Effective-Loading of Platinum–Chloroquine into PEGylated Neutral and Cationic Liposomes as a Drug Delivery System for Resistant Malaria Parasites

Abstract

The trans platinum–chloroquine diphosphate dichloride (PtCQ) is a new type of antimalarial drug used to fight parasites resistant to traditional drugs. PtCQ is synthesized by mixing platinum and chloroquine diphosphate (CQ). This study examines two efficient methods for forming a nanodrug, PtCQ-loaded liposomes, for use as a potential antimalarial drug-delivery system: the thin drug–lipid film method to incorporate the drug into a liposomal membrane, and a remote-loading method to load the drug into the interior of a cationic liposome. The membranes accordingly comprised PEGylated neutral or cationic liposomes. PtCQ was efficiently loaded into PEGylated neutral and cationic liposomes using the thin drug–lipid film method (encapsulation efficiency, EE: 76.1±6.7% for neutral liposomes, 1 : 14 drug-to-lipid weight ratio; 70.4±9.8% for cationic liposomes, 1 : 14 drug-to-lipid weight ratio). More PtCQ was loaded into PEGylated neutral liposomes using the remote-loading method than by the thin drug–lipid film method and the EE was maximum (96.1±4.5% for neutral liposomes, 1 : 7 (w/w)). PtCQ was encapsulated in PEGylated cationic liposomes comprising various amounts of cationic lipids (0–20 mol%; EE: 96.9–92.3%) using the remote-loading method. PEGylated neutral liposomes and cationic liposomes exhibited minimum leakage of PtCQ after two months’ storage at 4°C, and further exhibited little release under in vitro culture conditions at 37°C for 72 h. These results provide a useful framework for the design of future liposome-based in vivo drug delivery systems targeting the malaria parasite.

Malaria is a life-threatening infectious disease that remains a major cause of death, especially in tropical regions. The WHO estimated that 214 million new cases of malaria resulted in 438000 deaths in 2015.¹⁾ The mortality rate of malaria is higher than that caused by AIDS. Malaria is caused by a parasitic protozoan, Plasmodium falciparum, and is transmitted by infected Anopheles mosquitoes. The life cycle of Plasmodium parasites involves being bitten by an infected mosquito, during which the malaria parasite is injected into the bloodstream.^2,3) The parasite migrates into hepatocytes and establishes itself in the liver. After a few weeks of dormancy, the activated parasite enters the blood and then infiltrates erythrocytes (red blood cells; RBCs). The parasite grows and divides in the erythrocytes by absorbing the nutrients from the blood via transporters, and these transporters are currently a topic of investigation by medical researchers.

Most current antimalarial drugs are amphiphilic, distribute widely into body tissues after systemic or oral administration, and can be easily metabolized in the liver,²⁾ and thus a high dose that may cause toxic side effects is needed to provide an antimalarial effect. The need to lower the dose to avoid toxicity is likely a main factor contributing to the development of resistance; this low dose also means that an ineffective dose is delivered to Plasmodium-infected RBCs. There is therefore an urgent need to develop new strategies to treat malaria and overcome the resistance of the parasite to current antimalarial drugs, especially in regions where P. falciparum is endemic.

Lipid-based drug nano-carriers have various advantages for drug delivery.^4–7) For example, liposomal formulations can increase drug bioavailability and thus increase therapeutic activity, decreasing side effects due to reduced dosing. Plasmodium-infected RBCs are the main chemotherapeutic target because several life stages of the parasite occur in the blood and give rise to the symptoms and pathologies of malaria. Consequently, several liposomal formulations encapsulating antimalarial drugs have been developed over the past several years in order to test their utility in malaria chemotherapy.

The ability of cationic liposomes to be adsorbed onto the cell surface and fuse with the negatively charged cellular membranes and thus deliver their cargo has been previously reported.^8–13) Anionic lipids such as phosphatidylserine (PS) are typically located within the inner monolayer of erythrocytes. The infection of erythrocytes with the malaria parasite leads to erythrocyte apoptosis, and this suicidal erythrocyte death is called eryptosis. The main characteristic stage of eryptosis is lipid scrambling and exposure of negatively charged PS on the outer surface of erythrocytes,^14,15) which increase the ability of erythrocytes to fuse with cationic liposomes.⁹⁾ However, the use of cationic liposomes in vivo is limited due to their short circulation time, which leads to their recognition by the immune system including macrophages.^16–18) It is well known that polyethylene glycol (PEG) prevents the interaction with the biological in vivo environment and extend the circulation time of liposomes in blood. Cationic liposomes are therefore promising candidates for drug delivery targeting malaria-infected RBCs, but currently there is little information regarding the use of cationic liposomes.

The present study focuses on generating platinum chloroquine diphosphate dichloride (PtCQ)-loaded PEG-modified (PEGylated) cationic liposomes exhibiting high drug encapsulation and high drug retention. PtCQ is a trans platinum–chloroquine complex that shows therapeutic effects against the chloroquine-resistant malaria parasite,¹⁹⁾ but many fundamental properties of PtCQ, such as drug solubility, remain poorly investigated, although it was reported that PtCQ dissolves in dimethyl sulfoxide (DMSO). We therefore investigated methods for efficiently loading PtCQ into liposomes and first characterized the handling properties of PtCQ. Next, PtCQ was loaded with high encapsulation efficiency into cationic liposomes for the first time using the remote-loading method employing an ammonium sulfate gradient. The liposome formulations showed good drug retention, minimum leakage of PtCQ after two months’ storage at 4°C, and further exhibited little release under in vitro culture conditions at 37°C for 72 h.

MATERIALS AND METHODS

Reagents

Chloroquine diphosphate (CQDP), potassium tetrachloroplatinate (K₂[PtCl₄]), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dioleoyl-3-trimethylammonium-propane, 18 : 1 (DOTAP) and cholesterol (CHOL) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)₂₀₀₀] (DSPE-PEG₂₀₀₀) was generously donated by NOF Corporation (Tokyo, Japan).

