Arsonoliposomes for drug delivery

J. DRUG DEL. SCI. TECH., 17 (6) 377-388 2007

Arsonoliposomes for drug delivery S.G. Antimisiaris Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, Rio 26500, Patras, Greece *Correspondence: [email protected] Arsenic-containing liposomes or arsonoliposomes were recently prepared and characterized using novel arsenic-containing lipids, arsonolipids. Preliminary and follow up in vitro, and in vivo studies have shown that some of the arsonoliposome types studied have interesting anticancer and antiprotozoal activity. From a mechanistic point of view their activity (especially their anticancer activity) may be linked to the reduction of arsenic to its more toxic form (from As(V) to As(III)) which may be enhanced in areas where increased concentrations of thiol-containing compounds, such as glutathione, prevail, as is the case for some types of cancer cells. The physicochemical characteristics of arsonoliposomes; mean diameter, surface charge, morphology and membrane integrity are influenced by their composition. Indeed the specific arsonolipids and/or phospholipids used for their preparation, as well as the addition of cholesterol in their membrane and the relative amounts of each lipid type have been demonstrated to influence all the above mentioned vesicle characteristics. Accordingly, the antiprotozoal and in vivo distribution and kinetics were affected by arsonoliposome composition, while this is currently under investigation for anticancer activity. To conclude, the appropriate arsonoliposome type (in terms of lipid composition) should be selected when considering arsonoliposomes as nanocarriers for the delivery of drugs. Key words: Arsenic – Arsenolipid – Arsonolipid – Liposomes – Arsonoliposomes – Anti-cancer – Anti-parasitic – Drug delivery – Biodistribution – Pharmacokinetics.

From a pharmacological point of view, the potential of liposomal drug delivery systems to alter the pharmacokinetics and in vivo distribution of drugs is connected to increased drug activity (since more drug molecules can be directed to specific target cells where receptors that are connected with the allured drug action, reside) and decreased drug toxicity (since the concentration of drug at sites implicated with initiation of toxicity and adverse reactions could be minimized) [1-4]. The latter possibility (decreased toxicity), is of key importance for the delivery of toxic drugs (the toxicity of which restricts their broader use). Such drugs exist, mainly among anticancer agents, and several successful examples of drugs that have been improved in this aspect by liposomal formulations, as are the cases of doxorubicin [5, 6] and amphotericin-B [5-7], have been marketed. In line with this, we have been exploring the therapeutic potential of liposomes that contain arsenic compounds. Arsenic compounds have been known to be used as chemotherapeutics in the past. Arsphenamine (marketed under the trade name Salvarsan in 1910), which is a drug used to treat syphilis and trypanosomiasis, was the first modern chemotherapeutic agent discovered. This was in 1908 in Paul Ehrlich’s laboratory during a screening between many chemical derivatives of the dangerously-toxic drug atoxyl [8, 9]. In 1912, a more soluble (but slightly less effective) arsenical compound, Neosalvarsan (or neoarsphenamine), became available [10]. However, these arsenical compounds came with considerable risk of side effects, and for this reason they were replaced (for syphilis treatment) by penicillin in the 1940s. The recent approval of arsenic trioxide, marketed under the trade name Trisenox [11] has re-stimulated research of arsenic-compounds [12, 13]. Trisenox is indicated for induction of remission and consolidation in patients with acute promyelocytic leukemia (APL) when they are refractory to, or have relapsed from, retinoid and anthracycline chemotherapy, and whose APL is characterized by the presence of the t (15;17) translocation or PML/RAR-alpha gene expression [14]. Nevertheless, the high toxicity of this drug restricts dose increase and its use in other types of cancer, on which it is active [14]. Therefore, this specific drug would be a candidate for development of a liposomal formulation in order to decrease its toxicity and broaden its therapeutic applications. This has been investigated in our laboratory, however although the encapsulation in different liposome types was adequate, its retention in the vesicles was found to be inadequate for in vivo use

[15], leading us to consider the possibility of using arsenic-containing lipid compounds for the construction of arsenic-containing liposomes, in which arsenic would be stably incorporated. Nevertheless, lately, an active loading technique has been developed and the first results of this liposomal formulation of arsenic trioxide regarding its anticancer activity are encouraging [16]. Arsenolipids are naturally occurring arsenic-containing lipids that have been discovered and synthesized [17, 18]. Arsonolipids are analogs of phosphonolipids in which P has been replaced by As (Figure 1). The synthesis of arsonolipids has been explored [19, 20] and a simple one-pot method with high yield is currently available for the preparation of arsonolipid racemic mixtures [20]. After being synthesized and characterized it was anticipated that these lipids might have selective anticancer activity for cancer cells that are known to have increased thiol levels (in comparison to normal cells). This was based on the assumption that if arsonolipid arsenic (As(V)) is reduced to its more toxic form (As(III)) in the vicinity of such cancer cells [21], arsonolipids will be more toxic (towards such cells). Their anticancer activity was evaluated, after arsonolipids were dispersed in dimethylosulfoxide (DMSO), by screening tests performed by NCI, USA. However, in all cases of arsonolipids studied (C12-C18 arsonolipids) no or low activity was demonstrated and the suggestion was to halt further studies [22]. Some years ago, we started investigating the possibility of using arsonolipids for the preparation of arsenic containing liposomes, since even a low intrinsic anticancer activity could be beneficial for the delivery and targeting of preferably synergistically acting anticancer ROOO ROOO

ROOO

ROOO ROOO

O As

O

OH

As

OH

OH

OOOR O As OH OH

1

2

3

rac

(R)

(S)

R = C12 - C18

Figure 1 - Structure of arsonolipids. 377

OH

J. DRUG DEL. SCI. TECH., 17 (6) 377-388 2007

Arsonoliposomes for drug delivery S.G. Antimisiaris

drugs to target cancer cells. Furthermore, in line with the points mentioned above, perhaps the liposome structure would result in better biodistribution of the active moiety of the arsonoliposomes, providing a basis for their potential use in therapeutics. Indeed, the liposome structure was found to result in increased toxicity or arsonoliposomes towards cancer cell types, compared to normal cells [23, 24], and also in increased antiparasitic activity (substantially increased, compared to the corresponding activities of the dispersions of the same lipids in DMSO) [25, 26]. A number of investigations have therefore been carried out in the last seven years into arsonoliposomes and their potential in therapeutics [27]. The results and conclusions from these studies are reviewed below and our future directions are presented.

tioned above and explained previously [27]. For the preparation of arsonoliposomes with this technique, the lipid or lipids (arsonolipids C12-C18, PC or DSPC and in some cases Chol) (as powders) are mixed with the appropriate volume of aqueous solvent (water or Phosphate buffered saline [PBS], pH 7.40) and stirred vigorously on a magnetic hot plate, for 6-12  h [28]. High temperature must be used, higher than the transition temperature of the arsonolipid used in each case, throughout the whole liposome preparation process. Transition temperatures of arsonolipids range between 72-80°C (for rac, R and S, C12 Ars), 50-60°C (for rac, R and S, C14 Ars), and 65-72°C (for rac, R and S, C16 Ars), as published earlier for arsonolipid dispersion at pH 8.0 [20]. Recently [32], it was seen that the transition characteristics of the aqueous dispersions of plain arsonoliposomes in PBS at pH 7.4 change with the acyl chain length of the arsonolipid used (as found previously). In this later study phase transitions from 30°C for C12, up to 72°C for C18 arsonolipid-vesicles, were measured (at pH 7.4).

