Liposomal Systems as Nanocarriers for the Antiviral Agent Ivermectin

RNA virus infections can lead to the onset of severe diseases such as fever with haemorrhage, multiorgan failure, and mortality. The emergence and reemergence of RNA viruses continue to pose a significant public health threat worldwide with particular attention to the increasing incidence of flaviviruses, among others Dengue, West Nile Virus, and Yellow Fever viruses. Development of new and potent antivirals is thus urgently needed. Ivermectin, an already known antihelminthic drug, has shown potent effects in vitro on Flavivirus helicase, with EC50 values in the subnanomolar range for Yellow Fever and submicromolar EC50 for Dengue Fever, Japanese encephalitis, and tick-borne encephalitis viruses. However ivermectin is hampered in its application by pharmacokinetic problems (little solubility and high cytotoxicity). To overcome such problems we engineered different compositions of liposomes as ivermectin carriers characterizing and testing them on several cell lines for cytotoxicity. The engineered liposomes were less cytotoxic than ivermectin alone and they showed a significant increase of the antiviral activity in all the Dengue stains tested (1, 2, and S221). In the current study ivermectin is confirmed to be an effective potential antiviral and liposomes, as drug carriers, are shown to modulate the drug activity. All together the results represent a promising starting point for future improvement of ivermectin as antiviral and its delivery.


Introduction
Several emerging RNA viruses have been the cause of international epidemics within the past few years. The four Dengue virus (DENV) serotypes have considerably expanded their geographic distribution in recent years. With billions of people at risk, more than 50 million cases, and 12500-25000 deaths annually, DENV is considered an emerging pathogen in a growing number of countries [1]. In particular, the presence of four DENV (DENV 1, 2, 3, and 4) serotypes has complicated the design of vaccines because incomplete protection against one serotype may influence the disease outcome once infection is established by a different serotype, through a process referred to as antibody-mediated disease enhancement [2].
Since no specific antiviral treatment is available for flaviviruses, there is an urgent need for antiviral drugs.
Flaviviruses are enveloped viruses [3] with single-strand positive-sense RNA genome of about 11 kbs. There are more than 80 different flaviviruses [4] and most of them are important human pathogens, such as Yellow Fever virus (YFV), West Nile Virus (WNV) and its Australian variant Kunjin virus (KUNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), Saint Louis encephalitis virus (SLEV), and DENV. The C-terminal domain of nonstructural protein 3 (NS3) has an ATP-dependent helicase activity that maintains viral replication and for that reason is a good target for designing selective inhibitors of viral replication as therapeutic intervention [5]. 2 International Journal of Biomaterials Mastrangelo et al. [6] identified the widely used antihelminthic drug ivermectin as a molecule able to inhibit the NS3 helicase activity of several flaviviruses in vitro at submicromolar concentrations. Most importantly, ivermectin proved to be a selective inhibitor of the replication of YFV [6] and, although less efficiently, of other flaviviruses such as DENV, JEV, and TBEV. Ivermectin is now under clinical trials on humans, as a DENV therapeutic, at Mahidol University in Thailand [7].
Ivermectin is the common name of 22,23-dihydroavermectin B1, a semisynthetic derivative of avermectin family. It is a potent endo-and ectoparasitic agent with a broad spectrum of activity; its clinical use has been greatly recognized up the top award in science, being the Nobel Prize in Medicine and Physiology 2015 awarded to William C. Campbell and SatoshiŌmura for their discovery of avermectin [8].
The pharmacokinetic behavior of avermectin depends upon the route of administration, the formulation used, the animal species, and pathophysiological status of the host. It is established that subcutaneous injection is the most efficient route for ivermectin administration in terms of drug bioavailability in sheep, cattle, and goats when compared with oral and topical administration [9].
Pharmaceutical technology has been applied to develop different drug formulations and delivery systems to optimize the pharmacological availability of ivermectin. The most promising approach for improving formulation lies in innovative delivery systems using carriers with defined physicochemical properties, such as liposomes, microemulsion, and polymeric micelles.
Although the efficacy of ivermectin has been established in humans against several parasite diseases, the antiviral properties of this compound are not yet exploited, mainly due to its complex chemical structure which cannot be easily chemically modified. The lack of appropriate formulations which could improve cellular internalization of ivermectin reduces the unfavorable effect of the drug [10].
According to these considerations, the current paper reports the development of engineered liposome formulations for possible clinical use of ivermectin. In particular, this study describes the production and the characterization of liposomes with ivermectin followed by subsequent tests of their antiviral activity and cytotoxicity in different cell lines.
Our results show that ivermectin, when delivered through liposomes, reduces the cytotoxicity and, at the same time, inhibits DENV replication with EC 50 values in the micromolar range improving the performance of ivermectin alone.

