Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Dual Loading Of Primaquine And Chloroquine Into Liposome

Dual Loading Of Primaquine And Chloroquine Into Liposome Primaquine (PQ) has long been recognized as the only effective drug in the treatment of hepatic stage malaria. However, severe toxicity limits its therapeutical application. Combining PQ with chloroquine (CQ) has been reported as enhancing the former’s efficacy, while simultaneously reducing its toxicity. In this study, the optimal conditions for encapsulating PQ-CQ in liposome, including incubation time, temperature and drug to lipid ratio, were identified. Furthermore, the effect of the loading combination of these two drugs on liposomal characteristics and the drug released from liposome was evaluated. Liposome is composed of HSPC, cholesterol and DSPE-mPEG at a molar ratio of 55:40:5 and the drugs were loaded by means of the transmembrane pH gradient method. The particle size, ζ-potential and drug encapsulation efficiency were subsequently evaluated. The results showed that all liposome was produced with a similar particle size and ζ-potential. PQ and CQ could be optimally loaded into liposome by incubating the mixtures at 60 C for 20 minutes at a respective drug to lipid ratio of 1:3 for PQ and CQ. However, compared to single drug loading, dual-loading of PQ+CQ into liposome resulted in lower drug encapsulation and slower drug release. In conclusion, PQ and CQ can be jointly loaded into liposome with differing profiles of encapsulation and drug release. Keywords Dual loading – primaquine – chloroquine – liposome – release INTRODUCTION Globally, malaria ranks fourth on a scale of life-threatening metabolized by the liver into a carboxylic acid derivative infectious diseases (Mishra et al., 2017). Shortly after ultimately excreted in the urine. In order to treat malarial being bitten by a Plasmodium-infected female Anopheles infection and prevent relapse, PQ must be administered for a mosquito, the sporozoite accumulated in its salivary glands period of 14 days (Karyana et al., 2016). However, although it enters the liver leading to the hepatic phase of malarial demonstrates proven efficacy against hepatic phase malaria, infection. This stage is very important since it represents the PQ can cause methemoglobinemia and hemolysis in patients starting point of erythrocytic-stage malaria and fatal cerebral presenting glucose-6-phosphate dehydrogenase (G6PD) malaria (Prudêncio et al., 2006). In addition, the latent phase deficiency (Kedar et al., 2014; Marcsisin et al., 2016; Recht of hypnozoites in the liver often found in Plasmodium ovale et al., 2015). Furthermore, prolonged drug therapy can also and Plasmodium vivax infection can cause relapses in about induce abdominal cramps, nausea and vomiting (Jong and 50–80% of malaria sufferers (Chu and White, 2016). Nothdurft, 2001). Such side effects can potentially undermine Primaquine (PQ), recognized as the primary treatment the adherence of patients to the prescribed drug regime for the hepatic phase of malaria (Longley et al., 2016), is resulting in low PQ levels in the blood. It has been known an antimalarial pro-drug compound belonging to the that low doses administered in the cases of high parasitemia 8-aminoquinoline group that actively works against can induce drug resistance, which represents a significant sporozoites, hypnozoites, asexual phases and gametocytes problem in the control program relating to malaria (Gonzalez- through inhibition of the metabolic activity of mitochondrial Ceron et al., 2015). parasites and the production of reactive metabolites, which It has been previously reported that the administering of a are toxic to cells (Chu and White, 2016; Marcsisin et al., 2016). single dose of PQ combined with chloroquine (CQ) constitutes PQ constitutes a drug with a short half-life, which is rapidly an effective method of treating malaria (Gonzalez-Ceron et * E-mail: andang-m@ff.unair.ac.id © European Pharmaceutical Journal OR 18 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. al., 2015). CQ is a 4-aminoquinoline compound frequently of another study incorporating the use of Amodiaquine, employed in managing the er ythrocytic stage of malaria ( World a 4-aminoquinoline drug similar in structure to CQ, as the Health Organization, 2015). The combination of administering drug model. Amodiaquine demonstrated electrostatic and CQ tablets for three consecutive days and PQ tablets for 14 hydrophobic interactions with DPPC in the headgroup days proved effective in treating erythrocytic phase malarial region of the liposomal bilayer, thus increasing the lipid order infection and preventing its reoccurrence (Gonzalez-Ceron et (Barroso et al., 2015). These contradictory effects of PQ and al., 2015; World Health Organization, 2015). In addition, the CQ addition may affect their dual loading and the release of specific metabolite interaction between PQ and CQ reduced liposome. the toxicity of the former without compromising its efficacy It is generally accepted that, in order to achieve high drug against parasites (Fasinu et al., 2016). This study demonstrates accumulation in the target tissue, the drug should be stably that CQ can inhibit PQ metabolism by means of CYP2D6, thus encapsulated in liposome during distribution throughout the reducing the formation of active metabolites, which are toxic entire body, either by the use of a sturdy bilayer membrane to erythrocytes. (Barenholz, 2012; Kokkona et al., 2000) or the formation of Developing an effective anti-malarial treatment, especially drug aggregates in the intraliposomal phase (Barenholz, one countering hepatic phase infection, which could involve 2012; Lasic et al., 1992; Miatmoko et al., 2017). This study was the use of liposome to deliver PQ and CQ is important. aimed to determine the effect of the loading combination of Through the encapsulating of a combination of PQ and CQ in PQ+CQ compared to a single drug, on the physicochemical liposome, PQ will prove effective in treating acute infections characteristics and rate of release of PQ and CQ from caused by sporozoites and/or malaria relapse during the liposome. PQ and CQ were loaded into liposome consisting latent phase of hypnozoites in the liver, while the CQ loaded in of lipid with high rigidity, which was hydrogenated soy liposomes can provide prophylactic therapy for erythrocytic phosphatidylcholine (HSPC). It was found that dual loading phase infection. During hepatic phase infection, sporozoites PQ with CQ affected drug encapsulation efficiency and drug are known to specifically attack hepatocytes, rather than release from liposome. other non-parenchymal cells present in the liver. Therefore, MATERIALS AND METHODS the specific form of delivery intended for hepatocytes will prove useful in enhancing the efficacy and decreasing the toxicity of PQ and CQ during the treatment of malaria. Materials Liposome constitutes a vesicular formation consisting of a phospholipid bilayer surrounding an inner water phase, which For the purposes of this study, primaquine bisphosphate provides optimal protection for drugs against diffusion and (PQ) was purchased from Sigma-Aldrich Inc. (Rehovot, Israel), external factors (Kohli et al., 2014). Liposome with a particle while chloroquine diphosphate (CQ) was a product of Sigma- size within a 125–175 nm range can concentrate densely in Aldrich (Gyeonggi-do, South Korea). Hydrogenated soya hepar tissue because of the presence of an intercellular gap phosphatidylcholine (HSPC) and methoxy-(polyethylene- or fenestrae within endothelial cells in the liver sinusoid glycol)-distearylphosphatidyl-ethanolamine (mPEG-DSPE, (Baratta et al., 2009). Moreover, PEGylation of liposome can PEG mean molecular weight, 2000) were obtained from NOF minimize drug clearance from the body and produce drugs Inc. (Tokyo, Japan). The cholesterol constituted a product of that circulate for extended periods in the bloodstream Wako Pure Chemical Industries Inc. (Osaka, Japan). Potassium (Barenholz, 2012). Therefore, the drug will largely accumulate dihydrogen phsophate (KH PO ) and disodium hydrogen 2 4 in hepatocytes. phosphate (Na HPO ) were both products of Merck 2 4 The use of liposome as a carrier for PQ and CQ has been (Darmstadt, Germany), while Sephadex G-50 was obtained widely reported (Qiu et al., 2008; Stela Santos-Magalhães from Sigma-Aldrich Inc. (Steinhem, Germany). The organic and Carla Furtado Mosqueira, 2009; Stensrud et al., 2000), solvents, that is, chloroform and methanol, were products while, in contrast, no previous research on its application of Merck (Darmstadt, Germany). Deionized water (Otsuka to a combination of both drugs has been conducted. Inc., Lawang, Indonesia) was used as water solvent. All other Consequently, in this study, a dual drug loading of PQ and chemicals and reagents were of the highest quality available. CQ in liposome was prepared. However, it has been reported that PQ interacts strongly with the polar headgroup region Determination of optimal incubation for of dimyristoylphosphatidylcholine (DMPC) in the membrane preparation of liposome bilayer forming the space intercalation between the lipids (Basso et al., 2011). Turning to the results, perturbation in Liposome containing a single drug was generated by using the lipid order occurred, which increased the fluidity of CQ as a drug model to determine optimal conditions for drug the liposomal membrane. CQ has been reported to rigidify loading. Liposome was prepared in accordance with the thin- the dipalmitoylphosphatidylcholine (DPPC) liposomal film method (Miatmoko et al., 2016) at a molar ratio of 55:45:5 membrane by increasing molecular packing in the lipid for HSPC, cholesterol and DSPE-mPEG , respectively. (Ghosh et al., 1995). This observation is supported by that Each lipid compound was dissolved in chloroform before 19 20 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. Table 1: Formulation of liposome loading combination of PQ and CQ Formulation Component P1C0 P0C1 P1C1 P1C3 P1C5 PQ 1.00 mg - 1.66 mg 0.83 mg 0.55 mg CQ - 3.33 mg 1.66 mg 2.48 mg 2.78 mg HSPC 5.94 mg 5.94 mg 5.94 mg 5.94 mg 5.94 mg DSPE-mPEG 1.94 mg 1.94 mg 1.94 mg 1.94 mg 1.94 mg Cholesterol 2.13 mg 2.13 mg 2.13 mg 2.13 mg 2.13 mg Note: P1C0, weight ratio of PQ:total lipid (1:10); P0C1, weight ratio of CQ:total lipid (1:3); P1C1, weight ratio of PQ:CQ:total lipid (0.5:0.5:3); P1C3, weight ratio of PQ:CQ:total lipid (0.25:0.75:3); P1C5, weight ratio of PQ:CQ:total lipid (0.17:0.83:3) appropriate quantities were inserted into a round bottom flask. The chloroform was then completely removed by means of a vacuum rotary evaporator in a water bath (Buchi Rotavapor R-3, Flawil, Switzerland) at 60 C, leading to the Preparation of a liposome loading combination of formation of a thin dry film in the bottom of the flask. This PQ and CQ layer was hydrated with citrate buffer at pH 5.0. In order to prepare homogenous liposome suspension, the mixture Preparation of a liposome loading combination of PQ and was vortexed and subjected to sonication in a waterbath CQ involved processing the lipid components in the manner sonicator of approximately 15 minutes’ duration. The mixture described above. In order to prepare control liposome was passed through a polycarbonate membrane with a pore containing the drugs, PQ and CQ was added at respective size of 100 nm by means of an extruder (Avanti , Alabaster, drug:lipid weight ratios of 1:10 and 1:3, while for the liposome Alabama, US) in order to obtain a homogenous liposome loading combination of PQ and CQ, the drugs were added at particle size. a weight ratio of 1:3 for total PQ+CQ and lipid, respectively, The drug loading was conducted by transmembrane pH at a composition shown in Table 1. During the drug loading, gradient method, which involved eluting the liposome the drug-liposome mixtures was incubated at 60 C for 20 through a Sephadex G-50 column with phosphate buffer minutes. saline (PBS) at pH 7.4. The CQ solution in aquadest was then The entrapped PQ and CQ concentrations were measured added at a drug-lipid ratio of 1:5. The drug-liposome mixtures with a UV Spectrophotometer (Shimadzu, Kyoto, Japan) using were incubated at specific temperatures, which were 50 C a derivative order 1 method at λ = 280 nm or λ = 346 nm and 60 C, for various incubation periods of 10, 20 and 30 (data unpublished) for PQ and CQ respectively after lysing the minutes. liposomal vesicle with methanol (50% v/v). Determination of optimal drug to lipid ratios for the Determination of particle size and ζ-potential of preparation of liposome liposome In order to determine the optimal drug to lipid ratio for the In order to determine the particle size and ζ-potential preparation of liposome, the PQ or CQ was loaded as a single of liposome, the sample was diluted appropriately with drug component of the liposome. The drug loading was deionized water. The average particle size and ζ-potential completed by transmembrane pH gradient method, which of the liposomes were then measured using a cumulative involved eluting liposome hydrated with citrate buffer pH method and electrophoretic mobility with a light scattering 5.0 through a Sephadex G-50 column with phosphate buffer photometer (Delsa™ Nano C Particle Analyzer, Beckman saline (PBS) at pH 7.4. The PQ or CQ solution in aquadest was Coulter Inc., Indianapolis, US) at 25°C. subsequently added at a pre-determined drug-lipid ratio of 1:3, 1:5 or 1:10. The drug-liposome mixtures were incubated In vitro drug released from liposome at 60 C for 20 minutes. Separation of the liposomal drug from the free drug was achieved by eluting the mixture through a The in vitro study of PQ and CQ released from liposome was Sephadex G-50 column with PBS at pH 7.4. conducted by placing a liposome sample in dialysis tubing The concentration of entrapped PQ or CQ was measured with Spectra Por 7 with a molecular weight cut-off (MWCO) of a UV Spectrophotometer (Shimadzu, Kyoto, Japan) at λ= 282 3,500 (Spectrum Laboratories, Inc., Rancho Dominguez, CA, nm or λ= 330 nm after lysing with methanol (50% v/v). The USA). The dialysis media consisted of 50 mL of PBS at pH 7.4. encapsulation efficiency was calculated as follows: The study was performed through continuous agitation at a 19 20 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. Table 2: Characteristics of liposome loading CQ prepared at different temperature and period of incubation with drug loaded at a weight ratio of 1:5 for drug and total lipid, respectively Incubation Period of Particle size Polydispersity ζ-Potential Entrapment *) *) *) *) temperature incubation (nm) Index/PDI (mV) efficiency (%) 10 minutes 121.0 ± 6.5 0.30 ± 0.07 -11.3 ± 4.8 17.9 ± 3.2 50 C 20 minutes 123.1 ± 7.5 0.35 ± 0.11 - 5.6 ± 2.0 21.5 ± 4.6 30 minutes 126.2 ± 14.6 0.27 ± 0.05 -10.9 ± 6.1 15.0 ± 1.9 10 minutes 122.9 ± 21.4 0.31 ± 0.04 -16.9 ± 3.7 17.5 ± 2.1 60 C 20 minutes 123.4 ± 19.2 0.32 ± 0.08 -23.5 ± 12.2 18.2 ± 2.2 30 minutes 140.8 ± 30.5 0.26 ± 0.11 -19.8 ± 5.0 16.5 ± 2.8 *) Each value represents the mean ± S.D. (n = 3). speed of 400 rpm in a water bath at 37°C. of ζ- and potential of approximately -15 mV. There was no At determined sampling points, approximately 2 mL of significant difference in particle size or ζ-potential due to aliquots were drawn from the media and replaced with the the same components of liposome, that is, HSPC, DSPE- same volume of PBS at pH 7.4. The PQ and CQ concentration mPEG and cholesterol (Qiu et al., 2008; Yadav et al., 2011). was measured spectrophotometrically using a derivative Moreover, the implementation of this transmembrane pH order 1 method at λ = 280 nm or λ = 346 nm for PQ or CQ gradient method meant that only approximately 17–22% respectively. Dilution correction factor was used to calculate of the CQ could be loaded into the liposome. There were the cumulative amount of drug released (Aronson, 1993). no significant differences in the encapsulation efficiency of liposome CQ because of the use of varying temperatures in Statistical analysis different incubation periods, as shown in Table 2. For further experiments, the incubation of a drug mixture with liposome The data existed in triplicate and was presented as the mean will be performed at 60°C for 20 minutes, regarded as the ± S.D. The statistical analysis consisted of a