I. Arsonoliposome Preparation and Physicochemical Properties: Influence of Lipid composition

The preparation of liposomes from arsonolipids was not an easy task. Due to the arsonolipid high transition temperature and their high tendency to foam, many conventional liposome preparation techniques failed to produce vesicles, mainly because of the difficulty of dispersing these lipids in aqueous environments. After evaluating several techniques, the one step (bubble) technique was found to be the easiest for the preparation of arsonolipid-containing liposomes, or arsonoliposomes [28], especially in cases when arsonolipids were the only lipids used. Many types of arsonoliposomes have since been prepared and physicochemically characterized [29, 30], differing in: (i) The specific arsonolipid [Ars] used for their preparation (C12, C14, C16 and C18 arsonolipids were tested) [28]. (ii) The lipid composition of the vesicles. Initially plain arsonoliposomes consisting only of arsonolipids (without cholesterol [Chol]) were prepared, however due to their high instability that was improved by Chol incorporation in their membranes; in all the following experiments plain arsonoliposomes are those that consist of Ars and Chol [28]. Additionally, mixed arsonoliposomes, consisting of Ars mixed with phospholipids and cholesterol, and also pegylated-arsonoliposomes, which are pegylated (coated with polyethylene glycol molecules) mixed arsonoliposomes, were evaluated [29, 30]. Concerning the mixed arsonoliposomes, two phospholipids have been used thus far, egg-derived phospatidyl-choline (PC), and 1,2-distearoyl-sn-glycero3-phosphocholine (DSPC), and the arsonoliposomes produced are characterized as PC-based or DSPC-based arsonoliposomes, respectively. In the cases of mixed arsonoliposomes, the relative percentage of phospholipids and arsonolipids in arsonoliposomes may vary (most of the later studies have been carried out with 10 and 27 mol% Ars (of total lipids) arsonoliposomes). (iii) The size and lamellarity of the vesicles. Multilamellar vesicles (referred to as non-sonicated arsonoliposomes) have been produced by applying only the one-step technique for lipid dispersion and unilamellar ones (referred to as sonicated arsonoliposomes) after subjecting the first ones to sonication [28]. (iv) The surface properties of the vesicles. Pegylated-arsonoliposomes have been formulated by adding PEG-conjugated phosholipids (DSPE-PEG). PEG molecules with molecular weight 2000 have been used [27]. In the following subchapter sections, the preparation and physicochemical properties of all the arsonoliposome types that have been studied up to date will be discussed.

1.2. Sonicated arsonoliposomes After arsonoliposome formation using the one-step method, it was found that reduction of the vesicle size could be achieved by applying probe sonication to the initial suspensions [28]. Usually two 5-min cycles of sonication using a probe sonicator equipped with a tapered microtip were demonstrated to be sufficient for production of transparent arsonoliposome dispersions. On the contrary, preparation of sonicated arsonoliposomes by direct probe sonication of dry lipids mixed with buffer solutions (pH 7.40) or water was not possible. Proof that indeed vesicles were formed after the ‘one-step method’ and that they still exist after sonication was established by morphological observation of the various arsonoliposome dispersions formed using different types of electron microscopy (EM), as discussed below [27]. Additionally, their ability to encapsulate aqueous soluble markers as carboxyfluorescein or calcein served as proof that vesicular structures are present in most of the arsonoliposome dispersions prepared. At this point it should be mentioned that the encapsulation ability of arsonoliposomes was found to be comparable to that usually observed for conventional liposomes (i.e. composed of phospholipids) with approximately the same mean diameter range [29], meaning that arsonoliposomes (most of the structures formed) could be used as carriers of other drugs (that could be encapsulated in their aqueous spaces).

2. Arsonoliposome size distribution and morphology

The potential for application of liposomes in drug delivery is highly dependent on their physicochemical properties, and especially their mean size distribution and surface charge, which are both parameters that can modify their in vitro and in vivo performance [33-37]. Information about the morphology and size distribution of the different types of arsonoliposomes that have been formed, has been made available by size distribution measurements using DLS technique and by light microscopy (for general morphology) and different types of electron microscopy (scanning (SEM), transition (TEM) or cryo-EM) observations, and subsequent image analysis [27-30, 38]. From all the sizing measurements performed [28-30, 38] it was demonstrated (as seen in Table I) that arsonoliposomes range in size (mean diameter) from between 247 and 430 nm, in the case of nonsonicated vesicles and from between 63 and 120 nm, in the case of sonicated vesicles, depending on their lipid composition, the Ars used and the aqueous dispersion medium. In general, although the vesicles prepared by the one-step method (non-sonicated) were not considerably large for this type of preparation technique (compared to usual size of multilamellar vesicles formed by phospholipids, which have diameters > 1 um), sonication had a significant effect on their size, which was considerably reduced. Non-sonicated arsonoliposome vesicle sizes were slightly influenced by the length of the fatty acyl chain of the specific arsonolipid

1. Preparation techniques

1.1. Non-sonicated arsonoliposomes The “one-step method” is applied for arsonoliposome preparation [28, 31]. This method was found to be successful for the preparation of arsonolipid-containing liposomes, after evaluating several other techniques that were not successful for reasons that have been men378

Arsonoliposomes for drug delivery S.G. Antimisiaris

J. DRUG DEL. SCI. TECH., 17 (6) 377-388 2007

Table I - Diameter (nm) and zeta-potential (mV) of liposomes containing arsonolipids and prepared by the one step method followed or not by sonication. Liposomes were prepared in H2O or PBS, pH 7.4 (or other media if indicated), and measured immediately after their preparation (values were taken from [28-30] and [38]). Liposome type

Lipid composition

Ars

Ars PBS pH 7.4

Ars/Chol 1:1 mol/mol

Ars/DSPC 1:1 mol/mol

One-step

C12

251 ± 6* nm -46.4 ± 1.3 mV 329 ± 22 nm -44.1 ± 2.7 mV 273 ± 41 nm -57.7 ± 1.1 mV 265 ± 23 nm -38.5 ± 2.0 mV

270 ± 10* nm

247 ± 13 nm

346 ±19 nm

374 ± 27 nm

384 ± 36 nm

-

282 ± 17 nm

362 ± 18 nm

247 ± 13 nm

-

427 ± 61 nm

290 ± 22 nm

Ars

Ars/Chol 2:1 mol/mol

PC/Ars/Chol 12:8:10 mol/mol/mol

PC/Ars/Chol 17:3:10 mol/mol/mol

116 ± 10 nm -50.8 ± .6 mV 118 ± 15 nm -51.3 ± .8 mV 130.2 ± 9.6 nm -57.2 ± 1.3 mV -40.1 ± 2.1 mV

102.5 ± 8.5 nm -63.5 ± 6.3 mV 107.9 ± 8.7 nm -65.4 ± 1.5 mV 110.8 ± 8.7 nm -69.5 ± 2.3 mV 121.3 ± 6.8 nm -59.2 ± 3.1 mV

72.6 ± 8.4 nm -42.0 ± 2.8 mV 87.0 ± 6.8 nm -43.0 ± 2.6 mV 90.9 ± 6.6 nm -50.3 ± 1.0 mV 93.4 ± 5.4 nm -48.4 ± 1.2 mV

62.8 ± 7.5 nm -23.9 ± 1.9 mV 76.4 ± 3.1 nm -28.2 ± 1.1 mV 78.0 ± 5.7 nm -42.1 ± 2.9 mV 74.8 ± 6.7 nm -32.1 ± 2.3 mV

66,9 ± 2,4 nm -32,2 ± 0,23 mV

78,6 ± 1,8 nm -14,86 ± 0,90 mV

DSPC/Ars/Chol 12:8:10 mol/mol/mol

DSPC/Ars/Chol 17:3:10 mol/mol/mol

79,88 ± 0,93 nm -26,8 ± 0,80 mV

99,9 ± 1,1 nm -17,38 ± 0,75 mV

103,2 ± 1,7 nm -2,98 ± 0,98 mV

91,5 ± 2,8 nm -4,04 ± 0,86 mV

C14 C16 C18

Sonicated

C12 C14 C16 C18

Sonicated

C16 (in presence of calcein & EDTA)

