Liposomes for the treatment of intra-hepatic stage of malaria
To treat sporozoite-induced murine malaria, Alving et al
. [
163] engineered liposomes containing ceramide alone or neutral glycolipids such as cerebrosides (galactosylceramide, glucosylceramide, sulfogalactosylceramide), gangliosides (GM1, lactosylceramide) and phosphocholine ceramide (sphingomyelin) (see Table
2 for the different compositions). Interestingly, drug-free liposomes containing galactosyl-, glucosyl- or lactosylceramide interfere with the malarial life cycle during the liver stage and prevent the appearance of erythrocytic forms of malaria in mice infected with sporozoites [
163]. In fact, up to 85–95% of sporozoites-infected mice treated with these liposomes were cured, while the cure rate recorded for the untreated mice was about 20%. On the other hand, liposomes containing sphingomyelin, sulfogalactosylceramide, ganglioside GM1 or ceramide alone exhibited no anti-malarial efficacy in mice. Moreover, all the glycolipid-liposomes had no effect on intra-erythrocytic stages of malaria in mice. These phenomenon may be explained by the fact that certain membrane glycolipids are associated with mammalian cells via glycolipid-lectin interactions and fusion thereby likely resulting in competition of liposomes (lipid phase) with sporozoites for the same receptor [
163]. When primaquine was encapsulated in these galactosyl glycolipid-bearing liposomes for targeting hepatocytes via a galactose-binding lectin, its efficacy was enhanced by more than 46,000 times, thereby achieving a curative effect in mice at reduced doses [
164]
.Table 2
Composition of liposomes used as active targeting drug delivery systems in malaria therapy
– | DMPC: cholesterol: dicetylphosphate PC: sphingomyelin: cholestrol: dicetylphosphate | 1:0.7:0.11 0.8:0.2:0.75:0.11 | Glycolipids | |
– | EPC: cholesterol: gangliosides | 20:20:4 | Anti-rat erythrocyte F(ab’)2 | |
– | EPC: cholesterol | 1:1 | Tuftsin derivatives | |
Chloroquine | EPC: cholesterol: gangliosides | 20:20:4 | Anti-mouse erythrocyte F(ab’)2 | |
Chloroquine | PC: PS: cholesterol PC: PS: cholesterol: MPB-PE | 9.5:1:10 9.5:1:10:0.5 | Anti-mouse erythrocyte F(ab’)2 | |
Chloroquine | EPC: cholesterol: gangliosides | 20:20:4 | Anti-mouse erythrocyte F(ab’)2 | |
Chloroquine | EPC: cholesterol: gangliosides | 20:20:4 | MAbF10 MAbD2 | |
– | Lipid di22: 1-PC(1,2-dieucoyl-sn-3-PC), Lipid-PEG-conjugate di22: 1-PE-PEG5000(1,2-dierucoyl-sn-3-(PEG5000)-PE, Lipid-PEG-peptide-conjugate di22: 1-AP-PEG3400-peptide(1,2-dierucoyl-sn-3-(PEG3400-succinyl-peptide)-aminopropane, Fluorescent lipid di22: 1-AP-Bodipy-TR-X(1,2-dierucoyl-sn-3-(Bodipy-TR-X)-aminopropane) | 82:10:4:4b 87:7:2:4 91:4:1:4 86:10:4:0 | 19-Aminopeptide from the CSP of P. berghei | |
Chloroquine Fluorescent probe pyranine 655 ITK® carboxyl quantum dots | DOPC: cholesterol DOPC: cholesterol: MPB-PE | 80:20 77.5:20:2.5 80:20 | BM1234 | |
Chloroquine Fosmidomycin | DOPC: cholesterol DOPC: cholesterol: MPB-PE | 80:20 77.5:20:2.5 | BM1234 | |
Chloroquine | DOPC: cholesterol DSPC: cholesterol DOPC: cholesterol: DSPE-PEG-Mal DSPC: cholesterol: DSPE-PEG-Mal DOPC: cholesterol: MPB-PE | 80:20 90:10 75:20:5 85:10:5 65:20:15 | Anti-MAHRP121-40 Anti-HRP2 Anti-GPA | |
– | DSP: cholesterol: DSPE-mPEG | 2:1:0.1 | PSP | |
Lumefantrine | DSPC: cholesterol: DSPE-mPEG2000 | 85:10:5 | NTS-DBL1α N-terminal domain of a rosetting PfEMP1 | |
Chloroquine, 7c, 7d (4-aminoquinoline compounds) Primaquine Quinine, BCN-01, BCN-02 (aminoalcohol compounds) Tafenoquine | DSPC: cholesterol: DSPE-PEG2000-Mal DOPC: cholesterol: DSPE-PEG2000-Mal | 85:10:5 | Mouse monoclonal IgG2b anti-human GPA (SM3141P) Rat monoclonal IgG2b anti-mouse TER-119 (AM31858PU-N) | |
