Notes
Evaluation of Phosphatidylserine-Specific Peptide-Conjugated Liposomes Using a Model System of Malaria-Infected Erythrocytes
2015 Volume 38 Issue 10 Pages 1649-1651
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2015 Volume 38 Issue 10 Pages 1649-1651
Malaria is one of the most prevalent parasitic diseases and is most widespread in tropical regions. The malarial parasite grows and reproduces in erythrocytes during its life cycle, resulting in programmed erythrocyte death, termed eryptosis. Lipid scrambling, which occurs following the exposure of anionic lipids such as phosphatidylserine (PS) on the outer surface of erythrocytes, is a characteristic physical change that occurs early during eryptosis. Here, we prepared “PS specific peptide (PSP)”-conjugated liposomes (PSP-liposomes) and investigated whether PSP-liposomes hold promise as a novel strategy for actively targeting eryptosis. Eryptosis was induced by exposing red blood cells (RBCs) to ionomycin, a known calcium ionophore. When PSP liposomes were mixed with either RBCs or RBCs undergoing eryptosis (E-RBCs), the amount of PSP-liposome bound to E-RBCs was much higher than the amount bound to RBCs. However, the amount of PSP-liposome bound to E-RBCs was significantly inhibited by the presence of annexin V protein, which binds specifically to PS. These results suggest that PSP-liposomes could be an effective drug nanocarrier for treating E-RBCs and malaria-infected erythrocytes.
Malaria is a parasitic disease which occurs in more than 100 countries and is especially prevalent in Africa. More than 200 million people are infected, of which several percent die each year. The development of a drug delivery system for anti-malaria drugs is a promising strategy1) but is still in its infancy because of the complexity of the parasite’s life cycle. A promising methodology is active drug targeting against the malarial plasmodium, parasite-infected erythrocytes, hepatocytes, and macrophages. Immunoliposomes have been extensively investigated and shown to exhibit significant therapeutic effects.2) However, the development of immunoliposomes is hold: The antigens from different subspecies of malarial parasites are different. Therefore novel and universal targeting candidates which have effective targeting ability against various kinds of malarial parasites have been pursuing.
Eryptosis (erythrocyte apoptosis) is a term recently proposed by Lang’s group to describe programmed erythrocyte death.3) Eryptosis is induced by physical and chemical stress, and by infection of parasites, including the malarial parasite.4) Lipid scrambling of the cell membrane is the primary characteristic of early phase eryptosis.5) While anionic lipids such as phosphatidylserine (PS) are generally localized in the inner membrane of erythrocytes under normal conditions, after stimulation by eryptosis, anionic lipids are found on the outer surface of erythrocytes due to the inability of the cells to control lipid movement.
We hypothesized that the surface-exposure of PS resulting from eryptosis can be used for the novel and universal strategy of targeting malarial parasite-infected erythrocytes, but it is unclear whether PS specific peptides (PSPs) are effective against eryptosis. In this study we characterized PSP-conjugated liposomes (PSP-liposomes) and assessed their binding to erythrocytes undergoing eryptosis.
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from Avanti Polar Lipid (Avanti Polar Lipids, AL, U.S.A.). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG2000-DSPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide-conjugated (Mal-PEG2000-DSPE) were generously donated by NOF Corporation (Tokyo, Japan). Cholesterol (CHOL) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The lipophilic fluorescent dye, DiI, was purchased from Invitrogen (CA, U.S.A.). The sequence of PSP was LSYYPSYC as previously described with minor modifications6) was synthesized by Biologica Co. (Nagoya, Japan). All other reagents were of analytical grade.
Preparation of PSP-LiposomesThe liposomes were prepared by the thin film hydration method, followed by membrane extrusion to produce liposomes of uniform size, as described previously.7) In order to prepare fluorescently-labeled liposomes, 1 mol% of DiI was added. The mean diameters of liposomes were determined by dynamic light scattering with a particle sizer (ZetaSizer Nano-ZS; Malvern Instrument Ltd., Malvern, U.K.).
PSP-PEG-DSPE was inserted into liposomes by post-insertion methods, as described previously.8) The lipid ratio was DSPC/CHOL/PSP-PEG-DSPE=2/1/0.1 (molar ratio). A fluorescence dye (FAM)-conjugated PSP (synthesized by Biologica Co.) was used for the measurement of conjugation efficiency. The FAM-PSP-liposomal fraction and free FAM-PSP fraction was separated by gel filtration and the fluorescence intensity in each fraction was measured to calculate the conjugation efficiency of PSP into liposome.
AnimalsMale Std-ddY mice (aged 5–6 weeks, approximately 20 g) were purchased from Japan SLC (Shizuoka, Japan). All animal experiments were approved by the Animal Care Committee of Nagoya City University.
Preparation of Eryptosis-Induced Red Blood Cells (E-RBCs)Eryptosis was induced as described previously with some modification.4) Briefly, 100 µL of RBC pellet prepared was gently treated with 900 µL of Ringer’s solution in the presence of ionomycin solution (Sigma-Aldrich, MO, U.S.A.) or treated with high osmotic Ringer’s solution, then the mixture was incubated for 3 h at 37°C. The surface-exposure of PS was evaluated using an Annexin V-FLUOR staining kit (Roche Applied Science, IN, U.S.A.) with flow cytometry (FACScan; BD Biosciences, CA, U.S.A.).