C16 (in presence of calcein & EDTA)

Sonicated Sonicated + PEG 2000-DSPE

>>

Each value is the mean ± SD of measurements from at least four different samples. *Accuracy is questionable, since C12 was demonstrated to form tubular structures in a high proportion (Figure 2).

they contain, but practically unchanged when liposomes were prepared in PBS, pH 7.4, instead of water. On the contrary, acyl chain length of arsonolipids had no effect on the arsonoliposome size when DSPC or Chol was added (one at a time at 1:1 molar ratio) in their lipid bilayers [28]. In these cases the morphology of the structures is comparable, as detailed below. Also, for sonicated arsonoliposomes, the acyl chain length of the arsonolipid used for their preparation does not influence vesicle mean diameter, and there is no significant difference between the sizes of vesicles that have been prepared in water or buffer (PBS, pH 7.40) [28]. In some cases when arsonoliposomes (containing Ars mixed with PC and Chol) were prepared in PBS, pH 7.4, an increase in vesicle size was observed as their Ars content increased. This was attributed to the considerably larger head-group of arsonolipids, compared to that of phospholipids. However, when the same arsonoliposome preparations were prepared in the same buffer containing calcein and EDTA (1 mM), the complete opposite effect on vesicle size was observed (decrease in vesicle mean diameter, when more arsonolipid was included in the arsonoliposome compositions) [29, 30]. It was hypothesized that this effect may be related to the presence of EDTA, which forms complexes with residual amounts of divalent cations present in the vesicle dispersions preventing divalent cation-induce aggregation or arsonoliposomes. The presence of such aggregates in arsonoliposome dispersions would increase the mean vesicle diameter measured by DLS. Nevertheless, when higher quantities of Ars were included in arsonoliposome vesicles their surface charge was also modified (as discussed below) so most probably, depending on the dispersion medium used, it should be accepted that the overall effect of arsenic content on vesicle size is the final result of concurrent (and opposite in respect to their effect on vesicle size) events; increased electrical repulsion which results in vesicle size reduction, and arsonolipid

incorporation which results in vesicle size increase (due to the larger head group of arsonolipids compared to phospholipids). Concerning the effect of pegylation on arsonoliposome size (Table I), inclusion of 8mol% DSPE-PEG(MW-2000) resulted in a slight increase in vesicle size (from 80 to 103 nm) of Ars/DSPC/Chol when the Ars content was 27 mol% of total lipids, and a slight reduction of mean diameter (from 100 to 92 nm) when the Ars content was 10% of total lipids [30]. However, due to the differences between phospholipids and arsonolipids the mechanisms which are responsible for the PEG-lipid induced vesicle size modifications, which are not substantial in any case, could not be discussed in accordance with the theories proposed by others for different types of phospholipid-containing vesicles, such as sonicated liposomes [39, 40] or extruded liposomes [41]. One important conclusion that may be securely extracted from the size distribution results (together with the Cryo-EM data mentioned below) is that since arsonoliposome mean diameters were not substantially decreased by pegylation, the concentrations of PEG-lipids used (up to 8 mol%) do not induce solubilization of arsonoliposomes. As for vesicle morphology (Figure 2), when completely plain dispersions were prepared [28], consisting only of arsonolipids (without Chol), the different arsonolipids formed different types of structures, the most peculiar being those formed by C12 arsonolipid, which were not vesicles but thin, long rods or tubes the length of which was > 5 um and the diameter of which was ~ 50 nm. These tubes break into barrel-like structures or rods upon sonication and then rapidly re-shape into long tubes. The other arsonolipids (C14-C18) formed (plain arsonolipid) round-shaped vesicles, which were however very unstable (as will be discussed below). In all cases, when other lipids or cholesterol were added in the arsonoliposomes (mixed with arsonolipids) roundshape vesicles were formed. Also, all types of mixed arsonoliposomes consisting of C16-arsonolipid, phospholipids and Chol, were found to 379

J. DRUG DEL. SCI. TECH., 17 (6) 377-388 2007

Arsonoliposomes for drug delivery S.G. Antimisiaris

The effect of the dispersion media pH on the surface charge of non-sonicated plain and mixed arsonoliposomes (composed of mixtures of Ars with Chol and DSPC) was investigated in the pH range from 3 to 9 [43], and all values measured were found to be negative (in the full pH range investigated, not shown). This result is logical and explained when considering the ionization of arsonolipid head groups: Aliphatic arsonic acids are ionized (in the pH range studied) as shown below: O R

As (A)

OH OH

O

pKa 1 R

As (B)

OH O

O

pKa 2 R

As

O O

(C)

the pKa values being: pKa1 = 3.5-4.2 and pKa2 = 8.2-9.2 [44], and therefore it is expected that the “monomeric” arsonolipids will have pKa1 ~ 4, although this may not be absolutely accurate since it is known that for phosphatidic acid the pKa value changes when in bilayers [45, 46]. Nevertheless, it is expected that the zeta potential of plain and mixed (with Chol or neutral lipids) arsonoliposomes will become more negative as the pH increases, as demonstrated with the measurements performed. The addition of Chol or DSPC (in 1:1 molar ratio with Ars) in arsonoliposome membranes, results in moderate effects on the vesicle zeta potential, which were different for the different arsonolipids studied (C12-C16), but values were always negative [28, 29]. The only cases in which zeta potential values measured were not highly negative, were those of pegylated arsonoliposomes [30] (Table I). Indeed, under the conditions employed (measurements were performed in PBS buffer, pH 7.4, with 1 mM EDTA), pegylation minimizes and practically diminishes the negative surface charge of arsonoliposomes (values mentioned above). The fact that the surface charge of all pegylated arsonoliposomes was drastically affected proves that the vesicle surfaces were coated with PEG molecules [39].

Figure 2 - Cryo-electron microscopy of plain and mixed arsonoliposomes (A-B bar represents 500 nm). (A): Plain C12 arsonoliposomes made by the one-step method followed by probe sonication. Cube or barrel shaped structures were found directly after sonication. (B): The cubes and the barrels disappear progressively and transform into very long tubes (C-F bar represents 100 nm ). (C): C16/PC/Chol (8:12:10, mol/mol/mol) sonicated arsonoliposomes.(D & G): C16/DSPC/Chol (8:12:10, mol/mol/ mol) arsonoliposomes. (E): Pegylated C16/DSPC/Chol (8:12:10, mol/mol/ mol) arsonoliposomes as they are dispersed in H2O. (F): The previous arsonoliposomes after incubation in CaCl2 (1 mM final concentration) for 24 h. (H): C16/DSPC/Chol (8:12:10, mol/mol/mol) (DSPC-based, non-pegylated) arsonoliposomes after incubation in CaCl2 (1 mM final concentration) for 6 h and (I): after 24 h.

