In-vitro evaluation of nano-liposomal formulation of Fluconazole and Amphotericin B against visceral leishmaniasis

 

Atul Tripathi, Amber Vyas*

University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, India.

*Corresponding Author E-mail: ambervyas@gmail.com

 

ABSTRACT:

Objectives: The aim of the present study was to compare the efficacy of a dual and single drug loaded nano-liposomal formulation of Amphotericin B and Fluconazole for the treatment of visceral leishmaniasis with plain drugs. Methods: We have formulated nano-liposomes (200-250 nm) from Amphotericin B and Fluconazole using dry film hydration method and have tested their efficacy on promastigotes and amastigotes of Leishmania donovani strain. Physicochemical characterization, entrapment study, stability study, in-vitro release study, in-vitro macrophagic uptake studies (Confocal microscopy) and in-vitro antileishmanial activity were evaluated for various formulations containing Amphotericin B and Fluconazole. Results: The in-vitro cellular uptake confocal studies revealed that NR-loaded AmpB + Flu nanoliposomes have enhanced cellular uptake of formulation. The in-vitro inhibition of promastigotes and amastigotes with liposome containing both Amphotericin B and Fluconazole was significantly more than with liposomes containing individual drugs. The IC50 and CC50 of AmpB + Flu nanoliposomes against promastigotes was found to be 3.308μg/mL and 73.48μg/mL respectively, while the IC50 against axenic and intramacrophagic amastigotes was found to be 3.412 and 3.7028μg/mL respectively. Conclusion: In conclusion, Liposomal formulation containing both Amphotericin B and Fluconazole had significantly greater efficacy than conventional combination and other formulation with individual drugs. Current dual drug loaded formulation may have a favourable safety profile, and if production costs are low, it may prove to be a feasible alternative to currently available therapy after in-vivo testing.

 

KEYWORDS: Nano-liposomal formulation visceral Leishmaniasis intramacrophagic amastigotes.

 

 


INTRODUCTION:

Leishmaniasis is a dreadful disease that causes a lot of illness and deaths all over the world that includes visceral (also known as kala-azar, which is and the most serious form of the disease), cutaneous (the most common), and mucocutaneous.1,2. Visceral Leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani that are transmitted through the bites of infected female phlebotomine sandflies, those feed on blood to breed 3. According to World Health Organisation (WHO) fact sheet 2019, it was estimated that approximately 50,00090,000 new cases of VL occur globally each year 4. In India states such as Bihar, Jharkhand, Sikkim and Uttar Pradesh are endemic in which there are 19 regions identified by national programme, sporadic cases are reported from other states Assam, Delhi, Himachal Pradesh, Kerala, Madhya Pradesh, Punjab, Sikkim and Uttarakhand5. As, the majority of people infected by VL live in poverty, the cost of treatment for this disease is crucial4.

 

Liposomal Amphotericin Bis the currently available treatment for VL, but due to development of resistance effectiveness over the time is getting decreasing2,6-9. Amphoteric in B is basically fungicidal drug and has a lot of side effects such as nephrotoxicity, hepatotoxicity, hypokalaemia and hypomagnesemia10. In order to minimize the adverse effects of Amphotericin B, a medication made with sodium deoxycholate to minimize the adverse effects but no major change observed11,12. The surface of Lipid-based formulations such as liposomes can be easily altered to allow targeted delivery of therapeutic substances13,14. In this context studies suggested about Fluconazole as a good anti-leishmanial agent, synthetic triazole and approved antifungal15,16. Fluconazole works against amastigotes and promastigotes in the leishmania parasite's developmental stages17,18. Fluconazole is hydrophilic and thus penetrates very well into body fluids and tissues19,20. Therefore, we surmise that this novel combinatorial formulation has a promising anti-leishmanial potential.