Primaquine | DOPC: PE: cholesterol: DOTAP | 46:30:20:4 | Heparin | |
Pyronaridine + Atovaquone | DOPC: cholesterol: DSPE-PEG2000 POPC: cholesterol: DSPE-PEG2000 DSPC: cholesterol: DSPE-PEG2000 | 75:20:5 | Anti-GPA | |
Poupartone B | DOPC: cholesterol: DOTAP | 76:20:4 | Heparin | |
Taking into account the above-mentioned results, Gupta’s research group designed liposomes decorated with tuftsin and its derivative for the treatment of malaria liver stage [
107,
166]. The hydrophilic tuftsin, a natural tetrapeptide (threonine-lysine-proline-arginine) and its hydrophobic derivative (Thr-Lys-Pro-Arg-NH-(CH
2)
2-NH-CO-C
15H
31) are macrophage activators [
107,
166]. As previously reported, activated macrophages showed enhanced killing activity on intraerythrocytic malarial parasites [
25,
107,
144,
166,
183]. Interestingly, pre-treatment of
P. berghei-infected mice with the hydrophobic derivative of tuftsin (50–100 µg/animal, intravenous administration on days − 3 to 0) conjugated to EPC-cholesterol liposomes (1:1, molar ratio) exhibited higher prophylactic anti-malarial efficacy than that of free tuftsin derivative [
166]. Indeed, parasitaemia and mortality rate in the group of mice pre-treated with liposomal tuftsin decreased significantly, as compared to those pre-treated with either unloaded-liposomes or free tuftsin derivative. Surprisingly, curative treatment with these tuftsin-bearing liposomes (50–100 µg of ligand/animal on days 0 to + 3) did not confer much efficacy [
166].
Longmuir, Robertson and colleagues engineered dierucoyl phosphatidylcholine (DEPC)-based liposomes decorated with the 19-amino-acid sequence of the circumsporozoite protein (CSP) [
172]. The composition of these liposomes is shown in Table
2. They consisted of lipid, lipid-PEG-conjugate, lipid-PEG-peptide conjugate and fluorescent lipid (82:10:4:4, molar ratio). It should be remembered that the intra-hepatic malarial infections and the malaria recrudescence caused by sporozoites are, inter alia, attributed to the 19-amino-acid sequence of the circumsporozoite protein (CSP) exhibiting a liver-targeting specificity. Located at the surface of liver-stage parasite, this protein can bind to hepatocytes via the heparan sulfate proteoglycans (HSPGs) [
184,
185]. When administered intravenously into mice, the developed peptide-containing liposomes were rapidly cleared from the circulation and were recovered almost entirely in the liver (>80%) [
172]. The accumulation of these liposomes in the liver was several 100-fold higher compared to heart, lung and kidney, and more than tenfold higher compared to spleen [
174].
In another study, Longmuir, Robertson and co-workers evaluated the influence of composition of 19-aminopeptide-containing liposomes in their specificity to target liver. So, the authors systematically varied the mole fractions of lipid (82–91 mol%), lipid-PEG-conjugate and lipid-PEG-peptide conjugate (see Table
2) [
173]. The selected formulations exhibited effective liposome targeting to the liver, with approximately 80% of the total injected dose recovered in the liver within 15 min, in agreement with previous results [
174]. Moreover, uptake of these liposomes by liver cells was more than 600-fold higher than uptake by those in the heart, and more than 200-fold higher than uptake by lung or kidney cells. Additionally, this targeting to liver was effective upon repeated (up to three) administrations to the host at 14-day intervals [
173].