one-way ANOVA highest transition temperature (T ) of liposome component, followed by an LSD post-hoc test, which were performed to which is HSPC, at 55 C (Chen et al., 2013). However, it can be determine the significance of the difference. A P value less seen that the encapsulation efficiency of CQ at a drug:lipid than 0.05 was considered to be statistically significant. ratio of 1:5 was low. The optimal drug-to-lipid ratio for the entrapment of PQ RESULTS AND DISCUSSION and CQ in liposome was determined. A previous study reported that CQ was loaded into liposome at a drug-to-lipid The characteristics of liposomes are significantly influenced mass ratio of 1:80 (Qiu et al., 2008), while PQ was loaded at by several factors, including: length of the incubation period, one of 1:14 (Stensrud et al., 2000). It proved unfeasible to temperature during the incubation period and drug-to- achieve an efficient drug loading at a very low drug-to-lipid lipid ratio (Qiu et al., 2008). Moreover, the quantity of drug ratio. The optimum ratio of 1:5 adopted by other studies of released by liposomes depends predominantly on the liposome prepared by using the transmembrane pH gradient physicochemical properties of liposome membrane and its (Miatmoko et al., 2017) was modified to drug-to-lipid ratios encapsulated drugs (Liang, 2010). In this study, liposomes of 1:10 and 1:5. Decreasing the drug-to-lipid ratio enhanced were prepared for the loading of PQ and CQ. Dual loading the encapsulation efficiency of PQ in liposome. Compared to these drugs affected both encapsulation and the properties liposome PQ prepared at a drug-to-lipid ratio of 1:10, PQ1-L10 of drug release. demonstrated the highest encapsulation efficiency of 66.4%, The loading of PQ and CQ into liposome involved remote as shown in Table 3. In contrast, CQ could be optimally loading of a drug with a pH gradient using citrate buffer pH loaded at a high drug-to-lipid ratio of 1:3 (CQ1-L3) with an 5.0 as the intraliposomal phase and PBS pH 7.4 as the outer encapsulation efficiency of 60.1%. It has been reported that phase. The first step was to evaluate the effect of temperature the intravesicular loading capacity of liposome is limited and and the incubation period by using CQ as a drug model, since the significant addition of drugs will reduce the pH gradient – during clinical therapy – it will be at a higher dose than PQ between the intra- and extravesicular phases, thus reducing (World Health Organization, 2015), thus limiting the drug drug loading (Qiu et al., 2008). CQ will be protonated into two loading capacity of liposome. PQ has different properties to basic ionization states since it has pKa values of 8.10 and 9.94 CQ (Qiu et al., 2008; Stensrud et al., 2000), thereby probably (Qiu et al., 2008). On the other hand, PQ is an amphiphatic resulting in contrasting optimal loading conditions. However, drug with pKa values of 3.2 and 10.4 (Stensrud et al., 2000). these were undetermined by this study. As shown in Table Therefore, it produced a different profile of drug loading in 2, all liposomes were produced with a similar particle size of the same transmembrane pH gradient condition due to approximately 100–150 nm, with a slightly negative charge contrasting amounts of ionized and unionized drug fractions. 21 22 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. Table 3: Characteristics of liposome loading single PQ or CQ prepared by loading drugs at 60oC for 20 minutes Particle size Polydispersity ζ-Potential Entrapment Drug Component Formulation *) *) *) *) (nm) Index/PDI (mV) efficiency (%) PQ1-L3 175.8 ± 27.1 0.52 ± 0.33 -16.8 ± 5.2 40.0 ± 3.3 PQ PQ1-L5 162.1 ± 31.3 0.57 ± 0.25 -19.6 ± 5.4 48.5 ± 3.1 PQ1-L10 163.8 ± 41.4 0.34 ± 0.05 -17.8 ± 3.9 66.4 ± 8.2 CQ1-L3 149.1 ± 27.4 0.21 ± 0.02 -22.7 ± 5.3 60.1 ± 7.9 CQ CQ1-L5 123.4 ± 19.2 0.32 ± 0.08 -23.5 ± 12.2 21.5 ± 4.6 CQ1-L10 153.1 ± 23.1 0.15 ± 0.04 -22.2 ± 7.9 21.3 ± 9.2 *) Each value represents the mean ± S.D. (n = 3). PQ, primaquine; CQ, chloroquine; L, total lipid of liposome; PQ1-L3, one part of primaquine to 3 parts of total lipid of liposome (w/w) Based on these results, a 20-minute incubation at 60°C and within and pertubation to the bilayer membrane (Basso et al., PQ-to-lipid ratio of 1:10 and CQ to-lipid ratio of 1:3 (w/w) 2011). On the other hand, the positively charged amine of CQ were selected for loading drugs into liposome in further has been reported as interacting with negative phosphate experiments. groups of phosphatidylcholine and producing rigidification In order to prepare a liposome loading combination of PQ of the liposomal membrane (Barroso et al., 2015; Ghosh et and CQ, the liposome was added to PQ and CQ solution al., 1995). However, although dual loading produced low at a determined drug weight:lipid ratio, namely; 0.5:0.5:3; drug encapsulation, PQ could be delivered together with 0.25:0.75:3 and 0.13:0.87:3 for PQ:CQ:total lipid, as shown CQ, which may play an important role in drug metabolism in in Table 1. All liposomes were produced with particle sizes hepatocytes improving therapeutical efficacy of PQ as well as ranging from 100 to 175 nm as shown in Fig. 1A with a reducing its toxicity. polydispersity index of approximately 0.20–0.40 (Fig. 1B). The in vitro drug released from liposomes was evaluated by These liposomes had slightly negative ζ-potential charges immersing liposomes in PBS at pH 7.4 (Fig. 2). The results of -9.7 to -22.7 mV (Fig. 1C). Compared to single drug-loaded showed that both PQ and CQ were released more gradually liposome, combining PQ and CQ into liposome resulted in from dual drug-loaded (PICI) liposome than from P1C0 and lower drug encapsulation efficiency (Fig. 1D). The addition P0C1 liposomes. Approximately 63% of the initial dose of PQ of CQ into liposome affected PQ encapsulation, which stood was released from P1C0 liposome over a period of 48 hours, at 72% for the single drug-loaded PQ liposome (P1C0) and while this figure fell to 44% in the presence of CQ encapsulated 6% for dual drug-loaded liposome (P1C1). Moreover, PQ in P1C1 liposome. CQ displays a similar profile of liposomal also influenced liposomal encapsulation of CQ. Compared drug release indicating an approximate 50% reduction in to single-loaded CQ liposome (P0C1), dual drug loaded- the drug released by the P1C1 liposome compared to the liposome had a lower CQ loading, 56% and 31% for P0C1 and single CQ-loaded liposome (P0C1 liposome). These results P1C1 liposome, respectively. The PQ-CQ ratio also played an indicate that the liposome loading combination of PQ and important role in determining liposomal drug encapsulation, CQ produced slower drug release than single drug-loaded which decreases the proportion of CQ to PQ. This resulted liposome, suggesting that the CQ may produce powerful in lower encapsulation of PQ as achieved in P1C1 liposome. rigidifying effects on the liposomal bilayer since it contains In contrast, increasing the proportion of CQ to PQ did more numerous drug molecules entrapped within the not produce significant differences in CQ encapsulation. liposome than does PQ. It would be advantageous to avoid Although these two drugs were encapsulated within an premature PQ release during systemic circulation before the aqueous intraliposomal compartment of the same volume, liposome enters the hepatocytes. On the other hand, slow the addition of PQ and CQ probably affected the permeability release of CQ would also be important for the prophylactic of the bilayer during incubation in a contradictory manner. effect on the erythrocytic stage development. This produced a different optimal drug-to-lipid ratio required The dual drug loading of PQ and CQ into liposome, which was for the achieving of impressive encapsulation efficiency. In composed of HSPC, cholesterol and DSPE-mPEG , greatly liposome, drugs can be encapsulated within the hydrophobic influenced drug encapsulation efficiency and drug release. It bilayer or the hydrophilic aqueous phase, or may interact is important to produce high drug loading and tailor delivery with the polar headgroup region of the lipid bilayer. The for deliberate release of the drug in an appropriate manner in encapsulation efficiency is affected by many factors such as order to achieve high accumulation in liver tissue for treatment bilayer fluidity (Kulkarni et al., 1995). It has been reported that of hepatic stage malaria. However, further investigation is the positively charged amine of PQ interacts with the polar still required to evaluate PQ interaction with the liposomal headgroup region of phosphatidylcholine/PC. In contrast, membrane in the presence of CQ, pharmacokinetic profiles its quinolone ring indicates Van der Waals interaction with and activity for further exploration of dual-loaded PQ+CQ the hydrocarbon core of lipids resulting in fluidizing effects liposome as part of malaria therapy. 