4. Arsonoliposome stability

4.1. Membrane integrity of arsonoliposomes The integrity of vesicular systems during their journey in the body to reach the site of action is a very important parameter that determines – to a large extent – their success. The membrane integrity of arsonoliposomes was evaluated by measuring the release of small aqueous soluble molecules, which were initially entrapped in their aqueous interior during their preparation. Most studies were performed with calcein or 5, 6- carboxyfluorescein (CF) that offer the possibility of following their leakage from vesicles without needing to separate the amount released from that remaining entrapped, if quenched concentrations of these dyes (100 mM) are used [47]. From the experiments performed [28-30] it was demonstrated that although all compositions of non-sonicated arsonoliposomes (plain or mixed) composed of arsonolipids C12-C18, exhibited similar dye release kinetics when incubated in isotonic buffer, pH 7.40 at 37°C, the vesicles composed of C14 and C16 were significantly more stable compared to those from C12 and C18 (Figure 3). The addition of Chol in the plain arsonoliposomes significantly enhanced the stability of all arsonoliposomes, while the addition of DSPC significantly stabilized only the less stable C12-containing structures [28]. For comparative reasons we should mention at this point that conventional DSPC liposomes retain more than 80% of their dye content during 24-h incubation under the same conditions. However, the integrity of non-sonicated (one step) arsonoliposomes during incubation in serum proteins (80% v/v FCS [fetal calf serum]) was not found to be adequate for in vivo applications (even when containing Chol, these vesicles could not retain more than 50% of their dye content for 24 h [for all arsonolipids tested]). The latter conclusion, together with the fact that sonicated liposomes

have similar, round shaped morphology with a few occasional discshaped micelles (bicelles) present in their dispersions [29, 30], which indicate that in some cases the arsonolipids were not “well-fitted” nor equally distributed within the bilayers formed. An exception to this was the case of DSPC-based arsonoliposomes, which were seen to form disc-shaped vesicle with angles on their periphery (Figure 2D-G). This morphology is similar to that of other phospholipid-vesicle types, which are known to become round when more cholesterol is added to their membranes [42]. Nevertheless, when PEG-lipids were included in these DSPC-based arsonoliposomes (forming the so called pegylated-arsonoliposomes) their structure was again observed to be rounder, similar to the PC-based arsonoliposomes.

3. Arsonoliposome surface charge (zeta potential)

The zeta potential values (Table I) of non-pegylated/non-sonicated or sonicated arsonoliposomes [28-30] were always found to be highly negative (ranging between -70 to -17). The specific value of the zeta potential of arsonoliposomes was found to be dependant on the lipid composition (mainly the Ars content), the specific arsonolipid used and the dispersion media, as seen in Table I. Regardless of the arsonolipid used, all values are highly negative (with the exception of pegylated arsonoliposomes). For the pegylated arsonoliposomes zeta potentials of -4 and -3 mV were measured when the liposomes were dispersed in PBS buffer, pH 7.40. In all cases, as arsonolipid content of arsonoliposomes increases, zeta potential also increases, however the differences measured were also found to be influenced by the vesicle dispersion media used in each case as well as their lipid composition, as mentioned above. 380

Arsonoliposomes for drug delivery S.G. Antimisiaris

Calcein Latency (%) - 24 h

100

J. DRUG DEL. SCI. TECH., 17 (6) 377-388 2007

Furthermore, both types of mixed arsonoliposomes (PC-based and DSPC-based) have similar encapsulation efficiencies (calculated with CF [5,6 carboxyfluorescein] and HPTS) to those usually calculated for sonicated unilamellar liposomes of similar size [48, 49]. Indeed, while the trapping efficiency of conventional PC/Chol (2:1) and DSPC/ Chol (2:1) ranged between 4.2 and 5.5% (for both dyes evaluated) the equivalent values of the arsonoliposomes evaluated ranged between 5.8 and 10%. The results obtained after pegylation of sonicated arsonoliposomes [30] showed that arsonoliposomes composed of DSPC/Ars/Chol (lipid/Chol 2:1 mol/mol) and having arsonolipid contents higher than 27  mol% (of total lipid) can be highly stabilized by incorporating 8 mol% DSPE-PEG2000 into their membranes (Figure 5). This specific lipid composition resulted in the formation of arsonoliposomes with very high stability, comparable to that of sonicated DSPC/Chol (2:1) vesicles as well as sonicated DSPC/Chol (1:1) vesicles [47, 50, 51]. While addition of DSPE-PEG2000 in these arsonoliposomes resulted in increased vesicle stability (integrity) a similar effect was not achieved by DPPE-PEG2000 (same amount), perhaps due to the longer acyl-chain length of DSPE (compared to DPPE) in conjunction with the use of DSPC in the specific mixed arsonoliposomes (better lipid packing). In general, it was found that for this specific method of pegylation (adding 8 mol% of DSPE-PEG2000 in the lipid composition) of sonicated arsonoliposomes consisting of C16 arsonolipid, depending on the arsonolipid content of the vesicles – or the amount of arsonolipid used for liposome formation, two groups of pegylated arsonoliposomes are formed with substantially different stability (Figure 5): the low content arsonoliposomes (< 20 mol% arsonolipid) which are unstable and the high content arsonoliposomes (> 27 mol% arsonolipid) which are highly stable. In addition to high membrane integrity, the stable high content pegylated arsonoliposomes are morphologically perfectly roundshaped vesicles without the sharp edges observed in DSPC-containing arsonoliposomes [30] (as mentioned above).

PLAIN-Ars Ars/Chol (1:1) Ars/DSPC (1:1)

80 60 40 20 0

12 14 16 18 Arsonolipid Acyl-Chain Length Figure 3 - Carboxyfluorescein (CF) Latency (%) after 24-h incubation of different types (different arsonolipids were used for their formation) of CF-encapsulating plain and mixed arsonoliposomes in buffer at 37°C. Each value is the mean from at least three different experiments and bars represent the SD of the mean. The figure key is presented in the figure insert. Values were taken from [28].

are considered to be better-characterized systems, is the reason why most activity-related studies were performed with sonicated arsonoliposomes. Indeed, C14 sonicated arsonoliposomes demonstrated higher membrane integrity (compared to the corresponding non-sonicated vesicles) [28] during incubation in serum proteins (they retained more than 70% of the initially trapped dye (HPTS), under the same incubation conditions [24 hours, 80% FCS, 370C]). From all the studies performed with non-sonicated arsonoliposomes, the vesicles that contain C16 were demonstrated to be more stable (Figure 3) and smaller in diameter (Table I) [28, 29]. Therefore, C16 arsonolipid-containing sonicated arsonoliposomes in which arsonolipid was mixed with cholesterol only (plain arsonoliposomes) or also with egg-lecithin PC (PC-based, mixed arsonoliposomes) were studied extensively. In some experiments, PC was replaced with the synthetic phospholipid DSPC (DSPC-based arsonoliposomes) [29], and better membrane integrity was achieved, especially in the presence of serum proteins. And although the Ars content of PC-based arsonoliposomes was demonstrated to affect their integrity (decreased integrity with increasing Ars content), for the DSPC-based arsonoliposomes membrane integrity was demonstrated to be independent of Ars content in the range investigated (between 10 and 27 mol% arsonolipid [of total lipid]) [29] (Figure 4).

Calcein Retention (%)

100

6 hour - Calcein Latency (%)

100

80 60 40 20 0

80

0

10

20

30

40

50

60

Arsonolipid content (mol%) 60

Figure 5 - Retention of calcein (%) in pegylated DSPC-based arsonoliposomes with various arsonolipid contents, after 24-h incubation in the presence of serum proteins (80% FCS), All arsonoliposomes contain 8 mol% of DSPE-PEG2000. Each value is the mean from at least three different experiments and bars represent the SD of the mean. Values were taken from [30].