Synthesis of the trans-Pt(CQDP)₂(Cl)₂ Complex¹⁹⁾

A solution of K₂[PtCl₄] (100 mg, 0.24 mmol) in water (30 mL) was stirred until it completely dissolved and then CQDP (250 mg, 0.48 mmol) was added. Stirring was continued for 18 h at room temperature and a pink precipitate was obtained. This precipitate was collected by filtration, washed with water, and dried under vacuum.

Yield 87.8%; elemental analysis (%) Calcd for C₃₆H₆₄N₆Cl₄O₁₆P₄Pt (1297.65 g mol⁻¹): C 33.3; N 6.5; H 4.9. Found: C 31.7; N 6.7; H 4.4. IR ν(N–H) 3305 cm⁻¹; ν(C=C) 1616 cm⁻¹; ν(C=N) 1581 cm⁻¹; ν(Pt–Cl) 341 cm⁻¹; ν(Pt–N) 420 cm⁻¹. UV-Vis 238 and 344 nm. ¹H-NMR (DMSO-d₆; δ ppm): 9.05 (1H, d, J=6.09 Hz, NH), 8.82 (1H, d, J=9.13 Hz, H5), 8.58 (1H, d, J=7.01 Hz, H2), 8.02 (1H, d, J=1.83 Hz, H8), 7.8 (1H, dd, J₁=1.83, J₂=7.01 Hz, H6), 7.02 (1H, d, J=7.01 Hz, H3), 4.17 (1H, m, H1′), 3.15 (6H, m, H4′, H5′), 1.79 (4H, m, H2′, H3′), 1.3 (3H, d, J=6.39 Hz, H1″), 1.18 (6H, t, H6′).

Preparation of PtCQ-Loaded Liposomes Using the Thin Drug–Lipid Film Method

Liposomes were prepared by the thin drug–lipid film hydration method, followed by membrane extrusion as described previously, with minor modifications.²⁰⁾ Briefly, neutral PtCQ-loaded liposomes were prepared by dissolving each lipid powder (HSPC, CHOL and DSPE-PEG₂₀₀₀) with isopropanol and mixed at the molar ratio 55 : 45 : 5 (HSPC : CHOL : DSPE-PEG₂₀₀₀). PtCQ (1 mg) was dissolved in dichloromethane (DCM) and a trace of DMSO and then added to the lipid mixture. The drug–lipid mixture was evaporated to give a thin drug–lipid film that was placed under high vacuum for at least overnight to remove residual solvent. The dried drug–lipid film was hydrated in HBS (20 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 150 mM NaCl, pH 7.5) to form multilamellar vesicles (MLVs), sonicated for 10 min, and then extruded (Mini-extruder, Avanti Polar Lipids, Alabaster, AL, U.S.A.) 10 times through Whatman Nuclepore Track-Etched polycarbonate membranes (200 nm×10, 100 nm×10; GE Healthcare Life Sciences; Chicago, IL, U.S.A.) at 70°C to transform the MLVs into unilamellar vesicles. The formulation was cooled on ice for 15 min and unencapsulated PtCQ was removed by ultracentrifugation (21521 g, 4°C, 30 min) as previously reported.²¹⁾ Cationic PtCQ-loaded liposomes were prepared by dissolving the lipids (HSPC : DOTAP : CHOL : DSPE-PEG₂₀₀₀) in isopropanol, then mixing in the molar ratio 50 : 5 : 45 : 5, and 35 : 20 : 45 : 5. PtCQ (1 mg) was added to the lipid mixture at different weight ratios (1 : 7, 1 : 14) and then the vesicles were prepared as described above. The mean size and polydispersity index (PDI) were determined using a dynamic light scattering instrument (ZetaSizer Nano-ZS; Malvern Instrument Ltd., Malvern, U.K.). Zeta potentials were measured using Zetasizer Nano ZS90 (Malvern). The zeta potentials were measured by dispersing the liposome solution in 5% dextrose. The encapsulation efficiency (EE) was calculated as described in “Determination of the EE.”

Preparation of PtCQ-Loaded Liposomes Using the Remote-Loading Method

PtCQ-loaded liposomes were prepared as previously reported, with minor modifications.^22–25) Briefly, neutral PtCQ-loaded liposomes were prepared by mixing the lipids (HSPC : CHOL : DSPE-PEG₂₀₀₀) at the molar ratio 55 : 45 : 5. Cationic PtCQ-loaded liposomes were prepared by mixing the lipids (HSPC : DOTAP : CHOL : DSPE-PEG₂₀₀₀) with different molar ratios of the cationic lipid DOTAP (0, 5, 10, 15, 20 mol%) and dissolving in organic solvent. The solvent from the lipid mixtures was evaporated to provide thin lipid films that were placed under high vacuum for at least overnight to remove residual solvent. The thin films were hydrated with 250 mM ammonium sulfate (pH 4.0), sonicated for 10 min, and size-controlled liposomes were prepared using the extruder as described above. Next, the external liposomal phase was replaced with HBS via dialysis (Slide-A-Lyzer 10 kDa MWCO, Pierce Biotechnology, Rockford, IL, U.S.A.) for 24 h against 1000 volumes of HBS. The concentration of CHOL was determined using a cholesterol E-Wako kit (Wako Pure Chemical Industries, Ltd.) and a microplate reader (Wallac 4000 ARVO multi-label counter; PerkinElmer, Inc., Waltham, MA, U.S.A.) in order to estimate the concentrations of the various liposome preparations. Neutral PtCQ-loaded liposomes were prepared by mixing PtCQ solution in HBS (0.65 mg/mL) with the neutral liposomal solution at three drug-to-lipid ratios (1 : 2.5, 1 : 5 and 1 : 7 (w/w) and incubated at 60°C for 60 min for drug loading. The cationic PtCQ-loaded liposomes were prepared by using a drug–lipid ratio of 1 : 7 (w/w). The mean size, PDI, and zeta potential were measured using Zetasizer Nano ZS90 (Malvern) as described above and the EE was calculated as described in “Determination of the EE.”