Liposomes for the treatment of blood stage of malaria
In the 1980s, Gupta and his group initiated, for the first time, a vast project based on the development of antibody-mediated targeting of liposomes to red cells. In one of their first studies, they prepared anti-rat erythrocyte F(ab’)
2 bearing liposomes from EPC, cholesterol and gangliosides (see Table
2) [
165]. After intravenous injection in rats, the covalent attachment of F(ab’)
2 enhanced specifically the binding of liposomes to erythrocytes. Indeed, about 20% of these target-specific liposomes were associated with the erythrocytes [
165]. This binding was extended up to 3 h, reducing the blood clearance rate of the liposomes without affecting the survival time of the erythrocytes. Additionally, an appreciable decrease in the uptake of these F(ab’)
2-bearing liposomes by no target tissues (for example liver, spleen and plasma) was observed [
165].
As a result of these promising outcomes, Gupta and co-workers evaluated whether anti-mouse erythrocyte F(ab’)
2 bearing liposomes could be effective as vehicles for delivering chloroquine to erythrocytes in vivo [
167]. After administration to
P. berghei-infected mice, 15–20% of the injected dose of liposomes coupled to cell-specified antibody bound to the erythrocytes [
167], which agreed with previous results [
165]. Moreover, 20–30% of these cell-targeting liposomes deliver their contents to the target cells [
167]. Despite their poor antibody recognition of target cells, a single 5 mg/kg dose of chloroquine loaded anti-erythrocytes F(ab’)
2-bearing liposomes was found to be more effective than F(ab’)
2-free liposomes and free chloroquine in suppressing both chloroquine-sensitive and chloroquine-resistant
P. berghei infections in mice [
167,
171]. Additionally, this targeted liposomal formulation increased the therapeutic efficacy of chloroquine and prolonged the survival time of the treated animals at least up to day 12 post-chloroquine-resistant infection [
167,
171]. This confirmed the ability of these antibody-targeted liposomes to partly concentrate the drug in erythrocytes. Of note, Crommelin and colleagues have previously shown that encapsulation of chloroquine in non-targeted liposomes could increase the effectiveness of this drug against chloroquine-resistant
P. berghei infections, but this required a daily dose of 400 mg/kg (i.e. 8 mg/mouse of 20 g) for 3 consecutive days to achieve 90% survival with no recurrent infection [
117]; this corresponds to 80 times greater dose than the dose of chloroquine encapsulated in the immunoliposomes developed by Gupta and colleagues [
167,
171].
To further increase the cell-specifity of immunoliposomes, their fate in target cells (RBCs) and their pharmacological activity, Crommelin et al
. engineered drug-free immunoliposomes of the F(ab’)
2-MPB-PE-REV type that were made by covalently linking F(ab’)
2 fragments (from rabbit antimouse erythrocyte IgG) to reverse-phase evaporation vesicles (REV) via maleimido-4-(
p-phenylbutyrate)phosphatidylethanolamine) (MPB-PE) as anchor molecule (Table
2) [
168,
169]. Data revealed that, at equal protein doses (~ 130 µg), the unloaded F(ab’)
2-liposomes intravenously injected in rat induced a faster elimination of the RBCs from the bloodstream and higher uptake into the spleen than the free F(ab’)
2. Nevertheless, the fact that the doses of F(ab’)
2-liposomes and free F(ab’)
2 taken up into the liver (hepatocytes) was lower than those into the spleen constitute an important limitation suggesting an improvement of their specificity [
169]. In addition to this, chloroquine-loaded F(ab’)
2-MPB-PE-REV liposomes (0.8 mg of drug per rat) were significantly more effective than the non-targeted liposomes encapsulated chloroquine, free-MPB-PE-REV liposomes and free chloroquine in preventing or delaying a patent infection of non-synchronized pRBC in rats [
169,
170]. Indeed, parasitaemia became patent about 9.6 days, 6.4 days, 4.4 days and 6.2 days after infection in mice treated with the chloroquine immunoliposomes, chloroquine liposomes, free immunoliposomes and free chloroquine, respectively [
170]. By contrast, when the treatment was given to rats infected with synchronized pRBC (reticulocytes), the therapeutic efficacy of these chloroquine immunoliposomes was significantly improved [
170].
