Applying Taguchi Design for the Synthesis of Albumin Nanoparticles of Methotrexate by Desolvation Technique

Introduction

Treatment of rheumatoid arthritis by applying nanotechnology based ideas has produced promising new therapeutic strategies¹ . The nanoparticlesdriven approaches can be the predominant delivery method for rheumatoid arthritis treatment due to their higher selectivity, lower toxicity, and sustained release of drugs for longer period of time in comparison to the conventional mode of treatment² . Albumin has been examined extensively as ideal carrier for drug delivery. The major reported advantages are biodegradability, absence of toxicity and easy preparation by relatively simple methods. Because of their defined primary structure, albumin-based nanoparticles may offer various possibilities for surface modification and covalent bonding of active moieties and ligands. Further the albumin nanoparticles could allow the electrostatic adsorption of negatively or positively charged molecules without the requirements of other compounds³ . Furthermore, the high albumin uptake at the inflamed joint is because of high rate of metabolism of synovial cells and accumulation of nanoparticles due to enhanced permeability and retention effect (EPR).In EPR, due to inflammation in the joint, blood vessels of joint are dilated with leaky vasculature thereby facilitating the extravasate of nanoparticles of size less than 200nm^4,5. The size of the nanoparticles and polydispersity index are the parameters that will decide the fate of nanoparticles accumulation in inflamed joint. Hence in keeping the above facts in mind, the study was initiated to formulate the bovine serum albumin nanoparticles of methotrexate by desolvation technique and since the nanoparticle size is playing major role in accumulation of nanoparticles in inflamed joint, therefore the formulation and process parameters influencing nanoparticle size and polydispersity index was studied by Taguchi design.

Materials and Methods:

-Methotrexate was obtained from Mac chem private Ltd, Bovine serum albumin, glutaraldehyde and sodium hydroxide was procured from Hi-Media Ltd, India. All other chemicals used were of analytical grade. 

Preparation of methotrexate albumin nanoparticles by desolvation technique:-

The accurately weighed bovine serum albumin (BSA) is dissolved in distilled water. The solution is then titrated to pH 5.5-8.5 using 10mM NaoH respectively. The accurately weighed drug (methotrexate) is added to the solution of albumin. Then desolvation of BSA was achieved by addition of ethanol at a controlled rate under constant stirring. After the desolvation process, 10% glutaraldehyde in water was added as cross linker. Then the suspension was kept under constant stirring for next 24 hours to complete the process of cross-linking. All the formulations were freeze dried at -50 ˚C under reduced pressure for 12 hours to get a free flowing powder⁶ .

Screening of formulation and process variables by 4⁴ Taguchi design

Empirically to find the factors having most influential effect on particle size, large number experiments are required to be done. Therefore to minimise the efforts and time, statistical screening methods can be used to find out the independent factors affecting selected responses. Taguchi design is most effective screening design that provides systematic approach for performing experiments. 

Therefore, taguchi orthogonal array design was used to determine and select the most desirable conditions of independent parameters having the chief influence on the nanoparticle size and polydispersity index of methotrexate albumin nanoparticles. Signal- to-noise ratio (S/N) and orthogonal array (OA) are crucial key analyser of taguchi design. The (S/N) component evaluates quality characteristics and discrepancy from desired target. Signal refers to desirable and noise shows undesirable value for output characteristic. Therefore, title of smaller or bigger for (S/N) is used in analysis for better accuracy. Smaller the better type of (S/N) is suitable in our work, as we desired producing particle size less than 200nm^7-9.

Details of Experiment

From the preliminary screening study, the four independent factors affecting the methotrexate albumin NP’s size were ratio of albumin, pH, rate of addition of ethanol (desolvating agent) and the ratio of organic solvent/BSA solution. Taguchi’s orthogonal array table was obtained with independent parameters influencing the particle size and PDI. Table 1 shows the independent factors and their levels used in this experiment. Here, L₁₆ was selected, which represents 16 experiments with four four-level factors. Table 2 shows the structure of Taguchi’s orthogonal array design.

Evaluation Parameters

Particle Size& PDI

The particle size and polydispersity index of albumin nanoparticles of all the batches of methotrexate were measured by adding a small quantity of the sample in Malvern Nano S-90 zetasizer.

Results and Discussion

Taguchi Design

Screening studies were performed extensively for the selection of albumin concentration, pH, rate of addition of desolvating agent and volume of desolvating agent required for the formulation of drug loaded albumin nanoparticles by using Taguchi design (Minitab software, ver. 18). Using this design, graphs were constructed which showed maximum response for selecting the levels of the various independent parameters on the basis of signal to noise ratio.

Details of 4⁴ Taguchi design which was applied to the formulations of the methotrexate albumin nanoparticles using Minitab software (version. 18) are given in the Table 3.

