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Influence of Bioenhancers on the Release Pattern of Niosomes Containing Methotrexate
Address for correspondence: Narayana Charyulu R., Vice Principal and HOD, Department of Pharmaceutics, Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences, Deralakatte, Mangalore-575018, India Mobile: +919448164750 E-mail: charyulun@yahoo.co.in
This article was originally published by Thieme Medical and Scientific Publishers Private Ltd. and was migrated to Scientific Scholar after the change of Publisher.
Abstract
The aim of present study was to prepare sustained release formulations of niosomes of methotrexate (MTX) alone (N1 to N10) and along withbioenhancers (NB1 to NB9) by thin film hydration technique using span 60 as surfactant, cholesterol as membrane stabilizing agent, curcumin and piperine as bioenhancers and dicetyl phosphate (DCP) as charge inducing agent. All the formulations of niosomes were characterized on the basis of physical appearance and entrapment efficiency. The invitro release studies of optimized formulation of niosomes of MTX alone and along with bioenhancers were performed and compared with pure drug released. The entrapment efficiency of MTX in optimized formulation of niosomes containing MTX along with bioenhancers was found to 56.9 % and entrapment efficiency of bioenhancers curcumin and piperine was found to be 40.30 % and 69.1 % respectively. In vitro drug release of optimized formulations of niosomes of MTX without and with bioenhancers (F3) was found to be 98.89 % and 60.97 % at the end of 12 h respectively. Results concluded that Niosomes of MTX containing bioenhancers followed sustain release pattern.
Keywords
Methotrexate
Thin Film Hydration Technique
Entrapment efficiency
Invitro study
Introduction
Cancer still remains as one of the fatal disease inspite of outstanding improvements in molecular biology, genetics, and chemotherapy. Treatment of cancer involves the use of chemotherapy, radiation therapy, and surgery1,2,3. As antitumor agents have high potential to induce side -effects and toxicity, localization of the drug to the tumor site would certainly optimize the therapy. The concept of targeted drug delivery is designed for attempting to concentrate drug in the tissues of interest and thereby reducing the relative concentration of medication in the remaining tissues4. Certain carriers like liposomes, niosomes, microsphers, nanopaticles, cellular carriers like erythrocytes and lymphocytes may be used to ferry the drug to the required site. Ideally, such carriers should be targeted the pathological area to provide the maximum therapeutic efficacy. Niosomes have gained increasing importance as a means of targeting of drugs. Niosomes have received attention for their potential as drug delivery vehicles due to advantages like higher flexibility, better bioavailability, increased efficacy, and therapeutic index5,6. Bioavailability of drug encapsulated in niosome can be enhanced by encapsulating the drug along with bioenhancers in the niosomal vesicles. The coadministration of bioenhancer like 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 like curcumin increases the intracellular drug accumulation and restores the chemosensitivity7,8
MTX is an antimetabolite and antifolate drug
It acts by inhibiting the metabolism of folic acid. MTX is a standard chemotherapeutic agent which exhibits a dose dependent toxicity. 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.
The aim of the present study was to utilize 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 prolong the release of drug from niosones and also provided better stability to the formulation.
Materials and Methods
Materials
MTX was a gift sample from Khandelwal laboratories Pvt, Ltd. (Mumbai, India). Span 60(su//actant) was obtained from LobachemiePvt. 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 were obtained from Green Grover's Pvt Ltd. (Bangalore, India). DCPwas obtained from Sigma Aldrich Chemicals, (Bangalore, India). Sodium chloride, sodium hydroxide and disodium hydrogen phosphate were obtained fromCDH Laboratory Ltd. (Delhi, India). All chemicals used were of analytical grade
Methods
1. Preparation of Niosomes of MTX alone and along with Bioenhancers
Multilamellar vesicles of MTX alone and along with bioenhancers were prepared by thin film hydration technique using rotary flash evaporator as described method of Bangham, reported by Juliano and Daoud9 Accurately weighed quantity of cholesterol, span 6010,11 and DCP were dissolved in minimum quantity (about 3 ml) of a mixture of chloroform: methanol (2:1) in a 250 ml round bottom flask. Round bottom flask was then attached to a rotary evaporator. The organic solvent mixture was evaporated in a rotary flash evaporator under a vacuum of 25 inches of Hg at 60 ± 2°C and the flask rotated at 100 rpm until a very thin, smooth and dry film of surfactant was formed on the inner surface of the flask, The dry lipid film was slowly hydrated with 5 ml phosphate buffer saline (PBS) of pH 7.4 containing 10 mg MTX drug alone and with 10 ml PBS pH 7.4 containing 10 mg MTX drug and accurate quantity of bioenhancers at a temperature of 60 ± 2 °C for a period of 1h. It formed homogenous suspension of multilamellar vesicles (MLVs). The MLVs suspension was sonicated to form small unilamellar vesicles (SUVs)of niosomes by using probe sonicator. The final niosomal suspension was further hydrated at 4°C for overnightto stabilize the formulation. The amount of span, cholesterol and bioenhancers to be loaded was selected on the basis of entrapment efficiency of the vesicles. The compositions of different formulation of niosomes are given in Table 1a and 1b.