21 22 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. Figure 1: The characteristics of (A) particle size, (B) polydispersity index, (C) ζ-potential, (D) encapsulation efficiency of liposome encapsulating PQ (black), CQ (white) and the combination of PQ+CQ loaded by incubating the mixtures at 60 C for 20 minutes. Each value represents mean ± S.D. (n=3). *P< 0.05 compared with P1C0. #P< 0.05 compared with P0C1. Figure 2: Profiles of release of (A) PQ and (B) CQ from single drug-loaded liposome (P0C1 and P1C0) and dual drug-loaded liposome (P1C1) in phosphate-buffered saline (PBS), pH 7.4 at 37°C. 23 24 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. CONCLUSIONS CONFLICTS OF INTEREST In this study, liposomal containing dual drug loading, which The authors declare no conflict of interest or financial interests consisted of PQ and CQ, was prepared and subsequently such as grants, employment, gifts, stock holdings, honoraria, evaluated for drug loading and in vitro drug release. Nano- consultancies, expert testimony, patents and royalties, in any sized particles, high encapsulation for the PQ+CQ combination product or service mentioned in this article. and slow drug release were achieved by combinedly loading PQ and CQ at 1:1 weight ratio. This finding suggested that dual PQ+CQ-loaded liposome could potentially be used for comprehensive malaria therapy involving hepatic and erythrocytic stage malaria. ACKNOWLEDGEMENTS This research was supported by a Preliminary Research on Excellence in Higher Education Institutions (Penelitian Dasar Unggulan Perguruan Tinggi, PDUPT) Grant No. 200/UN3.14/ LT/2018 provided by the Ministry of Research, Science, and Technology of the Republic of Indonesia. References [1] Aronson, H.. Correction Factor for Dissolution Profile Calculations. O.L. Effectiveness of combined chloroquine and primaquine J. Pharm. Sc. 1993;82:3549. treatment in 14 days versus intermittent single dose regimen, in [2] Baratta, J.L., Ngo, A., Lopez, B., Kasabwalla, N., Kenneth, J., an open, non-randomized, clinical trial, to eliminate Plasmodium Robertson, R.T. Cellular organization of normal mouse liver: vivax in southern Mexico. Malar. J. 2015;14:426. A histological, quantitative immunocytochemical, and fine [11] Jong, E.C., Nothdurft, H.D. Current drugs for antimalarial structural analysis. Histochem Cell Bio. 2009;131:713–726. chemoprophylaxis: a review of efficacy and safety. J. Trav. Med. [3] Barenholz, Y. Doxil®-The first FDA-approved nano-drug: Lessons 2001;8:48–56. learned. J. Control. Release. 2012;160:117–134. [12] Karyana, M., Devine, A., Kenangalem, E., Burdarm, L., [4] Barroso, R.P., Basso, L.G.M., Costa-Filho, A.J. Interactions of the Poespoprodjo, J.R., Vemuri, R., Anstey, N.M., Tjitra, E., Price, R.N., antimalarial amodiaquine with lipid model membranes. Chem. Yeung, S. Treatment-seeking behaviour and associated costs for Phys. Lipids. 2015;186:68–78. malaria in Papua, Indonesia. Malar. J. 2016;15:536. [5] Basso, L.G.M., Rodrigues, R.Z., Naal, R.M.Z.G., Costa-Filho, A.J. [13] Kedar, P., Warang, P., Sanyal, S., Devendra, R., Ghosh, K., Colah, Effects of the antimalarial drug primaquine on the dynamic R. Primaquine-induced severe methemoglobinemia developed structure of lipid model membranes. Biochim. Biophys. Acta - during treatment of Plasmodium vivax malarial infection in an Biomembr. 2011;1808:55–64. Indian family associated with a novel mutation (p.Agr57Trp) in [6] Chen, J., Cheng, D., Li, J., Wang, Y., Guo, J., Chen, Z., Cai, B., the CYB5R3 gene. Clin. Chim. Acta 2014;437:103–105. Yang, T. Influence of lipid composition on the phase transition [14] Kohli, A.G., Kierstead, P.H., Venditto, V.J., Walsh, C.L., Szoka, F.C. temperature of liposomes composed of both DPPC and HSPC. Designer lipids for drug delivery: From heads to tails. J. Control. Drug Dev. Ind. Pharm. 2013;39:197–204. Release. 2014;190:274–287. [7] Chu, C.S., White, N.J. Management of relapsing Plasmodium vivax [15] Kokkona, M., Kallinteri, P., Fatouros, D., Antimisiaris, S.G. Stability malaria. Expert Rev. Anti. Infect. Ther. 2016;14:885–900. of SUV liposomes in the presence of cholate salts and pancreatic [8] Fasinu, P.S., Tekwani, B.L., Avula, B., Chaurasiya, N.D., Nanayakkara, lipases: effect of lipid composition. Eur. J. Pharm. Sci. 2000;9:245– N.P.D., Wang, Y.-H., Khan, I.A., Walker, L.A. Pathway-specific 252. inhibition of primaquine metabolism by chloroquine/quinine. [16] Kulkarni, S.B., Betageri, G. V, Singh, M. Factors affecting Malar. J. 2016;15:466. microencapsulation of drugs in liposomes. J. Microencapsul. [9] Ghosh, A.K., Basu, R., Nandy, P. Lipid perturbation of liposomal 1995;12:229–246. membrane of dipalmitoyl phosphatidylcholine by chloroquine [17] Lasic, D.D., Frederik, P.M., Stuart, M.C., Barenholz, Y., McIntosh, sulphate - a fluorescence anisotropic study. Colloids Surfaces B T.J. Gelation of liposome interior. A novel method for drug Biointerfaces. 1995;4:1–4. encapsulation. FEBS Lett. 1992;312:255–258. [10] Gonzalez-Ceron, L., Rodriguez, M.H., Sandoval, M.A., Santillan, [18] Liang, Y. Drug Release and Pharmacokinetic Properties of F., Galindo-Virgen, S., Betanzos, A.F., Rosales, A.F., Palomeque, Liposomal DB-67 [Theses]. University of Kentucky ;2010. 23 24 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. [19] Longley, R.J., Sripoorote, P., Chobson, P., Saeseu, T., Sukasem, C., Phuanukoonnon, S., Nguitragool, W., Mueller, I., Sattabongkot, J. High Efficacy of Primaquine Treatment for Plasmodium vivax in Western Thailand. Am. J. Trop. Med. Hyg. 2016;95:1086–1089. [20] Marcsisin, S.R., Reichard, G., Pybus, B.S. Primaquine pharmacology in the context of CYP 2D6 pharmacogenomics: Current state of the art. Pharmacol. Ther. 2016;161:1–10. [21] Miatmoko, A., Kawano, K., Yoda, H., Yonemochi, E., Hattori, Y. Tumor delivery of liposomal doxorubicin prepared with poly- L-glutamic acid as a drug-trapping agent. J. Liposome Res. 2017;27:99–107. [22] Miatmoko, A., Kawano, K., Yonemochi, E., Hattori, Y. Evaluation of Cisplatin-Loaded Polymeric Micelles and Hybrid Nanoparticles Containing Poly ( Ethylene Oxide ) -Block- Poly ( Methacrylic Acid ) on Tumor Delivery. Pharmacology & Pharmacy. 2016;7:1–8. [23] Mishra, M., Mishra, V.K., Kashaw, V., Iyer, A.K., Kashaw, S.K. Comprehensive review on various strategies for antimalarial drug discovery. Eur. J. Med. Chem. 2017;125:1300–1320. [24] Prudêncio, M., Rodriguez, A., Mota, M.M. The silent path to thousands of merozoites: the Plasmodium liver stage. Nat. Rev. Microbiol. 2006;4:849–56. [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. 2008;361:56–63. [26] Recht, J., White, N.J., Ashley, E., Safety of 8-Aminoquinoline Antimalarial Medicines. Geneva: World Health Organization, [27] Stela Santos-Magalhães, N., Carla Furtado Mosqueira, V. Nanotechnology applied to the treatment of malaria. Adv. Drug Deliv. Rev. 2009;62:560–575. [28] Stensrud, G., Sande, S.A., Kristensen, S., Smistad, G. Formulation and characterisation of primaquine loaded liposomes prepared by a pH gradient using experimental design. Int. J. Pharm. 2000;198:213–228. [29] World Health Organization. Guidelines for the treatment of malaria, Third Edit. ed. Geneva: World Health Organization, 2015. [30] Yadav, A. V., Murthy, M.S., Shete, A.S., Sakhare, S. Stability aspects of liposomes. Indian J. Pharm. Educ. Res. 2011;45:402–413. 25 OR http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

Dual Loading Of Primaquine And Chloroquine Into Liposome

Loading next page...
