How Can Immunosuppression After Organ Transplantation Be Made More Effective and Safer? – A Review on Liposomal Formulations With Consideration of Archaeal Tetraetherlipid

Immune-suppressive agents such as methylprednisolone and cyclosporine exert tremendous side effects, because of high dosage and long-term application required for immune suppression after organ transplantation. Major side effects of methylprednisolone include bleeding of the gastro-intestinal tract, hypertension, and osteoporosis, whereas cyclosporine is nephrotoxic. Liposomes are phospholipid particles that allow delivery of drugs preferentially to the reticuloendothelial system. They can be prepared from phospholipids, such as lecithin from soybean or egg yolk, other specific or modified lipids or from membrane-spanning tetraether lipid (TEL), which can be extracted and purified from archaeal cell membranes. One advantage in the use of liposomal application is reduced toxicity of many drugs. We report on various liposomal preparations of cyclosporine, methylprednisolone (L-MPL) and its palmitate derivative (L-MPLP). It has been documented that liposomal cyclosporine A (L-CsA), 1.75 mg/kg/ day for seven days has potential for use as an immune-suppressive agent in rats with increased efficacy and decreased nephrotoxicity compared to commercially available forms of intravenous CsA. Liposomal methylprednisolone (L-MPL) 2 mg/kg, intravenously (IV), twice a week shows significantly prolonged cardiac allograft survival in rats and tissue-selective sequestration of the drug in comparison with the same dosage regimen of methylprednisolone in solution, administered daily. We report on organ distribution of L-MPLP in rats after intraperitoneal (IP) administration. Conclusion: Liposomal preparations of immunosuppressants have significantly higher immune-suppressive potential and lower toxicity than non-liposomal preparations. Bipolar TEL can be extracted, fractionated and purified from archaea to form stable liposomes which are extremely resistant, even to gastric fluid. Hence, TEL liposomes allow (besides IV and IP) for oral administration of immunosuppressants after organ transplantation with pharmacological and toxicological advantages over common liposomal phospholipid bilayer preparations.

Organ transplantations are among the major health problems worldwide and especially in developing countries. The incidence of the organ rejection after transplantation is still high. Applying ineffective doses of immunosuppressive agents may be part of the problem. Immunosuppressive drugs such as methylprednisolone (MPL) and cyclosporine A (CsA) exert tremendous side effects, especially because of high dosage and longterm application required for immunosuppression after organ transplantation. During the episodes of acute rejection, 500-1000 mg of MPL are given intravenously for several days. Because of the high dosage of glucocorticoids required for immunosuppression, side effects are common, such as bleeding of the GI tract, hypertension, and osteoporosis. [1][2][3] Moreover, long-term medication with MPL will disturb the endogenous corticosteroid regulation. Complications related to systemic immunosuppression are major causes of morbidity and mortality following organ transplantation. The major unwanted side effect of high dose and longterm application of CsA is nephrotoxicity. [1][2][3] Incorporation of therapeutic compounds into liposomes depends on the physicochemical properties of both the compound and the lipid. The most effective way for the incorporation of sufficiently lipophilic compounds is to mix them with the lipids from the beginning of preparation, so that they are preferentially inserted into the liposomal membrane via hydrophobic interaction. Normally, therapeutic concentrations do not require amounts of a respective compound too high to be taken up into the liposomal membrane by approximately 100% with this method. This was also shown for methylprednisolone and cyclosporine; however, liposomes prepared without special stabilizers lack stability and exert rapid loss of incorporated compound(s). Hence, a special aspect of this review is the stabilization of liposomes with incorporated immunosuppressants by means of archaeal tetraether lipid. This part will be described in detail.

Cyclosporine
Cyclosporine A (CsA) is a cyclic peptide found in Tolypocladium inflatum, Gams. It is an extremely hydrophobic molecule. As an immunesuppressive agent, CsA has a highly selective ability to inhibit the activation of T-cells. Although its site of action has not been defined precisely, it inhibits an early cellular response to antigenic and regulatory stimuli, primarily in populations of Helper-T-cells. Cyclosporine also causes a general reduction in the production and release of lymphokines in response to an antigenic stimulus. At higher concentrations, CsA inhibits expression of receptors for IL-2. Although CsA can inhibit the activation of Helper-T-cells, it does not prevent the stimulation of their clonal expansion by IL-2. It is potentially significant that CsA allows the expression of suppressor cell activity at concentrations that inhibit the induction of cytotoxic T-cells. 4