Cytotoxicity
Test. 2 -c-Methylcytidine (2-cmc) was from Sigma-Aldrich, UK. All the other reagents and solvents were from Sigma-Aldrich, UK, having analytical grade.

Liposome Preparation and Characterization.
Liposomes were prepared by a minor modification of the ethanol injection procedure [11]. Typically, ivermectin was solubilized in ethanol (final concentration 50 mM) and different volumes were mixed with an ethanolic solution of PC 30-180 mM, plus cholesterol (10 mM) or DDAB (5-10 mM), in order to obtain the final required ivermectin concentrations. 500 L of the resulting ethanolic solution was injected by a syringe pump (500 L/min) into 4.5 mL of double-distilled water under magnetic stirring (300 rpm).
Liposome size analysis with photon correlation spectroscopy (PCS), the cryogenic transmission electron microscopy (cryo-TEM) analysis, and the determination of encapsulation efficiency were performed as previously described [11].

Cells and Viruses.
All the cell lines were grown at 37 ∘ C in a humidified atmosphere of 5% CO 2 with the addition in the medium of 100 U penicillin/mL and 100 g/mL streptomycin.
Dengue virus strains were obtained from the early Dengue infection and outcome (EDEN) study in Singapore [12] with the following GenBank accession numbers: DENV 1 (EU081230) and DENV 2 (EU081177) [13,14]. The mouse adapted strain of DENV 2 S221 was kindly provided by Dr. Sujan Shresta (La Jolla Institute for Allergy and Immunology, CA). All virus strains were grown in C6/36 cells. Virus stocks were titered by plaque assay and stored at −80 ∘ C.   stained with methylene blue, was examined microscopically. The wells were analysed giving a score to each well (high value: white; low value: blue).

CC 50
Calculation. The half maximal cytotoxicity concentration (CC 50 ) was defined as the compound concentration that reduces the number of viable cells by 50% and it was calculated based on the nonlinear regression dose response sigmoidal curve drawn in GraphPad Prism.

Antiviral Assay in Human Huh-7 Cells
Infected with DENV Viruses. 10 5 cells were infected with DENV 1, DENV 2, or DENV 2 mouse adapted S221 strain at MOI 0.3, in the presence of either ivermectin alone or ivermectin contained in liposomal formulations for 1 h. Inocula were discarded and replaced with maintenance media containing the same compounds and kept for 48 h. After 2 days, cell morphology was observed under microscope before collecting supernatant that was used for plaque quantitation assay. The virus titer without drug treatment routinely reached more than 10 5 pfu/mL, representing the standard infection level in Huh-7 cells [15].

EC 50 Calculation.
Half maximal effective concentration (EC 50 ) was defined as the compound concentration that reduces the virus effect by 50% and it was calculated based on the nonlinear regression dose response sigmoidal curve drawn in GraphPad Prism.

