1. Introduction
Cancer is one of the leading causes of mortality and morbidity across the world. To date, the most common treatments for cancer include radiation therapy, chemotherapy and surgery [
1]. However, their benefits are outnumbered by their disadvantages, such as renal toxicity, hepatic toxicity or lower availability of the drug at the target site [
2]. These problems can be addressed by using a target-specific and biocompatible drug delivery vehicle, such as human serum albumin [
3,
4,
5]. Human serum albumin (HSA) is the most abundant protein found in the human body with a molecular weight of 66.5 kDa. It is produced by the liver and has a half-life of around 19 days [
6]. As revealed by X-ray structure analysis, the structure of HSA comprises of three domains: I, II and III. Each of these domains consists of two subdomains (Ia, Ib, IIa, IIb, IIIa and IIIb), which are arranged together to form binding sites on the HSA molecules [
7]. HSA can bind to metabolic substrates, as well as therapeutic drugs, which include hydrophobic as well as hydrophilic drugs. HSA-NPs are formed by the aggregation of HSA molecules in solution forming intermolecular disulfide bonds [
8]. The properties of HSA-NPs include biocompatibility, biodegradability and non-immunogenicity [
4,
9]. The target specificity of HSA for the glycoprotein60 (gp60) receptor present on the surface of cancer cells, allows the delivery of various anti-cancer drugs, such as docetaxel [
10], paclitaxel [
11] and noscapine [
4], without inducing an immune response [
5,
9]. Paclitaxel is an anti-cancer drug, commercially available as Taxol
®, and has been widely used as a chemo-therapeutic agent for the treatment of different cancer types, such as breast, ovarian and lung cancer [
12,
13]. Due to the toxic effects of this formulation on normal cells, Paclitaxel was used in combination with HSA-NPs (Abraxane
®) for site-specific delivery [
12,
14,
15]. This has led to improved tumor targeting by enhancement of the enhanced permeability and retention (EPR) effect as opposed to administration of free drugs [
15].
The retention of these colloidal drug delivery systems within the body is highly influenced by the particle’s size, physical stability and surface characteristics [
16]. It is known that nanoparticles in the size range 10–100 nm enter the lymphatic capillaries and undergo clearance [
17]. Furthermore, particles in the size range of 250 nm–1 μm are identified by macrophages and removed by the reticuloendothelial system (RES) by the process of opsonization. This is a mechanism by which macrophages or monocytes identify and remove target cells or particles from the body by binding to them [
17,
18]. A lower surface curvature of the nanoparticles also lowers their chances of opsonization. This process is also influenced by the zeta potential of the particles. Negatively-charged particles prevent nanoparticle aggregation, whereas positively-charged particles promote binding to opsonin molecules, leading to their removal from blood circulation [
19]. Thus, it is essential to control the particle size and zeta potentials of HSA-NPs to prevent their removal and ensure maximum efficacy.
In order to develop HSA-NPs containing paclitaxel, the emulsion-solvent evaporation method is the most reliable for the production of nanoparticles with a smaller size, a lower polydispersity index, reproducibility and potential for scale up [
20]. The procedure is less complex, less time consuming and involves less use of chemicals for the preparation of HSA-NPs as compared to the pH coacervation technique or the microfluidics approach [
21]. A high pressure homogenizer is commonly used for the breakdown of particles by the generation of high shear, which disperses the hydrophobic drug into the HSA solution, forming homogeneously-dispersed nanoparticles [
14]. This technique was first demonstrated by Desai
et al. for the production of paclitaxel-bound HSA-NPs and can be used for improving the water solubility of most hydrophobic drugs [
14]. Kim
et al. later used this technique for the preparation of curcumin containing HSA-NPs [
22].