Liposomal Cyclosporine A
The first reports on liposomal CsA (L-CSA) go back to the eighties of last century. 5 In general, natural compositions of lecithin from soy beans or egg yolk were used, as well as defined lipid composition. [6][7][8] Liposomal CsA was reported less nephrotoxic. 9 In 1989, Vadiei et al. 10 compared two liposomal CsA formulations of dimyristoyl phosphatidylcholine (DMPC):stearylamine (SA) (7:1 molar ratio) and DMPC:dimyristoyl phosphatidylglycerol (DMPG) (4:1 molar ratio) for IV application, at optimal drug:lipid ratio of 1:20 (w/w) for both formulations. According to their evaluation, DMPC:SA is advantageous over the second formulation. 10 Later on, liposomes for CsA delivery have been prepared from more complex composition, phosphatidylserine/phosphatidylcholine, cholesterol sulfate, and lyso-phosphatidylcholine in a molar ratio of 3:4:2. 11 The authors reported higher efficacy and lower nephrotoxicity. Further characterization and pharmacokinetic studies including oral administration and intermembrane exchange were conducted. [12][13][14][15][16][17] The interaction and insertion of cyclosporine into biological and model membranes has widely been investigated and discussed. [18][19][20][21] Comparing the structure of cyclosporine, a lipophilic cyclic peptide with 11 amino acids with similar compounds that were investigated in bilayer and tetraether lipid (TEL) membranes, [22][23][24] valinomycin, a lipophilic cyclic peptide with 12 amino acids resembles cyclosporine most; however, alamethicin also exerts similar behavior, especially considering the interaction with cholesterol. Cyclosporine A interfered both with lateral lipid organization 19 and probably with cholesterol binding sites, because addition of cholesterol to liposomal membranes reduced their binding capacity from the maximum of one CsA molecule per 19 lipid molecules. 21 Although inserted deeply into the hydrophobic moiety of membranes, preferentially at the interface between fluid-crystalline and gel-analogous lipid domains, the single hydroxyl group of CsA may be oriented to and interact with the polar head groups of lipid molecules 20 and thus, with cholesterol binding sites. 25

Liposomal Preparations for Oral Administration
Liposomal preparations for oral cyclosporine delivery were investigated by several working groups. [26][27][28][29] Modified liposomes for the oral delivery of CsA have been tested in vitro and in rats. 30 Chitosan-modified liposomes (CS-Lip) were compared with Pluronic® F127-modified liposomes (PF127-Lip). Liposomes were prepared in a similar way as described above, finally extruded 6 times through polycarbonate filters of 200 nm pore size by means of a high-pressure homogenizer. [31][32] The composition was CsA, egg yolk phosphatidyl choline (EPC), and cholesterol at a 1:28:5 molar ratio. Pluronic® F127 (PF127), a non-ionic chemical polymer and cationic polysaccharide chitosan (CS), at a deacetylation degree of 94%, were added individually to yield hydrophilic non-ionic (PF127-Lip) and cationic (CS-Lip) polymer-modified liposomes. 30,33 The latter are known as mucoadhesive and the former as mucus-penetrating particles, 34 which both had been shown in animals to increase the intestinal absorption of loaded drugs versus the absorption of free drug and from unmodified liposomes.
Size stability of these particles was tested in artificially simulated gastric fluid (SGF, pH 1.2) for 2 hours and simulated intestinal fluid (SIF, pH 6.8) for 6 hours. Initial diameters were 165.25±9.28 nm of unmodified liposomes (Lip), 172.82±15.79 nm of PF127-Lip, and 207.81 ±12.21 nm of CS-Lip. In SGF, only PF127-Lip remained stable, whereas Lip significantly increased and CS-Lip significantly decreased in size. In SIF, Lip decreased in size, whereas PF127-Lip increased. Although significant these changes were still moderate within a range of less than 50 nm. On the contrast, CS-Lip changed tremendously in size, after 2 hours large aggregates precipitated and 40% of the liposome-loaded drug was released into the supernatant. 30 On the other hand, release profiles of all three preparations in ethanol-containing PBS within 24 hours were similar without burst phenomena indicating that CsA was well incorporated into the liposomes and not essentially influenced by the modifications. Entrapment efficiency was 85% in Lip, 82% in CS-Lip, and 90% in PF127-Lip, and release from all preparations was up to 85% of the incorporated CsA within 24 hours. 30 For the determination of the penetration into intestinal mucus and tissue in rats coumarin-6 was incorporated into the liposomes as hydrophobic model substance which allows for fluorophotometric measurement. Fluorescence was measured in the duodenum, jejunum and ileum and was highest in the mucus with CS-Lip and highest in the tissue with PF127-Lip. In other words, CS-Lip get stuck in the mucus, typical of mucoadhesive particles and don't reach enterocytes in intestinal tissue, which means that they are not suitable for effective oral drug delivery. In contrast, PF127-Lip are able to effectively deliver their drug load into the intestinal tissue. 30

