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Influence of bioenhancers on the in vivo fate of niosome-encapsulated methotrexate.

Introduction

Cancer presents one of the most formidable health problems worldwide. More than 10 million people are diagnosed with cancer every year [1,2]. Methotrexate (MTX) is one of the oldest and highly efficacious antineoplastic drugs; inhibit dihydrofolate reductase, blocking the conversion of dihydrofolic acid to tetrahydrofolic acid which is an essential coenzyme required for one carbon transfer reactions in denovo purine synthesis and amino acid inter conversion. The most common problem encountered with MTX is the development of resistance to tumors. Relatively small increase in drug resistance in cancer cells is thus sufficient to render the drug ineffective. Hence there is a need to improve its acceptability by minimizing the intensity of side effects and thus increasing the therapeutic efficacy of the drug.

Niosomes are the novel vesicular drug delivery system by which we can achieve the constant plasma drug concentration for the extended period of time. They are nonionic surfactant vesicles that have potential applications in the delivery of hydrophobic and hydrophilic drugs. They have received attention for their potential as drug delivery vehicles due to advantages like excellent stability, longer shelf life, higher flexibility, better bioavailability, increased efficacy, and therapeutic index [3,4]. Niosomes encapsulated drug does not come in contact with the blood resulting in no harmful effects and undergo less biodegradation. Bioavailability of drug encapsulated in niosome can be enhanced by encapsulating the drug along with bioenhancers in the niosomal vesicles. The bioenhancers such as piperine and curcumin enhance the theraputic efficacy of MTX. Co-administration of piperine with MTX inhibiting the P-glycoprotein and cytochrome p-450 enzymes enhances the efficacy of drug, makes drug more effective against cancer and transporter inhibitors (curcumin) increases the intracellular drug accumulation and restores the chemosensitivity [5,6].

The aim of this present study was to explore the utility of the principles of niosomal drug delivery systems to formulate a sustained release system for MTX alone and along with bioenhancers (a mixture of piperine and curcumin) by thin film hydration technique such that an increased entrapment, with increased storage stability and prolong release could be achieved.

Materials and Methods

Methotrexate was a gift sample from Khandelwal laboratories Pvt, Ltd. (Mumbai, India). Span 60 was obtained from Loba chemie Pvt. Ltd. (Mumbai, India). Methanol, hydrochloric acid and chloroform were obtained from Merck India Ltd, (Mumbai, India). Cholesterol and potassium dihydrogen phosphate were obtained from HiMedia Laboratories Pvt, Ltd. (Mumbai, India). Curcumin extract and piperine extracts (white) were obtained from Green Grover's Pvt Ltd. (Bangalore, India). Dicetyl phosphate was obtained from Sigma Aldrich Chemicals, (Bangalore, India). Sodium chloride, Sodium hydroxide and disodium hydrogen phosphate were obtained from CDH Laboratory Ltd. (Delhi, India). All chemicals used were of analytical grade.

Preparation of Niosomes of MTX alone and along with bioenhancers

Niosomes of MTX alone and along with bioenhancers were prepared by thin lipid film hydration technique using rotary flash evaporator. Weighed quantity of cholesterol, span 60 and dicetyl phosphate were dissolved in mixture of chloroform: methanol (2:1) in a 250 ml round bottom flask. Solvent mixture was evaporated under a vacuum of 25 inches of Hg at 60 [+ or -] 2[degrees]C and the flask rotated at 100 rpm until a very thin, smooth and dry film was formed. It is then slowly hydrated by 5 ml phosphate buffer saline (PBS) of pH 7.4 containing 10 mg MTX drug alone as well as with 10 ml PBS pH 7.4 containing 10 mg MTX drug and accurate quantity of bioenhancers at a temperature of 60 [+ or -] 2 [degrees]C for a period of 1h. The multilamellar vesicles (MLVs) suspension was sonicated to form small unilamellar vesicles (SUVs) of niosomes by using probe sonicator [7]. The compositions of different formulation of niosomes are given in Table 1.