In this study, the main aim was to develop and optimize the preparation process of HSA-NPs, following the emulsion-solvent evaporation method, in order to prepare reproducible and stable paclitaxel (PTX)-HSA-NPs with a particle size between 100 and 200 nm. The yield and encapsulation efficiencies of the particles were higher in comparison to other studies. By the optimization of the parameters, a high particle yield and high drug encapsulation efficiency was obtained. The nanoparticles were produced by high pressure homogenization (HPH) by optimizing parameters, such as the HSA concentration, organic solvent concentration, homogenization pressure, number of homogenization cycles and the Paclitaxel (PTX) concentration [
22]. These parameters were found to influence the final particles size, surface charge and morphology of the nanoparticles. The surface morphology of the nanoparticles was analyzed by using scanning electron microscopy (SEM). Further, to evaluate the effectiveness of the optimized parameters, different concentrations of PTX were added to prepare PTX-HSA-NPs. The safety effectiveness of the PTX-HSA-NPs was tested by performing an
in vitro drug release and cytotoxicity assay using human breast cancer cell line (MCF-7).
3. Discussion
The incidence of cancer is rapidly increasing throughout the world. It is thus important to develop more effective and efficient nanoparticle systems, such as HSA-NPs, for specific targeting of anticancer drugs like paclitaxel [
13,
28]. This study demonstrates the development of stable HSA-NPs using the emulsion-solvent evaporation method. This method is commonly used for improving the solubility of hydrophobic drugs by attachment to HSA-NPs or other polymeric nanoparticles [
29,
30]. As compared to other preparation methods, such as the pH coacervation method, this process is less complex, less time consuming and suitable for scale up [
31]. A high pressure homogenizer was used for the preparation of HSA-NPs and PTX-HSA-NPs of sizes in the range of 100–200 nm. High pressure homogenization is a technique used for the production of nano-sized emulsions [
25]. In this study, it was used to disperse paclitaxel, a water-insoluble anti-cancer drug, into the aqueous HSA solution. This emulsion on being subjected to repeated homogenization cycles is broken down into nano-sized emulsions, which ultimately form nanoparticles [
24]. This technique has also been applied to other therapeutic drugs, such as curcumin, doxorubicin and pirarubicin-paclitaxel for use in cancer therapy [
22,
32,
33]. It allows the dispersion of hydrophobic drug formulations into the aqueous HSA solution by facilitating their binding to the hydrophobic cavity on the HSA molecule [
34,
35]. Thus, the potential of such a methodology for the development of reproducible and stable drug containing HSA-NPs is greatly enhanced.
We optimized various parameters, such as the HSA starting concentration, organic solvent concentration, homogenization pressure, number of homogenization cycles and PTX concentration. Three different starting concentrations of PTX (0.5, 1 and 1.5 mg/mL) were tested by adding to the preparative HSA solution, which resulted in the formation of PTX-HSA-NPs. From the first experiment, results suggested that increasing the homogenization pressure from 10,000 psi to 20,000 psi led to a decrease in the HSA-NP size. This trend is expected since in order to reduce the particle size, it is necessary to overcome the minimum pressure, known as the Laplace pressure [
23]. The zeta potential of the particles showed a positive charge with a high standard deviation, which indicated the instability of the HSA-NPs (
Figure 1). Thus, further optimization was necessary to develop stable HSA-NPs, which was accomplished in the subsequent steps. It was observed that increasing the number of homogenization cycles led to a reduction in the size of the HSA-NPs. This was because, on increasing the applied homogenization pressure and number of homogenization cycles, the emulsion was repeatedly subjected to high shear, which further reduced the size of the emulsion droplets into nano-sized droplets [
23,
36]. The surface charge on the particles was not significantly different due to the high standard deviations (
Figure 2). Increasing the HSA concentration led to an increase in the size of the HSA-NPs with a decrease in the zeta potential (
Figure 3). HSA is a negatively-charged molecule, thus, increasing the amount of HSA in the starting solution leads to greater formation of intermolecular disulfide bonds. This further causes higher protein aggregation and the formation of larger-sized HSA-NPs with a more negative zeta potential [
37]. The effect of chloroform in the solution was also investigated. It was found that increasing the chloroform concentration led to a decrease in the size of the HSA-NPs (
Figure 4). The nanoparticles with the smallest size were formed when the chloroform concentration in the starting solution was 20%
v/
v of the starting HSA solution. Increasing the amount of organic solvent in the reaction mixture provides a larger surface area for the emulsion, undergoing repeated homogenization at high pressure, to be reduced to smaller droplets due to high shear. However, in order to minimize the exposure to higher amounts of chloroform, it was replaced with chloroform-ethanol in the ratio of 94:6. It is known that different organic solvents have different effects on the size of the emulsion droplets [
38]. It was noted that using 3%
v/
v CHCl
3-EtOH in solution resulted in HSA-NPs of a size comparable to those formed using 20%
v/
v CHCl
3. Increasing the concentration of CHCl
3-EtOH in the HSA solution reduced the size of the HSA-NPs further (
Figure 5) [
39]. On evaporating the organic solvent, the nanoparticles are retained in solution. It is important to evaporate the chloroform slowly under reduced pressure, keeping the bath temperature at 40 °C, in order to prevent immediate aggregation of the nanoparticles.