In another study, Crommelin et al
. evaluated the therapeutic efficacy of chloroquine loaded in immunoliposomes with rabbit anti-mouse RBCs (anti-mRBC) F(ab’)
2-liposomes in rats infected with early stage of
P. berghei infected cells (> 90% trophozoites). Due to the absence of mature schizonts in these synchronized parasitized reticulocytes, the latter are not able to release free parasites in the bloodstream. Interestingly, intravenous administration of chloroquine-loaded anti-mRBC F(ab’)
2-liposomes led to greater survival rate of the infected rats in comparison with free chloroquine: 33% versus 0%, respectively, while both used at a dose of chloroquine equivalent to ca. 12 mg/kg [
119].
Considering all the data presented above, researchers hypothesized that the therapeutic efficacy of anti-malarial drugs could be more pronounced if (i) the performance of targeted liposomes in specific pRBC recognition was better, (ii) the developed immunoliposomes could also bind to merozoites and free parasites (iii) the capacity of liposomes to encapsulate drugs as well as their endocytic activity at the surface of RBCs and pRBCs were increased [
167,
170].
Hence, to increase the survival rate and the survival time of the treated mice, Gupta and co-investigators looked for more specific ligands that could better deliver the drug in the biophase in comparison with anti-mouse erythrocyte F(ab’)
2 [
140]. For this purpose, they covalently attached F(ab’)
2 fragments of the monoclonal antibodies MAbF
10 and MAbD
2 to the surfaces of liposomes (see composition in Table
2). After assessing their binding specificities, it was found that MAbF
10-liposomes interacted in vitro with normal (< 3%) and
P. berghei-infected (~ 16%) mouse erythrocytes [
140]. By contrast, the maximum binding of MAbD
2-liposomes was 17–20% with both normal and
P. berghei-infected erythrocytes. These findings suggested that grafting of MAbF
10 on the liposome surface was more specific to
P. berghei-infected erythrocytes than that of MAbD
2-liposomes [
140]. After intravenous administration in mice infected with a chloroquine-resistant strain, MAbF
10 liposomes containing chloroquine exhibited considerably higher levels of anti-malarial activity than chloroquine-MAbD
2-liposomes. Indeed, at a dosage of 5 mg/kg per day on days 4 and 6 post-infection, MAbF
10-liposomes and MAbD
2-liposomes containing chloroquine cured, respectively, 75–90% and 40–50% of the animals on day 30 post-infection [
140]. These findings confirmed that the therapeutic efficacy of chloroquine was markedly increased through targeted liposomal drug delivery.
Fernàndez-Busquets and co-workers also engineered immunoliposomal nanovectors that were able to target more specifically pRBCs and release their contents inside these cells, but not in non-parasitized RBCs [
175]. For this purpose, the authors prepared liposomes using the mixture of 1,2-dioleoyl-
sn-glycero-3-phosphocholine (DOPC): cholesterol: MPB-PE (77.5:20:2.5). The resulting liposomes were covalently functionalized with about 5 molecules of BM1234, an oriented, specific targeting monoclonal antibody (which is also known as the membrane-associated histidine rich protein 1, MAHRP1) [
177]. This antigen is specific for pRBCs infected by the late forms of
Plasmodium species (trophozoites and schizonts), but not for ring-stage pRBCs. In living
P. falciparum cultures, these developed immunoliposomal nanovectors recognized 100% of late form-containing pRBCs (and 0% of non-infected RBCs) and infiltrated their content in the host cells in less than 90 min [
175]. Consequently, 2 nM chloroquine delivered inside targeted immunoliposomes cleared ~ 27% of pRBCs (versus 50% of pRBCs for free chloroquine at 20 nM) thereby suggesting an improvement in terms of drug efficacy. Liposomes not functionalized with antibodies were also specifically directed to pRBCs, although with less affinity than immunoliposomes [
175].