Entrapment efficiency

For determination of entrapment efficiency, unentrapped drug in the niosomal formulation was seperated using centrifugation at 20,000 rpm for 1 hour at 4 [degrees]C temperature. The supernatant contains unentrapped MTX was removed and the remaining pellet in the centrifuge tube resuspended in 0.1 N sodium hydroxide (as MTX is highly soluble in 0.1 N NaOH) and vortexed thoroughly for 3 min. After vortexing 1 ml of this suspension was taken in a micropipette and transferred to a test tube. To this add 5 ml of methanol and this was further vortexed for 2 min.The absorbance of resulting solution was measured using a shimadzu UVspectrophotometer at 292 nm after suitable dilution with methanol [8].

Average vesicle size and size distribution

The z-average diameter and polydispersibility index of optimised niosomal dispersions of MTX with or without bioenhancers were measured by Dynamic Light Scattering (DLS) using a Malvern 4700 (Malvern Ltd., Malvern, United Kingdom). All measurements were done at 25[degrees]C at an angle of 90[degrees] between laser and detector. Diluted the dispersions (20 [micro]l) to an appropriate volume with doubly filtered distilled water.The mean droplet size was calculated and the unimodal curves were recorded [9].

Zeta potential

Zeta potential was analyzed to measure the stability of niosome by studying its colloidal property. Zeta potential of formulations was determined using Zetasizer HSA 3000 (Malvern instrument Ltd., Malvern, U.K.). Samples were placed in clear disposable zeta cells and temperature was set as 25[degrees]C and results were recorded [10].

Optical photo microscopy

MLV suspension (100 [micro]l) was placed on a clean glass slide and viewed by MOTIC digital photogrphic microscope under 45X magnification. Size ([micro]) of the MLVs was also measured using the microscopic scale.

Transmission Electron Microscopy

The prepared niosomal formulation was characterized for their morphology using transmission electron microscopy (TEM) using JEOL 1200EX transmission electron microscope. To an aliquot of a suspension of prepared formulation (10 [micro]l),sufficient quantity of 1 % phosphotungstic acid was added and mixed gently. A drop of the mixture was placed on to the carbon-coated grid and drained off the excess. The grid was then allowed to dry, forming a thin film of stained vesicles which was then viewed at an operating voltage of 70kV [11,12].

Scanning Electron Microscopy

The surface morphology and the size distribution of niosomes were studied by scanning electron microscopy. Niosomes were sprinkled on to the double--sided tape that was affixed on aluminum stubs. The aluminum stubs was placed in vacuum chamber of scanning electron microscope (XL 30 ESEM with EDAX, Philips, Netherlands). The samples were observed for morphological characterization using a gaseous secondary electron detector [13]. (working pressure 0.8 torr, acceleration voltage: 30.00 KV) XL 30, (Philips, Netherlands).

In - vitro release study of optimized formulation

A volume of 1ml niosomal dispersion (encapsulation efficiency: 56.9%) was put in a dialysis bag (MWCO 12,000 Da, Sigma-Aldrich, USA.). The dialysis bag was suspended in 300 ml phosphate buffer pH 7.4 and maintained at 37[+ or -] 0.2 [degrees]C. The medium was stirred continuously during the release study. At predetermined time intervals of 15 min, 30 min, 45 min, 60 min, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h. 5 ml, aliquots were sampled and replaced with 5 ml fresh phosphate buffer pH 7.4. The concentration of MTX was determined by the UV spectrophotometer (Shimadzu UV 1700) at 303 nm [14,15].

In vivo studies

The swiss albino mices were obtained from animal housing facility, Nitte University, Mangalore. The 24 swiss albino mices weighing 20-25 g were taken and divided in four groups. 2 ml of Ehrlich Ascite Carcinoma (EAC) suspension containing 1 X [10.sup.6] EAC cells was injected each of 24 animals divided into 4 groups on the first day of experiment. After inoculation of EAC cell each group was treated with the following:

Group I: The saline treated control animals.