Lastly, the effects of varying the paclitaxel concentration (0.5, 1 and 1.5 mg/mL on the size and surface charge of the PTX-HSA-NPs was investigated. It was noted that increasing the PTX concentration from 0.5 mg/mL to 1.5 mg/mL led to an increase in the particle size and a more negative zeta potential (
Figure 6). The PDI of the nanoparticle solution was less than 0.2 in all of the samples. This result was contradictory to the results obtained by Desai
et al., who demonstrated that increasing the paclitaxel concentration leads to the formation of smaller-sized PTX-HSA-NPs [
14]. It is possible that this variation resulted due to the difference in organic solvents used for the preparation of the PTX-HSA-NPs or due to the optimized conditions in this study. It was observed that the encapsulation efficiency of the PTX-HSA-NPs was approximately 82%, 94% and 98%, which was much higher than those obtained by other research groups. Zhao
et al. and his team, who prepared paclitaxel-loaded HSA-NPs by a microfluidic technique and incorporating glutathione in HSA-NPs for additional stabilization, reported a maximum encapsulation efficiency of 11% for the PTX-HSA-NPs. Similarly, Gong
et al. co-encapsulated paclitaxel with pirarubicin to enhance the anti-tumor effect of the formulation and reported encapsulation efficiency of around 80% for paclitaxel [
33]. A high yield and high encapsulation efficiency allows greater entrapment of the drug molecules in the HSA-NP binding sites, and therefore, for lower nanoparticle concentrations, high drug release and cytotoxicity could be observed.
The
in vitro drug release of PTX from the PTX-HSA-NPs at time intervals 0, 1, 2, 9, 12, 18, 24 and 48 h was studied in triplicates (
Figure 8). This method of
in vitro drug release has previously been demonstrated in the literature [
4,
26,
27]. The PTX-HSA-NPs are dispersed in PBS at 37 °C and shaking conditions in order to simulate the dynamic
in vivo conditions. This is an indicator of the controlled release behavior of PTX-HSA-NPs. It was observed that the PTX-HSA-NPs prepared from a PTX concentration of 0.5 mg/mL provided a burst release of approximately 37.2% ± 2.1% of the drug within 12 h. Despite this initial burst release, the cumulative release reached 63.1% ± 6.8% within 48 h in a consistent manner. The size of the nanoparticles was 170.2 ± 1.4 nm; the charge was −17.44 ± 0.5 mV; and the drug encapsulation efficiency was approximately 82%. [
40]. The PTX-HSA-NPs prepared with a starting PTX concentration of 1.5 mg/mL released the drug slowly with only 27.2% ± 5.2% released in 48 h. Similarly, the cumulative release from PTX-HSA-NPs initially prepared from 1 mg/mL PTX concentration was 32.6% ± 5.1% at the end of 48 h. These results are comparable to a study using PTX-containing bovine serum albumin nanoparticles (BSA-NPs) showing an initial burst release and later consistency in the
in vitro drug release [
41]. It allows continuous targeting of the cancer cells with a decrease in cell viability over time, as opposed to complete release of the drug within 24 h. Contrarily, other studies demonstrate a quick cumulative release of approximately 80% drug from PTX-HSA-NPs within 12 h followed by a slow release [
11]. This condition is not suitable, since the half-life of paclitaxel lies between 3 and 52 h, after which, the drug will be removed by hepatic clearance without eluting a pronounced toxic effect on the cancer cells [
42].