The effect of independent parameters on nanoparticle size and polydispersity index

Effect of ratio of albumin on particle size and PDI

The influence of MTX/ BSA ratio on particle diameter and PDI of the resulted nanoparticles as indicated in table 3. It could be seen that the size of nanoparticle decreased with the rising content of BSA ranged from 1:1 to 1:6 (w/w). As a result, the formation of nanoparticles is feasible at MTX: BSA ratios between 1:1 to 1:6 (w/w). However when the BSA to MTX ratio exceeded 1:6, aggregates with large diameter appeared. Increasing BSA concentration the polydispersity of the samples was somewhat decrease. Increasing BSA concentration the polydispersity of the samples was increased from 0.2 to 0.59.

Effect of pH on particle size and PDI:

The pH value of the albumin solution plays a vital role in the desolvation process. The increasing pH of solution of albumin decreased both nanoparticle size and polydispersity index as indicated in Table 3. The isoelectric point of BSA is pH = 4.7. It indicates that the bovine serum has a positive charge below pH 4.7 and negative charge above pH 4.7. To prepare small particles, high repulsive forces need to be generated between the particles. From the study, it was discovered that with increasing pH of BSA solution, the particle size and PDI of the nanoparticles was reduced because of increased ionisation of the albumin at higher pH leading to repulsion of albumin molecules during particle formation. Thereby forming nanoparticles of desired size.^{10, 11}.

Effect of rate of addition of ethanol on particle size and PDI:

The ethanol addition rate (0.5–2mL/min) showed pronounced effect on nanoparticle size. The particle size was increased with increase rate of addition of ethanol. This noticed effect is due to the rapid albumin-phase separation rate caused by the added ethanol. The quick desolvation process might as well lead to large aggregates of BSA or precipitation of MTX. So the ethanol induced desolvation rate should be controlled in the proper range to obtain the desired particle size. Hence the nanoparticle size decreased with lower rate of addition of ethanol. The rate of ethanol addition also mainly influenced the particle size distribution range (PDI). The PDI was found to increase with the increased rate of addition of desolvating agent from 0.2 to 0.4 ^{11, 12}. 

Effect of the ratio of desolvating agent with respect to the albumin solution on particle size and PDI.

The addition of ethanol reduces the amount of water available to keep the BSA in solution, resulting in shrinkage of the hydrated BSA chains. At a particular point the degree of hydration will be low and the protein chains will precipitate as nanoparticles. Hence as the ratio of desolvating agent increased, the particle size was decreased. The PDI of the samples were found to be decreased from 0.45 to 0.25 with increasing ratio of the desolvating agent with respect to the albumin solution.⁹   

Taguchi design and ANOVA Approach:-

ANOVA is the statistical tool used to analyse the information contained in data and can deduce the extent of impact of the percentage contribution of each parameter analysed. Further can deduce which factor significantly affect the quality characteristic. Table 4 indicates all the parameters affecting the response (particle size) selected. The model F-value is traditionally used to find out the extent of impact of the each independent factor on the response selected. By comparing F-table (F0.05, 2,12) = 3.49 to F ratios with the level of significance = 5%, we found that F ratio of factor 2 is greater than 3.55 therefore, factor 2(i.e., pH) is the only parameter that is significantly affecting the particle size. pH has the biggest contribution in affecting particle size followed by BSA concentration, ratio of desolvating agent with respect to the albumin solution (v/v) and the least is rate of addition of desolvating agent.

Determination of optimal condition by taguchi design:-

The optimum condition can be determined through S/N ratio. The S/N ratio of all the controlling factor at each level was determined. Figure 1 & 2 depicts the response graph of S/N ratio for analysis of nanoparticle size and PDI.The S/N ratio selected for the study was smaller the better type, since the aim of the study is to prepare nanoparticles of size less than 200nm, therefore smaller the value of S/N ratio for the factor, better is the effect on selected parameter.Therefore it can be summarised that pH and the concentration of albumin is the most influential factors affecting the nanoparticle size and PDI.

Conclusion

BSA nanoparticles of methotrexate were successfully prepared by desolvation technique. The desolvation technique was selected because of the simple procedure, absence of toxic organic solvent in the procedure and can produce the desired particle size reproducibly. The optimum conditions for the formulation of BSA nanoparticles were determined by applying taguchi design with limited number of experiments. The NPs formulated from BSA was influenced by several process and formulation variables including BSA ratio with respect to the MTX, pH of the albumin solution, ratio of desolvating agent/ BSA and rate of addition of the desolvating agent. The application of taguchi design gave a systematic approach for the formulation of nanoparticles with desired size and PDI taking all the significant process and formulation variables into consideration at different levels.