Group II: The positive control animals (MTX 10 mg/kg body weight)

Group III: Niosomes of MTX (Equivalent to 10 mg/kg body weight)

Group IV: Niosomes of MTX along with bioenhancers (Equivalent to 10 mg/kg body weight)

On the 14th day, three animal of each group were sacrified and the peritoneal fluid was collected from them, measured to the nearest volume in ml and a smear prepared for detection of the status of EAC cells. The remaining animals from each group were monitored for determination of survival time. To determine the effect of MTX in different formulation, saw the changes in the status of inter-peritoneal tumor cells. Two indicators of EAC-cell activity were measured in both control and the MTX treated groups: (i) the volume of the fluids inside the peritoneal cavity; (ii) the numbers of EAC cells found in the fluid by using a haemocytometer. Percent tumor growth inhibition was calculated by comparing the total number of tumor cells present in the peritoneal cavity of the treated group and the control groups. Tumor cell growth in saline treated control group was taken as 100 percent cell growth [16].

Effect of MTX treatment on the mean survival time (MST) and the increase in the lifespan of EAC tumor-bearing mice were calculated by usinfg following formula:

MST = Survival time of each mouse (days)/Total mice

% ILS = [(MST treated group/MST control group) x 100] - 100

where, MST is mean survival time and ILS is increase in life span

Statistical analysis

All values are expressed as mean [+ or -] SD. The data were statistically analyzed by ANOVA. P values less than 0.05 were considered to be significant. In most cases only the highest-order significant interaction is presented because of the consequent problem in interpreting lower-order ones and main effects.

Histopathology studies of ascitic fluid drawn from the treated group and the control groups were also conducted by using tryptan blue dye exclusion test and observed under the microscope.

Stability studies

Stability studies were carried out according to the ICH guidelines. MTX loaded niosomal suspensions (5ml) were transferred into sealed glass vials and stored at refrigerated condition (RF;4-8 [degrees]C), room temperature (RT; 25 [+ or -] 2[degrees]C) and at 37[+ or -] 2[degrees]C. Ability of the vesicles to retain entrapped drug (i.e. drug retentive behavior) was assessed at weekly intervals for 3 monhs. Samples were also analyzed for size and polydispersibity index at 1 month intervals. Freeze dried niosomal suspension of MTX in combination with curcumin and piperine were also placed in the stability chamber maintained at 40 [degrees]C, 75% RH and analyzed for physical appearance, size, morphology and nature of drug in niosomal formulation at regular intervals of time (0,1,2 and 3 months)

Results and Discussion

Niosomes of MTX alone and along with bioenhancers such as curcumin and piperine were prepared by thin film hydration method by using span 60 and cholesterol as a surfactant. They were optimized on the basis of observation and maximum percent drug entrapment (PDE).

Entrapment efficiency

The percentage drug entrapment efficiency was found to be 56.9 [+ or -] 1.331 and 55.1 [+ or -] 0.49 respectively for niosomes of MTX alone and along with bioenhancers. The percentage entrapment efficiency of bioenhancers such as curcumin and piperine were found to be 40.30 [+ or -]0.67 and 64.31[+ or -] 0.96 respectively.

Average Vesicle Size and Size Distribution

The vesicle size analysis revealed that, the average size of vesicles for niosomal formulation of MTX was 155.2 nm and along with bioenhancers was 185.8 nm. The Vesicles less than 400 nm can cross the vascular endothelia and can accumulate in tumor site via the enhanced permeability retention effect. The formulated niosomes size was found to be less than 400 nm so these niosomes easily cross the vascular endothelia and can accumulate in tumor site. The polydispersibility index values were found to 0.002 and 0.051 for MTX without bioenhancers (Fig. 1a) and along with bioenhancers (Fig. 1b) respectively. The low value of polydispersibility index indicating high homogeneity of the vesicle populations.