In further studies, Fernàndez-Busquets and collaborators quantified the efficiency of these nanovectors bearing 5 BM1234 units in ameliorating the anti-malarial activity of both chloroquine and fosmidomycin (see composition in Table
2) [
176]. Liposomes containing either chloroquine (1.6 nM) or fosmidomycin (325 nM) reduced in vitro parasitaemia by 10% when added at the ring stage and by 26.5% when added at the trophozoite stage, which was in agreement with previous results [
175]. In contrast, free chloroquine (2 nM) or fosmidomycin (360 nM) reduced parasitaemia by 3–6% when added at either the ring or the trophozoite stage. On the other hand, 20 nM of free chloroquine were necessary for killing 28% of trophozoites [
176]. Consequently, liposomes covalently functionalized with an average of 5 half-antibodies BM1234 improved by tenfold the therapeutic effects of these two anti-malarial drugs [
175,
176]. In addition to this, immunoliposomes bearing 5, 50 or 250 BM1234 units and encapsulating 4 nM chloroquine led to a reduction of 30, 43 and 51% parasitaemia (trophozoite stage), respectively [
176]. All these results confirmed that the antibody-functionalized liposomal nanovectors for the targeted delivery of drugs specifically to pRBCs have shown complete discrimination in vitro for pRBCs vs. non-infected RBCs [
175,
176]. Of note, the best performing immunoliposomes were those added to
Plasmodium cultures having a larger number of late form-containing pRBCs (i.e. trophozoites and schizonts), consistent with the previous studies [
175]. These findings revealed also that increasing the number of antibodies on the surface of immunoliposomes may improve their in vivo performance [
175,
176].
Notably, the above-mentioned data on antibody-targeted liposomes for malaria therapy show tremendous progress in this field. However, the anti-malarial effect obtained was mainly attributed to the encapsulation of anti-malarial drugs in liposomes and partially to antibody targeting. To reverse this balance, Fernàndez-Busquets and his team tested the capacity of anti-glycophorin A (anti-GPA), anti-histidine-rich protein 2 (anti-HRP2) and anti-MAHRP1
21-40 as targeting agents for the functionalization of immunoliposomes (see composition in Table
2) [
177]. Anti-GPA and anti-HRP2 were respectively raised against GPA and HRP2 that are located on erythrocyte surfaces while anti-MAHRP1
21-40 was targeted against the intracellular MAHRP1 antigen. All the three selected antibodies were compared to BM1234 in terms of anti-malarial efficacy [
177]. To facilitate the coupling reaction of antigen to targeting antibodies and improve the targeting properties of the anti-GLA liposome formulations, a lipid bearing PEG 2000 linker terminated with a maleimide group (Mal) was included in their lipid bilayer. Depending on the chemical group (thiol, carbohydrate or primary amine) of the half-antibodies that binds to the maleimide group, three types of immunoliposomes were designed, namely immunoliposome-PEG-Mal-SH-Ab, immunoliposome-PEG-Mal-CHO-Ab and immunoliposome-PEG-Mal-NH
2-Ab. The results from in vitro studies indicated that free anti-GPA were able to recognize the entire RBC and pRBC populations whereas the 3 others antibodies bound less than 1% of pRBCs [
177]. Consistently with these results, anti-GLA immunoliposomes were able of targeting 100% RBCs and pRBCs after only 15 min exposure time to
P. falciparum cultures. The efficiency of cell targeting of these formulations was the highest with immunoliposome-PEG-Mal-NH
2-Ab and the lowest with immunoliposome-PEG-Mal-SH-Ab. Interestingly, when encapsulated with 50 nM of chloroquine, these immunoliposomes completely inhibited the in vitro growth of early stages of
P. falciparum 3D7 strain. In contrast, 200 nM of free chloroquine had no significant effect on the viability of this parasite [
177]. In addition to this, the immunoliposomes bearing antibodies against the RBC surface protein glycophorin A permitted to transfer efficiently the anti-malarial drug into non-parasitized RBCs. By doing so, the parasite that entered the host cells instantly encountered the drug, which compromised its growth and survival capacity. Intracellular delivery of the encapsulated cargo may mainly occur following a sustained release process that take place at the targeted cell surface [
177]. Finally, during preliminary in vivo studies, data demonstrated that, when administered intravenously at the dose of 0.5 mg/kg for 4 days, chloroquine loaded in immunoliposome-PEG-Mal-NH
2-Ab completely cleared
P. falciparum in mice grafted with human erythrocytes. This efficacy was 40- and 100-fold higher than that of free chloroquine (1.75 mg/kg × 4 days) and chloroquine in non-target liposomes (0.5 mg/kg × 4 days), respectively. Based on these data, the authors suggested that anti-GLA-immunoliposomes actively loaded with chloroquine may act as prophylactic and therapeutic agents by simultaneously delivering the drug to both infected and non-infected RBCs [
177].
