Application of Box–Behnken Design in the Optimization and Preparation of Salicylic Acid Nanopowder using Solvent-Free Green Mechanochemical Approach : Journal of Pharmacy and Bioallied Sciences

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Application of Box–Behnken Design in the Optimization and Preparation of Salicylic Acid Nanopowder using Solvent-Free Green Mechanochemical Approach

Javed, Shamama1,; Sultan, Muhammad H.1; Alam, M. Intakhab1; Sivadasan, Durgaramani1; Ahsan, Waquar2; Jabeen, Aamena1; Jaafari, Malak Haija A.3; Hawthan, Manal Hussain A.3; Byti, Azaheer H.3

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Journal of Pharmacy And Bioallied Sciences 15(1):p 29-34, Jan–Mar 2023. | DOI: 10.4103/jpbs.jpbs_577_22
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The number of active ingredient candidates with lower water solubility coming out of drug discovery and development has expanded over the past few decades. Despite the challenge, their formulation into dosage forms is advantageous in several ways. Preparation of drug nanoparticles (NPs) or nanopowder is one of the ways owing to the fact that the size reduction of drugs increases the specific surface area.[1] Based on the Noyes–Whitney equation, this nanoparticulate delivery system leads to the improvement in drug particle dissolution rate and therefore, improvement in their bioavailability.[2] Drug nanopowders are carrier-free NPs in which the solid drug is typically nanosized to a couple of hundred nanometers. Production of drug nanopowders can be achieved using two different approaches: (i) top-down and (ii) bottom-up techniques.[3] In top-down methods, particle size is reduced from coarse drug particles to NPs using different kinds of dry/wet milling or high-pressure homogenization techniques. In bottom-up techniques, drug NPs are built molecule by molecule using antisolvent precipitation or liquid atomization-based techniques. Nanomilling refers to the reduction of the drug particle size below 1000 nm using dry milling or wet media milling processes.[4]

To achieve this, a planetary ball mill, which belongs to the group of high-energy milling techniques, is used and which involves the application of mechanical energy to physically break down the coarser particles into finer ones and is regarded as a “top-down” approach in the production of fine particles. These fine drug particles are especially desired in the development of formulations intended for parenteral, respiratory, and transdermal applications.[5] The dry milling technique is usually employed for micronization yielding a particle size range of 1–2000 μm; however, to achieve dry nanonization, the application of certain additives and the prolonged milling time (2–4 h) would be required.[6] Processes inside planetary ball mills are complex and strongly depend on the processed material and, therefore, the optimum milling conditions have to be assessed and optimized for each individual system. Multiple milling process variables such as agitator speed, media load, media size, media type, and milling speed must be evaluated and optimized during the formulation development process.[7] Salicylic acid (SA) is a member of the group of salicylate compounds characterized as hydroxyl acids. It has been used as a skin topical agent to treat various skin disorders for many decades due to its prominent keratolytic properties. It is a lipid-soluble agent and is therefore miscible with epidermal lipids and sebaceous gland lipids in hair follicles. Presently, salicylates and their derivatives are present as a constituent in a variety of skin formulations.[8] This study aimed to identify various dry milling variables such as milling time, milling speed, and the number of balls, which affect the particle size reduction as well as the polydispersity index (PDI) of SA powder on a planetary ball mono mill using the Box–Behnken statistical design. The influence of different variables on the outcome of a controlled experiment was studied.


Chemicals and instruments

SA obtained from HiMedia (Mumbai, India) was used as a poorly soluble drug and matrix material in this study. Planetary ball monomill (Pulverisette 6 Classic line, Fritsch GmBH, Germany) was used for dry milling, and Malvern Zetasizer (Nano ZS90, UK) was used for estimation of size (nm) and PDI. Statistical designing software (Design Expert version 12.0) was employed for the statistical optimization of the milling process. Double-distilled water was prepared in our laboratory.

Optimization of dry milling parameters using the Box–Behnken statistical design

To obtain a uniform distribution of particle size in the nanometer range, the first step was to identify the independent variables or factors, which affect the final product or process, followed by the study of their effects on a dependent variable or response. Effects of three factors such as milling speed (A), milling time (B), and the number of balls (C) on three levels (−1, 0, +1) were observed on size (nm) and PDI. The Box–Behnken design was used in this study and 17 runs with different optimization conditions were designed and finalized.

