Tamoxifen Citrate Loaded Solid Lipid Nanoparticles- A Novel Approach In The Treatment of ER+ Breast Cancer.

 

Borkar Sudarshan*, Shende Vikas, Chatap Viveknand,  Sawant Vilas, R Suresh, Dama Ganesh

Sharadchandra Pawar College of Pharmacy, Otur, Pune-412409

 

 

ABSTRACT

Breast cancer is one of the most frequently occurring cancers in women and the second leading cause of cancer deaths in women. Biodegradable SLNs of Tamoxifen citrate (Tmx) can be used for the targeting of anticancer drugs to the organs, thereby achieving major benefits such as reduction in total dose and avoidance of systemic absorption. Solid lipid nanoparticles (SLNs) were prepared by O/W Microemulsion technique and characterized by various parameters such as particle size analysis, scanning electron microscopy, drug entrapment efficiency and in-vitro release studies. In-vitro release studies were performed in phosphate buffer of pH 7.4 along with 0.5% SLS for increasing the solubility of lipophilic drug in PBS using Franz diffusion cell by dialysis method. The kinetics of release was determined and fitted to an empirical equation. The Tmx-loaded tristearine SLNs shown maximum entrapment efficiency compared to the glycerol monostearate SLN. Percentage of tamoxifen citrate released from SLN formulations up to 8 hrs was in the range of 32.3 to 65.5% with Tristearine and 43.2 to 81.4% with Glycerol monostearate (GMS). Tristearine had shown slow release and maximum entrapment than GMS which can be attributed to the hydrophobic long chain fatty acids of the triglyceride that retain lipophilic drugs and also increased accommodation of lipophilic drugs. Thus the above mentioned solid lipid nanoparticles can be a beneficial system to deliver tamoxifen to cancer tissues through enhanced permeability and retention (EPR) effect.

 

KEYWORDS: Solid lipid nanoparticles; Tamoxifen citrate; Microemulsion Technique; Triglycerides.

 

 

INTRODUCTION

Breast cancer is a malignant cell growth in the breast. If left untreated the cancer spreads to other areas of the body. Excluding cancers of the skin, breast cancer is the most common type of cancer in women in the United States, accounting for one of every three cancer diagnoses. The most common type of breast cancer begins in the lining of the ducts and is called ductal carcinoma. Another type, called lobular carcinoma, arises in the lobules. When breast cancer spreads, it is called metastatic breast cancer 1.

 

The prevailing wisdom is that tamoxifen and other antiestrogens are active in preventing the development or progression of estrogen receptor positive (ER+) breast cancers only. Efficacy of tamoxifen for ER+ breast cancer has been clearly demonstrated in both metastatic and adjuvant settings. The benefit from adjuvant tamoxifen therapy was restricted to ER+ breast cancers in the Early Breast Cancer Trialists' meta-analysis.

 

Tamoxifen does not inhibit proliferation of estrogen receptor negative (ER-) breast cancer cell lines in tissue culture or in murine xenograft models2.

 

Women with breast cancer have a risk of developing contra lateral disease that is higher than the risk of developing a first breast cancer in the general population.

 

 

Spectra-1 FTIR spectra of (a) Tamoxifen citrate, (b) Drug loaded G.M.S SLNs; (c) Drug loaded Tristearine SLNs.

 

 

Spectra-2 DSC spectra of (a) Tamoxifen citrate, (b) Drug loaded G.M.S SLNs; (c) Drug loaded Tristearine SLNs.

 

The National Surgical Adjuvant Breast and Bowel Project (NSABP) has recently reported long-term follow-up from two large randomized trials (NSABP B-04 and NSABP B-06), in which the occurrence of contra lateral breast cancer as a first event among women with operable breast cancer ranged from 6% to 8.9%, with 50% to 60% of the cases occurring more than 5 years after treatment of the primary tumor. Risk factors for contra lateral breast cancer include lobular histology (invasive and in situ disease), family history, and age. Adjuvant tamoxifen reduces the risk of contra lateral breast cancer by 30%–50% for women with breast cancer. NSABP B14 demonstrated the survival benefits associated with 5 years of tamoxifen therapy in more than 2800 women with lymph node-negative, estrogen receptor (ER)-positive breast cancer, and a 37% decrease in the risk of contra lateral breast cancer after 10 years of follow-up. These data provided the rationale for the evaluation of tamoxifen for the prevention of breast cancer in healthy women. In the NSABP P-1 study, 13 388 women at high risk for developing breast cancer were randomly assigned to treatment either with tamoxifen. Tamoxifen statistically significantly reduced the risk of invasive breast cancer by 49% and the risk of ER-positive breast cancer by 69%. There was no effect on the risk for ER-negative disease3.

