A Novel Nanoformulation for Reducing the Toxicity and Increasing the Efficacy of Betulinic Acid Using Anionic Linear Globular Dendrimer

INTRODUCTION

Leishmaniasis is a parasitic disease which is transmitted to human through sandfly bite. The main problem for treatment of this disease is the limited number of therapeutics and their limited clinical application, thus development of novel therapeutics seems to be required [1]. The antileishmanial effects of betulinic acid (BA) have been shown in various studies [2-4].

BA (Fig. 1) is a pentacyclic triterpenoid com-pound derived from various natural plants such as bark of white birch (Betula species) [5]. It exists in different plant organs in form of aglycon and glycosyl derivatives [6]. Various pharmacological effects have been reported for this triterpenoid including anticancer, hepatoprotective, antimalarial, anti-inflammatory, anthelmintic, antileishmanial, and antioxidant effects [7]. However, it suffers from poor biopharmaceutics owing to low solubility and permeability, resulting in its low dosing for treatment [3].

There are various approaches to resolve the problem of low water solubility of therapeutics, such as using nanocarriers. In this regard, dendrimer nanoparticles have shown promising results [8]. Development of nanosized delivery systems causes fundamental changes in the field of pharmaceutical technology by improving the therapeutic effects and bioavailability of most drugs. In this context, nanoparticles have received increasing attention as drug carrier. They can cause an increase in the therapeutic effects and simultaneously cause a decrease in the side effects of drugs [9]. ALGD is a negatively charged nanoparticle which can be used as a powerful vehicle for drug delivery due to water solubility, biodegradability, biocompatibility, and low polydisprsity index. The negative charge of this molecule reduces the drug repulsion and cell surface junction leading to decreased intraction rate, enhanced physiological activity of cells to toxic material, and increased viability of cells [10].

To the best of our knowledge, no studies have been conducted to load BA into ALGD (Fig. 2) nanocarriers in order to decrease its toxicity and increase the solubility. Thus in the present study, we are going to use ALGD as nanocarrier to improve the solubility rate and toxicity of BA in order to provide an effective therapeutic dose. The formulation is being tested for the first time for the treatment of leishmania major (L. major) infected Balb/c mice.

MATERIALS AND METHODS

Materials

BA was purchased from Baoji Guokang Bio-Technology Co. Ltd (China). Chloroform, polyethylene glycol 600 (PEG600), N,N′-Dicyclohexylcarbodiimide (DCC) , MTT and acetic acid were purchased from Merck Company (Germany). The RPMI1640 medium, fetal bovine serum (FBS), and penicillin–streptomycin antibiotics were bought from Gibco Inc. (USA). Schneider culture medium, dimethyl sulfoxide (DMSO), dialysis membrane (cut off 6 KDa) and Polyethyleneglycol (PEG) were provided from Sigma Aldrich Company (USA). Iranian strain of L. Major (MRHO/IR/75/ER) was purchased from Pasteur Institute of Iran. All other materials used were of analytical grade. The double distilled water was used throughout the study.

Synthesis and drug loading into anionic linear globular dendrimer

ALGD was synthesized according to the method described by Zadeh Mehrizi et al. [11] using PEG, DMSO, DCC and citric acid. BA was loaded into dendrimer using adsorption procedure according to a literature method [11]. In addition, drug loading efficiency was measured using standard curve according to the method described in the literature [12] using the following formula:

100

In this formula, ED is equivalent to the primary drug used (mg) subtracted to the drug in supernatant (mg) and TD is the primary drug used.

Thin-layer chromatography (TLC)

The purity of BA and BD was evaluated using thin-layer chromatography (TLC). For this purpose, 2 µL of the samples were poured on a silica plate and left to dry at room temperature. Next, the plate was incubated at 45 ℃ in a chamber containing 10% methanol in chloroform for 45 min. Then, the plate was picked up, dried and analyzed to identify the components by means of iodine vapors to visualize the spots.

Determining the size, size distribution and zeta potential

Size, size distribution, and zeta potential of nanoparticles were determined using dynamic light scattering (DLS) technique according to a literature method [11]. In brief, the absorbance of nanoparticles suspension was read at 620 nm and then the suspension was introduced into the Zetasizer instrument (ZEN 3600, Malvern Instruments Ltd., Worcestershire, UK).