 

MATERIALS AND METHODS:

Chemicals

Medium 199, RPMI Medium 1640were purchased from Hi-media (Mumbai, India), Amphotericin B (Gift sample from Vhb Life Sciences Inc), Fluconazole (Gift sample from Eurolife Healthcare Pvt Ltd), Phosphatidylcholine (PC), Phosphatidylserine (PS), Cholesterol was purchased from SigmaAldrich (St. Louis, USA). Phosphate buffer saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Nile Red (NR) dye (Hi-media (Mumbai, India), Dimethyl sulfoxide (DMSO), Fetal calf serum (FCS) was purchased from Gibco (Life Technologies corporation, India). Chloroform was purchased from SD Fine Chemicals Limited, (Mumbai, India). Extruder set (mini) and polycarbonate membranes (100 nm) were purchased from Avanti polar lipids, USA. Amphotericin B solution (250 μg/mL in deionized water) was purchased from Thermo Fisher Scientific, and Fluconazole solution (2mg/ml in deionized water) was purchased from Torrent Pharmaceuticals Ltd.

 

Formulation of drug/s encapsulated nanoliposomes

Nano-liposomal formulations were prepared as described previously21 by dry lipid film hydration (DLFH) method using two drugs Amphotericin B (AmpB) and Fluconazole (Flu) alone and together, depicted image as shown in Figure 1(a). The phospholipid cholesterol and copolymers Phosphatidylcholine (PC), Phosphatidylserine (PS), Cholesterol were taken in ratio (10:1:4) and 0.1%w/w of Nile Red dye was added after drugs were dissolved in solution of chloroform and methanol (2:1).

 

Physicochemical characterization of nanoliposomes

Spectroscopic characterization of Amphotericin B, Fluconazole, AmpB-Liposome (LipoAmpB), Flu-Liposome (LipoFlu), AmpB+Flu-Liposome (LipoAmpB+Flu) were done by using UV Spectrophotometer Shimadzu 1800, Japan in a quartz cuvette. The size and shape of formulations were analysed by Scanning electron microscope (SEM), Carl Zeiss, Supra 55, Germany using thin layer of gold coating under vacuum operated at 10 kV followed by SEM imaging. Size, potential and polydispersity index were analysed on Malvern Zetasizer (NY, USA) in triplicate.

Stability Study

Stability studies of prepared nano liposomal formulations were performed for duration of 6 months stored at 4C 2C and 25C 2C according to the ICH guidelines. The nano liposomal formulations were stored in upright position in amber colour glass vials (5 ml) with rubber stoppers and aluminum-crimped tops. Aliquots were taken at predetermined time intervals (0, 0.5, 1, 2, 3, and 6 months) and different parameters like % Entrapment Efficiency, polydispersity index (P.I.), vesicle sizewere evaluated in triplicate.

 

Drug entrapment study

Percent of drug entrapment efficiency (E.E.) was determined using an ultra-centrifugation method to assess the amount of loaded AmpB and Flu in the nano liposomes. Nano liposomal dispersion (0.5 ml) was diluted with PBS (pH 7.2) and centrifuged in a cooling ultra-centrifuge (Beckman Coulter) at 80,000 rpm at 4C for 1 hr. The pellet of liposomal formulation settled at the bottom of the centrifugation tube was then dissolved using a mixture of methanol and Dimethyl sulfoxide (1:1). The clear solution after suitable dilutions was analysed by UV Spectrophotometer, Shimadzu 1800 at λmax of 405 nm for AmpB and 261nm for Flu and percentage entrapment efficiency was calculated.

 

Encapsulated Drug

Percent of Drug entrapment efficiency = ------------------------ 100

Total Drug

 

In vitro drug release study

In vitro drug release study was carried out at 7.2 & 5.0 pH by using dialysis membrane method (12 kDa dialysis membrane) at room temperature using modified USP type I apparatus for 24h at a rotation speed of 50 rpm. 1 mL of prepared nanoliposomes was placed into a dialysis bag and suspended in release media composed of 100ml PBS (pH 7.2 and 5.0) with 0.5% w/v of sodium lauryl sulphate (SLS). Aliquots of 2 ml were withdrawn at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h followed by replacing with equal volume of fresh release medium. The drug/s content in the aliquots were analysed by a UV Spectrophotometer method at 405 and 261 nm respectively and percent cumulative release was calculated.

