of Glutathione Pegylated Liposomal Doxorubicin (2B3-101), 877 PLoS ONE, 9 (2014) e82331. 878 [124] J. Rip, L. Chen, R. Hartman, A. van den Heuvel, A. Reijerkerk, J. van Kregten, B. van der Boom, C. 879 Appeldoorn, M. de Boer, D. Maussang, E.C.M. de Lange, P.J. Gaillard, Glutathione PEGylated liposomes: 880 pharmacokinetics and delivery of cargo across the blood–brain barrier in rats, Journal of Drug 881 Targeting, 22 (2014) 460-467. 882 [125] S.C. Semple, R. Leone, J. Wang, E.C. Leng, S.K. Klimuk, M.L. Eisenhardt, Z.-N. Yuan, K. Edwards, 883 N. Maurer, M.J. Hope, P.R. Cullis, Q.-F. Ahkong, Optimization and characterization of a 884 sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity, 885 Journal of Pharmaceutical Sciences, 94 (2005) 1024-1038. 886 [126] I.V. Zhigaltsev, N. Maurer, Q.-F. Akhong, R. Leone, E. Leng, J. Wang, S.C. Semple, P.R. Cullis, 887 Liposome-encapsulated vincristine, vinblastine and vinorelbine: A comparative study of drug loading 888 and retention, Journal of Controlled Release, 104 (2005) 103-111. 889 [127] M.J.W. Johnston, S.C. Semple, S.K. Klimuk, K. Edwards, M.L. Eisenhardt, E.C. Leng, G. Karlsson, 890 D. Yanko, P.R. Cullis, Therapeutically optimized rates of drug release can be achieved by varying the 891 drug-to-lipid ratio in liposomal vincristine formulations, Biochimica et Biophysica Acta (BBA) - 892 Biomembranes, 1758 (2006) 55-64. 893 [128] M.J. Johnston, K. Edwards, G. Karlsson, P.R. Cullis, Influence of drug-to-lipid ratio on drug release 894 properties and liposome integrity in liposomal doxorubicin formulations, J Liposome Res, 18 (2008) 895 145-157. 896 [129] L. Chernov, R.J. Deyell, M. Anantha, N. Dos Santos, R. Gilabert‐Oriol, M.B. Bally, Optimization 897 of liposomal topotecan for use in treating neuroblastoma, Cancer Medicine, 6 (2017) 1240-1254. 898 [130] J. Gubernator, D. Lipka, M. Korycińska, K. Kempińska, M. Milczarek, J. Wietrzyk, R. Hrynyk, S. 899 Barnert, R. Süss, A. Kozubek, Efficient Human Breast Cancer Xenograft Regression after a Single 900 Treatment with a Novel Liposomal Formulation of Epirubicin Prepared Using the EDTA Ion Gradient 901 Method, PLOS ONE, 9 (2014) e91487. 902 [131] I.V. Zhigaltsev, N. Maurer, K. Edwards, G. Karlsson, P.R. Cullis, Formation of drug–arylsulfonate 903 complexes inside liposomes: A novel approach to improve drug retention, Journal of Controlled 904 Release, 110 (2006) 378-386.

905 [132] L.R.C. Pedrosa, T.L.M. ten Hagen, R. Süss, A. van Hell, A.M.M. Eggermont, M. Verheij, G.A. Koning, 906 Short-Chain Glycoceramides Promote Intracellular Mitoxantrone Delivery from Novel Nanoliposomes 907 into Breast Cancer Cells, Pharmaceutical Research, 32 (2015) 1354-1367. 908 [133] L.D. Mayer, M.B. Bally, H. Loughrey, D. Masin, P.R. Cullis, Liposomal Vincristine Preparations 909 Which Exhibit Decreased Drug Toxicity and Increased Activity against Murine L1210 and P388 Tumors, 910 Cancer Research, 50 (1990) 575. 911 [134] R.P. Hertzberg, M.J. Caranfa, S.M. Hecht, On the mechanism of topoisomerase I inhibition by 912 camptothecin: evidence for binding to an enzyme-DNA complex, Biochemistry, 28 (1989) 4629-4638. 913 [135] J. Fassberg, V.J. Stella, A kinetic and Mechanistic Study of the Hydrolysis of Camptothecin and 914 Some Analogues, Journal of Pharmaceutical Sciences, 81 (1992) 676-684. 915 [136] T.G. Burke, X. Gao, Stabilization of Topotecan in Low pH Liposomes Composed of 916 Distearoylphosphatidylcholine, Journal of Pharmaceutical Sciences, 83 (1994) 967-969. 917 [137] P. Tardi, E. Choice, D. Masin, T. Redelmeier, M. Bally, T.D. Madden, Liposomal Encapsulation of 918 Topotecan Enhances Anticancer Efficacy in Murine and Human Xenograft Models, Cancer Research, 919 60 (2000) 3389. 920 [138] D. Zucker, Y. Barenholz, Optimization of vincristine–topotecan combination — Paving the way 921 for improved chemotherapy regimens by nanoliposomes, Journal of Controlled Release, 146 (2010) 922 326-333. 923 [139] T.