Determination of the EE

For PtCQ-loaded liposomes prepared using the thin drug–lipid film method, the sample was ultracentrifuged (21521 g, 4°C, minimum 30 min) and the pellet was resuspended in HBS buffer. The concentration of the encapsulated drug was measured by absorbance at 343 nm using a UV-Vis spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) after completely disrupting the liposomes using 2% Triton X-100. The absorbance was converted into drug concentration using a standard curve. The EE was calculated using the previously reported equation²⁶⁾:   

For PtCQ-loaded liposomes prepared using the remote-loading method, the samples were cooled and applied onto a gel filtration column (CL-4B, Sigma-Aldrich) equilibrated with HBS to remove unencapsulated PtCQ. The liposome fraction of the eluate was collected, the concentration of lipid was determined again, and the concentration of the encapsulated drug was measured as described above.

Measurement of Drug Release from PtCQ-Loaded Liposomes under Storage Conditions

All PtCQ-liposomal formulations were stored at 4°C. As a typical experiment, aliquots (100 µL) were withdrawn at specified time points (0, 1, 2, 4, 8 weeks), diluted to 1 mL with HBS buffer, and ultracentrifuged (21521 g, 4°C, 30 min) using Amicon filters (MWCO, 10K; Millipore, Bedford, MA, U.S.A.) to separate the released PtCQ. The released PtCQ was measured by absorbance at 343 nm as described above. The percentage of PtCQ released from the liposomes was calculated using the previously reported formula²⁷⁾:   

where “PtCQ released” indicates the concentration of released PtCQ collected at a specified time point t and “Total PtCQ” indicates the total amount of PtCQ entrapped in the liposomes.

Measurement of Drug Release from Liposomes under in Vitro Culture Conditions (Release Test)

As a typical experiment, 1 mL of liposomal solution was mixed with 1 mL of RPMI 1640 medium (Wako Pure Chemical Industries, Ltd.) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, U.S.A.) and the samples were incubated at 37°C in a humidified incubator in an atmosphere of 5% CO₂/95% air. Aliquots (100 µL) were withdrawn at specified time points (0, 1, 3, 6, 24, 48, 72 h), diluted to 1 mL with HBS buffer, and ultra-centrifuged to separate the released PtCQ (21521 g, 4°C, 30 min). The percentage of PtCQ released was determined as in “Measurement of Drug Release from PtCQ-Loaded Liposomes Under Storage Conditions.” The chloroquine absorbance at 343 nm was unaffected by the culture medium, and the standard curve was unaffected.

Statistical Analysis

All data shown here are represented in “mean value±standard deviation (S.D.).” An ordinary one-way ANOVA (followed by Tukey’s or Dunnett’s multiple comparison test), and a two-way ANOVA with Bonferroni post-test or Sidak’s multiple comparisons test were used to assess statistical significance by using GraphPad Prism (GraphPad Software Inc., CA, U.S.A.). p<0.05 was regarded as statistically significant.

RESULTS AND DISCUSSION

Synthesis and Characterization of the Pt(CQDP)₂(Cl)₂ Complex

The platinum–chloroquine diphosphate complex was synthesized and isolated in water at room temperature. A mixture of K₂[PtCl₄] and CQDP was prepared to displace two chloride ligands, leading to the new PtCQ complex that was isolated in good yield as a pink solid precipitate (Fig. 1). The complex was characterized by elemental analysis, and UV-Vis, IR and proton-NMR spectroscopy (for analysis data details, see “Synthesis of the trans-Pt(CQDP)₂(Cl)₂ Complex” and Fig. 2). The complex is slightly soluble in water and insoluble in organic solvents except DMSO. The elemental analysis of this complex is in agreement with the molecular formula proposed. The IR spectrum of the complex shows peaks clearly associated with the presence of coordinated CQDP. In the ¹H-NMR spectrum, the ¹H chemical shift variation of each signal with respect to that of the free ligand reported previously¹⁹⁾ was similar and used to deduce the mode of bonding of CQDP to the metal. The largest shift with respect to the free ligand (CQDP) was observed for NH and H1′ as they are the nearest protons to the N-atom bonded to the metal, indicating that CQDP is bound to platinum through the nitrogen of the secondary amine.

Fig. 1. Chemical Structure of Platinum Chloroquine Diphosphate Dichloride

Fig. 2. Spectral Characterization of the PtCQ Complex (A) UV-Vis, (B) IR, (C) Proton-NMR

PtCQ-Loaded Liposomes Prepared Using the Thin Drug–Lipid Film Method

Navarro et al. mentioned the high lipophilicity of metal chloroquine complexes²⁸⁾ and Khokhar et al. demonstrated the preparation of highly stable lipophilic cisplatin complex/liposomes with high encapsulation ratios.²⁹⁾ Consequently, in this study we succeeded in loading the PtCQ complex into neutral and cationic liposomes at reasonable drug-to-lipid ratios by incorporating the drug into the lipid phase using the thin drug–lipid film method. As shown in Table 1, the PtCQ-loaded liposomes had uniform sizes ranging from about 105 to 143 nm and a narrow size distribution (PDI<0.097). Furthermore, the zeta potential of PEGylated liposomes was changed from low negative (−7.1±0.5 mV) of the neutral liposomes (1 : 7) to low positive (10.6±0.9 mV, cat. 20) of the cationic liposomes (1 : 7). The negative charge of the neutral liposomes is due to the presence of negatively charged phosphate group in DSPE-PEG₂₀₀₀.³⁰⁾ In addition, Fig. 3 reveals that the cationic lipid content clearly affects the drug loading in the thin drug–lipid film method. Based on the statistical study, the addition of 5 mol% of the cationic lipid DOTAP at low liposome concentration (7 mg) has a significant decrease (p=0.0076) on the EE% of cat. 20 compared to cat. 5, however it has no significant effect on the EE% of cat. 5 and cat. 20 compared to neutral liposomes. On the other hand, at higher liposome concentration (14 mg), there is a significant decrease (p<0.0001) on the EE% of cat. 20 compared to cat. 5 and the neutral liposomes, however no significant effect was noticed in the EE% of cat. 5 compared to the neutral liposomes. The decrease in the EE% with the increase of the cationic lipid content could be due to the electrostatic repulsion between the cationic drug and the cationic lipid (DOTAP).