The cytotoxicity of the formulations including HSA-NPs, as well as PTX-HSA-NPs was tested on MCF-7 human breast cancer cells. The MCF-7 cells treated with PTX-HSA-NPs were incubated under static conditions as opposed to the shaking conditions for measuring the
in vitro drug release from PTX-HSA-NPs, because the cultured cells have enzymes that will subsequently degrade the nanoparticles over a period of time. However, the final goal is to test the PTX-HSA-NPs
in vivo, which is under dynamic conditions. The cell viability on incubation with HSA-NPs was approximately 88.5% ± 2.2% at 24 h and 85.3% ± 6.4% at 48 h and, hence, did not reduce significantly (
Figure 9). In the case of the PTX-HSA-NP formulations, the cells exhibited a concentration-dependent toxicity. Incubating the cells with the PTX-HSA-NPs with PTX concentrations of 31.4 μg/mL, 20.2 μg/mL and 8 μg/mL resulted in cell viability of approximately 61.3% ± 4.2%, 69.5% ± 5.4% and 72.4% ± 2.4%, respectively, at 24 h. The cell viability was reduced drastically to 22.6% ± 1.4%, 39.3% ± 3.9% and 52.1% ± 2.4%, respectively, at 48 h, as compared to the HSA-NPs. Assuming that approximately 62%, 32% and 27% of drug was released from the PTX-HSA-NPs containing 8 μg/mL, 20.2 μg/mL and 31.4 μg/mL of PTX, respectively, at 48 h, the expected cell viability is close in range with that measured using the dye 3-(4,5-dimtheylthiazol-2-yl)-2,5-diphenltetrazoliumbromide (MTT) assay. This study can be closely compared with a study in the literature by Bernaheu
et al., who have demonstrated the
in vitro performance of Abraxane on human breast cancer cells (MCF-7) and human breast cancer cells (MDA-MB-231) cells using similar cell culture conditions as in our study. Their cytotoxicity results revealed that an Abraxane concentration of 100 μg/mL resulted in cell viabilities of approximately 55% and 23% for MCF-7 and MDA-MB-231 cells, respectively [
43]. However, in our study, the optimized PTX-HSA-NPs with PTX 31.4 μg/mL cause a cytotoxic effect with only 22% cell viability at 48 h. Other studies in the literature have also studied the effect of Abraxane on non-small-cell lung cancer cells (A549) and prostate cancer cells (PC3) [
44,
45]. However, the results are not comparable due to the variation in experimental conditions and the difference in the
in vitro cell study.
Lastly, the dose-dependent response of the optimized PTX-HSA-NPs was studied in comparison with free PTX on the MCF-7 breast cancer cell line. After statistical analysis, an IC
50 of 4.9 μM for the optimized PTX-HSA-NPs and 2.1 μM for the free PTX was obtained. These values are lower than the >100 μM IC
50 values for Abraxane when tested in the MCF-7 breast cancer cell, as observed in a study by Bernaheu
et al. [
43]. Another study in the literature has demonstrated IC
50 values of 11.07 nM and 8.57 nM, for Abraxane and PTX, respectively, when tested in A549 non-small-cell lung cancer cells [
45]. The calculated IC
50 value of PTX was 12.4 μM when tested on 4T1 murine breast cancer cells [
33]. On testing in MDA-MB-231 breast cancer cells, IC
50 values for Abraxane and PTX were 2.7 nM and 2.5 nM, respectively, whereas in PC3 cells, 11.9 nM and 5.3 nM, respectively [
44]. Thus, the IC
50 values of the PTX-HSA-NPs and free PTX differ when evaluated in different cell lines.