Zeta Potential

The zeta potential of the niosomal formulation containing MTX was found to be -69.5mv and those of niosomal formulation of MTX along with bioenhancers was found to be -65.1 mV. The significance of zeta potential is that its value can be related to the stability of colloidal dispersions. A value of 25 mV (positive or negative) can be taken as the arbitrary value that separates low-charged surfaces from highly-charged surfaces. Higher the zeta potential value of the formulation more will be the stability of the formulation. When the zeta potential is low, attraction exceeds repulsion and the dispersion will break and flocculate. So, colloids with high zeta potential (negative or positive) are electrically stabilized while colloids with low zeta potentials tend to coagulate or flocculate. The inclusion of DCP increased the zeta potential values.

Optical Photomicrography

From optical photomicrography it was clear that vesicles formed were of spherical shape and multilamellar. Therefore multilamellar vesicles (MLVs) were obtained with small layers of interstitial water by thin lipid film hydration technique. Also some small unilamellar vesicles (SUV's) are formed (Fig. 2). Further confirmation of vesicle formation and integrity of bilayers was achieved by using TEM studies.

Transmission Electron Microscopy (TEM)

TEM images of niosomes of MTX show multilamellar vesicles prepared by thin film hydration technique were spherical in shape with a mean diameter of 200 nm (Fig: 3a). TEM images of niosomal suspension of MTX along with bioenhancers showed discrete spherical particles with a size less than 200 nm (Fig. 3b). Thus TEM images revealed the presence of well identified, nearly perfect spheres having a large internal space.

Scanning Electron Microscopy

Shape and surface characteristics of niosomes were examined by Scanning Electronic Microscopy (SEM) analysis. Scanning electron microscopy showed the porous surface and making more effective carrier for anticancer drugs like MTX. Surface morphology illustrated the smooth surface of niosomal formulation. The prepared vesicles were studied under 5000X magnifications to observe the formation of vesicles. The vesicles of niosomes of MTX had an average size of 5um. (Fig. 4a). The vesicles of niosomal formulation along with bioenhancers were studied under 8000X magnification showed a spherical shape and an average particle size of 2um. (Fig. 4b). Some unevenness of vesicles that observed under the study may be due to drying process under normal environment condition. The particles found to be uniform in size and shape.

In Vitro Release Study

The in vitro release study revealed that the release of the drug was sustained on encapsulation in niosomes. Approximately 98.77% of the free drug was released within 60 min, whereas the same percentage of drug release from niosomes of MTX was occurring at the end of 11 h (fig.5). Release of MTX from niosomes was biphasic with an initial faster release for 3 h followed by a period of slow release. Thus, the study revealed that initially there was a high rate of drug release, which may be due to the release of the adsorbed drug from the lipophilic region of niosomes, which help to achieve the optimum loading dose. This is necessary to give an initial burst to initiate the therapy. The most likely explanation for sustained release then after it was observed slower diffusion of the drug through the bilayer as the presence of cholesterol in the formulation affected the fluidity by making it more rigid. As the amount of cholesterol increased, they fill the pores of vesicular bilayers and abolish the gel-liquid phase transition of the niosomal systems and result was found to be markedly reducing of drug release from the niosomes. This confirms that addition of cholesterol acted as a membrane stabilizing agent that decreased the permeability and helped to sustain the release.

The maximum release of drug from niosomes containing MTX along with bioenhancers was 60.9% at the end of 12 h. The reason for slower release of the drug from noisome encapsulated complex may be the interaction of complex with the lipid/surfactant bilayers and bioenhancers. These results indicate that the release of MTX followed a slow release status, which suggests that it takes time for MTX to be released once encapsulated in the niosomes because the surfactant bilayers are stabilized by cholesterol.

In vivo study

In vivo studies were performed and result showed that formulation containing MTX and bioenhancers increased the survival rate of animals in cancer. Average volume of ascetic fluid was measured by sacrificing all the animals at the end of 14th day. Volume collected from group treated with niosomes of MTX along with bioenhancers was found to be less compared to other treatment groups. The number of tumor cells present in the ascetic fluid of each group was also counted using tryptan blue exclusion method. It was observed that the group treated with combination of MTX along with curcumin and piperine had only 56.70 x [10.sup.7] tumor cells as compared to 94.47 x [10.sup.7] cells in the saline treated control group. Finally the percent tumor growth inhibition was calculated and was found to be 12.17, 28.88 and 39.98% for free drug, niosomal of MTX alone and niosomes of MTX along with bioenhancers respectively.