 
/lp/de-gruyter/dual-loading-of-primaquine-and-chloroquine-into-liposome-xdQH0ocqHS
Publisher
de Gruyter
Copyright
© 2019 A. Miatmoko et al., published by Sciendo
ISSN
1338-6786
eISSN
2453-6725
DOI
10.2478/afpuc-2019-0009
Publisher site
See Article on Publisher Site

Abstract

Primaquine (PQ) has long been recognized as the only effective drug in the treatment of hepatic stage malaria. However, severe toxicity limits its therapeutical application. Combining PQ with chloroquine (CQ) has been reported as enhancing the former’s efficacy, while simultaneously reducing its toxicity. In this study, the optimal conditions for encapsulating PQ-CQ in liposome, including incubation time, temperature and drug to lipid ratio, were identified. Furthermore, the effect of the loading combination of these two drugs on liposomal characteristics and the drug released from liposome was evaluated. Liposome is composed of HSPC, cholesterol and DSPE-mPEG at a molar ratio of 55:40:5 and the drugs were loaded by means of the transmembrane pH gradient method. The particle size, ζ-potential and drug encapsulation efficiency were subsequently evaluated. The results showed that all liposome was produced with a similar particle size and ζ-potential. PQ and CQ could be optimally loaded into liposome by incubating the mixtures at 60 C for 20 minutes at a respective drug to lipid ratio of 1:3 for PQ and CQ. However, compared to single drug loading, dual-loading of PQ+CQ into liposome resulted in lower drug encapsulation and slower drug release. In conclusion, PQ and CQ can be jointly loaded into liposome with differing profiles of encapsulation and drug release. Keywords Dual loading – primaquine – chloroquine – liposome – release INTRODUCTION Globally, malaria ranks fourth on a scale of life-threatening metabolized by the liver into a carboxylic acid derivative infectious diseases (Mishra et al., 2017). Shortly after ultimately excreted in the urine. In order to treat malarial being bitten by a Plasmodium-infected female Anopheles infection and prevent relapse, PQ must be administered for a mosquito, the sporozoite accumulated in its salivary glands period of 14 days (Karyana et al., 2016). However, although it enters the liver leading to the hepatic phase of malarial demonstrates proven efficacy against hepatic phase malaria, infection. This stage is very important since it represents the PQ can cause methemoglobinemia and hemolysis in patients starting point of erythrocytic-stage malaria and fatal cerebral presenting glucose-6-phosphate dehydrogenase (G6PD) malaria (Prudêncio et al., 2006). In addition, the latent phase deficiency (Kedar et al., 2014; Marcsisin et al., 2016; Recht of hypnozoites in the liver often found in Plasmodium ovale et al., 2015). Furthermore, prolonged drug therapy can also and Plasmodium vivax infection can cause relapses in about induce abdominal cramps, nausea and vomiting (Jong and 50–80% of malaria sufferers (Chu and White, 2016). Nothdurft, 2001). Such side effects can potentially undermine Primaquine (PQ), recognized as the primary treatment the adherence of patients to the prescribed drug regime for the hepatic phase of malaria (Longley et al., 2016), is resulting in low PQ levels in the blood. It has been known an antimalarial pro-drug compound belonging to the that low doses administered in the cases of high parasitemia 8-aminoquinoline group that actively works against can induce drug resistance, which represents a significant sporozoites, hypnozoites, asexual phases and gametocytes problem in the control program relating to malaria (Gonzalez- through inhibition of the metabolic activity of mitochondrial Ceron et al., 2015). parasites and the production of reactive metabolites, which It has been previously reported that the administering of a are toxic to cells (Chu and White, 2016; Marcsisin et al., 2016). single dose of PQ combined with chloroquine (CQ) constitutes PQ constitutes a drug with a short half-life, which is rapidly an effective method of treating malaria (Gonzalez-Ceron et * E-mail: andang-m@ff.unair.ac.id © European Pharmaceutical Journal OR 18 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. al., 2015). CQ is a 4-aminoquinoline compound frequently of another study incorporating the use of Amodiaquine, employed in managing the er ythrocytic stage of malaria ( World a 4-aminoquinoline drug similar in structure to CQ, as the Health Organization, 2015). The combination of administering drug model. Amodiaquine demonstrated electrostatic and CQ tablets for three consecutive days and PQ tablets for 14 hydrophobic interactions with DPPC in the headgroup days proved effective in treating erythrocytic phase malarial region of the liposomal bilayer, thus increasing the lipid order infection and preventing its reoccurrence (Gonzalez-Ceron et (Barroso et al., 2015). These contradictory effects of PQ and al., 2015; World Health Organization, 2015). In addition, the CQ addition may affect their dual loading and the release of specific metabolite interaction between PQ and CQ reduced liposome. the toxicity of the former without compromising its efficacy It is generally accepted that, in order to achieve high drug against parasites (Fasinu et al., 2016). This study demonstrates accumulation in the target tissue, the drug should be stably that CQ can inhibit PQ metabolism by means of CYP2D6, thus encapsulated in liposome during distribution throughout the reducing the formation of active metabolites, which are toxic entire body, either by the use of a sturdy bilayer membrane to erythrocytes. (Barenholz, 2012; Kokkona et al., 2000) or the formation of Developing an effective anti-malarial treatment, especially drug aggregates in the intraliposomal phase (Barenholz, one countering hepatic phase infection, which could involve 2012; Lasic et al., 1992; Miatmoko et al., 2017). This study was the use of liposome to deliver PQ and CQ is important. aimed to determine the effect of the loading combination of Through the encapsulating of a combination of PQ and CQ in PQ+CQ compared to a single drug, on the physicochemical liposome, PQ will prove effective in treating acute infections characteristics and rate of release of PQ and CQ from caused by sporozoites and/or malaria relapse during the liposome. PQ and CQ were loaded into liposome consisting latent phase of hypnozoites in the liver, while the CQ loaded in of lipid with high rigidity, which was hydrogenated soy liposomes can provide prophylactic therapy for erythrocytic phosphatidylcholine (HSPC). It was found that dual loading phase infection. During hepatic phase infection, sporozoites PQ with CQ affected drug encapsulation efficiency and drug are known to specifically attack hepatocytes, rather than release from liposome. other non-parenchymal cells present in the liver. Therefore, MATERIALS AND METHODS the specific form of delivery intended for hepatocytes will prove useful in enhancing the efficacy and decreasing the toxicity of PQ and CQ during the treatment of malaria. Materials Liposome constitutes a vesicular formation consisting of a phospholipid bilayer surrounding an inner water phase, which For the purposes of this study, primaquine bisphosphate provides optimal protection for drugs against diffusion and (PQ) was purchased from Sigma-Aldrich Inc. (Rehovot, Israel), external factors (Kohli et al., 2014). Liposome with a particle while chloroquine diphosphate (CQ) was a product of Sigma- size within a 125–175 nm range can concentrate densely in Aldrich (Gyeonggi-do, South Korea). Hydrogenated soya hepar tissue because of the presence of an intercellular gap phosphatidylcholine (HSPC) and methoxy-(polyethylene- or fenestrae within endothelial cells in the liver sinusoid glycol)-distearylphosphatidyl-ethanolamine (mPEG-DSPE, (Baratta et al., 2009). Moreover, PEGylation of liposome can PEG mean molecular weight, 2000) were obtained from NOF minimize drug clearance from the body and produce drugs Inc. (Tokyo, Japan). The cholesterol constituted a product of that circulate for extended periods in the bloodstream Wako Pure Chemical Industries Inc. (Osaka, Japan). Potassium (Barenholz, 2012). Therefore, the drug will largely accumulate dihydrogen phsophate (KH