Morphology evaluation of Betulinic acid loaded dendrimer nanoparticles using Atomic Force Microscopy

BD was morphologically evaluated by Atomic Force Microscopy based on previously explained method [11]. For this purpose, a drop of the nanoparticle suspension was poured on a mica surface and left at room temperature to dry. Then Atomic Force Microscopy (AFM) imaging (NanoWizard II AFM, JPK Instruments, Berlin, Germany) was performed using a tapping-mode in air at room temperature on a silicon-nitride cantilever with a spring constant of Nm-1. The obtained images were analyzed by a JPK information processing software (Germany).

Betulinic acid loaded dendrimer nanoparticles evaluation by Fourier Transform Infrared Spectroscopy

Fourier Transform Infrared (FTIR) Spectroscopy was used to investigate the BD composition and determine the type of drug loading according to the previously described method [12]. Briefly, the suspension of drug-loaded nanoparticles was centrifuged (13000 r.p.m, 4 ℃, 30 min) and the pellet was dried at room temperature and mixed with potassium bromide at room temperature. The mixture was pressed to form a tablet and examined by the FTIR (Nicolet 740SX) device.

Betulinic acid loaded dendrimer nanoparticles evaluation using H-Nuclear Magnetic Resonance

BD was investigated in terms of the structure, molecular properties and chemical bonds using Proton Nuclear Magnetic Resonance (1HNMR) method, according to the procedure published elsewhere [11]. For this purpose, one mg of the powder form of BD nanoformulation was dissolved in DMSO deuterium and 1HNMR spectroscopy was performed in FT pulse mode at a resonance frequency of 300 MHz.

Cellular uptake and kinetic of drug release from betulinic acid loaded dendrimer nanoparticles

Cellular uptake of BD was determined using BD at the dose of 20 µg/mL according to the method defined in another study [11] using peritoneal macrophages obtained from the Balb/c mice. Pattern of BA release from ALGD nanoparticles was determined using dialysis membrane method according to a literature method [12].

In vitro viability and toxicity effects of the formulations

Peritoneal macrophages were obtained from Balb/c mice (from peritoneal lavage using cold RPMI-1640 medium) and in vitro viability effects of the formulations were evaluated according to the previous study [12]. Briefly, the peritoneal macrophages were cultured in 96-well plate (104 cells/well) containing 90% RPMI-1640 culture medium, 10% FBS and 1% Penicillin/Streptomycin. After 24 hours of incubation in a 5% CO2 incubator at 37 ℃, the culture medium was replaced with medium containing various concentrations of dendrimer, BA and BD formulations. The plate was then transferred to the incubator and after 48 hours, the culture medium containing different formulation was replaced with MTT solution (0.5 mg/mL in complete RPMI-1640 medium) and incubated for three hours in the incubator. Next, to dissolve the prepared formazan crystals, the MTT solution was replaced with 100 µl of acidic isopropanol and incubated for 15 minutes. After 15 minutes, the absorbance was read at 570 nm using microplate scanning spectrophotometer (ELISA reader; Organon Teknika, Boxtel, the Netherlands) and then the viability was measured by the formula below. The cells treated only with medium were considered as control.

In vivo toxicity of the formulations

Toxicity effects of the formulations were determined in vivo environment using female Balb/c mice according to previously described method in terms of mortality rate and enzymatic toxicity [11]. For enzymatic toxicity evaluation, the blood samples of the mice were obtained and serums were prepared. Next, serum concentrations of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine were measured.

Statistical analysis

After data evaluation and confirming their normal distribution, parametric statistics including two-way analysis of variance (ANOVA) and Tukey’s tests were used under the supervision of a statistician. Graphs were plotted using Graphpad Prism software version 6.

RESULTS AND DISCUSSION

Nanoparticles characterization

The drug loading efficiency was measured using the standard curve of BA and spectrophotometer at 254 nm, which was equal to 96%. Also, the results of DLS method demonstrated that monodispersed nanoparticles with the negative zeta potential were synthesized (Fig. 3, 156±15 nm, size distribution of 0.31±0.14 and zeta potential of -14.6±1.7 mV).

TLC

There are various methods for evaluating the purity of compounds such as TLC method [13]. TLC is the most effective method for the low-cost analysis of those samples that require minimal sample clean-up or sample preparation steps [14]. In the present study, the Rf values for BA and BD were 0.84 and 0.49, respectively, indicating the purity of the samples.