Liposomes Preparation and Characterization.
The preparation of liposomes was accomplished by ethanol injection since this method allows single-step formation of liposomes with a narrow size distribution, without degradation or oxidation of lipids or drugs. We used the "controlled" injection method in which the ethanolic solution of lipids is injected into the water phase via a syringe pump at a controlled rate, allowing precise ethanol diffusion in water. The mixing of ethanol with water is indeed the crucial step leading to lipid molecule self-assembly, driving liposome formation [11].
In order to optimize the formulation of liposomes for ivermectin ( Figure 1(a)), we explored the roles of different experimental parameters on liposome characteristics, such as (a) lipid composition, (b) type of cationic surfactant, (c) PC content, and (d) ivermectin content. Table 1 reports the complete composition of produced liposomes together with a unique identification code for each formulation.
The effect of lipid composition and drug concentration was also investigated. Figure 2 summarizes the results of photon correlation spectroscopy (PCS) measurements of liposomes composed of neutral lipids (PC/cholesterol (a)) and cationic lipids (PC/DDAB (b)). The effect of different amount of ivermectin (from 0.1 to 1.0 mM) on the dimension of liposomes is also shown in Figure 2.
The obtained results clearly indicate that cationic liposomes are smaller than respective neutral formulations; for instance, cationic liposomes containing 1.0 mM ivermectin have a -average of 72 nm, while for the neutral onesaverage is 193 nm. This effect is due to molecular or physical interactions in conjunction with an increase in membrane fluidity induced by DDAB. In fact cationic lipids could facilitate electrostatic interactions of charged head groups. These interactions could promote size reduction by decreasing the radius of curvature of lipid bilayer as a result of phospholipidcationic head group contact.
Since the performance of liposomes, such as drug release or cell uptake, is influenced by morphological characteristics,  an accurate microscopic characterization is mandatory before any biological evaluation. Results of PCS measurements were compared to those provided by cryo-TEM analysis. This technique enables the imaging of liposome architecture to evaluate its morphology together with an estimate of liposome size. The cryo-TEM images reported in Figure 3 demonstrate that both empty ((a), (c)) and ivermectin-loaded liposomes ((b), (d)), obtained by the "controlled" ethanol injection, had a spherical shape. The presence of a limited number of oligolamellar vesicles (typically 2-4 lamellae) in neutral liposome samples is evident ((a) and (b)). Microscopic images confirm that cationic liposomes are generally smaller than neutral ones and almost entirely composed of unilamellar vesicles with a size smaller than 100 nm in diameter (in agreement with PCS analysis).
As further characterization, the effect of PC content on liposome dimension in the produced ivermectin-loaded liposomes was considered. Figure 4 shows that an increase of International Journal of Biomaterials  Table 1. In the insets the macroscopic appearance of the formulation is also reported. PC from 3 mM to 9 mM causes the growth of the -average of neutral liposomes from 65 nm to 193 nm and, in case of cationic liposomes, from 30 nm to 72 nm.
The loading efficiency of liposomes for ivermectin was determined by size exclusion chromatography. As an example, the elution profile of ivermectin-loaded liposomes (#PC9-Ch1-ive1.0), obtained after Sepharose 4B chromatography, is shown in Figure 5. The elution profile shows a single well-resolved peak. The peak (indicated by the solid arrow) contains ivermectin-loaded liposomes; no peak was detected for free ivermectin, indicating that almost all ivermectin was associated with the liposomes. The presence of ivermectin in liposome peak was demonstrated by recording the UV spectrum of the corresponding fractions at 253 nm.
Therefore, the ivermectin loading yield was estimated by comparing the absorption at 253 nm of isolated liposomes with ivermectin standard solutions. The yield of ivermectin loading for liposomes prepared with both 3 mM and 9 mM PC was almost higher than 98% (data not shown). Such result indicates that the high liposome loading efficacy is achieved when ivermectin liposomes were produced by "controlled" ethanol injection method.  Table 2) and on Vero-118, Vero-F, and Huh-7 has higher CC 50s (Vero-118 CC 50 = 5.7 M, Vero-F CC 50 = 23.6 M, and Huh-7 CC 50 = 7.3 M, Table 2) (Figure 6(a)). In comparison to 2-cmc, already known viral polymerase inhibitor [16] (Figure 1(b)) ivermectin is 10 times more cytotoxic (Figure 6(b); Table 2).
The reduced cytotoxicity is the result of several possible factors not completely clear yet. The most important is the use of aqueous solvent to solubilise the liposomes instead of the organic solvent that is necessary for ivermectin solubilisation.