PO ) and disodium hydrogen 2 4 in hepatocytes. phosphate (Na HPO ) were both products of Merck 2 4 The use of liposome as a carrier for PQ and CQ has been (Darmstadt, Germany), while Sephadex G-50 was obtained widely reported (Qiu et al., 2008; Stela Santos-Magalhães from Sigma-Aldrich Inc. (Steinhem, Germany). The organic and Carla Furtado Mosqueira, 2009; Stensrud et al., 2000), solvents, that is, chloroform and methanol, were products while, in contrast, no previous research on its application of Merck (Darmstadt, Germany). Deionized water (Otsuka to a combination of both drugs has been conducted. Inc., Lawang, Indonesia) was used as water solvent. All other Consequently, in this study, a dual drug loading of PQ and chemicals and reagents were of the highest quality available. CQ in liposome was prepared. However, it has been reported that PQ interacts strongly with the polar headgroup region Determination of optimal incubation for of dimyristoylphosphatidylcholine (DMPC) in the membrane preparation of liposome bilayer forming the space intercalation between the lipids (Basso et al., 2011). Turning to the results, perturbation in Liposome containing a single drug was generated by using the lipid order occurred, which increased the fluidity of CQ as a drug model to determine optimal conditions for drug the liposomal membrane. CQ has been reported to rigidify loading. Liposome was prepared in accordance with the thin- the dipalmitoylphosphatidylcholine (DPPC) liposomal film method (Miatmoko et al., 2016) at a molar ratio of 55:45:5 membrane by increasing molecular packing in the lipid for HSPC, cholesterol and DSPE-mPEG , respectively. (Ghosh et al., 1995). This observation is supported by that Each lipid compound was dissolved in chloroform before 19 20 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. Table 1: Formulation of liposome loading combination of PQ and CQ Formulation Component P1C0 P0C1 P1C1 P1C3 P1C5 PQ 1.00 mg - 1.66 mg 0.83 mg 0.55 mg CQ - 3.33 mg 1.66 mg 2.48 mg 2.78 mg HSPC 5.94 mg 5.94 mg 5.94 mg 5.94 mg 5.94 mg DSPE-mPEG 1.94 mg 1.94 mg 1.94 mg 1.94 mg 1.94 mg Cholesterol 2.13 mg 2.13 mg 2.13 mg 2.13 mg 2.13 mg Note: P1C0, weight ratio of PQ:total lipid (1:10); P0C1, weight ratio of CQ:total lipid (1:3); P1C1, weight ratio of PQ:CQ:total lipid (0.5:0.5:3); P1C3, weight ratio of PQ:CQ:total lipid (0.25:0.75:3); P1C5, weight ratio of PQ:CQ:total lipid (0.17:0.83:3) appropriate quantities were inserted into a round bottom flask. The chloroform was then completely removed by means of a vacuum rotary evaporator in a water bath (Buchi Rotavapor R-3, Flawil, Switzerland) at 60 C, leading to the Preparation of a liposome loading combination of formation of a thin dry film in the bottom of the flask. This PQ and CQ layer was hydrated with citrate buffer at pH 5.0. In order to prepare homogenous liposome suspension, the mixture Preparation of a liposome loading combination of PQ and was vortexed and subjected to sonication in a waterbath CQ involved processing the lipid components in the manner sonicator of approximately 15 minutes’ duration. The mixture described above. In order to prepare control liposome was passed through a polycarbonate membrane with a pore containing the drugs, PQ and CQ was added at respective size of 100 nm by means of an extruder (Avanti , Alabaster, drug:lipid weight ratios of 1:10 and 1:3, while for the liposome Alabama, US) in order to obtain a homogenous liposome loading combination of PQ and CQ, the drugs were added at particle size. a weight ratio of 1:3 for total PQ+CQ and lipid, respectively, The drug loading was conducted by transmembrane pH at a composition shown in Table 1. During the drug loading, gradient method, which involved eluting the liposome the drug-liposome mixtures was incubated at 60 C for 20 through a Sephadex G-50 column with phosphate buffer minutes. saline (PBS) at pH 7.4. The CQ solution in aquadest was then The entrapped PQ and CQ concentrations were measured added at a drug-lipid ratio of 1:5. The drug-liposome mixtures with a UV Spectrophotometer (Shimadzu, Kyoto, Japan) using were incubated at specific temperatures, which were 50 C a derivative order 1 method at λ = 280 nm or λ = 346 nm and 60 C, for various incubation periods of 10, 20 and 30 (data unpublished) for PQ and CQ respectively after lysing the minutes. liposomal vesicle with methanol (50% v/v). Determination of optimal drug to lipid ratios for the Determination of particle size and ζ-potential of preparation of liposome liposome In order to determine the optimal drug to lipid ratio for the In order to determine the particle size and ζ-potential preparation of liposome, the PQ or CQ was loaded as a single of liposome, the sample was diluted appropriately with drug component of the liposome. The drug loading was deionized water. The average particle size and ζ-potential completed by transmembrane pH gradient method, which of the liposomes were then measured using a cumulative involved eluting liposome hydrated with citrate buffer pH method and electrophoretic mobility with a light scattering 5.0 through a Sephadex G-50 column with phosphate buffer photometer (Delsa™ Nano C Particle Analyzer, Beckman saline (PBS) at pH 7.4. The PQ or CQ solution in aquadest was Coulter Inc., Indianapolis, US) at 25°C. subsequently added at a pre-determined drug-lipid ratio of 1:3, 1:5 or 1:10. The drug-liposome mixtures were incubated In vitro drug released from liposome at 60 C for 20 minutes. Separation of the liposomal drug from the free drug was achieved by eluting the mixture through a The in vitro study of PQ and CQ released from liposome was Sephadex G-50 column with PBS at pH 7.4. conducted by placing a liposome sample in dialysis tubing The concentration of entrapped PQ or CQ was measured with Spectra Por 7 with a molecular weight cut-off (MWCO) of a UV Spectrophotometer (Shimadzu, Kyoto, Japan) at λ= 282 3,500 (Spectrum Laboratories, Inc., Rancho Dominguez, CA, nm or λ= 330 nm after lysing with methanol (50% v/v). The USA). The dialysis media consisted of 50 mL of PBS at pH 7.4. encapsulation efficiency was calculated as follows: The study was performed through continuous agitation at a 19 20 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. Table 2: Characteristics of liposome loading CQ prepared at different temperature and period of incubation with drug loaded at a weight ratio of 1:5 for drug and total lipid, respectively Incubation Period of Particle size Polydispersity ζ-Potential Entrapment *) *) *) *) temperature incubation (nm) Index/PDI (mV) efficiency (%) 10 minutes 121.0 ± 6.5 0.30 ± 0.07 -11.3 ± 4.8 17.9 ± 3.2 50 C 20 minutes 123.1 ± 7.5 0.35 ± 0.11 - 5.6 ± 2.0 21.5 ± 4.6 30 minutes 126.2 ± 14.6 0.27 ± 0.05 -10.9 ± 6.1 15.0 ± 1.9 10 minutes 122.9 ± 21.4 0.31 ± 0.04 -16.9 ± 3.7 17.5 ± 2.1 60 C 20 minutes 123.4 ± 19.2 0.32 ± 0.08 -23.5 ± 12.2 18.2 ± 2.2 30 minutes 140.8 ± 30.5 0.26 ± 0.11 -19.8 ± 5.0 16.5 ± 2.8 *) Each value represents the mean ± S.D. (n = 3). speed of 400 rpm in a water bath at 37°C. of ζ- and potential of approximately -15 mV. There was no At determined sampling points, approximately 2 mL of significant difference in particle size or ζ-potential due to aliquots were drawn from the media and replaced with the the same components of liposome, that is, HSPC, DSPE- same volume of PBS at pH 7.4. The PQ and CQ concentration mPEG and cholesterol (Qiu et al., 2008; Yadav et al., 2011). was measured spectrophotometrically using a derivative Moreover, the implementation of this transmembrane pH order 1 method at λ = 280 nm or λ = 346 nm for PQ or CQ gradient method meant that only approximately 17–22% respectively. Dilution correction factor was used to calculate of the CQ could be loaded into the liposome. There were the cumulative amount of drug released (Aronson, 1993). no significant differences in the encapsulation efficiency of liposome CQ because of the use of varying temperatures in Statistical analysis different incubation periods, as shown in Table 2. For further experiments, the incubation of a drug mixture with liposome The data existed in triplicate and was presented as the mean will be performed at 60°C for 20 minutes, regarded as the ± S.D. The statistical