Based on this approach, hydrophilic (pyronaridine) and lipophilic (atovaquone) anti-malarial drugs were encapsulated, alone or in combination, in GPA-targeted immuno-PEG-liposome [
181]. The developed immunoliposomes that consisted of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), cholesterol and DSPE-mPEG2000-Mal were obtained by coupling anti-GPA antibody to the maleimide group as mentioned above (Table
2) [
177]. While at concentration of 137.2 nM, the immunoliposomized pyronaridine resulted in 50% inhibition of the in vitro growth of the parasite, pyronaridine-loaded liposomes and free pyronaridine exhibited significantly lower antiplasmodial activity (16.2% and 2% inhibition, respectively). The same trend was observed for atovaquone which showed an IC
50 of 43.1 nM after incorporation in immunoliposomes [
181]. Nonetheless, the co-encapsulation of these two drugs in the immunoPEGliposomes required higher amount of drugs (379 nM for pyronaridine and 116 nm for atovaquone) for inhibiting 50% of growth of malaria parasite, suggesting that this particular drug combination is antagonistic and less active than when these individual drugs are used separately [
181].
Noteworthy, the outstanding performance of chloroquine-anti-GLA-liposomes was achieved in humanized and immunosuppressed mice infected with uncomplicated falciparum malaria (< 3% parasitaemia) [
177]. To overcome this feebleness, Fernàndez-Busquets and co-workers evaluated the ability of anti-GLA-targeted liposomes containing different aminoalcohol- and aminoquinoline-type drugs to display simultaneous prophylactic and therapeutic activities in fully immunocompetent mice infected with a lethal strain of
Plasmodium yoelii [
179]. Indeed, in comparison with the humanized mice, this murine malaria model offered a cost-effective alternative for the in vivo study of immunoliposome-based severe malaria therapies. The lipid phase of the developed liposomes contained DOPC, an unsaturated type of phosphatidylcholine or DSPC, which is a saturated-type of phosphatidylcholine that reduces membrane fluidity and permeability (see composition in Table
2). Based on their physicochemical properties, DOPC and DSPC allowed the encapsulation of lipophilic agents and hydrophilic drug, respectively. On the other hand, since considerable cell-agglutinating events were observed with immunoliposomes bearing mouse monoclonal IgG2b anti-human GPA (SM3141P) (> 10 µM lipid amounts), the authors also evaluated the potential of rat monoclonal IgG2b anti-mouse TER-119 (AM31858PU-N) antibodies to be used as ligand agents for generating immunoliposomes [
179]. The TER 119 marker was specifically expressed from the early proerythroblast to mature mouse RBC stages [
186,
187]. Data from in vitro studies indicated that, upon encapsulation into the GPA-immunoliposome targeting all RBCs, the compound 7c, a chloroquine analogue, was efficiently delivered to RBCs infected with chloroquine-sensitive and chloroquine-resistant
P. falciparum strains after only 15 min of exposure [
179]. With a high in vitro targeting efficacies to both naïve and
Plasmodium-infected-RBCs and retention yields of ~ 100% in agreement with previous reports [
177], these 7c-immunoliposomes exhibited IC
50 values of 48.6 nM (late forms of resistant parasites) and 100.7 nM (late forms of sensitive parasites), that were 12 to 27-fold lower than that of the free drug.
When intravenously administered to fully immunocompetent mice infected with
P. yoelii, 7c-anti-GPA-liposomes (20 mg/kg over 4 days) exhibited a significant parasitaemia reduction (45–55%). On the other hand, two minutes after its intravenous administration (5 mg/kg on day 1, 2.5 mg/kg on day 2 and 1 mg/kg on day 3), the 7c drug encapsulated in TER119-immunoliposomes targeted both RBCs and pRBCs, and reached retention efficacies of > 95% thereby decreasing blood parasitaemia from severe to uncomplicated malaria (i.e. from > 25% to < 5%) in
P. yoelii-infected immunocompetent mice [
179].
