Study of the efficacy of antimalarial drugs delivered inside targeted immunoliposomal nanovectors
© Urbán et al; licensee Springer. 2011
Received: 9 September 2011
Accepted: 7 December 2011
Published: 7 December 2011
Paul Ehrlich's dream of a 'magic bullet' that would specifically destroy invading microbes is now a major aspect of clinical medicine. However, a century later, the implementation of this medical holy grail continues being a challenge in three main fronts: identifying the right molecular or cellular targets for a particular disease, having a drug that is effective against it, and finding a strategy for the efficient delivery of sufficient amounts of the drug in an active state exclusively to the selected targets. In a previous work, we engineered an immunoliposomal nanovector for the targeted delivery of its contents exclusively to Plasmodium falciparum-infected red blood cells [pRBCs]. In preliminary assays, the antimalarial drug chloroquine showed improved efficacy when delivered inside immunoliposomes targeted with the pRBC-specific monoclonal antibody BM1234. Because difficulties in determining the exact concentration of the drug due to its low amounts prevented an accurate estimation of the nanovector performance, here, we have developed an HPLC-based method for the precise determination of the concentrations in the liposomal preparations of chloroquine and of a second antimalarial drug, fosmidomycin. The results obtained indicate that immunoliposome encapsulation of chloroquine and fosmidomycin improves by tenfold the efficacy of antimalarial drugs. The targeting antibody used binds preferentially to pRBCs containing late maturation stages of the parasite. In accordance with this observation, the best performing immunoliposomes are those added to Plasmodium cultures having a larger number of late form-containing pRBCs. An average of five antibody molecules per liposome significantly improves in cell cultures the performance of immunoliposomes over non-functionalized liposomes as drug delivery vessels. Increasing the number of antibodies on the liposome surface correspondingly increases performance, with a reduction of 50% parasitemia achieved with immunoliposomes encapsulating 4 nM chloroquine and bearing an estimated 250 BM1234 units. The nanovector prototype described here can be a valuable platform amenable to modification and improvement with the objective of designing a nanostructure adequate to enter the preclinical pipeline as a new antimalarial therapy.
The need to develop new strategies to treat malaria is urgent if one considers that the cases of resistance to current antimalarial agents increase, especially in zones in which P. falciparum is endemic, and calls for combined therapy approaches [5, 8]. Several drugs show different degrees of toxicity, which limits their use because current administration forms release the free compound in the blood and offer little specificity regarding the targeted cells . Consequently, to achieve therapeutic levels that extend over time, the initial concentration of the drug in the body should be high. On the other hand, if the administered chemical has unspecific toxicity, the low doses required contribute to the development of resistant parasite strains . The challenge of drug delivery is the liberation of adequate doses of therapeutic agents to a specific target site at the right time in a safe and reproducible manner . A number of mechanisms can provide controlled release, including transdermal patches, implants, inhalation systems, bioadhesive systems, and nanoencapsulation [12–14], but few of these have been designed for specificity. Taking into account the peculiarities of pRBCs, lipid-based nanocarriers have been one of the most promising approaches for the targeted delivery of antimalarial drugs .
Liposomes are synthetic lipid bilayer-enclosed structures up to several hundred nanometers in diameter that can improve the delivery of bioactive molecules by functioning as circulating microreservoirs for sustained release [16, 17]. Liposomes bearing cell-specific recognition ligands on their surfaces have been widely considered as drug carriers in therapy [18, 19], and liposome encapsulation has been assayed for the targeted delivery of compounds against murine malaria [20–22]. Liposomal nanovessels incorporating a P. berghei amino acid sequence have been shown to greatly increase their targeting to the liver [23, 24], suggesting that they can be an adequate vector to channel antimalarials towards the hepatocyte stages of the parasite.
In a previous work , we engineered an immunoliposomal nanovector for the targeted delivery of its contents exclusively to pRBCs. Two hundred-nanometer liposomes loaded with quantum dots were covalently functionalized with oriented, specific half-antibodies against P. falciparum late form-infected pRBCs. In less than 90 min, 100% of late form-containing pRBCs and 0% of noninfected RBCs in living P. falciparum cultures were infiltrated by the content of targeted immunoliposomes. Here, we present a quantitative study of the efficacy of this nanovector in ameliorating the activity of the antimalarial drugs chloroquine and fosmidomycin, and discuss its characteristics and room for improvement regarding future animal assays that might place this prototype on the threshold of clinical trials.