40 20 0

PC-based 0%

5%

4.2. Physical stability Self aggregation Vesicle dispersion turbidity has been successfully used as a measure of vesicle size distribution [41, 52]. Additionally vesicle size at various time points during incubation in different media can be used as a measure of the physical stability of liposomes. Both of these techniques were used for evaluation of arsonoliposome physical stability. By following vesicle dispersion turbidity during incubation in various media, arsonoliposome aggregation was measured, initially

DSPC-based 10%

27%

53%

Figure 4 - Calcein latency after 6-h incubation in the presence of serum proteins (80% FCS) of PC-based and DSPC-based arsonoliposomes, with various arsonolipid contents (mol% of arsonolipid). All arsonoliposomes contain Chol (lipid:Chol 2:1 mol/mol). Each value is the mean from at least three different experiments and bars represent the SD of the mean. The figure key is presented in the figure insert. Values were taken from [29]. 381

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in plain water (physical stability). Results showed that when the physical stability of the vesicles is considered: (i) plain arsonoliposomes (incorporating Chol) are more stable compared to mixed ones (composed of mixtures of PC with arsonolipids). (ii) When PC is replaced by DSPC in C16 containing arsonoliposomes, the physical stability of the mixed arsonoliposomes formed is comparable to that of plain arsonoliposomes and (iii) Arsonoliposomes prepared by the C18 arsonolipid are the least stable. However, although, plain arsonoliposomes were demonstrated to be more stable compared to the mixed ones, they were not selected for in vivo studies, since they were found to be very leaky in the membrane integrity studies performed [29]. When self-aggregation (physical instability) of pegylated-arsonoliposomes was studied, there were certain notable differences found between the two types of pegylated-arsonoliposomes formed [30]. For the low content arsonoliposomes, relative turbidity after 24-h incubation in water (24-h turbidity/0-h turbidity) was equal to 1.8 and 1.6, for the non-pegylated and the pegylated vesicles, respectively. Equivalent values for high content arsonoliposomes were significantly lower (1.4 and 1.04), indicating that high content arsonoliposomes demonstrated higher physical stability compared to low content ones, a fact that fully agrees with the substantially higher membrane integrity demonstrated for these arsonoliposomes in serum.

As a measurement of this, the integrity of vesicles was measured during incubation in the presence of glutathione. As anticipated, the ability of arsonoliposomes to retain the calcein encapsulated in their aqueous space during vesicle incubation in 10 mM glutathione decreased as the arsonolipid content of the arsonoliposomes increased (Figure 6), indicating that the interaction of glutathione with arsonolipid was responsible for the leakage of the vesicle-entrapped calcein [55]. Nevertheless, when more stable arsonoliposomes were tested (as DSPC-based and pegylated-arsonoliposomes) thiol-induced disruption of arsonoliposomes became less obvious. Indeed, the pegylated arsonoliposomes were found to be unaffected by glutathione. This may indicate that pegylation prevents the interaction between thiol groups and arsonoliposome arsonolipids, or that the rigidity of these specific arsonoliposomes is such that even though arsonolipids interact with thiol groups, this does not lead to vesicle disruption. Considering the fact that pegylated arsonoliposomes have been recently found to be also very toxic against cancer cells (recent unpublished results), the second theory seems more plausible. Nevertheless, although more studies may be needed to clarify this last point, the studies performed up to now show that arsonoliposomes are indeed thiol-sensitive and that this can be used as a “trigger” for site-specific release of anti-cancer agents from cancer cell targeted arsonoliposomes [56].

Calein Latency (%) [in Glutathione]

Divalent cation-induced aggregation Because of their negative surface charge, arsonoliposomes may be aggregated in the presence of divalent cations [47, 48, 53]. Indeed, it is known that when negatively charged lipids are included in liposome lipid membranes they tend to induce aggregation (and perhaps also fusion to larger particles) when divalent cations are present in the vesicle dispersion media [54]. Accordingly, vesicle aggregation was substantially enhanced for both, plain and mixed sonicated arsonoliposomes in the presence of Ca2+ ions [38]. A difference between mixed and plain arsonoliposomes in terms of Ca2+-induced vesicle aggregation is that in all cases of mixed arsonoliposomes (even the DSPC containing ones) aggregation is not EDTA-reversible. This indicated that fusion was the mechanism of the demonstrated increase of vesicle size. The fact that mixed arsonoliposomes fused to larger vesicles in the presence of calcium ions was also confirmed by morphological evaluation of the effect of calcium on mixed arsonoliposomes, using cryo-electron microscopy (as seen in Figure 2H and I) [38]. Sonicated mixed PCbased arsonoliposomes (one of the arsonoliposome types for which in vivo distribution was evaluated, as discussed below) fuse into larger vesicles after incubation in CaCl2. In fact, this phenomenon was linked to the low bioavailability (and distribution) realized for this specific type of arsonoliposomes, after in vivo administration. On the other hand, the stable pegylated arsonoliposomes (with the specific lipid compositions discussed above) were found to have high physical stability (no aggregation or fusion) during incubation in the presence of physiologically relevant concentrations of Ca2+ ions, as demonstrated by turbidity measurements and clarified by cryo-electron microscopy [30]. Indeed, 20 mol% arsonolipid arsonoliposomes were demonstrated to have increased turbidity as the concentration of CaCl2 increased, but after pegylation calcium ions had no effect on the vesicle turbidity. This confirms that pegylation inhibits the effect of calcium on arsonoliposomes.

PC-based (10%) DSCC-based (10%) Pegylated (10%)

100

(27%) (27%) (27%)

80 60 40 20 0 0

5

10

15

20

25

Incubation Time (h)

Figure 6 - Calcein latency (%) of various types of calcein encapsulating arsonoliposomes during incubation in the presence of 10 mM glutathione. Each value is the mean from at least four different experiments and bars represent the SD of the mean. The figure key is presented in the figure insert. Values were taken from [55].

II. Arsonoliposome in vivo Distribution and Pharmacokinetics

It is well known that the in vivo kinetics of any drug delivery system, or to be more precise, of the active moiety contained in the delivery system, is a major determining factor for its success. For most pharmacologic applications, the main disadvantage of conventional phospholipid-liposomal carriers is their fast clearance from circulation due to opsonization and RES macrophage uptake [47, 48]. Only when fast accumulation of drug in the RES macrophages is needed, as in some cases of parasitic diseases (parasites reside in RES macrophages), delivery via conventional liposomes is an efficient and easy method to achieve what is known as “passive targeting” [48]. In all other cases longer blood circulation times are required, and this can usually be achieved by surface modification of liposomes (or other particulate carrier systems) [5, 48].

4.3. Thiol sensitivity of arsonoliposomes The theory that arsonolipid arsenic will be reduced in the presence of thiol-groups and that AS(V) will be converted into the more toxic As(III) (as shown in the reaction presented below), was investigated by evaluating the sensitivity of various types of arsonoliposomes towards thiol groups.