analysis consisted of a one-way ANOVA highest transition temperature (T ) of liposome component, followed by an LSD post-hoc test, which were performed to which is HSPC, at 55 C (Chen et al., 2013). However, it can be determine the significance of the difference. A P value less seen that the encapsulation efficiency of CQ at a drug:lipid than 0.05 was considered to be statistically significant. ratio of 1:5 was low. The optimal drug-to-lipid ratio for the entrapment of PQ RESULTS AND DISCUSSION and CQ in liposome was determined. A previous study reported that CQ was loaded into liposome at a drug-to-lipid The characteristics of liposomes are significantly influenced mass ratio of 1:80 (Qiu et al., 2008), while PQ was loaded at by several factors, including: length of the incubation period, one of 1:14 (Stensrud et al., 2000). It proved unfeasible to temperature during the incubation period and drug-to- achieve an efficient drug loading at a very low drug-to-lipid lipid ratio (Qiu et al., 2008). Moreover, the quantity of drug ratio. The optimum ratio of 1:5 adopted by other studies of released by liposomes depends predominantly on the liposome prepared by using the transmembrane pH gradient physicochemical properties of liposome membrane and its (Miatmoko et al., 2017) was modified to drug-to-lipid ratios encapsulated drugs (Liang, 2010). In this study, liposomes of 1:10 and 1:5. Decreasing the drug-to-lipid ratio enhanced were prepared for the loading of PQ and CQ. Dual loading the encapsulation efficiency of PQ in liposome. Compared to these drugs affected both encapsulation and the properties liposome PQ prepared at a drug-to-lipid ratio of 1:10, PQ1-L10 of drug release. demonstrated the highest encapsulation efficiency of 66.4%, The loading of PQ and CQ into liposome involved remote as shown in Table 3. In contrast, CQ could be optimally loading of a drug with a pH gradient using citrate buffer pH loaded at a high drug-to-lipid ratio of 1:3 (CQ1-L3) with an 5.0 as the intraliposomal phase and PBS pH 7.4 as the outer encapsulation efficiency of 60.1%. It has been reported that phase. The first step was to evaluate the effect of temperature the intravesicular loading capacity of liposome is limited and and the incubation period by using CQ as a drug model, since the significant addition of drugs will reduce the pH gradient – during clinical therapy – it will be at a higher dose than PQ between the intra- and extravesicular phases, thus reducing (World Health Organization, 2015), thus limiting the drug drug loading (Qiu et al., 2008). CQ will be protonated into two loading capacity of liposome. PQ has different properties to basic ionization states since it has pKa values of 8.10 and 9.94 CQ (Qiu et al., 2008; Stensrud et al., 2000), thereby probably (Qiu et al., 2008). On the other hand, PQ is an amphiphatic resulting in contrasting optimal loading conditions. However, drug with pKa values of 3.2 and 10.4 (Stensrud et al., 2000). these were undetermined by this study. As shown in Table Therefore, it produced a different profile of drug loading in 2, all liposomes were produced with a similar particle size of the same transmembrane pH gradient condition due to approximately 100–150 nm, with a slightly negative charge contrasting amounts of ionized and unionized drug fractions. 21 22 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. Table 3: Characteristics of liposome loading single PQ or CQ prepared by loading drugs at 60oC for 20 minutes Particle size Polydispersity ζ-Potential Entrapment Drug Component Formulation *) *) *) *) (nm) Index/PDI (mV) efficiency (%) PQ1-L3 175.8 ± 27.1 0.52 ± 0.33 -16.8 ± 5.2 40.0 ± 3.3 PQ PQ1-L5 162.1 ± 31.3 0.57 ± 0.25 -19.6 ± 5.4 48.5 ± 3.1 PQ1-L10 163.8 ± 41.4 0.34 ± 0.05 -17.8 ± 3.9 66.4 ± 8.2 CQ1-L3 149.1 ± 27.4 0.21 ± 0.02 -22.7 ± 5.3 60.1 ± 7.9 CQ CQ1-L5 123.4 ± 19.2 0.32 ± 0.08 -23.5 ± 12.2 21.5 ± 4.6 CQ1-L10 153.1 ± 23.1 0.15 ± 0.04 -22.2 ± 7.9 21.3 ± 9.2 *) Each value represents the mean ± S.D. (n = 3). PQ, primaquine; CQ, chloroquine; L, total lipid of liposome; PQ1-L3, one part of primaquine to 3 parts of total lipid of liposome (w/w) Based on these results, a 20-minute incubation at 60°C and within and pertubation to the bilayer membrane (Basso et al., PQ-to-lipid ratio of 1:10 and CQ to-lipid ratio of 1:3 (w/w) 2011). On the other hand, the positively charged amine of CQ were selected for loading drugs into liposome in further has been reported as interacting with negative phosphate experiments. groups of phosphatidylcholine and producing rigidification In order to prepare a liposome loading combination of PQ of the liposomal membrane (Barroso et al., 2015; Ghosh et and CQ, the liposome was added to PQ and CQ solution al., 1995). However, although dual loading produced low at a determined drug weight:lipid ratio, namely; 0.5:0.5:3; drug encapsulation, PQ could be delivered together with 0.25:0.75:3 and 0.13:0.87:3 for PQ:CQ:total lipid, as shown CQ, which may play an important role in drug metabolism in in Table 1. All liposomes were produced with particle sizes hepatocytes improving therapeutical efficacy of PQ as well as ranging from 100 to 175 nm as shown in Fig. 1A with a reducing its toxicity. polydispersity index of approximately 0.20–0.40 (Fig. 1B). The in vitro drug released from liposomes was evaluated by These liposomes had slightly negative ζ-potential charges immersing liposomes in PBS at pH 7.4 (Fig. 2). The results of -9.7 to -22.7 mV (Fig. 1C). Compared to single drug-loaded showed that both PQ and CQ were released more gradually liposome, combining PQ and CQ into liposome resulted in from dual drug-loaded (PICI) liposome than from P1C0 and lower drug encapsulation efficiency (Fig. 1D). The addition P0C1 liposomes. Approximately 63% of the initial dose of PQ of CQ into liposome affected PQ encapsulation, which stood was released from P1C0 liposome over a period of 48 hours, at 72% for the single drug-loaded PQ liposome (P1C0) and while this figure fell to 44% in the presence of CQ encapsulated 6% for dual drug-loaded liposome (P1C1). Moreover, PQ in P1C1 liposome. CQ displays a similar profile of liposomal also influenced liposomal encapsulation of CQ. Compared drug release indicating an approximate 50% reduction in to single-loaded CQ liposome (P0C1), dual drug loaded- the drug released by the P1C1 liposome compared to the liposome had a lower CQ loading, 56% and 31% for P0C1 and single CQ-loaded liposome (P0C1 liposome). These results P1C1 liposome, respectively. The PQ-CQ ratio also played an indicate that the liposome loading combination of PQ and important role in determining liposomal drug encapsulation, CQ produced slower drug release than single drug-loaded which decreases the proportion of CQ to PQ. This resulted liposome, suggesting that the CQ may produce powerful in lower encapsulation of PQ as achieved in P1C1 liposome. rigidifying effects on the liposomal bilayer since it contains In contrast, increasing the proportion of CQ to PQ did more numerous drug molecules entrapped within the not produce significant differences in CQ encapsulation. liposome than does PQ. It would be advantageous to avoid Although these two drugs were encapsulated within an premature PQ release during systemic circulation before the aqueous intraliposomal compartment of the same volume, liposome enters the hepatocytes. On the other hand, slow the addition of PQ and CQ probably affected the permeability release of CQ would also be important for the prophylactic of the bilayer during incubation in a contradictory manner. effect on the erythrocytic stage development. This produced a different optimal drug-to-lipid ratio required The dual drug loading of PQ and CQ into liposome, which was for the achieving of impressive encapsulation efficiency. In composed of HSPC, cholesterol and DSPE-mPEG , greatly liposome, drugs can be encapsulated within the hydrophobic influenced drug encapsulation efficiency and drug release. It bilayer or the hydrophilic aqueous phase, or may interact is important to produce high drug loading and tailor delivery with the polar headgroup region of the lipid bilayer. The for deliberate release of the drug in an appropriate manner in encapsulation efficiency is affected by many factors such as order to achieve high accumulation in liver tissue for treatment bilayer fluidity (Kulkarni et al., 1995). It has been reported that of hepatic stage malaria. However, further investigation is the positively charged amine of PQ interacts with the polar still required to evaluate PQ interaction with the liposomal headgroup region of phosphatidylcholine/PC. In contrast, membrane in the presence of CQ, pharmacokinetic profiles its quinolone ring indicates Van der Waals interaction with and activity for further exploration of dual-loaded PQ+CQ the hydrocarbon core of lipids resulting in fluidizing effects liposome as part of malaria therapy. 