 

Scanning Electron Microscopy (SEM):

Graph-1 Graphical representation of average particle sizes of Tamoxifen citrate loaded SLNs

 

Tamoxifen is most widely used drug for the treatment of estrogen receptor positive breast cancer and is the only drug approved for prevention of breast cancer in healthy women at high risk of breast cancer. Following long-term therapy, tamoxifen has some major side effects (These side effects were reported to be dose and concentration dependent), including higher incidence of endometrial cancer, liver cancer, thromboembolic disorders, and development of drug resistance. Tamoxifen resistance has been shown in a variety of cells in vitro as well as in vivo. These unwanted side effects of tamoxifen, as well as various barriers to the delivery of the drugs to tumor, call for targeted delivery to the tumor site and enhanced uptake by the tumor cells. One approach to overcome the undesirable side effects of tamoxifen includes the use of biodegradable nanoparticles for tumor-targeted drug delivery4. Conventionally administered cytotoxic agents often extensively and indiscriminately bind to body tissues and serum protein in a highly unpredictable manner. Only a small fraction of the drugs reach the tumor site. This may both reduce the therapeutic efficacy and increase systemic drug toxicity. Moreover, even though cytotoxic drugs ideally should only kill cancer cells, in reality they are also toxic to non-cancerous cells, especially to rapidly dividing cells, e.g. bone marrow cells and cells of the gastrointestinal tract. As a result submicron-sized particulate matter may preferentially extravasate into the tumor and be retained there. This is often referred as the “enhanced permeability and retention” (EPR) effect. This EPR effect can be taken advantage of by a properly designed nanoparticle system such as SLN to achieve passive tumor targeting. By doing so, the aforementioned poor tissue specificity problem can be partly solved5.

 

The major disadvantages of polymeric nanoparticles are there relatively slow biodegradability (up to 3-4 weeks), which might cause systemic toxicity by impairment of reticuloendothelial system as well as cytotoxicity towards macrophages, presence of residual toxic agent (organic solvents) employ during preparation and reproducibility. Polymeric nanoparticles may not be sterilized by autoclaving. They have been sterilized by γ radiation. However, this treatment causes the formation of unacceptable toxic reaction products large scale production of polymeric nanoparticles is problematic therefore; this carrier system has so far not relevant for pharmaceutical market In the middle of 1990s, the attention of different research group has focused on alternative nanoparticles made from solid lipids, the so called solid lipid nanoparticles (SLN or lipospheres or nanospheres).The SLN combine the advantages of other innovative carrier systems (e.g. physical stability, protection of incorporated labile drugs from degradation, controlled release, excellent tolerability) while at the same time minimizing the associated problems. SLN formulations for various application routes (Parenteral, oral, dermal, ocular, pulmonary, rectal) have been developed and thoroughly characterized in vitro and in vivo. A first product has recently been introduced to the Polish market (Nanobase, Yamanouchi) as a topically applied moisturizer 6.

 

Solid lipid nanoparticles (SLN, also referred to as lipospheres or solid lipid nanospheres) are a relatively new class of drug carrier. They are particles of submicron size (50 to 1000 nm) made from lipids that remain in a solid state at room temperature and body temperature. SLN can be conveniently prepared using a wide variety of lipids including lipid acids, mono-, di-, or triglycerides, glyceride mixtures or waxes, and stabilized by the biocompatible surfactant(s) of choice (non-ionic or ionic). Because of numerous advantages SLN can offer, this relatively new drug carrier is emerging in the field of anticancer drug delivery 7.

 

MATERIALS AND METHODS:

Tamoxifen citrate was gift sample from Biochem pharmaceuticals Mumbai, Tristearine, Tween-80 and Dialysis membrane was purchased from Himedia Mumbai, Glycerol monostearate was purchased from Lobachem Mumbai, Soya lecithin was gift sample from sun pharma Ahmedabad. Remaining chemicals used were of analytical grade.

 

Graph-2 Graphical representation of Tamoxifen citrate entrapment efficiencies with both lipid carriers

 

Compatibility studies of drug and polymers: 8, 9

1. FT-IR spectra were recorded with a Thermo Nicolet. Japan In the range 450–4000 cm−1 using a resolution of 4 cm−1 and 16 scans. Samples were diluted with KBr mixing Powder, and pressed to obtain self-supporting disks. Liquid samples formulations were analyzed to form a thin liquid film between two KBr disks.