Materials and methods
Except where otherwise indicated, all reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). The commercial monoclonal antibody BM1234 against P. falciparum pRBCs was obtained from Acris Antibodies GmbH (Herford, Germany).
Liposome and immunoliposome preparation
Liposomes were prepared by the lipid film hydration method , with the lipid formulation 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine [DOPC] (Avanti Polar Lipids, Inc., Alabaster, AL, USA)/cholesterol 80:20, as described previously . The dry lipids were hydrated at 37°C in phosphate-buffered saline [PBS] containing the antimalarial drug to obtain a concentration of 10 mM lipid. For the coupling of targeting antibodies to liposomes, we followed established protocols  that use 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] [MPB-PE] (Avanti Polar Lipids, Inc.) to incorporate proteins into liposomes (DOPC/cholesterol/MPB-PE 77.5:20:2.5, 10 mM lipid) through the reaction of the maleimide group in the lipid with a thiol group from the ligand .
Quantification of chloroquine and fosmidomycin
The amount of chloroquine and fosmidomycin encapsulated inside liposomes was determined using high-performance liquid chromatography with tandem mass spectrometry [HPLC-MS/MS]. Prior to analysis, lipids were extracted following stardard protocols  in order to release the entrapped drug. Briefly, 100 μl of liposome sample was mixed with 225 μl of cold methanol and 125 μl of chloroform. Phase separation was achieved by adding 125 μl of 0.1 M HCl and 125 μl of chloroform. After mixing thoroughly, the samples were spun down at 7,500 × g for 5 min. The upper water-methanol layer, containing the drug that had been encapsulated inside liposomes, was collected and stored at -20°C until analysis. HPLC analysis was performed in an Alliance 2695 chromatographic system (Waters Corporation, Milford, MA, USA) using an Atlantis dC18 analytical column (internal diameter [i.d.] 150 × 2.1 mm, 5 μm, Waters Corporation) for chloroquine and a Luna C18 analytical column (i.d. 150 × 2.0 mm, 5 μm, Phenomenex, Torrance, CA, USA) for fosmidomycin, with mobile phase A (0.2% formic acid in water) and B (acetonitrile). The linear gradient for the determination of chloroquine at a flow rate of 0.3 ml/min was (% mobile phase B/min) 0/0, 10/2, 40/17, and 90/22 whereas for the determination of fosmidomycin at a flow rate of 0.4 ml/min, it was 0/0, 0/5, and 95/10. The HPLC system was coupled to a SCIEX API 365 Triple Quadrupole mass spectrometer equipped with a Turbo Ion Spray ion source (PerkinElmer, Waltham, MA, USA). For quantification purposes, data were collected in the multiple reaction monitoring mode with positive-ion detection for chloroquine and negative-ion detection for fosmidomycin, tracking the transition of the parent and product ions specific for each compound: 320.3/141.9 and 320.3/246.7 for chloroquine and 182.0/79.0 and 182.0/136.0 for fosmidomycin.
For cryo-electron microscopy analysis of the preparations of liposomes, a thin aqueous film was formed by dipping a glow-discharged holey carbon grid in the liposome suspension and then blotting the grid against a filter paper. The resulting thin sample films spanning the grid holes were vitrified by plunging the grid (kept at 100% humidity and room temperature) into ethane, which was maintained at its melting point with liquid nitrogen, using a Vitrobot (FEI Company, Eindhoven, The Netherlands). The vitreous films were transferred to a Tecnai F20 transmission electron microscope [TEM] (FEI Company) using a cryotransfer (Gatan, Inc., Pleasanton, CA, USA), and the samples were observed in a low-dose mode. Images were acquired at 200 kV at a temperature between -170°C and -175°C, using low-dose imaging conditions not exceeding 20 e -/Å2, with a 4,096 × 4,096 pixel CCD Eagle camera (FEI Company), and digitized with the Tecnai Image Acquisition program.