Therefore, on comparing the in vitro performance of the optimized PTX-HSA-NPs with Abraxane and similar PTX-containing nanoparticle formulations at the given experimental conditions, from the above discussion, it can be concluded that this study demonstrates higher encapsulation efficiency, improved drug release characteristics and improved cytotoxicity. Therefore, the optimized PTX-HSA-NPs are anticipated to exhibit enhanced anticancer characteristics.
4. Materials and Methods
4.1. Reagents and Chemicals
Human serum albumin (lyophilized powder, ≥96%) was obtained from Sigma Aldrich (Oakville, ON, Canada). Paclitaxel (powder) was obtained from LC Laboratories (Woburn, MA, USA). Chloroform and all other chemicals were obtained from VWR International (Mississauga, ON, Canada).
4.2. High Pressure Homogenizer
The Avestin C-5 High Pressure Homogenizer (Avestin Inc., Ottawa, ON, Canada) was used for the preparation of HSA-NPs by the application of high pressure (5,000–60,000 psi) to break the HSA-containing emulsion into nano-sized emulsion droplets. The working solution volume for the preparation of HSA-NPs was 10 mL.
4.3. Process Optimization and Preparation of PTX-HSA-NPs
The HSA-NPs were prepared by the emulsion-solvent evaporation method using a high pressure homogenizer. The starting HSA concentrations of 10 mg/mL, 20 mg/mL, 30 mg/mL and 40 mg/mL were prepared in 10 mL of deionized water. To the preparative HSA solution, chloroform (CHCl3) (3% v/v, 5% v/v, 10 and 20% v/v) was added to the starting HSA solution. On further optimization, the chloroform addition step was replaced by the addition of a mixture of chloroform and ethanol (EtOH) in the ratio of 94:6. The concentrations of CHCl3-EtOH in the starting HSA solution were 3%, 5% and 10% v/v. This emulsion was first subjected to primary homogenization for 3.5 min, using a hand-held D1000 Benchmark homogenizer (Benchmark Scientific, Edison, NJ, USA) followed by high pressure homogenization. The homogenization pressure (10,000 psi, 15,000 psi and 20,000 psi) was applied to the emulsion, and the number of homogenization cycles (6, 9, 12 and 15 cycles) was also optimized. The emulsion subjected to various homogenization cycles was passed through the homogenizer valve and collected through a connecting tube at the base of the assembly, thus forming nano-sized emulsion droplets.
For the preparation of paclitaxel-containing HSA-NPs, paclitaxel (0.5, 1 and 1.5 mg/mL) was dissolved in a 3% v/v CHCl3-EtOH mixture (94:6) and then mixed with the HSA solution volume. This emulsion was subjected to primary homogenization for 3.5 min using the hand-held D1000 Benchmark homogenizer. After primary homogenization, the emulsion was subjected to 12 cycles of high pressure homogenization at 20,000 psi pressure per cycle.
Following high pressure homogenization, the resulting colloidal solution was transferred to a round-bottomed flask and subjected to rotary evaporation at 90 rpm by applying a vacuum pressure of 400 mm Hg for 30 min at 40 °C. This process ensured complete removal of the organic solvent from the emulsion, which led to the formation of HSA-NPs.
4.4. Nanoparticles Size Measurement, Zeta Potential Analysis and Surface Morphology
The average particle size of the HSA-NPs, both with and without paclitaxel, was measured by dynamic light scattering (DLS) using a particle size analyzer (Brookhavens Instruments Corporation, Holtsville, NY, USA). The samples were diluted with deionized water and measured at a scattering angle of 90° and temperature of 25 °C. The polydispersity index (PDI) gave an estimate of the size distribution of the HSA-NPs. The zeta potential was measured by a zeta potential analyzer (Malvern Instruments, Worcestershire, UK) using electrophoretic laser Doppler anemometry. The size, shape and surface morphology of the HSA-NPs were examined by scanning electron microscopy (Hitachi S-4700 FE-SEM, Tokyo, Japan).