The number of dead cells in the peritoneal fluid of mice increased fourfold by treating with the combination therapy (niosomes of MTX + bioenhancers) compared to control. These results substantiate the niosomal formulation along with bioenhancers exhibited a significantly higher tumor cell necrosis. Thus the formulation of niosomes along with bioenhancers proved to be more efficient against cancer as shown in Table 3.

Histopathological studies also supported the above studies and by comparing with control (Fig. 6a), it was found that slight decrease in number of viable tumor cells in case of mice treated with niosomes of MTX (Fig. 6b). A significant decrease in cell size and a large number of dead cells were noticed in mice treated with niosomal formulation of MTX along with bioenhancers (Fig. 6c), recognized due to the increased uptake of the stain. The result revealed that efficacy of niosomal formulation of MTX along with bioenhancers was enhanced.

The mortality rate was also observed in tumor-induced mice. The untreated mice which served as tumor control survived for 23 days, while the mice treated with free drug survived for 30 days, showing an increase in life span of 31%. The mice treated with niosomal formulation containing MTX alone showed a enhanced MST of 32 days indicating an increase in life span of 38% and those treated with niosomes of MTX + bioenhancers survived for 40 days with a significant increase in ILS of about 71.61%. An enhanced life span by 40.59% over free drug administration was observed with niosomal formulation of MTX with Bioenhancers as shown in Table 4.

Stability Studies

The stability of niosomes is a great issue and a major challenge in commercializing the formulations. The niosomes storage under refrigerated condition showed promising results of 71.42% of drug retained at the end of 12 weeks whereas at room temperature the percent of drug retained was 56.72% and at 37 [degrees]C drug retained was 32.93% (Fig. 7). The loss of drug from niosomal vesicles stored at higher temperature may be due to the effect of temperature on the gel to liquid transition of the bilayers together with degradation of phospholipids leading to destabilize the membrane packing. The fatty acid chain of the surfactant tends to adopt a structural conformation other than all the Trans straight chain conformation.

The niosomes stored at lower temperature (4[degrees]C) did not show any appreciable change in vesicle size distribution. This may be due to decreased mobility of the bilayer at lower temperature and providing the stability in the membrane. As a result leakage of drug from niosomals vesicle was reduced. But curcumin and piperine which were used as a bioenhancers are unstable on long term storage at refrigerated condition and hence freeze drying is the viable option for long term stability so niosomes along with bioenhancers were freeze dried using 5% sucrose as cryoprotectant. After 3 months of storage at accelerated condition, freeze dried suspension was found to be stable without any collapse or shrinkage of dried cake. The vesicle size, Polydispersibility index (PDI), amount of MTX, curcumin and piperine retained were measured (Table 5).

Conclusion

The use of various pharmaceutical nanocarriers has become one of the most important areas of nanomedicine. Currently, niosomes are drug delivery system with greater potential for targeted and controlled release. All the formulations of niosomes were characterized on the basis of entrapment efficiency, scanning electron microscopy, size distribution and zeta potential. The present formulation study on methotrexate is an attempt to prepare niosomal drug delivery system and evaluate its in vitro and in vivo performance. The in vitro study revealed that the release pattern of the drug was sustained in niosomes and it was further significantly sustained by addition of bioenhancers. In vivo studies revealed that formulation containing MTX along with bioenhancers increased the survival rate of animals as compared to other formulation. Stability studies showed that the freeze dried niosomal suspensions of MTX in combination with bioenhancers were able to withstand the ICH accelerated stability conditions. In conclusion, niosomal formulation along with bioenhancers could be a promising delivery system for methotrexate with prolonged drug release profile and extention of this research work may be useful in the targeted delivery of methotrexate.