Using the Box–Behnken statistical design tool, the equation used for size was as follows:

Size range = +5.44 + 0.4189A-0.0394B-0.4216-0.3505AB + 0.8395AC-0.0238BC

whereas, the equation used for PDI was as below:

PDI = +0.4251-0.1217A + 0.0148B + 0.0789C - 0.0797AB - 0.0693AC - 0.0215BC

Method of preparation of SA nanopowder

A planetary ball monomill was used for dry milling due to its simplicity and capability of altering the speed of rotation, whereas other types of mills are not equipped with this feature. The methodology used here was based on the optimization of independent milling variables to study their effects on dependent variables. The factors influencing the dependent variables and the milling conditions used for optimization are shown in Table 1. To prevent the overheating of the grinding medium, the milling process was interrupted at proper intervals after each milling round, until room temperature was achieved. Different milling conditions employed were milling speed (100, 200, and 300 rpm), milling time (5, 10, and 15 min), and the number of balls (1, 2, and 3) to study their effects on the dependent variables such as particle size and PDI of the nanopowder.[7]

Table 1:
Optimization of variables in Box–Behnken statistical design

The planetary ball mono mill used consisted of a single working station based on the principle of impact force. The grinding bowl size was 500 mL and was made up of stainless steel (Ni and Cr). The materials used for balls and bowls should be similar, and therefore, we chose the bowl with 17–19% Cr + 8–10% Ni material and balls of 12.5–14.5% Cr + 1% Ni material. Moreover, the diameter of the balls used should always be the same to achieve proper milling and to avoid higher abrasion of balls.

For the preparation of drug NPs, 10 g of SA powder was placed in the container of the planetary mono mill and was operated according to the set conditions as shown in Table 1. With the help of Box–Behnken statistical design, 17 runs were performed [Table 2] to study the effect of independent variables such as milling time, milling speed, and the number of balls on fabricated nanoparticles on particle size in the nanometer range and PDI. Optimization by the Design Expert software suggested 17 runs by combining the three variables rpm, milling time, and the number of balls. For each run, first, the accurately weighed quantity of SA powder was placed in the pan and the milling conditions were changed. The rpm was changed to either 100, 200, or 300 and the milling time was set to either 5, 10, or 15 min. An appropriate number of balls was placed in the container with SA. One by one, SA was milled using the conditions mentioned in Table 2, and the sample was transferred to a clean container for further analysis. All 17 samples were thus obtained using 17 different conditions, and the particle size analysis and PDI determination were performed using the dynamic light scattering method by employing the Malvern Zetasizer instrument.

Table 2:
Effect of milling parameters on the size and PDI of the powder

Analysis of particle size and zeta potential

The SA sample was suspended in double-distilled water and 1 mL of the resulting suspension was placed in the sample cell. Double-distilled water was used in the reference cell, and the analysis was performed using the instrument. Peaks corresponding to different molecular sizes with their relative intensities were obtained using the Malvern Zetasizer software version 7.01. The particle size (nm) and PDI for each sample were determined and compared.


Mechanical attrition produces nanostructures by the structural decomposition of coarser particles as a result of plastic deformation.[9] Commonly used in mechanical alloying purposes, recently, ball milling techniques have received much attention as a powerful tool for the fabrication of NPs. For this purpose, various high-energy mills such as planetary ball mills, vibrating ball mills, and attrition ball mills are employed. Planetary mono mill offers fast and fine grinding of samples, is operator friendly, and produces consistent and reproducible results.[10] Herein, the centrifugal forces are exerted due to the rotation of the disc and turning of the vial, milling balls, and powder roll on the inner wall of the container and are thrown off across the bowl at high speed.[11] The overlapping of the centrifugal forces causes the sample material and grinding balls to bounce off the inner wall of the grinding bowl. The grinding balls cross the bowl diagonally at an extremely high speed and grind the sample material on the opposite wall of the bowl. The application of the Design of Experiment tools in milling improves process understanding as well as facilitates the monitoring and control of the particle size reduction process. This enables the production of fine particulates with predictable and controllable physicochemical characteristics.[12]

Parameters such as milling/grinding time, milling speed, and the number of balls have a remarkable effect on the size and PDI of the prepared nanopowder. A longer grinding time increases the fine fraction. Similarly, increasing the speed shortens the grinding time and increases the percentage of fine particles, whereas lower speeds increase the grinding time and lower the temperature of the mill. To reduce the grinding time, balls and bowls with higher density should be used. The milling speed should also be optimized carefully to obtain desired particle size range. Lower rotational speeds lead to longer milling periods and greater non-homogeneity in powders because of inadequate kinetic energy output. Similarly, higher rotational speeds can cause the balls to be pinned to the inner walls of the vial and not to fall to exert any impact force, therefore, lowering the fracturing efficiency in such cases. The milling speed as a function of milling duration should also be set carefully and optimized for a given set of experimental conditions. Additionally, the number of balls also has a major impact on the grinding process as a higher number of balls reduces the grinding time. The use of many small balls increases the fine fraction if the milling time is increased. It is not advisable to mix balls of different diameters as it might result in increased wear and damage to the grinding elements.[13]