Pharmacological Parameters of CsA and L-CsA
Oral bioavailability of CsA varies from 20 to 50 %. About 60-70 % of the drug in whole blood is contained in erythrocytes, 10-20 % contained in leukocytes. Cyclosporine has a plasma half-life of 5.6±2 hours and a plasma clearance of 5.9±1.0 mL/min/kg. The volume of distribution is 1.2±0.2 L/kg. Oral treatment with a dose of 15 mg/kg BW is initiated 4 to 24 hours prior to transplantation, once daily and continued for one to two weeks after transplantation. Thereafter, the dosage is reduced each week until a maintenance dose of 3 to 10 mg/ kg/day is reached. Dosage is generally guided by signs of renal toxicity, as judged from changes in creatinine clearance. 4 In the study of Chen et al., 30 intestinal absorption and bioavailability of liposomal CsA after oral administration of 10mg/kg BW were determined by the measurement of C max [µg/mL], T max [h], and AUC 0→t [µg . h/mL] in the blood of Sprague-Dawley rats. The values of CsA in plasma differed between PF127-Lip and CS-Lip roughly by a factor of two; C max (1.37 ± 0.15 vs. 0.79 ± 0.10) and AUC 0→t (11.59 ± 0.7 vs 6.30 ± 097) were significantly higher and T max (1.68 ± 0.29 vs. 3.67 ± 0.58) significantly lower in PF127-Lip than in CS-Lip; the values with unmodified liposomes were in between. 30 Cyclosporine encapsulation using glyceryl monooleate-poloxamer 407 nanoparticles (poloxamer 407 is identical with Pluronic® F127) yielded 85% encapsulation efficacy and higher relative oral bioavailability in beagle dogs of about 178% versus Sandimmun Neoral as reference. 40 The relative oral bioavailability of L-CsA encapsulated in soy phosphatidylcholine (SPC) liposomes containing sodium deoxycholate instead of cholesterol was slightly more than 120% as compared with cholesterol-containing SPC liposomes or Sandimmun Neoral. 31 If CsA cannot be administered orally, it must be infused slowly over a period of 2 to 6 hours or longer. The daily dose should be only onethird the oral dose (usually 5-6 mg/kg BW). 4 Two hours after IV injection of L-CsA in rats at a single dose of 2 mg/kg, the distribution was increased two-fold in liver and spleen tissues compared to CsA in saline. Intravenous administration of 10 mg/kg CsA in saline was demonstrated to exhibit exaggerated nephrotoxicity in renal ischemia induced by contralateral nephrectomy in rats, whereas serum creatinine in animals, which had received liposomal CsA, returned to control levels within 96 hours. In liver transplant models in rats, the dose of 1.75 mg/kg of L-CsA resulted in significantly prolonged survival rates for about 93 days as compared to the group, which had received the same dose of CsA in saline. 11

Dexamethasone
Dexamethasone is a glucocorticoid, which has the capacity to suppress immune responses. Although considered to be immune suppressive, therapeutic doses of glucocorticoids do not significantly decrease the concentration of antibodies in the circulation. The immune response is initiated by the interaction of an antigen with macrophages and with antibodies on the surface of B-lymphocytes. Glucocorticoids interfere with the function of macrophages in several ways: firstly, they inhibit the action of MIF (migration-inhibitory factors), thereby promoting the emigration of macrophages from affected areas; secondly, they inhibit the processing and display of antigens by interfering with the facilitating actions of a-interferon; thirdly, they inhibit the synthesis and release of IL-1. More importantly, IL-1 participates in the activation of resting T-lymphocytes when they come in contact with processed and histocompatibility antigens displayed on the surface of activated macrophages. Hence, glucocorticoids suppress the activation of T-cells by several mechanisms. Recently, dexamethasone has come into the focus with SARS-CoV2 infections and COVID-19, 41 but this is not the topic to be discussed here.

Liposomal Dexamethasone
Tanaguchi et al. 42 compared the incorporation rates of dexamethasone (DM), dexamethasone acetate (DA), dexamethasone valerate (DV), and dexamethasone palmitate (DP) into liposomes at a ratio of 16 mM lipid and 0.5 mM steroid (or its respective derivative), which is equivalent to 3.125 mol% steroid. They determined the water solubility in physiological buffer pH 7.4 at 37 o C as 80.5, 29.6, 1.3, and 0.052 mg/L, respectively. Chloroform/water partition coefficients (PC) were 9, 657, 8780, and not measurable for DP, which values are comparable to DM logP 0.95, DA logP 2.8, DV logP 3.9 and a higher, not measurable, value for DP. 42 The authors used sonication and subsequent filtration through a polycarbonate filter with one µm pore width. Incorporation rates were increasing (slightly depending on sonication time between 2.5 and 20 min), 90.5-92.0 % (DM), 94.7-96.0 % (DA), 99.5-99.6 % (DV), and 100% for DP. Various lipid compositions comprised egg yolk lecithin (EYL), dioleoyl-phosphatidylcholine (DOPC), stearylamine (SA), diacetyl-phosphate (DCP) with similar incorporation rates; only dipalmitoyl-phosphatidylcholine (DPPC) had lower DM incorporation rates of 78.5%. 42 The authors also investigated the influence of cholesterol on the incorporation of steroids (DM and derivatives): cholesterol up to 4 mM concentration increased free steroid in the preparations concentration-dependently from 20 to 40 mg/L (DM); from slightly above 10 to 20 mg/L (DA); much less with DV (below 3.5 mg/L); and not at all with DP (0 mg/L). 42 It was concluded that steroids interact differently from DP with lipids in the membrane and that cholesterol competes with them. 43 Steroids may be oriented horizontally, so that the hydroxyls remain hydrated, whereas DP may be inserted into the membrane perpendicularly via its palmitate side chain, similar to cortisone-21palmitate. 44,45