Table 1. PtCQ-Liposomes Loaded by Thin Drug–Lipid Film Method

Drug–lipid (w/w) neutral and cationic liposomes	Particle size (nm)	Polydispersity index	Zeta potential (mV)
1 : 7 (HSPC/CHOL/DSPE-PEG2000) 55 : 45 : 5 (neut.)	133.0±10.6	0.055±0.006	−7.1±0.5
1 : 7 (HSPC/DOTAP/CHOL/DSPE-PEG2000) 50 : 5 : 45 : 5 (cat. 5)	133.9±10.6	0.065±0.008	3.6±0.6
1 : 7 (HSPC/DOTAP/CHOL/DSPE-PEG2000) 35 : 20 : 45 : 5 (cat. 20)	120.7±6.4	0.043±0.044	10.6±0.9
1 : 14 (HSPC/CHOL/DSPE-PEG2000) 55 : 45 : 5 (neut.)	142.6±6.9	0.082±0.019	−6.4±0.8
1 : 14 (HSPC/ DOTAP/CHOL/DSPE-PEG2000) 50 : 5 : 45 : 5 (cat. 5)	132.4±4.0	0.097±0.010	5.1±0.7
1 : 14 (HSPC/DOTAP/CHOL/DSPE-PEG2000) 35 : 20 : 45 : 5 (cat. 20)	105.1±3.8	0.032±0.017	10.0±0.6

Neutral and cationic formulations with drug–lipid (1 : 7, 1 : 14 w/w) have been prepared. The neutral lipids have been mixed with the molar ratio (55 : 45 : 5). The cationic lipids have been mixed with the molar ratio (5 : 50 : 45 : 5) and (35 : 20 : 45 : 5). Data represent the mean±S.D. (n=3).

Fig. 3. The EE of Liposomes Prepared Using the Thin Drug–Lipid Film Method

Three formulations were prepared, at drug to lipid ratios of 1 : 7, 1 : 14 (w/w). Data represent the mean±S.D. (n=3), (**** p<0.0001, ** p=0.0076). The information about liposomal composition is written in Table 1.

PtCQ-Loaded Liposomes Prepared Using the Remote-Loading Method

To our knowledge, this is the first report describing the encapsulation of the PtCQ complex into liposomes using an ammonium sulfate gradient and the results are promising. The rate of loading and the stability of the loaded drug in the intra-liposomal medium mainly depend on the properties of i) the loaded drug, ii) the intra-liposomal medium, and iii) the liposome bilayer, as discussed below.

i) Properties of the Loaded Drug

Chemical compounds can be categorized as hydrophilic compounds, amphiphilic compounds, and hydrophobic compounds. Hydrophilic compounds do not interact with the liposome bilayer and can be encapsulated in the aqueous phase of the liposome interior. Hydrophobic compounds integrate into the liposome bilayer. Amphiphilic compounds may be amenable to high loading due to their possible high affinity for the liposomal membrane or may be remotely loaded into the intra-liposomal aqueous phase by using a pH gradient. CQ is a diprotic amphiphilic weak base with two proton-binding groups: the side chain terminal amine (pK_a1=10.2) and the quinoline nitrogen atom (pK_a2=8.4). Accordingly, unprotonated CQ can be driven by a pH gradient across a parasite’s food vacuole membrane and thus accumulate in the acidic food vacuole.

CQ can be actively encapsulated by applying a pH gradient,^25,31) during which it is protonated to the diproton form following exposure to the intra-liposomal acidic medium, then captured inside the liposomes due to the low permeability of the liposomal bilayer membrane to the protonated drug.²⁵⁾ In the present study we used the ammonium sulfate gradient to load PtCQ into liposomes composed mainly of neutral or cationic saturated phospholipids with an EE of almost 100%, as shown in Figs. 4 and 5.

Fig. 4. Effect of Increasing the Liposomal Content on the EE of PtCQ Loaded Inside Neutral Liposomes Prepared Using the Remote Loading Method

Three formulations were prepared, at drug to lipid ratios of 1 : 2.5, 1 : 5 and 1 : 7 (w/w). Data represent the mean±S.D. (n=3). Statistical data infers that each group is significantly different (**** p<0.0001, *** p=0.0008). The information about liposomal composition is written in Table 2.