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In an initial in vivo study arsenic distribution and kinetics were evaluated, by measuring the concentration of arsenic in the tissues of mice at various time points after intraperitoneal (i.p.) injection of arsonoliposomes [25, 57] in order to understand whether surface modification would be needed for extending the potential applications of arsonoliposomes. The i.p. route was chosen for the first in vivo administration of arsonoliposomes because higher amounts could be injected (reliably) in small animals and also because data on the distribution of arsenic after ip administration were available in the literature; therefore comparison of our results with those data would easily demonstrate if and to what extent, the construction of arsenic in the form of arsonoliposomes modifies its distribution. Sonicated PC-based arsonoliposomes (C16/PC/Chol at 8:12:10 mol/mol/mol) that were previously found to demonstrate acceptable in vitro stability (as discussed above) were used. An arsonoliposome dose, corresponding to 5 mg arsenate/kg was administered by i.p. injection in balb-c mice and at various time points post-injection, the mice were sacrificed and distribution of arsenic in the organs was measured using atomic absorption spectroscopy [57] after nitric acid digestion of the biological samples. The details of the technique used for arsenic concentration measurement in tissues are mentioned in detail elsewhere [57]. It should be understood that the arsenic levels measured follow intact liposomes as well as arsenic that has been released from arsonolipids or already metabolized. Experimental results revealed that at the first time point, 1-h postinjection, the distribution of arsenic was greater in the carcass and skin samples, followed by – in descending order –: intestine, liver, stomach, kidney, spleen, lung, and heart. Nevertheless, at 1-h post-injection only about 30% of the total dose administered is cumulatively present in the animal tissues. After this rapid initial elimination of most of the arsenic administered, the elimination of the remaining portion of the dose is a very slow process. The elimination rate constant calculated for this second phase of arsenic elimination (0.023 h-1) is for a half-life of 30 h. The demonstrated fast clearance of arsenic from the body shows that after 1 h, any amount of liposomes absorbed in the circulation was already cleared very fast by the RES and ended up in the liver and spleen. Indeed the 1-h distribution in both of these tissues was high [57] implying that opsonization of arsonoliposomes was taking place, although the fact that the i.p. route of administration was used may be also implicated. Another possibility, however, is that this fast clearance could be facilitated by aggregation of arsonoliposomes immediately after their absorption and dispersion in blood [48, 49, 53] since it was found that these arsonoliposomes aggregate and fuse in the presence of calcium ions as discussed above. Perhaps the Ca2+ ion concentration present in the blood stream is enough to cause such an effect, since it is known that adhesion energies resulting from Ca2+-induced aggregation are so significant that they result in fusion [58, 59]. This problem urged the consideration of grafting sonicated arsonoliposomes with PEG moieties as a method to increase their in vivo stability. Nevertheless, it should be understood that the distribution of arsenic in tissues is a complex mechanism not fully elucidated, a combination of the metabolism of the arsenic-containing compound and the relative kinetics of each metabolite. Additionally, arsonolipid metabolism may be completely different from that of inorganic arsenic, as seen for arsenosugars [60]. By comparing the results of arsenic distribution after administration of PC-based arsonoliposomes with those of disposition of inorganic arsenic from the literature [61], we concluded that for all the tissues measured, the concentration of arsenic was substantially higher after arsonoliposome administration (arsenic levels from 2 to 10 times higher in tissues), a fact which indicated increased retention of arsenic in the abdominal tissues when it was administered in the form of a lipid incorporated in vesicles. Therefore, before arsonoliposome ap-

plications in therapeutics could be considered, toxicity studies in the tissues where high arsenic disposition was found should be carried out. In our latest in vivo study [62], we evaluated the in vivo distribution of arsenic after administration (by the same route) of the arsonoliposome compositions that were found to be more stable (compared to PCbased arsonoliposomes): DSPC-based and pegylated-arsonoliposomes [30, 38]. These results are essential for evaluating the potential of the various arsonoliposome types as anticancer or antiprotozoal therapeutics. This latest study showed that arsonoliposome stability has a significant effect on the vesicle distribution after i.p. administration. Indeed the levels of arsenic not only in abdominal tissues, but also in blood, were significantly higher compared to those measured previously after in vivo administration of the PC-based arsonoliposomes. The pharmacokinetics of arsenic was furthermore influenced, when pegylated-arsonoliposomes were administered. In general the arsenic pharmacokinetic profile after pegylated-arsonoliposome administration seemed to be influenced by the i.p. route of administration as well as the PEG coating applied on the vesicles. By comparing our latest results with those of a previous study, in which the tissue distribution of adriamycin was evaluated after i.v. and i.p. injection of conventional and pegylated drug-encapsulating liposomes [63, 64], since adriamycin distribution pattern was similar to the blood and organ distribution observed for arsenic, it was concluded that the distribution of arsenic after administration of arsonoliposomes by ip injection is mainly governed by the vesicle distribution and that any effect of arsonoliposome disruption and subsequent metabolism of arsonolipids should be minimal. At this point, it is important to mention that no acute toxicity was observed during the in vivo distribution investigations carried out up to date [57, 62], and that the body weight and organ weight of the mice receiving this high dose of As(V) were not altered. To summarize, in vivo studies indicate that arsonoliposome lipid composition has a significant effect on the in vivo distribution of these vesicles, at least after i.p. administration. The in vivo distribution of arsenic after intravenous administration of the various types of arsonoliposomes formed is currently being studied.

III. Arsonoliposome anticancer activity 1. Cytotoxicity studies

In order to investigate whether arsonoliposomes have anti-cancer activity a series of studies were performed with different types of cells (normal cells and cancer cells) in culture, [23, 24, 65]. As explained above, based on the results of size, zeta potential and membrane integrity measurements, sonicated arsonoliposomes were evaluated. Studies with liposomes containing C16 arsonolipid at different molar ratios (plain arsonolipids, Ars/Chol 20:10, Ars/PC/Chol 3:17:10 and Ars/PC/Chol 8:12:10) were performed and their effect on the viability of cells was studied [23, 24]. In particular two types of normal cells, HUVEC (human umbilical cord endothelial cells) and RAME (rat adrenal medulla microvacsular endothelial) cells and three types of cancer cell lines, the HL-60, C6 and GH3, were initially used, and in all cases, the effect of conventional (phospholipid) PC/Chol (2:1) liposomes on the same cells under the same incubation conditions was also investigated, for comparison. Arsonoliposomes containing the C12, C14 and C18 side chain arsonolipids (with different lipid compositions) were also studied under identical conditions (with the C16-containing arsonoliposomes) [24]. In all cases a dose-dependent inhibition of survival of the three malignant cell lines studied was demonstrated. In addition, the corresponding toxicity against normal cells was again much lower for all arsonoliposomes, except for the C12 side chain arsonoliposomes which proved to be relatively toxic towards normal cells, especially RAME. IC50 values were calculated for each preparation (Table II) and in general ranged between 0.7 and 14.6 × 10-5 M for the cancer 383

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Table II - 50% growth inhibition concentrations (IC50) of sonicated mixed (PC-based) arsonoliposomes (expressed as the arsonolipid content of liposomes in each case) for the various cell types studied. The values were taken from our previous studies [23, 24]. Arsonoliposome composition

IC50 (x 105 M)2 HL-60 cells

C6 cells

GH3 cells

HUVEC cells

RAME cells

1.23 (.22)a,b 1.90 (.41)

85 (23)b ND

12.3 (3.5) ND

13.6 (3.4) * ND

ND ND

ND ND

5.5 (1.1)b,* ND

ND > 200 ND

147 (84)* ND

6.4 (1.7) * ND

ND > 200 ND

ND > 300 ND

C12 arosnolipid C12/PC/Chol (8:12:10) C12/PC/Chol (3:17:10)

4.2 (1.1)a,b 11.3 (2.7)

4.8 (1.7)a,b ND

C14/PC/Chol (8:12:10) C14/PC/Chol (3:17:10)

0.71 (.12) * 0.71 (.18) *

5.2 (1.1) ND

C16/PC/Chol (8:12:10) C16/PC/Chol (3:17:10)

2.9 (0.7) 6.1 (1.2)

14.6 (3.7)b,* ND

C18/PC/Chol (8:12:10) C18/PC/Chol (3:17:10)

10.6 (3.5) ~9.7

6.2 (1.7) 5.5 (1.3)