Prednisolone and Methylprednisolone Liposomal Prednisolone
Prednisone and Prednisolone are mainly used for the treatment of rheumatic and atherosclerotic diseases. Liposomal preparations have been reported with prednisolone, 46 liposomal preparations have not been applied for immunosuppression and are therefore not discussed here.

Liposomal Methylprednisolone (L-MPL)
Methylprednisolone is a glucocorticoid with immunosuppressive activity similar to what was already described for dexamethasone. Mishina et al. considered the optimum formulation being EPC and phosphatidylglycerol (PG) at molar ratio of 9:1 and 5 mol% MPL. 49 The authors refer to [50][51][52] and consider 10% of negatively charged lipid in the EPC membrane best compromise for incorporation of MPL into and its retention in liposomal membranes. Liposomes were stable overnight, but lost 70% of MPL within one week. 49 Therefore, it was decided to stabilize L-MPL with archaeal tetraether lipid. Liposomal MPL was prepared from EPC (= EYL) and phosphatidylglycerol (9:1 molar ratio) with 5 mol% of MPL. 53,54 Ethanolic solutions of these mixtures (20mg/mL) were evaporated under reduced pressure (250 mbar) in a round-bottom flask at 40 o C on the water bath of a Büchi Rotavapor. The dry lipid film was stored at RT for 12 hours in a vacuum desiccator in order to remove residual moisture. Subsequently, the lipid film was suspended in PBS, pH 7.4, at an amount to result in 15-20 mg of lipid mixture per mL of buffer. This suspension was shaken by hand with two glass beads added into the flask. The resulting suspension of large multilamellar vesicles (LMV) was sonicated and extruded (5 to 7 times) through Liposofast® polycarbonate filters (pore size 100 nm). 55 To separate free (not encapsulated) drug, the liposomal suspension was applied to an open Sephadex G-75 chromatography column. All liposomal preparations were routinely checked in a Malvern Particle Sizer and several representative preparations in addition by electron microscopy. 24 The polycarbonate filter extrusion resulted in unilamellar vesicles of roughly 100 nm in diameter, different preparations varied from above 50 to 120 nm, but each single preparation had to be uniformly size-distributed in order to be used for further investigation.
For the incorporation of MPL into liposomes, phosphatidylglycerol (PG) was admixed to EPC at 10 mol% or (instead of PG) TEL extracted and purified from Thermoplasma acidophilum (T.a.) at 2.5 or 10 mol%; in addition, pure TEL liposomes (100 mol%) were used. In all cases, 5 mol% MPL were applied for liposomal incorporation resulting in molar liposomal ratios of 9.4-9.5:0.5-0.6 (lipid:MPL), equivalent to incorporation ratio of about 100% in all cases and 0.5-0.7 mg MPL per mL of liposomal suspension. Since liposomes prepared according to this method lack stability (they must be freshly prepared for each test series), Oertl et al. 56,57 and Bräutigam et al. 58,59 suggested stabilization of L-MPL preparations with TEL according to Freisleben et al. 60 (Table 1). The steroid MPL is effective when given orally; its bioavailability is 82 ± 13 %. In the plasma, 90% or more are reversibly bound to proteins (albumin and globulin) under normal conditions. MPL clearance is 6.2 ± 0.9 mL/min/kg. 4 MPL has a short plasma half-life, but the liposomal formulation L-MPL markedly prolonged plasma circulation time and led to sequestration of the steroid into the lymphatic tissues. Terminal halflife was dramatically extended from 1.5-2.5 hours for MPL to more than 30 hours for L-MPL (Table  2). 49 The distribution volume of L-MPL 1-2 hours after intravenous injection of a 2 mg/kg bolus in rats was significantly increased from 1.2 ± 0.2 L/ kg of MPL in lymphatic tissues: in spleen 77-fold; in thymus 27-fold and in liver 9-fold. In liver and spleen MPL remained detectable for 26 days after L-MPL injection confirming the tissue-selective sequestration of the drug. 49 Male Lewis RT1 rats served as recipients of cardiac allografts from Lewis x Brown Norway F 1 (LEWxBN)F1 hybrids. 53,54,56,57,61 Hearts were anastomosed to the abdominal great vessels using standard microvascular techniques. Ventricular contractions were assessed daily by palpation, and rejection was defined as the day of cessation of heartbeat. 53,56,57,59,61 The dosage of L-MPL of 2 mg/kg BW, intravenously twice a week led to cardiac allograft survival of 20.8 ± 6.5 days (maximum 30 days), whereas with the same dose of non-liposomal MPL (from a conventional commercial preparation) the allograft was rejected after 7.8 ± 1.0 days, similar to the untreated control group (9.2 ± 1.2 days). The dosage of 4 mg/kg BW, intravenously once a week, also significantly prolonged cardiac allograft survival in rats to 17.5 ± 2.8 days, which was comparable with daily administration of 2 mg/kg BW of MPL in solution (17.0 ± 2.7 days). Only the daily injection of 4 mg/kg BW of nonliposomal MPL (i.e., seven times higher dosage) led to results comparable to the treatment with liposomal MPL. 53,61 Bilaterally nephrectomized LEW rats were recipients of BN kidney transplantation (Tx). 54,58,59 L-MPL was administered IV, 2 mg/kg BW, twice a week or 4 mg/kg BW, once a week. Distribution of L-MPL was determined in liver, spleen and thymus between one and two hours after injection. 49 Renal allograft survival with 4 mg/ kg BW of L-MPL (IV once a week) significantly prolonged allograft survival to 20.2 ± 7.4 days (comparable with daily administration of 4 mg/ kg MPL in solution: 19.0 ± 6.8 days) vs. acute rejection of 8.5 ± 0.5 days (p < 0.001). MPL + empty liposomes injected separately once a week was ineffective (8.8 ± 0.5 days). 54