Fig. 5. Effect of Increasing the Cationic Lipid Content (DOTAP) on the EE of PtCQ Loaded Inside Cationic Liposomes Prepared Using the Remote Loading Method

Five formulations were prepared (0, 5, 10, 15, 20 mol% as cationic lipid). The drug-to-lipid ratio is 1 : 7 (w/w). Data represent the mean±S.D. (n=4). The information about liposomal composition is written in Table 3.

ii) Intra-liposomal Medium Properties

Various chemical gradients have been used to create a trans-membrane potential as a driving force to facilitate the loading of a drug through an active loading mechanism. For example, ammonium sulfate²²⁾ induces a potential gradient for weak bases and acids and this gradient remains stable for over six months for liposomal formulations stored below the transition temperature of the phospholipids. Furthermore, lipophilic amino-containing drugs can be partitioned into the lipid bilayer, resulting in a drug gradient through the lipid bilayer that aids the influx of the drug from the outside to the inside.³²⁾

In the present study, we loaded the PtCQ complex into neutral liposomes using a pH gradient and the trans-membrane potential generated by an ammonium sulfate gradient as driving forces. Following hydration of the PtCQ-loaded liposomes using an ammonium sulfate solution, an equal concentration of ammonium sulfate is found outside and inside the liposomes. Removal of the outside ammonium sulfate solution by dialysis leads to an ammonium sulfate gradient in which the concentration of ammonium sulfate inside the liposomes is greater than that outside and the gradient is due to the large difference in the permeability coefficient through lipid bilayer as mentioned before.²²⁾ The higher concentration of ammonium inside the liposome causes the efflux of neutral ammonium, leaving the protons inside the liposome. This ammonium gradient results in the influx of the PtCQ into the acidic intra-liposomal medium and thus its encapsulation. The drug may be tetra-protonated due to the presence of two chloroquine molecules in the complex. The outside-inside pH gradient was 7.5–4 to give a pH gradient equal to 3.5 units (ΔpH=(external pH−internal pH)=3.5) since this produces a much higher inside concentration of the weak base compared to the outside concentration. As shown in Fig. 4 and Table 2, PtCQ-loaded liposomes were prepared with different drug-to-lipid weight ratios and the maximum EE was obtained at a drug-to-lipid ratio of 1 : 7 (w/w). In a preliminary experiment, the loading of PtCQ was compared using ammonium sulfate or citrate buffer and the EE was found to be higher with ammonium sulfate, perhaps due to the high permeability coefficient of the NH₃ molecule, which causes fast diffusion of neutral ammonia to the external medium of the liposomes. Each diffusing NH₃ molecule leaves one proton inside the liposome, thus acidifying the intra-liposomal medium and resulting in a pH gradient and influx of the drug.²²⁾ The pH gradient had a greater effect on CQ, which has two amino moieties, than on a weak base with one amino moiety.²⁵⁾

Table 2. PtCQ-Liposomes Loaded by Ammonium Sulfate Gradient

Drug–lipid (w/w), neutral liposomes	Particle size (nm)	Polydispersity index	Zeta potential (mV)
1 : 2.5	109.1±2.9	0.047±0.014	−7.2±1.8
1 : 5	108.6±2.8	0.055±0.017	−7.8±1.2
1 : 7	107.6±0.7	0.036±0.004	−7.9±1.0

The neutral lipids have been mixed with the molar ratio (HSPC/CHOL/DSPE-PEG₂₀₀₀=55 : 45 : 5). Three different formulations with drug–lipid (1 : 2.5, 1 : 5, 1 : 7 w/w) have been prepared. Data represent the mean±S.D. (n=3).

iii) Liposome Bilayer Properties

It was previously demonstrated²²⁾ that drug-loaded liposomes prepared by the remote loading method and comprising saturated lipids can be maintained at 4°C for at least six months without deterioration. Liposomes composed of saturated lipids can maintain a pH gradient due to the relatively high transition temperature of the bilayer. In the present work, we used the saturated lipid HSPC to construct the membrane of PtCQ-loaded liposomes generated using the remote loading method. The demonstrated stability of the PtCQ-loaded liposomes under storage and culture conditions verifies the ability of this saturated lipid bilayer to maintain a specified pH gradient and retain the drug in the interior phase of the liposome as long as the temperature does not exceed the transition temperature of the lipid, thus preventing thermal mobility that could disrupt the membrane required for maintaining the pH gradient.²⁵⁾ We studied the effect of temperature on drug loading at two temperatures below the transition temperature 60°C, after incubation for one hour and obtained maximum loading at 60°C.

Cationic PtCQ-Loaded Liposomes Produced Using the Remote-Loading Method

Five types of cationic PtCQ-loaded liposomes with a drug-to-lipid ratio of 1 : 7 (w/w) but differing in the ratios of the constituent lipids were prepared and characterized, and the results are shown in Table 3. The PtCQ-loaded cationic liposomes has uniform sizes about 130 nm and a narrow size distribution (PDI<0.05). It was reported that the cationic liposomes demonstrate an enhanced cellular uptake compared to the neutral liposomes, it could be due to the electrostatic interaction between the positive charge and the cell.¹²⁾ Additionally, the increase of cationic lipids in liposomes could lead to a higher affinity for the anionic sites, however it could result in their aggregation in the bloodstream through electrostatic interactions with the anionic species in the blood and thus increase their uptake by the reticuloendothelial system (RES).¹²⁾ Liposomes are typically modified with PEG (PEGylation) to form a water layer that could prevent protein adsorption onto the liposome surface and decrease the uptake of PEG-liposomes by macrophages and thus help in prolonging the lifetime of liposomes in the blood circulation.³³⁾ The partial coating of cationic particles with PEG masks the values of zeta potential (electric potential across a double membrane surface), however it can’t prevent them from binding to the cells and the biodistribution completely. Moribe et al. reported that the inclusion of 0 to 10% PEG sharply decreases zeta potential of the liposome.³⁴⁾ For this reason typically 5–10 mol% of PEG is enough to delay the recognition of liposomes by immune system cells. This concentration could retain a significant cationic charge and binding to the cells. Consequently, All formulations prepared in this study were PEGylated with 5 mol%. Additionally, it has been reported that the adhesion of the liposome membrane to the cell depends on the ratio of the liposome–cell charge, thus the probability of adhesion is maximized at a finite polymer layer thickness in case of the cationic liposomes are overcharged compared to the cell.³⁵⁾ Accordingly, in this study, several PtCQ-loaded liposomes with different molar ratio of the cationic lipid (DOTAP, 0, 5, 10, 15, 20 mol%) were prepared to provide stable cationic PtCQ-loaded liposomes that could selectively bind to erythrocytes. Table 3 reveals that the zeta potential of PEGylated cationic liposomes changes from low negative (−7.5±1.3) for (cat. 0) to neutral (−0.4±0.7, cat. 10), and to low positive (10.7±3.0, cat. 20).