Plasmodium falciparum cell culture and growth inhibition assays
The P. falciparum 3D7 strain was grown in vitro in group B washed human RBCs prepared as described elsewhere  using previously described conditions . Briefly, parasites (thawed from glycerol stocks) were cultured at 37°C in Petri dishes containing RBCs in RPMI complete medium under a gas mixture of 92% N2, 5% CO2, and 3% O2. Synchronized cultures were obtained by 5% sorbitol lysis , and the medium was changed every 2 days maintaining 3% hematocrit. For culture maintenance, parasitemias were kept below 5% late forms by dilution with washed RBCs. For standard growth inhibition assays, parasitemia was adjusted to 1.5% with more than 90% of parasites at the ring stage after sorbitol synchronization. For modified growth inhibition assays, synchronized cultures were incubated for 24 h before addition of the drug to allow for the appearance of late forms presenting the epitope recognized by BM1234 bound to immunoliposomes. Two hundred microliters of these living Plasmodium cultures were plated in 96-well plates and incubated for 48 h at 37°C in the presence of free drugs and drugs encapsulated in liposomes and immunoliposomes at a final concentration of 100 μM lipid. Parasitemia was determined by microscopic counting of blood smears or by fluorescence-assisted cell sorting [FACS]. Smears were fixed in methanol for a few seconds and then stained for 10 min with Giemsa (Merck Chemicals, Darmstadt, Germany) diluted 1:10 in Sorenson's buffer, pH 7.2. After washing with distilled water and drying, the ratio of the infected vs. noninfected RBCs was determined by microscopic analysis. For FACS analysis, noninfected RBCs and samples containing pRBCs were diluted to a final concentration of 1 to 10 × 106 cells/ml. The cell suspension was stained with SYTO 11 (0.5 mM stock in DMSO, Molecular Probes, Eugene, OR, USA) to a final concentration of 0.5 μM, and samples were incubated for 5 to 10 min prior to analysis in a Cytomics FC 500 MPL (Beckman Coulter, Inc., Fullerton, CA, USA) set up with the standard configuration. Excitation of the sample was done using a 488-nm, air-cooled, argon-ion laser at 15-mW power using forward and side scatter to gate the RBC population, and SYTO 11 green fluorescence (525 nm) was collected in a logarithmic scale. The single-cell population was selected on a forward-side scattergram, and the green fluorescence from this population was analyzed. Parasitemia was expressed as the number of parasitized cells per 100 erythrocytes.
Data are presented as the mean ± standard error of at least three independent experiments, and the corresponding standard errors in histograms are represented by error bars. Statistical analyses were performed using the Statgraphics Centurion XVI.I data analysis and statistical software. The parametric Student's t test was used to compare two independent groups when data followed a Gaussian distribution. Otherwise, the nonparametric Mann-Whitney test was used. Differences were considered significant when the p value was ≤ 0.05.
Confocal fluorescence microscopy
For immunofluorescence assays, pRBC smears were fixed in ice for 2 min in acetone/methanol (90:10) and incubated with 2 μg BM1234 monoclonal antibody/ml PBS containing 0.75% w/v BSA for 90 min at 37°C. After PBS washing steps, primary antibodies were detected with a secondary fluorescent antibody Alexa Fluor 488 goat anti-mouse F(ab')2 (Molecular Probes). Parasite nuclei were stained with 4'6-diamino-2-phenylindole [DAPI] (Invitrogen Corporation, Carlsbad, CA, USA) during the secondary antibody incubation, and the RBC membrane was labeled with a wheat germ agglutinin [WGA]-tetramethylrhodamine conjugate (Molecular Probes). After the corresponding incubations and PBS washing steps, the samples were finally mounted with Mowiol (Calbiochem, Merck Chemicals) following standard protocols . The samples were imaged with a TCS SP5 laser scanning confocal microscope equipped with an acoustic optical beam splitter (Leica Microsytstems Inc., Buffalo Grove, IL, USA), a DMI6000 inverted microscope (Leica Microsytstems Inc.), blue diode (405 nm), Ar (458/476/488/496/514 nm), diode-pumped solid-state (561 nm), and HeNe (594/633 nm) lasers, and APO ×63 oil (NA 1.4) or glycerol (NA 1.3) immersion objective lenses. DAPI, Alexa Fluor 488, reflection (for hemozoin detection), and WGA-rhodamine images were acquired sequentially using 405, 488, 488, and 561 laser lines and emission detection ranges 415 to 480 nm, 500 to 550 nm, 480 to 500 nm, and 571 to 625 nm, respectively, with the confocal pinhole set at 1 airy unit. Bright field transmitted light images were acquired simultaneously at 400 Hz in a 512 × 512 pixel format, ×8 zoom, and a pixel size of 60 × 60 nm.