Liposomal Methylprednisolone Palmitate (L-MPLP)
Methylprednisolone-palmitate (MPL-P) is a prodrug synthetized for the same reason as the derivates of dexamethasone (DA, DV, DP), in order to increase and stabilize their incorporation into liposomal membranes; in case of EPC alone MPL-P was incorporated at a maximum of 79%. The addition of 2.5 mol % of TEL from Sulfolobus acidocaldarius (TEL-S.a.) increased the incorporation rate to approximately 95% (range 94-97%) at concentrations of 4-10 mol% added to the original suspension. TEL itself was incorporated at a rate of 85-86% (Table 3). 62 In detail, the incorporation of 2. Particle size stability of the liposomes was determined; L-MPLP is stable at 20°C for 21 days, although the size tends to become larger and size distribution broadens. EPC-MPLP liposomes are less stable (Table 4, data collected from 63,64 ).

Pharmacological Parameters
Organ distribution of MPL/MPLP was measured as µg/g tissue after IP injection of L-MPLP (data collected from 65 ).   Suppression of TNFa levels was determined in in-vitro and ex-vivo lymphocyte cultures. 66 The percentages of TNFa levels in each group were compared to controls (= 100%); control groups: Tris buffer 5 ml/kg BW; MPL at 0.005, 0.05, and 0.5 mM MPL sodium succinate concentrations; L-MPLP at 0.005, 0.05, and 0.5 mM MPLP concentrations in liposomes; For the in-vitro cultures, the TNFa levels were measured using the splenic lymphocyte cultures. For the ex-vivo cultures, experimental CH3-mice were sacrificed 48 hours after the drug administration, spleens removed and used for lymphocyte cultures. 66

Liposome Technology
Liposomes as vehicles or carriers for pharmaceuticals already brought about   68 because the liposomal application of lipophilic antimycotic amphotericin B is much better tolerated and is more effective than IV administered non-liposomal preparations. 69 Other liposomal formulations are already successful with doxorubicin in cancer therapy. [70][71][72][73] Moreover, liposomes exert also immunoadjuvant properties for several antigens in vaccine design 74 in that they are capable to induce both lymphokine production and potent humoral response characterized by increased production of IgM and IgG2a. 75 There are various mechanisms of interaction between liposomes and cells or cell membranes. The most promising and effective one appears to be intermembrane exchange, [18][19][20][21]76 but also fusion processes between liposomes and cell membranes and total uptake of liposomes via endocytosis play important roles in liposomal delivery systems. The surface of liposomes can be modified and adjusted to cell or tissue targeting or therapeutic aims. 22 Liposomes from archaeal lipid are extremely stable towards acidic environment; hence, they could well be used for oral or local application in the GI tract, 60,77 or instillation into the urinary tract. 60 Archaeosomes are liposomal formulations based on natural archaeal 78 or synthetic TEL. 79,80