Table 3. PtCQ-Liposomes Loaded by Ammonium Sulfate Gradient

Drug–lipid=1 : 7 (w/w), cationic liposomes	Particle size (nm)	Polydispersity index	Zeta potential (mV)
HSPC/DOTAP/CHOL/DSPE-PEG2000=55 : 0 : 45 : 5 (cat. 0)	127.8±6.5	0.029±0.017	−7.5±1.3
HSPC/DOTAP/CHOL/DSPE-PEG2000=50 : 5 : 45 : 5 (cat. 5)	112.2±3.6	0.048±0.013	−3.3±0.5
HSPC/DOTAP/CHOL/DSPE-PEG2000=45 : 10 : 45 : 5 (cat. 10)	129.0±5.0	0.037±0.018	−0.4±0.7
HSPC/DOTAP/CHOL/DSPE-PEG2000=40 : 15 : 45 : 5 (cat. 15)	115.0±1.6	0.046±0.029	8.1±1.2
HSPC/DOTAP/CHOL/DSPE-PEG2000=35 : 20 : 45 : 5 (cat. 20)	114.1±4.4	0.047±0.013	10.7±3.0

The cationic lipids have been mixed with different molar ratios. Five different formulations with HSPC–DOTAP (55 : 0, 50 : 5, 45 : 10, 40 : 15, 35 : 20) have been prepared. Data represent the mean±S.D. (n=3, 4).

The addition of negatively-charged lipids to liposomes generated using the passive loading method can increase EE due to electrostatic interaction between the positively charged chloroquine and the negatively charged liposomes. However, characterization of the PtCQ-loaded liposomes prepared using the thin drug–lipid film method presented in “PtCQ-Loaded Liposomes Prepared Using the Thin Drug–Lipid Film Method” confirmed that the EE of cationic liposomes decrease with increasing the cationic lipid content and this could be due to the electrostatic repulsion between the cationic drug and the cationic lipid. In contrast, Fig. 5 reveals that the different cationic PtCQ-loaded liposomes prepared by ammonium sulfate gradient (cat. 5, cat. 10, cat. 15, and cat. 20) have no significant effect on EE% compared to cat. 0 (96.9±5.0%). This result confirms that the most of PtCQ loaded by ammonium sulfate gradient is not bilayer-associated, in good agreement with a previous report.²²⁾

Drug Release from Cationic PtCQ-Loaded Liposomes under Storage Conditions (Storage Test)

The stability of cationic PtCQ-loaded liposomes (cat. 0, cat. 5, cat. 10, cat. 15, cat. 20) loaded using the ammonium sulfate gradient (drug-to-lipid ratio 1 : 7 (w/w)) was studied during eight weeks’ storage at 4°C. Figure 6 reveals that there is no significant release of the drug at different cationic PtCQ-loaded liposomes after eight weeks’ storage at 4°C, compared to cat. 0 (5.7±4.9%). This demonstrates the utility of the ammonium sulfate gradient method for incorporating amphiphilic drugs into stable liposomes containing a saturated lipid (HSPC). It was reported that the addition of PEG stabilizes liposomes due to repulsive barrier resulted from steric pressure forces and charged phosphate moieties.^36,37) Additionally, the presence of CHOL stabilizes liposomal formulations, as reported previously.^38,39)

Fig. 6. Release Pattern of PtCQ from Cationic Liposomes, Prepared by Remote Loading Method, in HEPES Buffer during Storage at 4°C

PtCQ was loaded into the liposomes at a drug-to-lipid ratio of 1 : 7 (w/w). Data represent the mean±S.D. (n=3).

Drug Release from Cationic PtCQ-Loaded Liposomes under Culture Conditions (Release Test)

A drug release study was carried out using medium containing FBS to mimic in vivo conditions. Figure 7 reveals that the PtCQ liposomes loaded by thin drug–lipid film method (drug to lipid ratio 1 : 7 (w/w)) have initial burst release equal to 8.9±0.5% of neutral liposomes and equal to 13.2±2.1% of cationic liposomes (cat. 5) at 0 h. The cause for initial burst release may be due to the adsorption of some drug molecules on the liposome surface during formation of the thin film. Furthermore, after 72 h, there is a significant drug release (p=0.0024) equal to 25.8±0.6% of cationic liposome compared to neutral liposomes’cumulative release equal to 17.8±2.6%. The cationic liposomes showed a maximum release which is slightly higher than that of neutral liposomes and this could be due to electrostatic repulsion between the cationic drug and the cationic lipid.

Fig. 7. Release Pattern of PtCQ from Neutral and Cationic Liposomes, Prepared by Thin Drug–Lipid Film Method, under Culture Conditions

PtCQ-loaded liposomes were incubated in medium containing 10% FBS at 37°C. PtCQ was loaded into the liposomes at a drug-to-lipid ratio of 1 : 7 (w/w). Data represent the mean±S.D. (n=3). (**** p<0.0001, *** p=0.0007, ** p=0.0024) compared to neutral liposomes.