 

In vitro studies

Maintenance of Parasitic culture

The L. donovani Ag83 promastigotes were cultured in M199 containing 10% heat inactivated FCS supplemented with Gentamicine. The cultures were grown in incubator at 37C and maintained 95% relative humidity and routinely sub-cultured when cell density reached 107 cells/ ml.

In vitro Cellular Uptake Studies

In the present study, inverted fluorescence microscope (IFM) (Carl Zeiss LSM 880, JENA/Germany) was used to study the cellular uptake of formulations using Nile red (NR) dye as a fluorescence marker that was performed on isolated murine peritoneal macrophage/s (MPM) cells by injecting 0.5ml of 2% of starch in the peritoneal cavity. The NR-loaded formulations were added separately to MPM cells (5106/well) at 1:50 dilution and incubated for 60mins at 37C, 5% CO2, and 95% relative humidity. Then, observed at 0min and post 60mins of incubation using IFM with excitation and emission wavelength of 485 and 525 nm, respectively.

 

In vitro Macrophages cytotoxicity assay using MTT assay

Cells were seeded in 96 well plates at a density of 0.2106 and were allowed to adhere overnight. The medium was replaced and cells incubated for different time intervals (6, 48 and 72 h) with different formulations. Cells were then washed thrice and incubated in the presence of 5 mg/mL MTT for 4 h at 37C. DMSO (50 ml) was added to each well to solubilize formazan crystals and optically measured using a multi-well scanning spectrophotometer (iMARK Microplate Absorbance Reader, US) at 550 nm.

 

In vitro activity against promastigotes

The promastigote of Leishmania donovani (Ag83) type strain were maintained in M-199 medium supplemented with 25 mM HEPES buffer, 80mg Gentamicin, 2g Sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (FBS) at 26C in a dark environment under an atmosphere of 5%CO2. Parasites under their logarithmic growth phase were used for all experiments. Promastigotes were grown at 37C in 5% CO2 atmosphere for 24 hours before treatment for differentiation. After 24 hours & differentiation of promastigotes, the axenic amastigotes were generated22,23. Then to compare the efficacy of various formulations on promastigotes and axenic amastigotes different concentrations of AmpB and/or Flu (5, 10, 15 and 20 mg/mL of either drug/formulation) were generated by adding free drug/s or formulation/s. The cells were incubated for 48 h at 25C after treatment. The promastigotes were counted using a Neubauer Haemocytometer under a light microscope.

Avg. no. of cells Avg. no. of cells in control (+)

% Cell viability = --------------------------------------------------- 100

Avg. no. of cells in control (-)

 

Evaluation of in-vitro activity against intramacrophagic amastigotes

For evaluation Murine Peritoneal Macrophage (MPM) cells (1105 cells/well) were taken in16-well chamber slides and maintained inRPMI medium 1640 under a final volume of 100 μL/well and allowed to adhere for 24 h at 37 C in a 5% CO2with 95% air mixture. Adherent macrophages were infected with late log phase Ag83 promastigotes at a macrophages-to-parasite ratio of 1:10. After 12 hrs of infection, the Lab-Tek Chamber Slide were washed thrice with PBS (pH 7.2) to remove extracellular promastigotes and medium replaced. Different concentrations of either drug/formulation (5, 10, 15, and 20 mg/mL) were added to the wells in triplicate and incubated for 48 h. Cells were fixed in absolute methanol, stained with Giemsa stain, and examined under oil immersion objective (Zeiss Axiovert 25C) with at least a counting of 100 macrophages per well. Untreated infected macrophages were used for comparison with the number of infected cells and amastigotes present per cell. The parasite load was determined in triplicate using light microscopy by counting at least 200 cells per slide. The 50% inhibitory concentration (IC50) was calculated for intramacrophagic amastigotes by fitting the values to a curve analysis and percentage.

 

RESULTS:

Physicochemical characterization of nanoliposomes

SEM image of the formulation were found to be spherical in shape as shown in Figure 1(b). The vesicle size of AmpB-loaded, Flu-loaded, and AmpB+Flu-loaded liposome (AmpB+Flu-Lipo) was found to be 206.9 10.59, 209.8 12.26, 215.6 11.44, and 245.1 11.67 nm with P.D.I. of 0.212 0.062, 0.216 0.047, 0.219 0.026 and 0.239 0.041 respectively. The surface potential value of AmpB, Flu, and AmpB+Flu loaded liposomes was found to be −25.65, −24.25 and −28.85 mV, respectively.