Determination of Fluorescently-Labeled Liposomes Bound to ErythrocytesDiI-labeled liposome suspension (10 µL) was gently mixed with 965 µL of Ringer’s solution and 25 µL of RBCs or E-RBC suspension, then the mixtures were incubated for 0.5–3 h at 37°C. The erythrocyte samples with bound DiI-labeled liposomes were analyzed by flow cytometry (BD Biosciences). The fluorescence intensity was normalized by the mean fluorescence intensity of E-RBCs with bound PSP-liposomes.
Statistical AnalysisAll data are mean values±standard deviation (S.D.). A two way ANOVA with Bonferroni post-test was used to assess statistical significance by using GraphPad Prism (GraphPad Software Inc., CA, U.S.A.).
The PSP-liposomes was characterized in Table 1. The induction of eryptosis was evaluated by detecting exposed PS on the surface of the erythrocytes. Approximately 50% of the erythrocytes were PS-positive in the presence of ionomycin (at least 1 µM), a calcium ionophore which could induce eryptosis by the influx of calcium ion into erythrocytes (Supplementary Fig. 1). Brand et al. reported that an influx of calcium ion was induced by the growth of the parasite, followed by PS exposure.4) These results are in agreement with our observations. Although our experimental model does not completely reproduce the infection of erythrocytes by the malarial parasite, they do mimic PS exposure caused by eryptosis. Similar eryptosis was observed by high osmotic pressure (900 mOsm).
| Diameter (nm) | 132.6±7.7 |
| PDI | 0.053±0.024 |
| Zeta potential (mV) | −33.9±0.3 |
| Conjugation efficiency (%) | 62.0±7.1 |
Data represent mean±S.D. (n=3).
The amount of liposome bound to RBCs or E-RBCs was evaluated using fluorescence dye (DiI)-labeled liposomes. PSP-liposomes bound significantly to E-RBCs (Fig. 1) compared with RBCs. In contrast, PEGylated liposomes did not bind to RBCs or E-RBCs. Unexpectedly, maleimide-group-conjugated PEG liposomes, in which the maleimide group was used to conjugate PEG and PSP, non-specifically bound with E-RBCs. Various proteins containing a thiol group can be exposed during eryptosis, suggesting that Mal-PEG conjugated liposomes could interact with them. We previously have demonstrated the experiment by using cationic liposomes in our earlier trials. The liposomal composition containing cationic lipid and PEG-lipid could affect the binding amount of E-RBC. By testing various incubation times after mixing PSP-liposomes and erythrocytes (Fig. 2), we demonstrated that specific binding of PSP-liposomes with E-RBCs occurred within 3 h. We speculate that PEGylated liposome could bind with neither E-RBC nor RBC, in contrast, Mal-PEG-liposome might bind E-RBC at some extent.
White column, RBC; Black column, E-RBC. After the induction of eryptosis in the presence of ionomycin (1 µM) for 3 h, the E-RBCs were incubated with DiI-labeled liposome for 3 h. Data represent mean±S.D. (n=3); *** p<0.005 vs. control E-RBC; †† p<0.01, ††† p<0.005, RBC vs. E-RBC; ‡‡ p<0.01 vs. Mal-PEG-liposome treatment group for E-RBC.
White column, RBC; Black column, E-RBC. After the induction of eryptosis in the presence of ionomycin (1 µM) for 3 h, the E-RBCs were incubated with DiI-labeled liposome for 0, 0.5 h, 1 h or 3 h. Data represent mean±S.D. (n=3); * p<0.05, *** p<0.005 vs. control E-RBC; ††† p<0.005, RBC vs. E-RBC.
To confirm the specific binding of PSP, PSP-liposomes were incubated with E-RBCs and RBCs in the presence of annexin V. As shown in Fig. 3, the fluorescence of bound PSP-liposomes to E-RBCs was significantly inhibited by annexin V, whereas the fluorescence of bound PSP-liposomes to RBCs was unchanged. In the point of incomplete inhibition of the binding of PSP-liposome in the presence of excess amount of annexin V, we do not have clear reasons. PSP-liposome has possibility to bind with other components which are induced by eryptosis. We speculate that non-fluorescence-labeled PSP-liposome also can inhibit the binding of DiI-labeled PSP-liposome.
White column, RBC; Black column, E-RBC. After the induction of eryptosis in the presence of ionomycin (1 µM) for 3 h, the E-RBCs were incubated with DiI-labeled liposome in the presence of Annexin V for 3 h. Data represent mean±S.D. (n=3); *** p<0.005 vs. PSP-liposome treatment group for E-RBC; ††† p<0.005, RBC vs. E-RBC.
We have demonstrated a new strategy for delivering drugs to eryptosis-induced erythrocyte which is a model of malaria parasite-infected erythrocytes. PS is likely a universal target for erythrocytes undergoing eryptosis. Although further improvements are necessary for the development of efficient and specific nanocarriers, the present study provides insights into the design of an effective and practical drug delivery system for malaria-infected erythrocytes.
This research was supported in part by a Grant from SENSHIN Medical Research Foundation and by a Grant-in-Aid for Research from Nagoya City University, Japan. According to the measurement of zeta potentials of liposomes, we express our appreciation to Dr. Noriko Ogawa, Dr. Chisato Takahashi who are the members of Dr. Hiromitsu Yamamoto’s lab (Aichi-Gakuin University).
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