All three factors (A, B, and C) showed a remarkable impact on the particle size reduction as well as the PDI of prepared nanopowders; however, amongst all factors, the number of balls (factor C) was found to have the maximum effect on the size reduction. From the model term and their powers, milling speed had a power of 68.8%, milling time had a power of 62.1%, and the number of balls had a power of 72.2%, respectively, which was concordant with our results too. When the milling speed was set to 100 rpm, milling time was kept to 10 min and the number of balls was 1, the particle size of SA reduced to 441.1 nm from the initial size of 776.3 nm. Furthermore, the size was reduced to 218 nm at speed of 100 rpm, milling time of 15 min with 2 balls. The lowest size of powder (16.32 nm) was achieved at speed of 100 rpm, milling time of 10 min when the number of balls was 3. This clearly showed that the number of balls had the greatest impact on the particle size reduction of SA powder. At higher milling speeds (200 and 300 rpm) and milling times (10 and 15 min), no further significant size reduction was observed. This can be due to the amorphous nature of the SA powder, which limited the particle size reduction to a maximum value. The particle size ranged between 200 and 400 nm in all such experimental conditions. The most optimized conditions for the size reduction process of 10 g SA powder were speed = 100 rpm, time = 5 min, and the number of balls = 1 to achieve the optimum particle size in the range of 205.0 nm and PDI (0.383) for further pharmaceutical applications. We were able to reduce the particle size of neat SA powder from 776.3 nm to 205.0 nm by dry milling efficiently.

The PDI is defined as the square of the standard deviation/mean diameter. It is a parameter used to define the size range of nanoparticles. The PDI values generally range from 0 to 1 and the values from 0 to 0.08 indicate a nearly monodisperse sample, values from 0.08 to 0.7 indicates a mid-range sample, whereas the values between 0.7 and 1.0 show a broad distribution of particles. In the case of pharmaceutical nanoparticles, PDI values below 0.3 are desirable. Because of the larger size, the aggregated particles may interfere with cellular uptake as compared to single particles. Values around 0.1–0.3 may be considered good for monomodal dispersion, whereas higher values (more than 0.7) are observed in the case of multimodal dispersion.[14] In the 17 runs conducted by us during the experimental procedure, the effect of various milling factors on PDI values was recorded. At low milling speeds of 100 and 200 rpm, increased time, and with more than one ball, the PDI values were found to be the highest. For example, at Run no. 10 (100 rpm, 15 min, and 2 balls) PDI was found to be 0.666. Similarly, at Run no. 12 (100 rpm, 10 min, and 3 balls) PDI value was found to be 0.907 and at Run no. 13 (200 rpm, 10 min, and 2 balls), the PDI value went up to 1. All these results showed that these factors had an impact on PDI values. Thus, the aggregation of particles was happening at a low speed and increased the number of balls. The effects of various factors on the size and PDI of SA particles were studied using the Zetasizer instrument and the responses are summarized in Table 2. The untreated powder before milling showed a particle size of 776.3 nm and a PDI value of 0.60 [Figure 1], which was used for further size reduction in the planetary mono mill. After the milling processes, the optimized nanopowder showed a remarkable reduction in the particle size as well as PDI values to 205.0 nm and 0.383; respectively [Figure 2]. The effects of the three factors on the size and PDI of the prepared nanopowder were studied using statistical design and the three-dimensional (3D) Box–Behnken plots as shown in Figures 3 and 4.