 

Stability Studies

At 4C, all the nano liposomal formulations showed no significant change in their entrapment efficiency with 6%-7% leakage of drugs and 13% - 15% variation in vesicle size whereas at 25C, there was 11%-13% leakage of drugs and 14% - 16% variation in vesicle size at the end of 6 months. All formulations also showed significant increase (p < 0.05) in the vesicle size at 25C signifying temperature-induced increase in size of the nano liposomal formulations.

 

Figure 1:(a) Co-encapsulated nanoliposome (b)SEMimage of prepared formulation

 

Drug entrapmentand in-vitro drug release study

The E.E. of AmpB and Flu in nano liposomal formulation was found to be >89%, >85% when loaded individually and >85% and >81% when loaded simultaneously indicating excellent association of AmpB and Flu with the liposomes. Comparative release profile of the AmpB solution, Flu solution, AmpB-loaded, Flu-loaded, and AmpB+Flu-loaded liposomal formulation in PBS (pH 7.2 and 5.0) with 0.5%w/v of SLS, respectively was studied for 24hrs. At the end of 24 h, maximum release of around 50% was observed from both AmpB and Flu solutions at pH 7.2 whereas AmpB-loaded, Flu-loaded, and AmpB+Flu-loaded liposomal formulations showed 22.6%, 24.3%, 26.7% respectively. On the other hand, at pH 5.0, AmpB and Flu solution showed ~58% and 66% release whereas AmpB-loaded, Flu-loaded, and AmpB+Flu-loaded liposomal formulations showed 43.3%, 44.8%, 46.4% respectively at the end of 24 hrs.

 

In vitro cellular uptake studies

Fluorescent observation cellularuptake of NR loaded AmpB + Flu nanoliposomes revealed increased fluorescence compared to NR-loaded AmpB and NR-loaded Flu after 60mins post addition indicating enhanced cellular uptake of nanoliposomes.

 

Macrophage cytotoxicity assay

In vitro macrophage cytotoxic activity was performed to evaluate the safety of the drugs concentration used on promastigotes and amastigotes was whether toxic or not on macrophages themselves. Nano-formulations are expected to be more biocompatible because entrapment of drug inside the vesicles would minimize the direct exposure of AmpB and Flu to the normal cells. However, the assay revealed that the cytotoxic values for macrophages of AmpB Solution, Flu Solution, AmpB+Flu Solution, LipoAmpB, LipoFlu and LipoAmpB+Flu was found 10.69, 79.57, 28.69, 36.5, 45.59, 73.48 μg/ml, respectively as shown in Table 1. The comparison of CC50 values indicated that there was 39.19, 42.70, 53.01% reduction in CC50 of LipoAmpB, LipoFlu and LipoAmpB+Flu in comparison to AmpB Solution, Flu Solution, AmpB+Flu Solution as shown in Table 1.

 

In-vitro activity against promastigotes

The anti-promastigote activity of formulations (solution/ liposomal) containing drug/s single or dual against L. donovani was primarily screened for reducing the toxicity of already well-established treatment. The liposomal formulations were evaluated for anti-promastigote activity in comparison with plain drugs. The obtained comparative results against promastigotes at various concentrations of formulations are presented in Table 1, respectively. As evidenced from data Table 1, the formulated nano-liposomal formulation Lipo AmpB+Flu showed more reduced IC50in comparison to lesser cytotoxicity then other formulations.

 

In vitro activity against intramacrophagic amastigotes

Treatment resulted significant decrease in the number of intramacrophage amastigotes. The IC50 of plain AmpB and Flu solution and liposomal formulations (single and dual) was considerably lower than their CC50 value. Several studies showed that vesicles enhanced the anti-leishmanial activity of AmpB when compared with plain AmpB solution against Leishmania. Table 1 shows that the IC50 of intramacrophagic amastigotes of AmpB, Flu, AmpB+Flu, LipoAmpB, LipoFlu and LipoAmpB+Flu was 2.628, 26.2948, 6.003, 1.24, 12.625, 3.7028 μg/ml respectively with CC50 10.69, 79.57, 28.69, 36.5, 45.59, 73.48 μg/ml. The obtained comparative results against intramacrophagic amastigotes at various concentrations of formulations are presented in Table 1 respectively.