C14 arsonolipid

C16 arsonolipid b

C18 arsonolipid

SD values for the IC50 values were not calculated, since these values were estimated from graphical interpolations. Significantly different (p < 0.05) with the corresponding IC50 value for HUVEC. b Significantly different (p < 0.05) with the corresponding IC50 value for RAME cells. *Significantly different (p < 0.05) with the corresponding IC50 value of the same cells incubated with the C12-arsonoliposomes (same type of liposomes). ND: not determined. 1 a

cell types and between 91 and 300 × 10-5 M for normal cells (with the exception of the C12 arsonoliposomes that had lower IC50 values (7.1 to 85 × 10-5 M) for the normal cells). Considering the effect of the incubation period, it was demonstrated that cancer cell viability was significantly decreased after treating cells for 24  h with any arsonoliposome type. Time dependence of arsonoliposome effect (containing 27% of arsonolipid), on cell viability was also studied in two human cell lines HL-60 (cancer) and HUVEC (normal), for treatment periods of up to 96 h [23] and it was demonstrated that there was a significant effect of the duration of cell exposure on the viability of both cell-types studied. The rate of decrease in cell viability was found to be higher for the cancer cells (compared to normal). Currently [65], more cell types are being evaluated and the cytotoxicity of the more stable DSPC-based and pegylated arsonoliposomes is being evaluated. Arsonoliposomes have been found cytotoxic towards PC3 (prostate cancer) and NB4 (leukemia cells). The first results of these studies [65] showed that depending on the cell types the different types of arsonoliposomes are more or less cytotoxic, however, no substantial differences between PC-based, DSPC-based or Pegylated arsonoliposome-induced cytotoxicity for the same cancer cell type have been, up to this point, demonstrated. Therefore, it seems that lipid composition is not very important for the anticancer activity of arsonoliposomes, however more experiments are currently being carried out to verify the initial results.

of arsonoliposomes. In the HL-60 cells significant cell and nucleus swelling (a preliminary step to membrane rupture during cell necrosis) was observed (Figure 7C), whereas in some of the C6 cells, acridine fluorescence of the cell nucleus was significantly higher when compared with control cells (that were not incubated with arsonoliposomes-not shown here). On the other hand, no obvious effect on cell morphology was observed in the HUVEC (Figure 7D-F) and RAME cells (not shown here), with the exception of some small changes in the shape of the RAME cells that was possibly related to the way they adhere to the slide surface. In contrast to this, high concentrations (10-4 M) of arsenic trioxide were clearly demonstrated to cause apoptosis to HL-60 cells after staining the cells under the same conditions (not shown here) [23]. In the case of the C12-containing arsonoliposomes, microscopy studies showed that these vesicles possibly cause apoptosis of most cell types studied [24] (not shown here). This, together with the findings that these arsonoliposomes demonstrated a different rate of cytotoxicity against cancer cells and are also more toxic against normal cells, implies that the mechanism of action of the C12-arsonoliposomes is different from the other arsonoliposomes studied (C14-C18) [24]. This difference may be related to the different physicochemical properties

2. Cell interaction studies

In order to elucidate the mechanism of the demonstrated arsonoliposome toxicity towards cancer cells, morphological observations of the treated cells using acridine orange as a cell staining agent were performed [23]. In addition, the uptake of arsenic by the cells after incubation with arsonoliposomes was measured, in order to understand whether a correlation between the amount of arsenic taken up by cells and arsonoliposome-induced cell toxicity exists. The morphological studies (Figure 7) revealed that no obvious morphologic changes that are characteristic of apoptosis, such as cell shrinkage or cell membrane blebbing, were visible in most cases, when C16 arsonoliposomes were incubated with the cells [23]. Nevertheless, in the cases of the cancer cells studied (HL-60 and C6 cells) it was obvious that their morphology was significantly affected after incubation with the highest dose

Figure 7 - Morphologic characteristics of HL-60 (A, B, C), HUVEC (D, E, F), before (A, D) and after treatment with various concentrations of sonicated (C16) arsonoliposomes for 24 h, and staining with acridine orange. The lipid composition of arsonoliposomes used was Ars/PC/ Chol 8:12:10 (mol/mol/mol) and the arsonolipid concentration in each case was: B = 2 x 10-5 M, C = 10-4 M, E = 5 x 10-4 and F = 10-3 M (from [23]). 384

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and/or stability of the vesicles (arsonoliposomes that consist of different arsonolipids). The intensity of arsonoliposome-cell interactions was evaluated by measuring the uptake of arsonoliposome-lipid [66] or arsenic [67] in the cells, after incubation of the cells with arsonoliposomes. These studies showed a significantly higher interaction between arsonoliposomes and HL-60 cells compared to that measured for HUVECs [23]. However, in a separate study preformed under the same conditions, the opposite results were obtained when conventional liposomes (PC/ Chol and PS/Chol) were incubated with the two types of cells (higher interaction with HUVEC compared to HL-60 cells) [66]. Therefore, the demonstrated increased affinity of arsonoliposomes (compared to phospholipid liposomes) for cancer cells is attributed to the presence of arsenic on the liposome membrane that causes a different type of interaction between arsonoliposomes and cancer cells (compared to conventional phospholipid liposomes), and results in higher uptake of arsenic by the cells. However, it has not yet been proved (by thiol concentration measurements) whether this demonstrated increased interaction between arsonoliposome and cancer cells is definitely connected with the higher thiol contents of these cells and is a logical explanation for the higher toxicity of arsonoliposomes towards cancer cells (compared to normal). In addition, and since it was demonstrated in the screening studies performed with plain arsonolipid dispersions (in DMSO) that the anti-cancer activity of these arsonolipids is low [22] (and thus characterized as not being worth further exploitation), it may be concluded that the specific type of formulation (liposome structure) modifies the interaction between the lipids and the cancer cells, thus resulting in significant anti-cancer activity. Currently, the mechanism of cell uptake of the various types of arsonoliposomes is being investigated by the HPTS (8-hydroxypyrene1,3,6-trisulfonic acid trisodium salt) technique [67] although the results of these studies are not yet available.

the same Ars content. It was postulated that the reason for the higher antileishmanial activity of mixed arsonoliposomes compared to plain ones, was the higher arsonolipid amount present on the outer bilayer of the first (for equal total arsonolipid content), that possibly initiated improved interaction between infected cells and arsonoliposomes. On the other hand, no activity was demonstrated for the plain (dispersed in DMSO) arsonolipids. Therefore, it was demonstrated that arsonoliposome activity is related to the liposomal structure, as was found for their anti-cancer activity. When the same formulations were tested against T. brucei brucei [25], they showed trypanocidal activity in the same manner as before (with Leishmania), with C16-containing vesicles (MEC at 0.20-0.24 uM) performing better than those containing C12 (MEC at 0.58-0.87 uM), compared to 2.20  uM for the positive control pentamidine. Once again the plain lipid dispersions were totally inactive. However the presence of PC in the membrane of arsonoliposomes did not have any effect on their trypanocidal activity (no significant difference between the activity of plain and mixed arsonoliposomes) indicating that the total amount of arsonolipid included in the vesicles was active for the specific formulation tested. The general conclusion from the results of these experiments was that arsonoliposomes may have potential as therapeutics for parasitic diseases. However, the in vivo distribution of arsonoliposomes [57, 62] which, as mentioned above, was found to be influenced by their lipid composition (stability), should also be compatible with any desired application in therapeutics. Therefore, after the in vivo kinetics of the different types of arsonoliposomes were realized, we continued investigations in order to evaluate the effect of each arsonoliposome type on their antiparasitic activity (in vitro, as well as in animal models) [26]. The in vitro trypanocidal activity of DSPC-based and pegylatedarsonoliposomes was similar against both Trypanosoma brucei brucei strains that were evaluated. Their minimum effective concentration (MEC), however, was found to be 5 mM (for both DSPC-based and PEG-arsonoliposomes) on both strains after a 24-h incubation period. Pentamidine was used as reference compound and exhibited a MEC at 2.2  ± 0.2  mM. As mentioned above, the MEC of the PC-based arsonoliposomes was determined equal to 0.2 mM (on both fast and chronic strains) [25]. The comparison of the two sets of results leads to the conclusion that the PC-based arsonoliposomes were approximately 25 times more active against trypanosomes in vitro compared to the DSPC-based and pegylated-arsonoliposomes. Since the size of the particles was found to be equivalent, the higher rigidity of the DSPC-based and pegylated-arsonoliposomes should be implicated in the observed activity differences. It was postulated that the high rigidity of the first vesicle types leads to internalization of liposomes within the parasite via its flagellar pocket, which is not followed by the disruption of the (highly rigid) vesicles and subsequent release of arsenic in the parasite. Thus, it seems that arsenic remains sequestrated in the parasite pocket due to the high stability of the specific arsonoliposome types and could not therefore act to kill the parasite.