 

2. Differential Scanning Calorimetry (DSC) studies are also a qualitative identification of substance in the pure form and in combination. DSC was carried by the action of Argon purging with 80ml/min, where it is hermetically sealed with Aluminium Pans, from this Sample of 40μl is used. The program is run at 25.0-250.00C/min. The onset, endset and the peaks are recorded for individual pure drug, polymer, lecithin and in combination.

 

 

Graph 3:  In-vitro Dissolution Profile of Tmxg-me-01 to Tmxg-me-08

 

 

FORMULATION DESIGN:

Procedure for preparation of Tamoxifen Citrate loaded SLN’S by Microemulsion Technique: 10

Tamoxifen Citrate SLNs were prepared from o/w microemulsion technique containing [glycerol monostearate (GMS) and Tristearin]   as lipid carrier, Soya lecithin as surfactant and tween 80 as co- surfactant. In brief drug was disperse in molten lipid (70oC) this dispersion was added carefully drop wise into ice cold water (2-3oC) contains surfactants with continuous stirring (IKA-Ultra Turrax T25 USA) to form nanosuspension.

 

Evaluation of SLN’S:

1. Particle Size Analysis: 11

v    Procedure:

The prepared nanoparticles were analyzed by CIS-L50 Particle Size Analyzer. The particles were scanned from 0-150μm using lens A. The suspension is taken in a cuvette and diluted with distilled water to give a concentration of 10-9 particles with a Standard normalizing factor (SNF) value of 1. The cuvettes are madeup of polystyrene of 1cm path length. The particles are analysed for its size (length x breadth x volume) by using laser channel beam. The mechanism of working of CFS-L50 is TOT (time of transition),

 

2. Scanning Electron Microscopy (SEM): 12

v    Procedure:

Surface morphology of the specimens will be determined by using a scanning electron microscope (SEM), Model JSM 840A, JEOL, Japan. The samples are dried thoroughly in vaccum desicator before mounting on brass specimen studies, using double sided adhesive tape. Gold-palladium alloy of 1200A knees was coated on the sample using sputter coating unit (Model E5 100 Polaron U.K.) in Argon at ambient of 8-10 Pascal with plasma voltage about 20MA. The sputtering was done for nearly 5 minutes to obtain uniform coating on the sample to enable good quality SEM images.

The SEM was operated at low accelerating voltage of about 15KV with load current of about 80MA.

The condenser lens position was maintained between 4.4-5.1. The objective lens aperture has a diameter of 240 microns and the working distance WD=39mm.

 

3.  Total content: 13

Tamoxifen loaded SLNs (1 ml) were diluted to 25 ml of methanol. Final dilution was made with methanol with in its beeres range. And total drug content was determined by using UV spectrophotometer (Jasco V-530 Japan) at 275nm by taking methanol as blank.

 

Graph 4:  In-vitro Dissolution Profile of Tmxt-me-01 to Tmxt-me-08

 

4. Entrapment Efficiency:  14

Entrapment efficiency of TMX-SLNs was determined by centrifugation of samples at 10,000 rpm for 10 min. The amount of free drug was determined in the clear supernatant by UV spectrophotometer (Jasco V-530 Japan) at 275nm using supernatant of non loaded nanoparticles on basic correction. The entrapment efficiency (EE %) could be achieved by the following equation.

 

EE (%) = W initial drug – W free drug Χ100

               W initial drug

 

IN-VITRO RELEASE STUDY:

1. Dialysis method: 15, 16

In vitro release studies were performed using modified Franz diffusion cell. Dialysis membrane having pore size 2.4 nm; molecular weight cut off 12,000–14,000, was used (Membrane was socked in double-distilled water for 12h before mounting in a franz diffusion cell). A volume equivalent to 5 mg of Tamoxifen Citrate (Practically calculated) loaded SLN formulation was placed in the donor compartment and the receptor compartment was filled with 50 ml of 7.4 PBS solution containing 0.5 %( w/v) sodium lauryl sulfate (SLS). SLS was used to increase the solubility of tamoxifen in the buffer solution and prevent absorption of the tamoxifen on the surface of the tube.  The content of the cell was stirred with the help of magnetic stirrer at 370C. An aliquot was withdrawn from receiver compartment through side tube at hourly based time intervals up to 8 hours. Fresh medium of SLS-PBS solution was replaced each time to maintain constant volume. Samples were analyzed by UV visible spectroscopy at 275nm.