-X. Xiang, B.D. Anderson, Influence of Chain Ordering on the Selectivity of 924 Dipalmitoylphosphatidylcholine Bilayer Membranes for Permeant Size and Shape, Biophysical journal, 925 75 (1998) 2658-2671. 926 [140] S. Mitragotri, M.E. Johnson, D. Blankschtein, R. Langer, An Analysis of the Size Selectivity of 927 Solute Partitioning, Diffusion, and Permeation across Lipid Bilayers, Biophysical journal, 77 (1999) 928 1268-1283. 929 [141] J. Shen, D.J. Burgess, In Vitro Dissolution Testing Strategies for Nanoparticulate Drug Delivery 930 Systems: Recent Developments and Challenges, Drug delivery and translational research, 3 (2013) 931 409-415. 932 [142] U. Bhardwaj, D.J. Burgess, A novel USP apparatus 4 based release testing method for dispersed 933 systems, International Journal of Pharmaceutics, 388 (2010) 287-294. 934 [143] K.D. Fugit, T.-X. Xiang, D.H. Choi, S. Kangarlou, E. Csuhai, P.M. Bummer, B.D. Anderson, 935 Mechanistic model and analysis of doxorubicin release from liposomal formulations, Journal of 936 Controlled Release, 217 (2015) 82-91. 937 [144] G. Moreno-Bautista, K.C. Tam, Evaluation of dialysis membrane process for quantifying the in 938 vitro drug-release from colloidal drug carriers, Colloids and Surfaces A: Physicochemical and 939 Engineering Aspects, 389 (2011) 299-303. 940 [145] C. Xiao, X. Qi, Y. Maitani, T. Nagai, Sustained release of cisplatin from multivesicular liposomes: 941 Potentiation of antitumor efficacy against S180 murine carcinoma, Journal of Pharmaceutical Sciences, 942 93 (2004) 1718-1724. 943 [146] D. Cipolla, H. Wu, S. Eastman, T. Redelmeier, I. Gonda, H.K. Chan, Development and 944 Characterization of an In Vitro Release Assay for Liposomal Ciprofloxacin for Inhalation, Journal of 945 Pharmaceutical Sciences, 103 (2014) 314-327. 946 [147] S. D'Souza, A Review of In Vitro Drug Release Test Methods for Nano-Sized Dosage Forms, 947 Advances in Pharmaceutics, 2014 (2014) 12. 948 [148] S.J. Wallace, J. Li, R.L. Nation, B.J. Boyd, Drug release from nanomedicines: selection of 949 appropriate encapsulation and release methodology, Drug Delivery and Translational Research, 2 950 (2012) 284-292. 951 [149] W. Yuan, R. Kuai, Z. Dai, Y. Yuan, N. Zheng, W. Jiang, C. Noble, M. Hayes, F.C. Szoka, A. 952 Schwendeman, Development of a Flow-Through USP-4 Apparatus Drug Release Assay to Evaluate 953 Doxorubicin Liposomes, The AAPS Journal, 19 (2017) 150-160.

954 [150] E. Csuhai, S. Kangarlou, T.-X. Xiang, A. Ponta, P. Bummer, D. Choi, B.D. Anderson, Determination 955 of Key Parameters for a Mechanism-Based Model to Predict Doxorubicin Release from Actively Loaded 956 Liposomes, Journal of Pharmaceutical Sciences, 104 (2015) 1087-1098. 957 [151] K.D. Fugit, B.D. Anderson, The role of pH and ring-opening hydrolysis kinetics on liposomal 958 release of topotecan, Journal of Controlled Release, 174 (2014) 88-97. 959 [152] K.D. Fugit, A. Jyoti, M. Upreti, B.D. Anderson, Insights into accelerated liposomal release of 960 topotecan in plasma monitored by a non-invasive fluorescence spectroscopic method, Journal of 961 Controlled Release, 197 (2015) 10-19. 962 [153] L.M. Russell, M. Hultz, P.C. Searson, Leakage kinetics of the liposomal chemotherapeutic agent 963 Doxil: The role of dissolution, protonation, and passive transport, and implications for mechanism of 964 action, Journal of Controlled Release, 269 (2018) 171-176. 965 [154] J.-U.A.H. Junghanns, R.H. Müller, Nanocrystal technology, drug delivery and clinical applications, 966 International Journal of Nanomedicine, 3 (2008) 295-310. 967 [155] A.A. Noyes, W.R. Whitney, THE RATE OF SOLUTION OF SOLID SUBSTANCES IN THEIR OWN 968 SOLUTIONS, Journal of the American Chemical Society, 19 (1897) 930-934. 969 [156] C.J. Porter, N.L. Trevaskis, W.N. Charman, Lipids and lipid-based formulations: optimizing the 970 oral delivery of lipophilic drugs, Nature reviews. Drug discovery, 6 (2007) 231-248. 