The five formulations of cationic PtCQ-loaded liposomes (cat. 0, cat. 5, cat. 10, cat. 15, cat. 20) loaded using an ammonium sulfate gradient (drug to lipid ratio 1 : 7 (w/w) described above did not show initial burst release and showed no significant release of the drug after 72 h compared to cat. 0 (4.0±2.3%), as shown in Fig. 8. This demonstrates that the ammonium sulfate gradient is a promising method for the encapsulation of amphiphilic drugs into liposomes containing a saturated lipid (HSPC). Furthermore, the amount of drug released was sustainably released indicating that liposomes loaded with amphipathic weak bases using an ammonium sulfate gradient can provide controlled release of the loaded molecule. Increasing the cationic lipid from 0 to 20% mole percent has no significant effect on the release of the drug, showing that the surface charge has no effect on the release of amphipathic weak bases loaded using ammonium sulfate and pH gradients. The slow drug release observed in case of ammonium sulfate gradient loaded PtCQ-liposomes is necessary for long storage and to avoid drug leaking before reaching their target.⁵⁾ Afterward the liposomes would be adsorbed on the cell surface followed by a depletion of the liposomal proton gradient due to the body temperature or liposomes-cell interaction episodes. This depletion leads to accumulation of the weak basic drugs inside the RBC due to the electrochemical gradient resulted from the phospholipid asymmetry in RBC membranes, which maintains negatively charged membranes.

Fig. 8. Release Pattern of PtCQ from Cationic Liposomes, Prepared by Remote Loading Method, under Culture Conditions

PtCQ-loaded liposomes were incubated in medium containing 10% FBS at 37°C. PtCQ was loaded into the liposomes at a drug-to-lipid ratio of 1 : 7 (w/w). Data represent the mean±S.D. (n=3).

CONCLUSION

This paper described the synthesis and characterization of the trans-platinum–chloroquine complex (PtCQ) and the application of two methods to generate PtCQ-loaded liposomes: the thin drug–lipid film and the remote loading methods. Liposomes of uniform diameter were prepared using the thin film hydration and extrusion technique and the PtCQ was encapsulated in the liposomes. The most efficient encapsulation was obtained at a drug-to-lipid ratio of 1 : 7 (w/w) using the ammonium sulfate gradient active loading method. This approach provided liposomal formulations capable of sustaining a proton gradient for the weak basic drug PtCQ and the encapsulation efficiency was approximately 100%. High intra-liposomal retention levels of the drug inside different cationic liposomes for several hours under culture and storage conditions were maintained, suggesting that such liposomes hold promise for the future treatment of malaria by targeting P. falciparum. We will study the in vitro and in vivo effects of cationic PtCQ-loaded liposomes on this parasite and the encapsulation of other metal–CQ complexes in the future.