Received 21 February 2013; Accepted 16 June 2013; Available online 10 October 2013

Acknowledgement

The authors are expressing their sincere gratitude to the Principal, NGSMIPS, Mangalore and also to Nitte University, Mangalore for providing necessary facilities to carry out this research work.

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R. Narayana Charyulu, B. Gandhi Kinjal, Jobin Jose, Sneh Priya, Nimmy Chacko

Department of Pharmaceutics, N.G.S.M. Institute of Pharmaceutical Sciences, Paneer, Deralakatte, Mangalore 575 018

Table 1: Optimized formulation composition of niosomes of MTX alone
and along with bioenhancers

Formulatio   MTX    Span 60   Cholesterol   Curcumin   Piperine
Code         (mg)   (mg)      (mg)          (mg)       (mg)

NM           10     150       20            --         --
BNM          10     150       20            50         10

Formulatio   DCP            Organic     Hydration
Code         ([micro]mol)   solvent     Volume
                            (ml)        (ml)

NM           7              3           5
BNM          7              3           10

Table 2: Percentage entrapment efficiency, average particle size and
zeta potential of niosomes of MTX

Batch   % Drug              % Curcumin          % Piperine
name    entrapped           entrapped           entrapped

NM      56.9[+ 0r -]1.33    --                  --
BNM     55.1[+ 0r -]0.49    40.30[+ 0r -]0.67   64.31[+ 0r -]0.96

Batch   Average particle    Zeta
name    size(nm)            potential(mV)

NM      155.2               -69.5
BNM     185.8               -65.1

Table 3: Percentage tumor growth inhibition after treatment

                     Average
                     volume       Average no. of   %Tumor   %Tumor
                     of ascitic   tumor cells x    cell     growth
Groups               fluid (ml)   [10.sup.7]       growth   inhibition

Control              7.64         94.47            100      0.00
Positive control     7.01         82.97            87.83    12.17
(MTX 10mg/kg)
Niosomes of MTX      9.88         75.28            71.12    28.88
equivalent to
(10mg/kg)
Niosomes of          3.41         56.70            60.02    39.98
MTX + bioenhancers
equivalent to
(10mg/kg)

Table 4: Mean Survival Time values after treatment

Group and Dose                   MST(Days)            ILS (%)

Control                          23.50[+ or -]1.23    --
Positive Control MTX(10mg/kg)    30.79[+ or -]0.78    31.02
Niosomal drug delivery of MTX    32.48[+ or -]1.08    38.21
(10mg/kg body weight)
Niosomal drug delivery of MTX    40.33[+ or -]0.34    71.61
along with bioenhancers
(10mg/kg body weight)

MST = Mean Survival Time. ILS = Increase in Life Span. n = 6 animals
in each group.

*** p<0.05 in all the groups when compared with control. Values
are expressed as Mean [+ or -] SEM

Table 5: Stability data of freeze dried niosomal formulation
of MTX along with bioenhancers at accelerated condition

Time      Vesicle             Polydispersibility   Amount of curcumin
point     Size(nm)            index                retained *

0         185[+ or -]1        0.051[+ or -]0.03    40.39[+ or -]0.2
1 month   187[+ or -]1        0.052[+ or -]0.06    38.55[+ or -]0.3
2 month   189[+ or -]1        0.052[+ or -]0.08    36.67[+ or -]0.3
3 month   194[+ or -]1        0.07[+ or -]0.02     35.55[+ or -]0.3

Time      Amount of piperine   Amount of drug
point     retained *           retained *

0         69.1[+ or -]0.19     56.66[+ or -]0.577
1 month   67.24[+ or -]0.02    42.66[+ or -]1.157
2 month   54.45[+ or -]0.43    38.00[+ or -]1.000
3 month   44.24[+ or -]0.67    34.6[+ or -]1.157

* Data are expressed as Mean[+ or -]SD. (n = 3)
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Title Annotation:Original Article
Author:Charyulu, R. Narayana; Kinjal, B. Gandhi; Jose, Jobin; Priya, Sneh; Chacko, Nimmy
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Oct 1, 2013
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