971 [157] R. Schubert, K. Beyer, H. Wolburg, K.H. Schmidt, Structural changes in membranes of large 972 unilamellar vesicles after binding of sodium cholate, Biochemistry, 25 (1986) 5263-5269. 973 [158] A. Hildebrand, K. Beyer, R. Neubert, P. Garidel, A. Blume, Solubilization of negatively charged 974 DPPC/DPPG liposomes by bile salts, Journal of Colloid and Interface Science, 279 (2004) 559-571. 975 [159] M. Anderson, A. Omri, The Effect of Different Lipid Components on the In Vitro Stability and 976 Release Kinetics of Liposome Formulations, Drug Delivery, 11 (2004) 33-39. 977 [160] F. Nacka, M. Cansell, B. Entressangles, In vitro behavior of marine lipid-based liposomes. 978 Influence of pH, temperature, bile salts, and phospholipase A2, Lipids, 36 (2001) 35-42. 979 [161] J. Khan, T. Rades, B.J. Boyd, Lipid-Based Formulations Can Enable the Model Poorly Water- 980 Soluble Weakly Basic Drug Cinnarizine To Precipitate in an Amorphous-Salt Form During In Vitro 981 Digestion, Molecular Pharmaceutics, 13 (2016) 3783-3793. 982 [162] K.L. Hennenfent, R. Govindan, Novel formulations of taxanes: a review. Old wine in a new bottle?, 983 Annals of Oncology, 17 (2006) 735-749. 984 [163] K. Egbaria, N. Weiner, Liposomes as a topical drug delivery system, Advanced Drug Delivery 985 Reviews, 5 (1990) 287-300. 986 [164] H.A.E. Benson, Transfersomes for transdermal drug delivery, Expert Opinion on Drug Delivery, 3 987 (2006) 727-737. 988 [165] G. Cevc, Transfersomes, liposomes and other lipid suspensions on the skin: permeation 989 enhancement, vesicle penetration, and transdermal drug delivery, Critical reviews in therapeutic drug 990 carrier systems, 13 (1996) 257-388. 991 [166] M.J. Choi, H.I. Maibach, Liposomes and niosomes as topical drug delivery systems, Skin 992 pharmacology and physiology, 18 (2005) 209-219. 993 [167] G.M. El Maghraby, B.W. Barry, A.C. Williams, Liposomes and skin: From drug delivery to model 994 membranes, European Journal of Pharmaceutical Sciences, 34 (2008) 203-222. 995 [168] M.M.A. Elsayed, O.Y. Abdallah, V.F. Naggar, N.M. Khalafallah, Lipid vesicles for skin delivery of 996 drugs: Reviewing three decades of research, International Journal of Pharmaceutics, 332 (2007) 1-16. 997 [169] M. Kirjavainen, J. Monkkonen, M. Saukkosaari, R. Valjakka-Koskela, J. Kiesvaara, A. Urtti, 998 Phospholipids affect stratum corneum lipid bilayer fluidity and drug partitioning into the bilayers, 999 Journal of controlled release : official journal of the Controlled Release Society, 58 (1999) 207-214. 1000 [170] M. Kirjavainen, A. Urtti, I. Jääskeläinen, T. Marjukka Suhonen, P. Paronen, R. Valjakka-Koskela, J. 1001 Kiesvaara, J. Mönkkönen, Interaction of liposomes with human skin in vitro — The influence of lipid 1002 composition and structure, Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1304 1003 (1996) 179-189.

1004 [171] V. Patel, O.P. Sharma, T. Mehta, Nanocrystal: a novel approach to overcome skin barriers for 1005 improved topical drug delivery, Expert Opin Drug Deliv, 15 (2018) 351-368. 1006 [172] J. Hecq, M. Deleers, D. Fanara, H. Vranckx, K. Amighi, Preparation and characterization of 1007 nanocrystals for solubility and dissolution rate enhancement of nifedipine, International Journal of 1008 Pharmaceutics, 299 (2005) 167-177. 1009 [173] B. Van Eerdenbrugh, J. Vermant, J.A. Martens, L. Froyen, J.V. Humbeeck, G. Van den Mooter, P. 1010 Augustijns, Solubility Increases Associated with Crystalline Drug Nanoparticles: Methodologies and 1011 Significance, Molecular Pharmaceutics, 7 (2010) 1858-1870. 1012 [174] K.C. Bhol, P.J. Schechter, Topical nanocrystalline silver cream suppresses inflammatory cytokines 1013 and induces apoptosis of inflammatory cells in a murine model of allergic contact dermatitis, The 1014 British journal of dermatology, 152 (2005) 1235-1242. 1015 [175] K. Mitri, R. Shegokar, S. Gohla, C. Anselmi, R.H. Muller, Lutein nanocrystals as antioxidant 1016 formulation for oral and dermal delivery, Int J Pharm, 420 (2011) 141-146.

1017