21 22 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. Figure 1: The characteristics of (A) particle size, (B) polydispersity index, (C) ζ-potential, (D) encapsulation efficiency of liposome encapsulating PQ (black), CQ (white) and the combination of PQ+CQ loaded by incubating the mixtures at 60 C for 20 minutes. Each value represents mean ± S.D. (n=3). *P< 0.05 compared with P1C0. #P< 0.05 compared with P0C1. Figure 2: Profiles of release of (A) PQ and (B) CQ from single drug-loaded liposome (P0C1 and P1C0) and dual drug-loaded liposome (P1C1) in phosphate-buffered saline (PBS), pH 7.4 at 37°C. 23 24 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. CONCLUSIONS CONFLICTS OF INTEREST In this study, liposomal containing dual drug loading, which The authors declare no conflict of interest or financial interests consisted of PQ and CQ, was prepared and subsequently such as grants, employment, gifts, stock holdings, honoraria, evaluated for drug loading and in vitro drug release. Nano- consultancies, expert testimony, patents and royalties, in any sized particles, high encapsulation for the PQ+CQ combination product or service mentioned in this article. and slow drug release were achieved by combinedly loading PQ and CQ at 1:1 weight ratio. This finding suggested that dual PQ+CQ-loaded liposome could potentially be used for comprehensive malaria therapy involving hepatic and erythrocytic stage malaria. ACKNOWLEDGEMENTS This research was supported by a Preliminary Research on Excellence in Higher Education Institutions (Penelitian Dasar Unggulan Perguruan Tinggi, PDUPT) Grant No. 200/UN3.14/ LT/2018 provided by the Ministry of Research, Science, and Technology of the Republic of Indonesia. References [1] Aronson, H.. Correction Factor for Dissolution Profile Calculations. O.L. Effectiveness of combined chloroquine and primaquine J. Pharm. Sc. 1993;82:3549. treatment in 14 days versus intermittent single dose regimen, in [2] Baratta, J.L., Ngo, A., Lopez, B., Kasabwalla, N., Kenneth, J., an open, non-randomized, clinical trial, to eliminate Plasmodium Robertson, R.T. Cellular organization of normal mouse liver: vivax in southern Mexico. Malar. J. 2015;14:426. A histological, quantitative immunocytochemical, and fine [11] Jong, E.C., Nothdurft, H.D. Current drugs for antimalarial structural analysis. Histochem Cell Bio. 2009;131:713–726. chemoprophylaxis: a review of efficacy and safety. J. Trav. Med. [3] Barenholz, Y. Doxil®-The first FDA-approved nano-drug: Lessons 2001;8:48–56. learned. J. Control. Release. 2012;160:117–134. [12] Karyana, M., Devine, A., Kenangalem, E., Burdarm, L., [4] Barroso, R.P., Basso, L.G.M., Costa-Filho, A.J. Interactions of the Poespoprodjo, J.R., Vemuri, R., Anstey, N.M., Tjitra, E., Price, R.N., antimalarial amodiaquine with lipid model membranes. Chem. Yeung, S. Treatment-seeking behaviour and associated costs for Phys. Lipids. 2015;186:68–78. malaria in Papua, Indonesia. Malar. J. 2016;15:536. [5] Basso, L.G.M., Rodrigues, R.Z., Naal, R.M.Z.G., Costa-Filho, A.J. [13] Kedar, P., Warang, P., Sanyal, S., Devendra, R., Ghosh, K., Colah, Effects of the antimalarial drug primaquine on the dynamic R. Primaquine-induced severe methemoglobinemia developed structure of lipid model membranes. Biochim. Biophys. Acta - during treatment of Plasmodium vivax malarial infection in an Biomembr. 2011;1808:55–64. Indian family associated with a novel mutation (p.Agr57Trp) in [6] Chen, J., Cheng, D., Li, J., Wang, Y., Guo, J., Chen, Z., Cai, B., the CYB5R3 gene. Clin. Chim. Acta 2014;437:103–105. Yang, T. Influence of lipid composition on the phase transition [14] Kohli, A.G., Kierstead, P.H., Venditto, V.J., Walsh, C.L., Szoka, F.C. temperature of liposomes composed of both DPPC and HSPC. Designer lipids for drug delivery: From heads to tails. J. Control. Drug Dev. Ind. Pharm. 2013;39:197–204. Release. 2014;190:274–287. [7] Chu, C.S., White, N.J. Management of relapsing Plasmodium vivax [15] Kokkona, M., Kallinteri, P., Fatouros, D., Antimisiaris, S.G. Stability malaria. Expert Rev. Anti. Infect. Ther. 2016;14:885–900. of SUV liposomes in the presence of cholate salts and pancreatic [8] Fasinu, P.S., Tekwani, B.L., Avula, B., Chaurasiya, N.D., Nanayakkara, lipases: effect of lipid composition. Eur. J. Pharm. Sci. 2000;9:245– N.P.D., Wang, Y.-H., Khan, I.A., Walker, L.A. Pathway-specific 252. inhibition of primaquine metabolism by chloroquine/quinine. [16] Kulkarni, S.B., Betageri, G. V, Singh, M. Factors affecting Malar. J. 2016;15:466. microencapsulation of drugs in liposomes. J. Microencapsul. [9] Ghosh, A.K., Basu, R., Nandy, P. Lipid perturbation of liposomal 1995;12:229–246. membrane of dipalmitoyl phosphatidylcholine by chloroquine [17] Lasic, D.D., Frederik, P.M., Stuart, M.C., Barenholz, Y., McIntosh, sulphate - a fluorescence anisotropic study. Colloids Surfaces B T.J. Gelation of liposome interior. A novel method for drug Biointerfaces. 1995;4:1–4. encapsulation. FEBS Lett. 1992;312:255–258. [10] Gonzalez-Ceron, L., Rodriguez, M.H., Sandoval, M.A., Santillan, [18] Liang, Y. Drug Release and Pharmacokinetic Properties of F., Galindo-Virgen, S., Betanzos, A.F., Rosales, A.F., Palomeque, Liposomal DB-67 [Theses]. University of Kentucky ;2010. 23 24 Eur. Pharm. J. 2019, 66(2), 18-25 Dual Loading Of Primaquine And Chloroquine Into Liposome Miatmoko A. et al. [19] Longley, R.J., Sripoorote, P., Chobson, P., Saeseu, T., Sukasem, C., Phuanukoonnon, S., Nguitragool, W., Mueller, I., Sattabongkot, J. High Efficacy of Primaquine Treatment for Plasmodium vivax in Western Thailand. Am. J. Trop. Med. Hyg. 2016;95:1086–1089. [20] Marcsisin, S.R., Reichard, G., Pybus, B.S. Primaquine pharmacology in the context of CYP 2D6 pharmacogenomics: Current state of the art. Pharmacol. Ther. 2016;161:1–10. [21] Miatmoko, A., Kawano, K., Yoda, H., Yonemochi, E., Hattori, Y. Tumor delivery of liposomal doxorubicin prepared with poly- L-glutamic acid as a drug-trapping agent. J. Liposome Res. 2017;27:99–107. [22] Miatmoko, A., Kawano, K., Yonemochi, E., Hattori, Y. Evaluation of Cisplatin-Loaded Polymeric Micelles and Hybrid Nanoparticles Containing Poly ( Ethylene Oxide ) -Block- Poly ( Methacrylic Acid ) on Tumor Delivery. Pharmacology & Pharmacy. 2016;7:1–8. [23] Mishra, M., Mishra, V.K., Kashaw, V., Iyer, A.K., Kashaw, S.K. Comprehensive review on various strategies for antimalarial drug discovery. Eur. J. Med. Chem. 2017;125:1300–1320. [24] Prudêncio, M., Rodriguez, A., Mota, M.M. The silent path to thousands of merozoites: the Plasmodium liver stage. Nat. Rev. Microbiol. 2006;4:849–56. [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. 2008;361:56–63. [26] Recht, J., White, N.J., Ashley, E., Safety of 8-Aminoquinoline Antimalarial Medicines. Geneva: World Health Organization, [27] Stela Santos-Magalhães, N., Carla Furtado Mosqueira, V. Nanotechnology applied to the treatment of malaria. Adv. Drug Deliv. Rev. 2009;62:560–575. [28] Stensrud, G., Sande, S.A., Kristensen, S., Smistad, G. Formulation and characterisation of primaquine loaded liposomes prepared by a pH gradient using experimental design. Int. J. Pharm. 2000;198:213–228. [29] World Health Organization. Guidelines for the treatment of malaria, Third Edit. ed. Geneva: World Health Organization, 2015. [30] Yadav, A. V., Murthy, M.S., Shete, A.S., Sakhare, S. Stability aspects of liposomes. Indian J. Pharm. Educ. Res. 2011;45:402–413. 25 OR

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Nov 1, 2019

Keywords: Dual loading; primaquine; chloroquine; liposome; release

References