 

DATA ANALYSIS: 17

The Colloidal systems were reported to follow the zero order release rate by the diffusion mechanism for the release of the drug. To analyse the mechanism for the release and release rate kinetics of the dosage form, the data obtained was fitted in to Zero order, First order, Higuchi matrix and Krosmeyer and Peppas model. Using

 

 

Table 1 : Formulation Design of GMS SL Ns. by microemulsion techniques

Formulation codes

Polymer % W/V

Drug (mg)

Conc. of Surfactant/Co-Surfactant

W/V (1:1)

Stirrer

Speed

(rpm)

Tmxg-me-01

0.5 %

200 mg

1%

Ultra Stirrer

6,500

Tmxg-me-02

1.0 %

200 mg

1%

Ultra Stirrer

6,500

Tmxg-me-03

1.5 %

200 mg

1%

Ultra Stirrer

6,500

Tmxg-me-04

2.0 %

200 mg

1%

Ultra Stirrer

6,500

Tmxg-me-05

0.5 %

200 mg

1%

Ultra Stirrer

9,500

Tmxg-me-06

0.5 %

200 mg

1%

Ultra Stirrer

13,500

Tmxg-me-07

0.5 %

200 mg

1%

Ultra Stirrer

17,500

Tmxg-me-08

0.5 %

200 mg

1%

Ultra Stirrer

21,500

 

Table 2 : Formulation Design of Tristearin SL Ns. by microemulsion techniques

Formulation codes

Polymer % W/V

Drug (mg)

Conc. of Surfactant/Co-Surfactant

W/V (1:1)

Stirrer

Speed

(rpm)

Tmxg-me-01

0.5 %

200 mg

1%

Ultra Stirrer

6,500

Tmxg-me-02

1.0 %

200 mg

1%

Ultra Stirrer

6,500

Tmxg-me-03

1.5 %

200 mg

1%

Ultra Stirrer

6,500

Tmxg-me-04

2.0 %

200 mg

1%

Ultra Stirrer

6,500

Tmxg-me-05

0.5 %

200 mg

1%

Ultra Stirrer

9,500

Tmxg-me-06

0.5 %

200 mg

1%

Ultra Stirrer

13,500

Tmxg-me-07

0.5 %

200 mg

1%

Ultra Stirrer

17,500

Tmxg-me-08

0.5 %

200 mg

1%

Ultra Stirrer

21,500

 

PSP-DISSO – v2 software.  Comparing the r-values obtained, the best-fit model was selected.

 

RESULT AND DISSCUSSION:

Compatibility Studies:

1.   Drug polymer compatibility studies were carried out using IR-200 (FT-IR) to establish the possible interaction in the formulations. It was found that there was no possible interaction in between drug and lipid carrier in their individual form and in formulations too. With different surfactants such as lecithin and tween-80 when kept for one month in different conditions. (Spectra-1).

2.   Compatibility studies were also carried by Differential Scanning Calorimetry, which is a qualitative analytical tool for assessing the interactions. The pure drug and the formulations were studied for DSC. It was found that the thermal peaks of drug are identical in formulations with lipid carrier and surfactants. This indicates that, there is no interaction between drug, lipid carrier and surfactants. (Spectra-2).

 

Formulation of Tamoxifen citrate loaded SLNs:

Tamoxifen citrate loaded SLNs were successfully prepared by a o/w microemulsion technique. The SLNs were obtained immediately when dispersing the warm microemulsion into cold water with the aid of a homogenizer. The cold water facilitated rapid lipid crystallization and prevented lipid aggregation10. Different lipid carriers were used like GMS and Tristearine along with mixtures of surfactants. Formulations were design by variation of concentrations of lipid carrier from 0.5 to 2.0% at constant speed and concentration of surfactants. And variation of speed at constant lipid concentration and concentration of surfactants. (Table-1and2) The prepared SLNs were subjected for the further evaluation parameters.

 

Evaluation Parameters:

1. Particle size Analysis:

The formulations with GMS such as Tmxg-me-01 to Tmxg-me-08 showed wide distribution in particle size from 740nm to 980nm respectively; likewise formulation with tristearine such as Tmxt-me-01 to Tmxt-me-08 showed particle size from 740nm to 780nm respectively the particle size analysis reveals that the size reduction was with varying speeds and size increment with varying lipid carrier concentrations. The reported reasons for changing the particle size with formulation designs are as follows the decreasing of the particle size with the increasing of stirring rate can be explained by the intensification of the micromixing (i.e. mixing on the molecular level) between the multi-phases. Hence, higher stirring rate favored the formation of the smaller and more uniform drug particles 8. But there is a slight increase of particle size with the increase of the stirring rate. Maybe the high stirring rate might result in the formation of the small particles and then the small particles could aggregate to form a large nanoparticle because of the absence of enough surfactants 17. When the concentration of the lipid exceeded 1.0% with a fixed concentration of Surfactants, there were insufficient surfactants available to coat the surface of all the lipid droplets, resulting in particle aggregation and an increase in particle size 18. The surface morphology of the SLNs had not been altered by the type of lipid carrier, concentration of lipid carrier and speed. (Graph-1).