Miscibility of TEL with Other Components
In differential scanning calorimetry (DSC) and differential thermo-analysis (DTA) experiments with highly purified TEL from T.a., it was found that -except for methyl-branched ether bond diphytanylglucosylglycerol which exerts unlimited miscibility 81 -it depends on the phase status of the lipids whether they are able to form uniform mixed phases or undergo some phase separation with domains of higher and lower contents of TEL. 82 TEL and bilayer-forming lipids with this kind   86 Mixed phases of DPPC with TEL at higher ratios of DPPC:TEL appear more homogeneous than higher ratios of TEL:DPPC. 82 This is consistent with observations of the stability of mixed liposomes; higher ratios of EPC (= EYL) or SBL:TEL form more stable liposomes than higher ratios of TEL:lecithin. [87][88][89] It was postulated that TEL should not exceed 25 % in EPC/SBL liposomes, equivalent to 12 mol%. 60,90 Moreover, liposome stability increases with higher purity of TEL, [87][88][89] as was demonstrated with the polar   membrane fraction extracted from T.a. and main (glycol)-phospholipid highly purified from this fraction. 91 Stabilization of lecithin liposomes with 2.5 mol% up to 5 mol% phosphatidic acid or other negatively-charged phospholipids has widely been used in liposome technology. 92 Ten mol% of negatively charged lipid was found best for the incorporation and retention of MPL in lecithin liposomes. 49 Similarly, 2.5% and 10% of negatively-charged TEL results in stable liposomes not differing much between each other.

Liposomal CsA
Many liposomal preparations and modifications have been tested in experimental animals for both, IV and oral administrations.
So far, tetraether lipid has not been applied for liposomal encapsulation of CsA, but for oral delivery of other therapeutic peptides. 77 Hence, future studies on L-CsA should include TELstabilized liposomes.

Liposomal MPL
Optimal results were obtained with EPC, 2.5mol% or 10mol% TEL and 5mol% MPL, with respect to the incorporation rate and stability as measured through loss of MPL within one week. Stability as measured through size distribution was best with liposomes of highly purified TEL from T.a. according to Antonopoulos et al. 91 Since highly purified TEL is expensive, it was suggested to use 2.5 mol% for stabilization instead of 10 mol%. Highly purified TEL-T.a. from strain DSM 1728 was tested for its toxicity and mutagenicity and considered safe; it was neither cytotoxic nor mutagenic, 93 nor toxic to animals. 94 Archaeosomes from total polar lipid extracts of archaea were also shown not to be toxic and well tolerated in mice. 95

Liposomal MPLP
Addition of TEL-S.a. to stabilize EPC liposomes also improves the incorporation of MPLP. This MPL derivative inserts differently into liposomal membranes compared to the original compound MPL, the fatty acid residue functioning like an anchor into the hydrophobic depth of the membrane. The insertion into and orientation in the membrane resembles rather cholesterol. Among all liposomal formulations investigated in this study, the mixture of MPLP with EPC and TEL-S.a. resulted in the most stable pharmaceutical formula. [62][63][64] Pharmacological Data Table 8 compares liposomal and nonliposomal formulations of CsA and MPL including a dextran-MPL formulation in animal transplantation experiments.
Non-liposomal MPL was injected alone and combined with the simultaneous, but separate injection of empty liposomes in the same dosage (4 mg) and frequency (weekly) as the liposomal formulation. The survival time of kidney allograft was the same with or without the simultaneous injection of empty liposomes (8.8 ± 0.5 days). Only the daily injection of 4 mg MPL resulted in longer survival of the allograft (19.0 ± 6.8 days), however with the seven-fold dosage as compared with the liposomal preparation, which exerted allograft survival of 20.3 ± 7.4 days with a maximum survival of even more than 80 days. 54 summary and Highlights I n c e l l c u l t u r e a n d a n i m a l experiments, liposomal preparations improve both pharmacokinetic and pharmacodynamic parameters.
• Liposomal MPLP reduces TNFa in lymphocyte cultures better than non-liposomal MPL, both in vitro and ex vivo experiments.
• The pharmacokinetic profile of L-MPL is essentially improved over non-liposomal MPL.
• Allograft survival is considerably prolongated with liposomal preparations as compared to nonliposomal drugs.