Ivermectin and Liposomes Antiviral Activity on DENV.
The antiviral activity of ivermectin, empty liposomes, and ivermectin liposomes was assayed in vitro on different virus strains, namely, DENV 1 (EU081230), DENV 2 (EU081177), and the mouse-adapted strain DENV 2 S221 [13, 14]. Since Dengue virus causes systemic infection and in particular the liver is implicated in humans, for the in vitro test, the Huh-7 cells were employed, representing one of the standard cell lines that are routinely used for the cell-based assay for Dengue infection.
Bassissi et al. [17] demonstrated that slight differences in formulation could change the plasma kinetics and efficacy of ivermectin in rabbits, subcutaneously given 0.3 mg/kg of ivomec (a commercial formulation of ivermectin) or ivermectin in liposomes. In order to test this finding, the in vitro antiviral activity of free ivermectin was compared to that of different liposomal formulations containing the drug. The antiviral assay was determined by adding the virus and ivermectin or ivermectin in liposomes at the same time. Particularly, it is important to underline that the routine setup commonly used for antiviral assays is indeed cotreatment (i.e., adding the compound to be tested concomitantly with the virus); thereafter drug/virus inoculum was removed and a further addition of the compound was made for 48 h incubation.
By this experimental approach, information about the antiviral activity of the compound (both direct and indirect acting) would be obtained. A different order of addition would provide preliminary insights into whether the compound is acting as a viral entry inhibitor (pretreatment) or virus replication inhibitor (posttreatment). Since ivermectin has been previously shown to act on viral helicase NS3 [6] and/or NS5-importin / [18], it would be ideal to test the different formulations of ivermectin by cotreatment assay as described in this paper.
Notably the administration of ivermectin by a liposomal formulation yielded higher max and significantly faster absorption. This finding suggested that the use of liposomes could improve the in vivo efficacy of ivermectin [17]. Figures  11 and 12 report the activity of liposome preparations against DENV 1, 2, and S221 on Huh-7 cell line.
In order to analyse a higher concentration of ivermectin (1.0 mM), we tested liposomes #PC9-Ch1-ive1.0 and #PC9-Br1-ive1.0 and their respective empty liposomes (without ivermectin inside) ( Figure 12 Table 1. Data represent the average of 2 determinations.   Table 1. Data represent the average of 2 determinations. In conclusion we tentatively attribute the increase of antiviral activity (measured in terms of EC 50 on DENV 2) from 2.6 M to 0.3 M to the fact that liposomes containing ivermectin could merge with cell membranes, facilitating the intracellular uptake of the drug. This hypothesis is supported by the fact that positively charged liposomes (i.e., those containing the cationic surfactants DDAC and DDAB) such as #PC9-Cl1-ive0.1 and #PC9-Br1-ive0.1 are the formulations displaying the highest antiviral activity (EC 50 0.3 and 0.6 M, resp.).

Conclusions
We demonstrate that ivermectin, when delivered through liposomes, reduced cytotoxicity up to 5 times. They can effectively inhibit DENV replication with EC 50 values in the same International Journal of Biomaterials  Table 1. Data represent the average of 2 determinations. range of ivermectin alone and even improve its activity in several formulations. The possibility of dissolving ivermectin into an aqueous solution thanks to the use of liposome as drug carriers is a new step towards the solution of its pharmacokinetics problems, in particular its high cytotoxicity. This could also amplify the spectrum of ivermectin activities.
The important improvement of ivermectin activity loading in liposomes with ions compositions creates a promising starting point for a future development of these nanocarriers which one day could be the possible solution to the lack of drugs against flaviviruses. The following liposomal formulations were tested: #PC3-Ch1-ive0.1 (e), #PC3-Cl1-ive0.1 (f), and #PC3-Br1-ive0.1 (g). For comparison, data relative to the free ivermectin are reported in (h). The related empty liposomes do not have any effect on the cells. The phosphatidylcholine concentration (top -axis) is related to empty liposomes while the ivermectin concentration (bottom -axis) is related to ivermectin liposomes. Details on the identification codes and liposomal compositions are included in Table 1. Details on the EC 50 values are in Table 3. Data represent the average of 2 determinations ± SD.  Table 1. Details on the EC 50 values are in Table 3. Data represent the average of 2 determinations ± SD. for the provision of DENV 2 S221 stand. Thanks go to Professor Johan Neyts and Dr. Dirk Jochmans (KU Leuven, Leuven, Belgium) for hospitality in their virology laboratories and precious advice. Thanks go to Dr. Isha Raj for language editing and critical reading of the paper.