| Batch name | MTX (mg) | Span 60 (mg) | Cholesterol (mg) | DCP (μmol) | Organic solvent (ml) | Hydration Volume (ml) |
|---|---|---|---|---|---|---|
| N1 | s10 | 50 | 10 | 7 | 3 | 5 |
| N2 | 10 | 75 | 10 | 7 | 3 | 5 |
| N3 | 10 | 100 | 10 | 7 | 3 | 5 |
| N4 | 10 | 125 | 10 | 7 | 3 | 5 |
| N5 | 10 | 150 | 10 | 7 | 3 | 5 |
| N6 | 10 | 175 | 10 | 7 | 3 | 5 |
| N7 | 10 | 150 | 20 | 7 | 3 | 5 |
| N8 | 10 | 150 | 30 | 7 | 3 | 5 |
| N9 | 10 | 150 | 40 | 7 | 3 | 5 |
| N10 | 10 | 150 | 50 | 7 | 3 | 5 |
| Batch name | MTX (mg) | Span 60 (mg) | Cholesterol (mg) | Curcumin (mg) | Piperine (mg) | DCP (μmol) | Organic solvent (ml) | Hydration Volume (ml) |
|---|---|---|---|---|---|---|---|---|
| Nb1 | 10 | 150 | 20 | 5 | 10 | 7 | 3 | 10 |
| Nb2 | 10 | 150 | 20 | 10 | 10 | 7 | 3 | 10 |
| Nb3 | 10 | 150 | 20 | 20 | 10 | 7 | 3 | 10 |
| NB4 | 10 | 150 | 20 | 30 | 10 | 7 | 3 | 10 |
| NB5 | 10 | 150 | 20 | 40 | 10 | 7 | 3 | 10 |
| NB6 | 10 | 150 | 20 | 50 | 10 | 7 | 3 | 10 |
| NB7 | 10 | 150 | 20 | 50 | 20 | 7 | 3 | 10 |
| NB8 | 10 | 150 | 20 | 50 | 30 | 7 | 3 | 10 |
| NB9 | 10 | 150 | 20 | 50 | 40 | 7 | 3 | 10 |
2. Characterization of Niosomes of MTX alone and along with Bioenhancers
2.1. Entrapment efficiency
For determination of entrapment efficiency, unentrapped drug in the niosomal formulation was seperated using centrifugation at 20,000 rpm for 1 h at 4 °C. 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 the suspension was taken in a micropipette and transferred to a test tube. To this added 5 ml of methanol and was further vortexed for 2 min. The absorbance of resulting solution was measured using a shimadzu UV Spectrophotometer(1700) at 292 nm after suitable dilution with methanol10
2.2. 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±0.2°C. The medium was stirred continuously during the release study. At predetermined time intervals of15 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 nm11,12.
Result and Discussion
Niosomes of MTX without and with bioenhancers such as curcumin and piperine were prepared by thin film hydration method by using span 60 and cholesterolas a surfactant. They are optimized on the basis of observation and maximum percentage drug entrapment (PDE).
Entrapment efficiency
Theoptimization ofniosomes ofMTX without bioenhancers depends on the basis of entrapment efficiency. The entrapment efficiency was varied as concentration of surfactant varied. The amount of span 60 was increased by keeping drug and cholesterol concentration constant. As the amount of span 60 increased, the PDE of drug was also increased upto the formulation N6 (1:15 ratioof drug and span60) and further increasing the amount of span 60 did not change encapsulation efficiency. The higher entrapment of span 60 may be due to their high phase transition temperature and hydrophobic in nature.