Morphology evaluation of betulinic acid loaded dendrimer nanoparticles using Atomic Force Microscopy

Results of AFM Microscopy indicated that BD nanoparticles were formed as swollen particles owing to BA loading into nanoparticles, resulting in a novel topology (Fig. 4). Also, results demonstrated that dendrimer nanoparticles were formed as spherical particles (Fig. S1, supplemental file). Both BD and dendrimer nanoparticles were formed as smooth shapes which can cause a decrease in the tissue irritation compared to the crystalline and irregular particles [15]. Overall, AFM results depicted that BA was loaded well into nanoparticles.

Betulinic acid loaded dendrimer nanoparticles evaluation by Fourier Transform Infrared Spectroscopy

FTIR results of BD showed the presence of common spectra related to BA (regions of 1451, 1043 and 855 cm-1) and ALGD (regions of 3380, 2920, 1250 cm-1) into formulation and confirmed the loading of BA into ALGD nanoparticles (Table 1, Fig. 5). The band at 1451 is assigned to symmetrical deformation of -CH2 of the cyclopentane functional group of BA, the peak at 1043 cm-1 is related to the C–O stretching of alcohol group of BA and the peak at 855 cm-1 is attributed to the R2C=CH2 functional group of BA. Overall, results illustrated the decent loading of BA into nanoparticles.

Betulinic acid loaded dendrimer nanoparticles evaluation using H-Nuclear Magnetic Resonance

ALGD nanoparticles were synthesized using divergent method and BA was loaded into the nanoparticles. The particles were evaluated using 1HNMR method. Results demonstrated that BD showed the peaks related to BA and ALGD in which the peaks of carboxylic acid groups appeared at 12 ppm and double bonds at 4 and 5 ppm. In the obtained result, the peak of 2.5 ppm was related to the functional group of citrate, while the peak of 3.5 was corresponded to the PEG molecule (Table 2, Fig. 6). Overall, results of 1HNMR indicated that BA was loaded well into the nanoparticles.

Cellular uptake and kinetic of drug release from betulinic acid loaded dendrimer nanoparticles

Results of cellular uptake measurement using flow cytometry demonstrated that BD had proper cellular uptake equal to 94.6% (Fig. 7) [16]. BA was released from BD formulation in a slow and sustained manner over a period of 48 hours in which 90% of the loaded drug was released in this period of time, confirming that BD released BA in a controlled manner (Fig. 8) [16]. These results indicated that BD nanoparticles could be utilized for BA release in a prolonged and controlled manner, resulting in a reduction in the fluctuations of the BA plasma concentrations and improvement of BA therapeutic effects [17].

In vitro viability and toxicity effects of the formulations

The viability effects of BD and BA formulations were compared to that of control using MTT test [16]. In the present study, BD at a dose of 20 µg/ml was found to be perfectly non-toxic compared to that of BA (P˂ 0.001) (Fig. 9). Then the therapeutic efficacy of BD was evaluated on the promastigote and amastigote of L. major showing that BD was more potent than BA to kill the parasite. One of the main feature of nanoparticles is their ability to improve the efficacy of drugs which was demonstrated in this study [18-21].

In vivo toxicity of the formulations

BD at a dose of 40 mg/kg was found non-toxic in terms of liver enzymes (AST, ALT and ALP) and kidney markers (BUN and creatinine) compared to BA at the same dose which was toxic and increased the enzymes and markers compared to the control group (Fig. 10). Also, the mortality rate in BD (40 mg/kg) receiver mice was found to be 0%, while BA (40 mg/kg) was toxic and caused the mortality rate of 30% (Table 3). BD at the dose of 40 mg/kg increased the solubility rate of BA by more than 700-fold which cause an increase in the efficient dose, resulting in efficient treatment of L. major infected mice [13, 16, 18, 22]. Overall, the results indicated that BD at the dose of 40 mg/kg did not cause any toxicity compared to same dose of BA. Table 4 represents the promising results of our study in terms of in vivo and in vitro effects compared to other published articles.

ACKNOWLEDGMENTS.

The authors would like to thanks Pasteur Institute of Iran.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interests regarding the publication of this manuscript.