Lipids and Liposome Technology
Since part of the original reports on MPL and MPLP in TEL-containing liposomes have been published in Indonesian language or are otherwise not easily accessible, materials and methods used in these experiments are presented here in detail.
Methylprednisolone and all other chemicals were purchased from Sigma-Aldrich, Singapore or Munich, Germany and from Merck-Darmstadt, Germany or from their Indonesian subsidiary, each at highest purity available. Methylprednisolone palmitate was synthetized by Bernina GmbH Munich, Germany.
Polar archaeal tetraether lipid (TEL) used for encapsulation of MPL and MPLP was obtained from different sources: i) from Sulfolobus acidocaldarius (S.a., Bernina GmbH, Munich), ii) from T.a. stem 1728, 97 purified yielding 99% pure 2,3,2',3'-tetra-O-dibiphytanyl-di-snglycerol-1'-gulosyl-1-phosphoryl-3''-sn-glycerol = gulopyranosyl-(â1-1)-caldarchaetidyl-glycerol. 91 In preliminary studies, TEL was also used from "crude" extract of the polar membrane fraction of T.a. mainly containing glycopyranosylcaldarchaetidyl-glycerol after one column passage to remove brownish membrane dyes. 91,98 The polar (liposome-forming) lipid fraction E (PLFE) from S. a. contains 90% of caldito-glycerocaldarchaeol (glycerol dibiphytanyl calditol tetraether, GDNT*) with phosphatidylmyoinositol ester at the glycerol of one end and ß-D-glucopyranose at the calditol moiety of the other end and 10% of glycerol dibiphytanyl glycerol tetraether (GDGT) with phosphatidylmyoinositol ester at the glycerol of one end and ß-D-galactopyranosyl-ß-D-glucopyranose at the glycerol of the other end. [99][100][101][102] The number of pentacycles varies in all these tetraether lipids. (*Note: GDNT = glycerol dibiphytanyl nonitol tetraether was the original term, until it was proven that the chemical structure of this residue is not nonitol but calditol. However, the original abbreviation is still used in scientific work). [99][100][101][102] Liposomes are spherical phospholipid particles that allow for drug delivery. These vesicles can be prepared from defined phospholipids in various formulations. Irrespective of the preparation method, the lipid mixture and the respective drugs were dissolved in chloroform/methanol (2:1 v/v). The solvent was removed by a rotary evaporator at about 40 o C-50 o C. The dry lipid film was subjected to a high vacuum (18)(19)(20) in a desiccator at room temperature for at least twelve hours to remove chloroform, methanol and water residues. In the next step, the dry film was suspended in PBS (phosphate buffered saline, pH 7.4) by handshaking or vortexing to a final lipid concentration of 10-20 mg/mL. 92,103 To obtain smaller liposomes and more homogenous dispersion, in a subsequent step, the hand-shaken suspension was extruded through 100 nm polycarbonate filters 55,104 which served also as sterile filtration, because heat sterilization is limited to heat-stable TEL.
For experimental reasons, extrusion was conducted between 5 and 21 times. In addition, size reduction of the liposomes could be obtained by sonication 105 prior to extrusion. In general, the preferred preparation was extrusion through polycarbonate filter (without sonication) for 5-7 times, if not mentioned differently.
After preparation, the liposomal suspension was centrifuged at 3000 rpm for 10 minutes to remove non-liposomal material. The liposomes were contained in the supernatant which was subsequently passed through Sephadex G-75 column (gel filtration chromatography, GFC). In general, there were no significant differences between centrifuged and non-centrifuged preparations after GFC. Hence, the centrifugation step was skipped as a normal procedure and only carried out exceptionally, in case of suspected nonliposomal contamination.
Mixed liposomes were also prepared by various methods 24,92 in order to achieve optimal incorporation of the components, such as TEL, MPL, and MPLP. In all cases, liposomes were extruded through polycarbonate filters with 100 nm pore size. Subsequently, liposomes were passed and fractionated through Sephadex-75 (GFC) into 0.5 mL or 1.0 mL fractions. Table 9 presents three measurements of EPC liposomes and mixed EPC:TEL liposomes with or without sonication and with extrusion through a polycarbonate filter with 100 nm pore size. Shelf stability of liposomes from egg phosphatidylcholine (EPC) = egg yolk lecithin (EYL), TEL and various mixtures from both had been compared, 24,90 at 20 o C EPC liposomes are only stable for less than a week, at 4-8 o C slightly longer, between one week and one month (depending on the conditions; apart from confluence to larger size EYL liposomes tend to be contaminated within this time), under very cautious germ reduced storage conditions at 4 o C the longest observation of EYL liposomes with initially 159 nm in diameter was size stability up to 66 days (final size 149 nm). 24 Shelf stability of TEL liposomes of about 150 nm in diameter was 109 weeks at temperatures up to 37 o C (size variation between 155 and 165 nm) and from 60-100 o C TEL liposomes were stable for 10 weeks (size variation between 150 and 177 nm). 90 Liposomal stability in terms of leakiness was also determined by carboxyfluorescein release within 24 hours at pH 7.4 and elevated temperatures (Table 11). 56,57,76 Liposomal swelling rates, which can be considered as osmotic stability, were determined at 30 o C by absorbance measurement at 570 nm, under addition of isotonic glycerol-containing buffer medium. Within 15 min, liposomes of TEL-T.a. had a swelling rate of 3%, EPC liposomes of 68%, and EPC liposomes containing 10 mol% of TEL-T.a. had a swelling rate of 16%. 106