 

In vitro activity against Axenic amastigotes

Treatment resulted in a significant decrease in the number of axenic amastigotes. The IC50 of plain AmpB and Flu solution and liposomal formulations (individually and in combination) was considerably lower than their CC50value. Table 1 shows that the IC50 of axenic amastigotes of AmpB, Flu, AmpB+Flu, LipoAmpB, LipoFlu and LipoAmpB+Flu was 2.153, 24.247, 5.165, 1.074, 11.162, 3.412μg/ml respectively with CC50 10.69, 79.57, 28.69, 36.5, 45.59, 73.48μg/ml.


 

Table 1: In vitro evaluation of the cytotoxicity and the anti-leishmanial activity on L. donovani (Promastigotes andAmastigotes)

Formulations

IC50(IA)

(μg/ml) SD

IC50(AA)

(μg/ml) SD

CC50 (CM)

(μg/ml) SD

SI=

CC50/IC50

IC50 (PM)

(μg/ml) SD

AmpB

2.628 0.129

2.153 0.18

10.69 0.269

4.067732116

1.95 0.01

Flu

26.2948 2.36

24.247 1.59

79.57 3.54

3.026073596

74.977 4.63

AmpB+Flu mixture

6.003 0.369

5.165 0.89

28.69 3.69

4.779277028

6.265 0.33

Lipo AmpB

1.24 0.027

1.074 0.019

36.5 2.69

29.43548387

1.26 0.01

Lipo Flu

12.625 0.105

11.162 0.958

45.59 2.26

3.611089109

10.519 1.69

Lipo AmpB + Flu

3.7028 0.0142

3.412 0.023

73.48 3.59

19.84444204

3.308 0.93

IC50, 50% Inhibitory concentration; S.I., Selectivity Index = [CC50murine cells/IC50L. (L) donovani amastigotes]; AmpB, free Amphotericin B; Flu, Fluconazole; LipoAmpB, Liposomal Amphotericin B; LipoFlu, Liposomal Fluconazole; LipoAmpB+FLU Liposome containing Amphotericin B & Fluconazole both; SD, standard deviation; IC50Inhibitory Concentration values are representative of three independent assays.

Intramacrophage amastigotes (IA), Axenic amastigotes (AA), Cytotoxicity macrophages (CM), Selectivity index (SI), Promastigotes (PM)

 


DISCUSSION:

The drawbacks of available therapeutic options are aggravated by the increased resistance towards the drug, and dose dependent toxicity24. Therefore, combinatorial optimization of antifungal therapy could be an option for effective treatment. Some researchers investigated the antifungal potential of Fluconazole and found that the combination of Fluconazole with Amphotericin B is more efficient than monotherapy25. The characterization data revealed that the developed LipoAmpB+Flu are 200 nm in diameter and stable in aqueous suspension for several weeks. The drug release kinetics pattern showed that 25% and 50% release at pH 7.2 and 5.0, respectively within 24 h. Further, from cell viability study, it can be concluded that the LipoAmpB+Flu exposure to promastigotes and amastigotes (Intramacrophagic and Axenic) of L. donovani (Ag83) leads to significant decrease in number of viable parasites. Confocal studies revealed the increase in cellular uptake of nanoliposomes and delivery of active drugs inside the macrophages which ultimately causes the effective treatment. Thus, the findings of this report indicate that the combined liposomal formulation of AmpB and Flu can offer better therapeutic efficacy. Although the use of LipoAmpB+Flu showed exciting results under invitro experimental conditions, in vivo experimental investigation is needed to establish the developed of formulation as an effective antileishmanial treatment in near future.

 

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Received on 17.03.2021 Modified on 19.06.2021

Accepted on 26.08.2021 RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(9):4929-4933.

DOI: 10.52711/0974-360X.2021.00857