 

2. Scanning Electron Microscopy:

This was performed to study the surface morphology of the particles, although the particles were abundantly found and they were spherical in their shape. Thus, both types of surfactants produced better surface characteristics. The surface morphology of the SLNs had not been altered by the type of lipid carrier, concentration of lipid carrier and speed. (Fig-1 to 4).

 

3. Total Drug content and Entrapment Efficiency:

The total drug content was not altered with experimental variable is almost in the range of 80-90% for SLNs made from different lipid carriers. But it was not so far in the case of entrapment efficiency. The experimental results indicate that the concentration of lipid, speed had critical effects on the tamoxifen incorporation efficacy

The entrapment efficiencies of SLNs made from different concentrations of lipid carrier were come up in the ascending order the entrapment efficiency is lower for the sample with lower lipid concentration. It has to be noticed that during the cooling process, the lipid solidifies and the drug is distributed into the shell of the particles, if the concentration of the drug in the melted lipid is well below its saturation solubility. As a result, SLN show a drug-enriched shell model. A drug-enriched core model is formed when the drug in the melted lipid is closed to its saturation solubility. The cooling process leads to supersaturation of the drug and subsequently to drug crystallization prior to lipid crystallization 14. The little bit reduction in entrapment efficiencies was observed with the varying speed. Among the lipid carriers formulations prepared with glyceryl monostearate had shown less entrapment efficiencies (In the range of 39.26% to 53.08%) compared with those of prepared with tristearine (In the range of 42.31% to 72.37%) respectively.(Graph-2) The entrapment efficiencies of the SLNs for tamoxifen was in the order of Tmxt-me>Tmxg-me. The higher entrapment efficiency with TS is attributed to the high hydrophobicity due to the long chain fatty acids attached to the triglyceride resulting in increased accommodation of lipophilic drugs 13.

 

4. In-vitro Dissolution Studies:

In-vitro drug release data from the SLNs were carried out for 8hrs and graphically represented as % cumulative drug release v/s time profile. For all eight formulations of G.M.S showed Cumulative Percent drug released after 8hrs for Tmxg-me-01 to 08 was 43.2 to 81.4% respectively. And all eight formulations of Tristearine showed Cumulative Percent drug released after 8hrs for Tmxt-me-01 to 08 was 32.3 to 65.5 % respectively (Graph 3and4). Interestingly, the particle size had no influence on the in vitro release of Tamoxifen citrate. The release of a drug from the SLN can be influenced by nature of the lipid matrix and its concentration. The burst release was observed at the initial hour (In all formulations) and released nearly 20-25% of the drug from the SLN. After that, a prolonged release was obtained and released 5-8% of drug from the SLN at every hour, Among the glycerides, Tristearine had shown slow release than GMS then Stearic acid which can be attributed to the hydrophobic long chain fatty acids of the triglyceride that retain lipophilic drugs 13.

 

5. Kinetic Study:

The release study was further investigated for the kinetic studies. Various kinetic models were applied. All formulations (with different lipid carrier) were found to follow the Matrix model. From the n values obtained it can be said that the diffusion followed Fickian mechanism.

 

CONCLUSION:

Tamoxifen citrate loaded SLNs can be successfully formulated from microemulsion technique to enhance the efficacy of cytotoxic drug at the target area by reducing the side effects from the dose. Tristearine had shown controlled release and maximum entrapment than others lipid carriers which can be attributed to the hydrophobic long chain fatty acids of the triglyceride that retain lipophilic drugs and also increased accommodation of lipophilic drugs. Thus, from the above studies it can be concluded that the current investigation illustrates the effect of lipid nature on the entrapment efficiency and in vitro release of lipophilic drug.

 

ACKNOWLEDGMENT:

The authors wish to thanks Biochem Pharmaceuticals, Mumbai for the kind gift of the Tamoxifen citrate.

 

 

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Received on 13.07.2009

Accepted on 10.08.2009     

© A and V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 1(2): Sept.-Oct. 2009, 143-149