Results and discussion
We have calculated that the protocol used for the preparation of the samples from Figure 4A results in an average number of five antibody molecules per liposome , and the data presented here indicate that this is the minimal antibody/liposome ratio that significantly improves the activity of immunoliposomal drugs. This ratio was sufficient to provide a complete pRBC targeting specificity according to the delivery of immunoliposome-contained quantum dots , but the same amount of targeting antibody in immunoliposomes does not result in a 100% elimination of parasitemia at the low antimalarial drug concentrations assayed. Because the role of targeting antibodies is most likely to anchor liposomes to pRBCs when a random collision occurs, the in vitro efficacy of immunoliposomes as drug delivery vessels can likely be increased by incorporating more antibodies on their surfaces. As shown in Figure 4B, C, increasing the antibody concentration in immunoliposomes containing 4 nM chloroquine results in a corresponding significant increase of nanovector performance, reaching a clearance of 50% parasitemia for liposomes studded with an estimated 250 antibody molecules. However, keeping the antibody concentration as low as possible will contribute to reduce the immune response towards plasma-circulating immunoliposomes. Thus, when designing in vivo assays, a balance has to be reached between a sufficiently high density of targeting antibody for efficient drug delivery and levels below those triggering fast lymphocyte uptake. Since another factor affecting the number of random encounters between liposomes and pRBCs is the movement of both particles in solution, we expect that the turbulent mixing in the bloodstream will permit the use of low targeting antibody amounts. As mentioned above, BM1234 immunoliposomes deliver their contents to all pRBCs present in a sample, but when loaded with drug, they are not able to eliminate all Plasmodium parasites. It is possible that an increased performance can be achieved even with a few targeting antibody molecules per liposome if the amount of the encapsulated drug could be increased. In this regard, Ashley et al.  have assembled liposome-enclosed silica nanoparticles termed protocells, able to carry high anticancer drug payloads, with a single protocell having the capacity to kill a cancer cell. It would be worth exploring if such protocells can also be used as antimalarial drug-containing structures.
The remarkable capacity of liposomes to inject their contents into pRBCs presumably has its basis on alterations of the pRBC plasma membrane, rendering it less elastic and thus limiting the rebounding of colliding liposomes. The resulting slightly longer interactions, a phenomenon exacerbated if targeting antibodies are present, likely allow enough time for the physical phenomenon of lipid bilayer fusion to occur. This hypothesis could also explain why liposomes devoid of the antibody do perform better when added at the trophozoite stage since the membrane of ring-containing pRBCs is more similar to uninfected erythrocytes than that of pRBCs infected by later forms.
Next in our agenda is to advance towards a nanovector-based antimalarial delivery strategy suitable to enter preclinical trials. Liposomal nanovectors are adequate for a parenteral delivery, indicated in cases of complicated malaria, those at risk of developing severe disease, or if the patient is vomiting and unable to take oral antimalarials. Parenteral treatment can also be required in the last mile of a malaria eradication protocol for the single-dose, individualized administration of highly toxic drugs specifically targeted to pRBCs with good accuracy with the objective of eliminating remaining multiresistant strains. The nanovector prototype described here can improve ca. tenfold the activity of hydrosoluble antimalarial drugs, and it is amenable to improvement or adaptation through modification of some of its parts, e.g., better antibodies or targeting molecules, different nanocapsule structures, or new antimalarial drugs. Finally, BM1234 immunoliposomes can also be an efficient drug delivery vessel for the administration of liposoluble drugs against P. falciparum; unlike chloroquine and fosmidomycin, the solubility of hydrophobic compounds will be low when free in plasma, but can be significantly increased when incorporated in the lipid bilayers of liposomes.
This work was supported by grants BIO2008-01184, and CSD2006-00012 from the Ministerio de Ciencia e Innovación, Spain, which included FEDER funds, and by grant 2009SGR-760 from the Generalitat de Catalunya, Spain. A fellowship of the Instituto de Salud Carlos III (Spain) is acknowledged by Patricia Urbán. We acknowledge the support of the Scientific and Technological Centres from the University of Barcelona (CCiT-UB: cytometry, confocal microscopy, cryo-electron microscopy, and liquid chromatography units).
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