Figure 1:
Particle size analysis of untreated salicylic acid powder
Figure 2:
Particle size analysis of the optimized nanosized salicylic acid powder
Figure 3:
Three-dimensional (3D) graphs showing the effect of factors (AB, AC, and BC) on the size of SA nanopowder
Figure 4:
Three-dimensional (3D) graphs showing the effect of factors AB, AC, and BC on PDI of SA nanopowder

The properties of nanoparticles greatly depend on their size. Upon increase in the particles’ size, their surfaces to volumes ratios decrease and their properties change; thus, plenty of biological mechanisms elicit at the nanoscale. The particle size of SA was aimed to be reduced using the dry milling technique so that the prepared nanosized powder can further be formulated into a nanoformulation suitable to be delivered transdermally. The transdermal route is the most convenient route of drug administration for the application of the formulation to the intact skin at a controlled rate locally or to systemic circulation. Through a transdermal drug delivery system (TDDS), bioactive compounds can be targeted to the site of infection/disease and systemic side effects can be kept at a minimum. With respect to other factors influencing the mechanisms of TDDS, the particle size of the nanocarrier is foremost important. For drug delivery through the transdermal route, the approximate particle size range should be between 10 and 600 nm. Nanocarriers with a diameter of 600 nm or above cannot deliver the material into deep layers of the skin. Nanovesicles with 300 nm or below are able to deliver their contents to some deeper layers of skin. However, nanocarriers with a diameter of 70 nm or below have shown increased disposition of contents in both viable dermal and epidermal layers. Nanoparticles below 36 nm can be absorbed through aqueous pores, whereas nanoparticles below 6–7 nm in size can be absorbed through the lipidic trans-epidermal routes. Particles in the range of 10–210 nm, however, may preferentially penetrate through the trans-follicular route.[14]

Nanoparticle formulation technologies have provided the pharmaceutical industry with new strategies to solve the solubility issues related to poorly soluble drugs. Preparation of nanopowders using a top-down size reduction technique is one such strategy, which can further be developed into various dosage forms providing maximal drug exposure, permeation enhancement, and bioavailability. The process of particle size reduction of a poorly soluble drug from micro-scale to nano-scale using a planetary ball mono mill is a very feasible and acceptable physical method, “Top-down approach,” in the formulation of nanoparticles.[15] Through the principles of mechanical forces such as abrasion, compression, impact, and attrition, the particle size of the drug can be reduced considerably, which leads to the increased surface area of the particle and thus increased solubility profile. This method can be categorized under the Green nanotechnology approach for being solvent-free, environmentally friendly, cost-effective, sustainable, and easily scalable in making dry drug nanoparticles for enhanced drug solubility and bioavailability.[16]

The present method is advantageous over other methods of preparation of nanoparticles in being free from the use of any toxic and hazardous chemicals or solvents, which are often used in nanoparticle production using other methods. Also, if properly optimized, this method produces very fine powder and varying particle sizes can be achieved depending on the requirement. The size of the nanopowder obtained can easily be controlled by controlling the milling conditions. Another advantage associated with this technique is that it is suitable for decreasing the size of toxic materials also as the toxic materials can only be processed in enclosed containers without human exposure. Also, this technique is mechanical and therefore can be used continuously and a wide range of samples can be used simultaneously as it offers a simple and non-tedious procedure. Both bath and continuous operations can be performed using this technique. This method is also suitable for highly abrasive as well as explosive materials and can be used for materials of all degrees of hardness. This approach has good potential to be scaled up and can be utilized in pharmaceutical industries for the production of nanopowders of various active pharmaceutical ingredients in a rapid, inexpensive, and efficient manner.


In this study, we achieved the nanonization of SA using the dry milling technique on a planetary ball mono mill and successfully produced its nanopowder under optimized conditions. Because SA is a topical anti-inflammatory and a keratolytic agent, smaller particle size will result in better skin absorption as compared to a larger size when applied topically for various skin-related diseases. The main advantage of using a planetary ball mono mill in the preparation of nanoparticles is that it does not require hazardous organic solvents and chemicals and has proven to be an environment-friendly approach. However, comminution and mechanochemical processes that take place in the planetary ball mono mill are complex and are mainly dependent on the type of material to be milled. All parameters affecting the milling process should be optimized carefully to achieve the desired outcomes. The extent of size reduction depends on high-stress energies, which can be achieved by larger ball diameters and higher rotational and revolution speeds. However, to avoid wear and tear and higher energy consumption, it is always recommended to operate at medium revolution speed, especially for sensitive materials, which need controlled temperature conditions.

Data availability

Data related to the study are available with the authors and can be provided upon request

Financial support and sponsorship

This work was funded by the Deanship of Scientific Research, Jazan University, Jazan under the Future Scientist Program No (FS10-013).

Conflicts of interest

There are no conflicts of interest.


The authors acknowledge the Deanship of Scientific Research, Jazan University, Jazan for providing financial assistance to carry out this work.


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Dry milling; nanopowder; planetary ball mill; poorly water-soluble; salicylic acid; top-down approach

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