The amount of cholesterol was increased by keeping drug and surfactant ratio constant. The ratio1:15:2 gave highest encapsulation efficiency due to stabilizing effect of cholesterol. The cholesterol in niosomes greatly affects the membrane properties of the bilayers by reducing the rotational freedom of hydrocarbon chains. Cholesterol also eliminates the gel to liquid phase transition of the vesicle bilayers and induces permanent transition of the gel-state bilayer to an ordered liquid crystalline state. Both these mechanisms make the bilayers more stable leading to increase in entrapment efficiency. Further increase in cholesterol concentration, decreased the fluidity of the bilayers by filling empty spaces among the surfactant molecules and results of the membrane become more rigid and ultimately decreased the encapsulation efficiency (Table 2a).
Optimization of niosomes of MTX with bioenhancers was done on the basis of entrapment efficiency. The formulation in which bioenhancers were added (40mg of curcuminand 10mg of piperine) produced a uniform dispersion with lower drug entrapment. Further increase in curcumin concentration (50mg) by keeping piperine concentration constant (10mg) produced a uniform suspension with an acceptable PDE. Further increase in concentration of piperine (>10mg)reduce the entrapment efficiency. So the formulation NB6 containing curcumin (50mg) and piperine (10 mg)as a bioenhancers was found to be an optimized formulation which gave highest drug entrapment (55.1±0.49 %) (Table 2b).
| Batch name | Observation | % Drug entrapped* |
|---|---|---|
| N1 | Flaking | 22.17±1.667 |
| N2 | Flaking | 29.89±1.025 |
| N3 | Flaking | 38.49±1.351 |
| N4 | Flaking | 43.35±0.920 |
| N5 | Uniform dispersion | 50.73±0.714 |
| N6 | Non-uniform dispersion | 21.35±0.840 |
| N7 | Uniform dispersion without flaking | 56.9±1.331 |
| N8 | Uniform dispersion lower PDE | 53.85±0.818 |
| N9 | Uniform dispersion Lower PDE | 51.03±0.512 |
| N10 | Uniform dispersion lower PDE | 49.48±0.918 |
| Batch | Observation | % Drug | %Curcumin | % Piperine |
|---|---|---|---|---|
| name | entrapped* | entrapped* | entrapped* | |
| NB1 | Flaking | 18.90±0.21 | 27.66±0.66 | 10.05±0.36 |
| NB2 | Flaking | 23.46±0.39 | 31.66±0.67 | 23.58±0.21 |
| NB3 | Flaking | 25.89±0.44 | 32.78±0.12 | 24.30±0.64 |
| NB4 | Uniform dispersion | 27.67±0.62 | 35.94±0.51 | 32.10±0.26 |
| NB5 | Uniform Dispersion | 32.24±0.51 | 39.01±0.21 | 39.00±0.82 |
| NB6 | Uniform Dispersion | 55.1±0.49 | 40.30±0.67 | 64.31±0.96 |
| NB7 | Non-uniform Dispersion | 49.89±0.53 | 33.87±0.54 | 29.00±0.38 |
| NB8 | Non-uniform Dispersion | 47.77±0.55 | 31.33±0.86 | 27.13±0.27 |
| NB9 | Non-Uniform Dispersion | 46.98±0.34 | 28.00±0.96 | 26.18±0.91 |
In Vitro Release Study
The invitro release study revealed that the release of the drug was sustained on encapsulation in niosomes. The free drug released approximately 98.77 % of the drug within 60 minwhereas the same percentage of drug release from niosomes of MTXwas occurring at the end of 11 h. 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 lipophilicregion of niosomes, which help to achieve the optimum loading dose. The drug diffuses slowly after 3 h due to the presence of cholesterol in the formulation which affects the fluidityby making it more rigid. As the amount cholesterol increased, they filled the pores of vesicular bilayers and abolished the gel-liquid phase transition of the niosomal systems. 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 niosomes 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 sustain release pattern (Figure: 1).
- In vitro release of pure drug, niosomes of MTXalone and niosomes of MTX along with bioenhancers
Conclusions
The use of various pharmaceutical nanocarriers has become one of the most important areas of nanomedicine. Niosomes of methotrexate alone and along with bioenhancers such as curcumin and piperine were prepared by thin film hydration method by using surfactant span 60 and cholesterol that were optimized on the basis of entrapment efficiency. The in vitrostudy revealed that the release pattern of the drug was sustained in niosomes and it was further significantly sustained by addition of bioenhancers. Further in vivo and stability studies can be performed to see the pharmacological activity as well as the stability of the formulation because the stability of niosomes is a great issue and a major challenge in commercializing the formulations.
Acknowledgment
The authors are expressing their sincere gratitude to the Nitte University for providing necessary facilities to carry out this research work.
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