IV. Arsonoliposome antiprotozoal activity 1. In vitro studies

Antimonial drugs (as pentamidine and amphotericin B) have been used for the treatment of visceral leishmaniasis extensively however the increased drug resistance that usually develops very fast, in combination with the toxicity of therapeutic doses are the main drawbacks of these agents [68]. When liposomes are used as carriers of anti-leishmanial agents, lower amounts of drugs are needed (compared to free drugs) for equivalent biological effects [69-71] and thus liposomal drug formulations are connected with considerably lower toxicity (compared to other types of formulation). On this theoretical basis, the potential of arsonoliposomes as an antiprotozoal therapy (against Leishmania and Trypanosoma) was evaluated [25, 26, 72]. Sonicated arsonoliposomes containing C12 or C16 Ars, Chol and mixed or not with PC, were incubated for 72 h with wild-type promastigote forms of Leishmania donovani [25]. Additionally, two drug-resistant lines of leishmania were used, one resistant to amphotericin B and the other to miltefosine. The antiparasitic activities of plain arsonolipids (dispersed in DMSO) were also studied under identical conditions. Antileishmanial activity was observed (IC50 ranged between 0.21 and 11.60 uM of arsonolipid) for all the formulations tested against all Leishmania strains used. It is mentioned here that the IC50 for pentimidine (reference compound) on L. domovani strain was 7.7 uM. The resistant strains were significantly more sensitive (IC50 was between 0.2 and 2.33 uM) to the arsonoliposomes compared to the wild types (IC50 between 0.40 and 11.60). Arsonoliposomes prepared from C16 arsonolipid (IC50 between 0.21 and 5.88 uM) were found to be approximately twice as active as the liposomes made of C12 arsonolipid (IC50 between 0.43 and 11.60 uM). Furthermore, mixed arsonoliposomes (containing PC) demonstrated higher antileishmanial activity (IC50 0.21-11.60 uM) compared to the plain arsonoliposomes (IC50 0.97-9.25 uM) that had

2. In vivo trypanocidal activity studies

The in vivo trypanocidal activity of PC-based arsonoliposomes was evaluated in an acute trypanosomiasis model (Trypanosoma b. brucei CMP mouse model) [26]. The corresponding activity of potassium melarsonyl (used as a reference control) was also measured. PC-based arsonoliposomes were active at 25  mg/kg after a single intraperitoneal administration (all mice were cured). Although the PC-based arsonoliposomes were less active than potassium melarsonyl (reference compound), no toxicity was detected at the effective dose, and such an activity detected after single dose administration was considered as promising and merited further exploitation. Therefore, after this result was found it was decided to continue the study by 385

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evaluating the activity of the more stable (in the presence of serum proteins) arsonoliposome types. The latter activity evaluations were carried out on a chronic disease mouse model that is more relevant to the human situation. Unfortunately both types of stable arsonoliposomes were found to be inactive and even after a repeated (i.p.) dose of 50 mg/kg, no activity was detected. The administration of a repeated dose, four days after the administration of the first, had no effect on the activity suggesting that this kind of arsonoliposome was not able to cross the blood brain barrier. However, no toxicity was detected even at the high doses administered. The in vivo trypanocidal activity studies were in line with the comparative results of the relevant in vitro studies, indicating the usefulness of in vitro studies for evaluation of arsonoliposome activity. Indeed, although the PC-based arsonoliposomes demonstrated interesting trypanocidal activity that prompted our group to continue experiments with the more stable DSPC-based and pegylated arsonoliposomes on a chronic trypanosome model, the latter revealed that the more stable vesicles were inactive.

The challenge of constructing drug carriers with the ability to pass the blood-brain barrier (BBB) is currently a very hot issue in nanomedicine-related research. Therefore, since arsenic was not detected in the brain of the animals receiving the different arsonoliposome types studied as expected, due to the fact that BBB-penetration is not an easy task, an interesting additional challenge for future exploitation would be to coat arsonoliposomes with ligands that will improve their ability to cross the blood brain barrier in order to either kill parasites (activity on the late stage of the African trypanosomiasis could be developed) in the central nervous system or act as an anticancer agents against brain tumors.

References 1.

2.

V. Summary, Conclusions and future prospects

Arsonolipids, are analogs of phosphonolipids, in which P has been replaced by As [19, 20, 22], but although they posses interesting biophysical and biochemical properties their anticancer or antiparasitic activity was not considered adequate for therapeutic applications. Nevertheless, when arsonolipids were incorporated in liposomes [2830], the vesicles formulated were found to have interesting properties [23-27]. In cell culture studies arsonoliposomes showed increased toxicity against cancer cells (compared to that of arsenic trioxide) while at the same time being less toxic or non-toxic for normal cells [23, 24]. Furthermore, arsonoliposomes demonstrated antiparasitic activity in vitro [25] and in vivo [26]. If used as nanocarrier systems for the delivery of other anticancer drugs, in addition to the per se anticancer activity arsonoliposomes may provide a cancer-cell specific trigger [56], which is a very interesting asset for any nanocarrier. It has indeed been recently demonstrated in in vitro studies that arsonoliposomes are “thiol sensitive” and encapsulated molecules are released faster from the arsonoliposomes when they are incubated in a thiol-rich (glutathione) environment [55]. Nevertheless, drug encapsulation in arsonoliposomes has not been studied so far, although the encapsulation efficiencies obtained for the various types of labels (as HPTS, CF and calcein) used in the studies performed (which were equivalent to those obtained with conventional liposomes of similar size) make us confident that many types of drugs can be easily entrapped in this type of carriers. Arsonoliposome lipid composition was recently found to have considerable influence not only on their in vitro and in vivo stability [27-31, 38], as anticipated, but also on their in vivo kinetics [57, unpublished results] and trypanocidal activity [26]. However, the first results of currently running investigations show that the anti-cancer activity of arsonoliposomes is not substantially influenced by their lipid composition. Therefore, it becomes obvious that depending on the intended therapeutic application, different aspects should be considered as a priority when designing arsonoliposome formulations. To conclude, the investigations carried out so far prove that arsonoliposomes have the potential to find many applications in therapeutics, as antiprotozoal or anticancer systems, when used alone or as carriers of other preferably synergistically acting drugs. However, when designing arsonoliposome formulations for usage as antiprotozoal or anticancer therapeutics, in addition to the relative activities demonstrated by the different arsonoliposome types (lipid compositions) their biodistribution profiles should also be taken into account in order to fine tune their structure with the aim of obtaining the best activity in conjunction with the in vivo distribution required for the specific application.

3.

4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16.

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Acknowledgements The Research discussed in this review article was partly supported by the General Secretariat of Research and Technology, Athens Greece (PENED 95). The author would like to thank all the scientists who have contributed to this on-going research, particularly Prof. P.V. Ioannou, Prof. D. Fatouros, Prof. P. Klepetsanis, Prof. P. Loiseau, Prof. P. Frederik, as well as all (former and recent) students who have contributed with their work and dedication.

Manuscript Received 23 May 2007, accepted for publication 30 August 2007.

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