To prevent microvascular sequestration of pRBC, which is strongly linked to their cytoadherence (i.e. adherence on vasculature endothelium) and rosetting (i.e. adherence on non-parasitized RBCs), Fernàndez-Busquets et al
. developed lumefantrine-loaded immunoliposomes functionalized with a polyclonal anti-rosetting antibody against the NTS-DBL1α N-terminal domain of a rosetting
P. falciparum erythrocyte membrane (PfEMP1) variant. The composition of immunoliposomes is shown in Table
2. After 30 min of incubation in
P. falciparum cultures, the immunoliposomes containing 2 µM of lumefantrine showed a 60% growth inhibition for rosettes (i.e. pRBCs with a rosetting phenotype) and a reduction in ca. 70% of pRBCs containing all the parasitic forms [
178]. This approach is of interest since rosettes facilitate the occlusion of microvascular blood vessels, which obstructs blood flow leading to hypoxia, metabolic acidosis and other phenomena involved in the pathophysiology of malaria [
21,
188,
189].
Because anionic lipids such as phosphatidylserine (PS) move from the inner membrane of erythrocytes to their outer surfaces during eryptosis, Tagami et al
. [
24] engineered liposomes conjugated to a PS-specific peptide (PSP). The developed PSP-liposomes were composed of DSPC: cholesterol and PSP-DSPE-mPEG2000 (2:1:0.1, molar ratio). Interestingly, the binding of PSP-liposomes to eryptosis-induced RBCs (eRBCs) was significantly higher than those of RBCs. The specific binding of PSP-liposomes to eRBCs occurred within 3 h post-incubation. These findings suggest that PSP-conjugated liposomes could be an effective targeted nanocarrier drug delivery system for treating eRBCs and different malaria-infected RBCs (unlike liposomes coupled with monoclonal targeting antibodies) [
24].
Heparin acts as an anti-malarial drug that blocks merozoite invasion of erythrocytes [
190], but do not influence the clinical course in human falciparum malaria [
144,
191]. Even though no anti-malarial resistance to heparin has been described so far, it has been abandoned in therapeutic because of its strong anticoagulant action that was associated with intracranial bleeding [
190]. Nevertheless, heparin and others glycosaminoglycans like heparan sulfate and chondroitin sulfate have specific binding affinity for pRBC (vs. non-infected RBCs), which make them interesting alternatives to antibody-mediated targeting [
190,
192‐
194]. This is why Fernàndez-Busquets and colleagues engineered liposomes bearing heparin or its related polysaccharides (see composition in Table
2) [
180]. Although the heparin-related compounds are recognized as pRBC-binding molecules, their attachment on the liposome surface requires careful optimization to achieve good reproducibility and stability [
180,
194‐
196]. In contrary, heparin that targets mainly trophozoite- and schizont-infected RBCs stages was successfully adsorbed onto positively charged liposomes via electrostatic interactions [
180,
197,
198]. Used at non-anticoagulating concentrations, these heparin-bearing liposomes enhanced three-fold the antiplasmodial activity of encapsulated primaquine in in vitro
P. falciparum cultures, and provided potent parasiticidal activity in mice [
180]. Apart from these significant targeting and pharmacological activities, the heparin-based approach is very interesting in economical point of view since heparin is about 10 times more affordable than monoclonal antibodies [
180].
Recently, poupartone B, a promising anti-malarial compound isolated from
Poupartia borbonica, was encapsulated in liposomes coated with heparin and tested on artemisinin-resistant
P. falciparum isolate [
182]. The composition of this liposomal formulation is shown in Table
2. The poupartone B-engineered liposomes exhibited IC
50 value of 0.41 µg/ml (versus 0.69 µg/ml for the free drug). Moreover, the liposomal formulation improved the selectivity index 2 times in vitro and proved to be 3 times less toxic than the unloaded poupartone B in a zebrafish (
Danio rerio) model [
182].