incorporation of MPLP
Liposome suspensions were formed with input concentrations of 1.7 mM and 3.4 mM EPC, 2.5 mol% TEL, and 2-10 mol% MPLP. Best results were obtained with 4-5 mol% of MPLP. In liposome suspensions containing MPLP in 1.7 mM EPC, the highest amounts of EPC and MPLP were found in 1.0 mL GFC fractions 2-4 with the distribution value of MPLP in fraction 3 around 40-43% (43.1% at MPLP 4 mol%, and 40.0% at MPLP 5 mol%) and in liposomal mixtures containing TEL, distribution value in fraction 3 was 47% at MPLP input of 4-5 mol%. 63,64 Figure 2 shows thin layer chromatography (TLC) of TEL-and MPLPcontaining mixed liposomes before and after GFC.
Loss of liposomal components through the polycarbonate filter extrusion was determined by 11-21 filter passages. Extrusion through the filter of 100 nm pore size and subsequent GFC through Sephadex G-75 column revealed that the amount of lipid, EPC (without TEL) and MPLP incorporated into the liposomal membrane decreased by 40-46%, while in the combination of EPC with 2.5 mol% TEL components were only reduced by up to 12% after 21 extrusion steps and after 11 extrusion steps even less than 10% (2.5-8%). Fractions collected after GFC were either 0.5 or 1.0 mL, each. The concentrations of MPL, MPLP and TEL were subsequently detected by TLC (Fig.  2), EPC was determined spectrophotometrically at wavelength l= 490 nm after enzymatic reaction. [62][63][64] Percentage of MPLP distribution in EPC liposomes (Table 12) and EPC/TEL liposomes (Table 13) was determined in 0.5 ml fractions 5-7 after GFC fractionation. [62][63][64] In liposomes containing EPC and TEL 2.5 mol%, the input composition of EPC, TEL, and 5 mol% MPLP was 3.4:0.08:0.17 mM concentration; after extrusion, 11 times through the 100 nm polycarbonate filter, the composition was reduced by 2.9% to 3.28:0.077:0.16 mM concentrations. Distribution after GFC fractionation was 48.2% for EPC, 41% for TEL, and 47.1% for MPLP in fraction 6 (fraction volume 1 mL).
In EPC:TEL:MPLP input composition of 3.4:0.08:0.34 mM, the content of each component was reduced by 8% to 3.13:0.074:3.128 mM after extrusion and the distribution of the components after GFC fractionation was EPC 54.8%, TEL 52.8%, and MPLP 45.5% in fraction 6. Halving the same ratio of components to 1.7:0.04:0.17 mM input concentrations reduced the liposomal components by 2.7% after 11 times of extrusion through the polycarbonate membrane. The distribution after GFC fractionation was EPC 49.6%, TEL 44.9% and MPLP 47.5% in fraction 6. [62][63][64] Incorporation of TEL and MPLP in fractions with the highest amount of EPC (determined enzymatically) before and after GFC fractionation ranged for TEL before 0.05-0.08 mM, after fractionation 0.03-0.06 mM, with an incorporation rate of 50-82% and MPLP before 0.16-0.19 mM, after fractionation 0.12-0.18 mM with an incorporation rate of 73.6-96.8%. Incorporation could be improved to TEL before 0.1-0.14 mM, after fractionation 0.07-0.12 mM with an incorporation rate of 65-88%; MPLP before 0.48-0.54 mM, after fractionation 0. 46

COnCLusiOns
Various nanoparticles have been developed for effective delivery of hydrophobic immunosuppressants such as abovementioned chitosan particles or silica-coated iron oxide nanoparticles. 107 However, liposomes or modified liposomes appear the most frequently used formulations for modern delivery of immunosuppressive drugs.
Liposomal CsA, MPL, and MPLP were shown to exert pharmacological advantages in immunosuppression in lymphatic tissue cultures 66 and in experimental animals, in particluar after organ transplantation. Lymphatic tissue selectivity 49 plays certainly a major role in liposomal drug delivery formulations. For liposome-delivered MPL extended receptor binding adds to enhanced immunosuppression. 49 Liposomes by themselves appear to exert adjuvanticity. 75 Moreover, liposomal drug transfer to transplants (possibly even prior to transplantation in terms of preconditioning) could directly modify or shelter MHC and surface antigens in order to prevent immunogenic attack without general immunosuppression. Liposomal preconditioning might also be a possibility to prevent reperfusion injury in transplants.
Improved pharmacokinetics of TELcontaining liposomes or pure TEL-liposomes 59 have not yet been demonstrated versus freshly prepared EPC liposomes, but can be expected for oral administration of immunosuppressants, which may also have a positive impact on the pharmacodynamic efficacy. 108 The already proven advantages of TEL-containing liposomes are better incorporation rates of MPLP 62 and higher stability with incorporated MPL 57,59 compared to EPC liposomes.

Author Contributions
Conceptualization This review received no external funding.

Conflicts of Interest
The authors declare no conflict of interest. The abovementioned funders had no influence on the writing of the manuscript, or on the decision to publish this review.