Acknowledgments

This work was partially supported by the Joint Usage/Research Center on Tropical Disease, Institute of Tropical Medicine, Nagasaki University (27-Ippan-3). According to the measurement of zeta potentials, authors are grateful to Dr. Noriko Ogawa and Dr. Chisato Takahashi who are the members of Dr. Hiromitsu Yamamoto’s lab (Aichi-Gakuin University).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) WHO: World Malaria Report. World Health Organization (2015).
• 2) Greenwood BM, Fidock DA, Kyle DE, Kappe SH, Alonso PL, Collins PLFH, Duffy PE. Malaria: progress, perils, and prospects for eradication. J. Clin. Invest., 118, 1266–1276 (2008).
• 3) Miller LH, Ackerman HC, Su X-Z, Wellems TE. Malaria biology and disease pathogenesis: insights for new treatments. Nat. Med., 19, 156–167 (2013).
• 4) Urbán P, Estelrich J, Cortés A, Fernàndez-Busquets X. A nanovector with complete discrimination for targeted delivery to Plasmodium falciparum-infected versus non-infected red blood cells in vitro. J. Control. Release, 151, 202–211 (2011).
• 5) Moles E, Urbán P, Jiménez-Díaz MB, Viera-Morilla S, Angulo-Barturen I, Busquets MA, Fernàndez-Busquets X. Immunoliposome-mediated drug delivery to Plasmodium-infected and non-infected red blood cells as a dual therapeutic/prophylactic antimalarial strategy. J. Control. Release, 210, 217–229 (2015).
• 6) Peeters PAM, Huiskamp CWEM, Eling WMC, Crommelin DJA. Chloroquine containing liposomes in the chemotherapy of murine malaria. Cambridge Journals, Parasitology, 98, 381–386 (1989).
• 7) Santos-Magalhães NS, Mosqueira VCF. Nanotechnology applied to the treatment of malaria. Adv. Drug Deliv. Rev., 62, 560–575 (2010).
• 8) Li LH, Hui SW. The effect of lipid molecular packing stress on cationic liposome-induced rabbit erythrocyte fusion. Biochim. Biophys. Acta, 1323, 105–116 (1997).
• 9) Stebelska K, Wyrozumska P, Sikorski AF. PS exposure increases the susceptibility of cells to fusion with DOTAP liposomes. Chem. Biol. Interact., 160, 165–174 (2006).
• 10) Obata Y, Tajima S, Takeoka S. Evaluation of pH-responsive liposomes containing amino acid-based zwitterionic lipids for improving intracellular drug delivery in vitro and in vivo. J. Control. Release, 142, 267–276 (2010).
• 11) Campbell RB, Ying B, Kuesters GM, Hemphill R. Fighting cancer: from the bench to bedside using second generation cationic liposomal therapeutics. J. Pharm. Sci., 98, 411–429 (2009).
• 12) Zhao W, Zhuang S, Qi XR. Comparative study of the in vitro and in vivo characteristics of cationic and neutral liposomes. Int. J. Nanomedicine, 6, 3087–3098 (2011).
• 13) Abu Lila AS, Kizuki S, Doi Y, Suzuki T, Ishida T, Kiwada H. Oxaliplatin encapsulated in PEG-coated cationic liposomes induces significant tumor growth suppression via a dual-targeting approach in a murine solid tumor model. J. Control. Release, 137, 8–14 (2009).
• 14) Tagami T, Yanai H, Terada Y, Ozeki T. Evaluation of phosphatidylserine-specific peptide-conjugated liposomes using a model system of malaria-infected erythrocytes. Biol. Pharm. Bull., 38, 1649–1651 (2015).
• 15) Föller M, Bobbala D, Koka S, Huber SM, Gulbins E, Lang F. Suicide for survival—Death of infected erythrocytes as a host mechanism to survive malaria. Cell. Physiol. Biochem., 24, 133–140 (2009).
• 16) Levchenko TS, Rammohan R, Lukyanov AN, Whiteman KR, Torchilin VP. Liposome clearance in mice: the effect of a separate and combined presence of surface charge and polymer coating. Int. J. Pharm., 240, 95–102 (2002).
• 17) Johnstone SA, Masin D, Mayer L, Bally MB. Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly(ethylene glycol) liposomes by mouse macrophages. Biochim. Biophys. Acta, 1513, 25–37 (2001).
• 18) Campbell RB, Fukumura D, Brown EB, Mazzola LM, Izumi Y, Jain RK, Torchilin VP, Munn LL. Cationic charge determines the distribution of liposomes between the vascular and extravascular compartments of tumors. Cancer Res., 62, 6831–6836 (2002).
• 19) Navarro M, Castro W, Higuera-Padilla AR, Sierraalta A, Abad MJ, Taylor P, Sánchez-Delgado RA. Synthesis, characterization and biological activity of trans-platinum(II) complexes with chloroquine. J. Inorg. Biochem., 105, 1684–1691 (2011).
• 20) Isacchi B, Arrigucci S, la Marca G, Bergonzi MC, Vannucchi MG, Novelli A, Bilia AR. Conventional and long-circulating liposomes of artemisinin: preparation, characterization, and pharmacokinetic profile in mice. J. Liposome Res., 21, 237–244 (2011).
• 21) Laouini A, Jaafar-Maalej C, Limayem-Blouza I, Sfar S, Charcosset C, Fessi H. Preparation, characterization and applications of liposomes: state of the art. Journal of Colloid Science and Biotechnology, 1, 147–168 (2012).
• 22) Haran G, Cohen R, Bar LK, Barenholz Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim. Biophys. Acta, 1151, 201–215 (1993).
• 23) Tagami T, Kubota M, Ozeki T. Effective remote loading of doxorubicin into DPPC/Poloxamer 188 hybrid liposome to retain thermosensitive property and the assessment of carrier-based acute cytotoxicity for pulmonary administration. J. Pharm. Sci., 104, 3824–3832 (2015).
• 24) Tagami T, Ernsting MJ, Li S-D. Efficient tumor regression by a single and low dose treatment with a novel and enhanced formulation of thermosensitive liposomal doxorubicin. J. Control. Release, 152, 303–309 (2011).
• 25) Qiu L, Jing N, Jin Y. Preparation and in vitro evaluation of liposomal chloroquine diphosphate loaded by a transmembrane pH-gradient method. Int. J. Pharm., 361, 56–63 (2008).
• 26) Ramana LN, Sethuraman S, Ranga U, Krishnan UM. Development of a liposomal nanodelivery system for nevirapine. J. Biomed. Sci., 17, 57 (2010).
• 27) Aditya NP, Chimote G, Gunalan K, Banerjee R, Patankar S, Madhusudhan B. Curcuminoids-loaded liposomes in combination with arteether protects against Plasmodium berghei infection in mice. Exp. Parasitol., 131, 292–299 (2012).
• 28) Navarro M, Castro W, Madamet M, Amalvict R, Benoit N, Pradines B. Metal-chloroquine derivatives as possible anti-malarial drugs: evaluation of anti-malarial activity and mode of action. Malar. J., 13, 471 (2014).
• 29) Khokhar AR, Al-Baker S, Brown T, Perez-Soler R. Chemical and biological studies on a series of lipid-soluble (trans-(R,R)- and -(S,S)-1,2-diaminocyclohexane) platinum(II) complexes incorporated in liposomes. J. Med. Chem., 34, 325–329 (1991).
• 30) Woodle MC, Collins LR, Sponsler E, Kossovsky N, Papahadjopoulos D, Martin FJ. Sterically stabilized liposomes reduction in electrophoretic mobility but not electrostatic surface potential. Biophys. J., 61, 902–910 (1992).
• 31) Madden TD, Harrigan PR, Tai LCL, Bally MB, Mayer LD, Redelmeier TE, Loughrey HC, Tilcock CP, Reinish LW, Cullis PR. The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: a survey. Chem. Phys. Lipids, 53, 37–46 (1990).
• 32) Harrigan PR, Wong KF, Redelmeier TE, Wheeler JJ, Cullis PR. Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients. Biochim. Biophys. Acta, 1149, 329–338 (1993).
• 33) Kraft JC, Freeling JP, Wang Z, Ho RJY. Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. J. Pharm. Sci., 103, 29–52 (2014).
• 34) Moribe K, Maruyama K, Iwatsuru M. Estimation of surface state of poly(ethylene glycol)-coated liposomes using an aqueous two-phase partitioning technique. Chem. Pharm. Bull., 45, 1683–1687 (1997).
• 35) Dan N. Effect of liposome charge and PEG polymer layer thickness on cell-liposome electrostatic interactions. Biochim. Biophys. Acta, 1564, 343–348 (2002).
• 36) Woodle MC, Lasic DL. Sterically stabilized liposomes. Biochim. Biophys. Acta, 1113, 171–199 (1992).
• 37) Needham D, Mclntosh TJ, Lasic DD. Repulsive interactions and mechanical stability of polymer-grafted lipid membranes. Biochim. Biophys. Acta, 1108, 40–48 (1992).
• 38) Briuglia M-L, Rotella C, McFarlane A, Lamprou DA. Influence of cholesterol on liposome stability and on in vitro drug release. Drug Delivery and Translational Research, 5, 231–242 (2015).
• 39) Lee S-C, Lee K-E, Kim J-J, Lim S-H. The effect of cholesterol in the liposome bilayer on the stabilization of incorporated retinol. J. Liposome Res., 15, 157–166 (2005).