4.5. Yield and Encapsulation Efficiency of HSA-NPs
The yield of the HSA-NPs was measured by the UV-spectrophotometric method. A standard curve of HSA dissolved in a solution of phosphate buffered saline (PBS), containing 0.02%
v/
v Tween 20%
v/
v and 10%
v/
v acetonitrile, was used as a reference. The absorbance values for HSA were measured at 280 nm. For the calculation of the yield, the following equation was used.
To calculate the encapsulation efficiency of paclitaxel in HSA-NPs, the PTX-HSA-NPs were spin concentrated using Amicon centrifugal filters (Cedarlane, Burlington, ON, Canada) with a molecular weight cut off (MWCO) of 10,000 Da. This allowed the non-encapsulated paclitaxel drug to be eluted out in the collection tube. The concentration of non-encapsulated PTX was determined by the UV-spectrophotometric method. A standard curve of PTX in a mixture containing MeOH/PBS (30:70) and 1%
v/
v sodium dodecyl sulfate (SDS) was used as a reference [
46]. The absorbance values were measured at 230 nm. The encapsulated paclitaxel in the PTX-HSA-NPs was calculated using the following equation.
4.6. Measuring In Vitro Paclitaxel Drug Release
The
in vitro drug release was measured by the UV-visible spectrophotometric method as published previously [
4,
26,
27]. In brief, PTX-HSA-NPs were spin concentrated using Amicon centrifugal filters (Cedarlane, Burlington, ON, Canada) with MWCO 10,000 Da. The nanoparticles were re-suspended in 5 mL PBS and placed in a shaker at 37 °C at 120 rpm. At pre-determined time intervals (0, 1, 2, 9, 12, 18, 24 and 48 h), 0.5 mL of the PTX-HSA-NPs solution was withdrawn starting from 0 h for the absorbance measurement at 230 nm and re-substituted with 0.5 mL of fresh PBS. From the absorbance measurements, the cumulative amount of PTX released into the solution at the different time intervals was determined. This study was performed in triplicates.
4.7. Cell Viability Due to PTX-HSA-NPs
MCF-7 breast cancer cell lines were received as a kind gift by Dr. Jose Teodoro (Biochemistry, McGill University). The MCF-7 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM). The medium was supplemented with 10% fetal bovine serum (FBS) and 1.5 g/L sodium bicarbonate. The cells were cultured in a humidified incubator at 37 °C with 5% CO2.
To determine the cell viability due to PTX-HSA-NPs, the MTT assay was performed. The MTT assay is a commonly-used colorimetric assay using the MTT for the rapid determination of biomaterial cell toxicity [
4]. MCF-7 cells were seeded at an initial concentration of 5,000 cells/mL in 96-well culture plates. After 24 h of incubation in a humidified incubator at 37 °C with 5% CO
2, the media of the adherent cells was replaced with serum-free culture media and treated with HSA-NPs and PTX-HSA-NPs with a final concentration of 0.2 mg/mL. The cells were further incubated for 24 and 48 h, followed by replacing the treatment medium with 100 μL of fresh cell culture media. To assess the cytotoxicity of nanoparticles, 10 μL of MTT reagent was added to each well containing 100 μL of cell culture medium and incubated in a humidified incubator for 4 h at 37 °C and 5% CO
2. After incubation, 100 μL of lysis buffer (99.4% dimethylsulfoxide (DMSO) and 0.6% acetic acid) was added to the wells and incubated for another 15 min at room temperature. The absorbance was measured at 570 nm using a Victor3V 1420 Multilabel Counter spectrophotometer (Perkin Elmer, Woodbridge, ON, Canada). For the dose-response study, different concentrations of PTX-HSA-NPs and free PTX were diluted with the cell culture medium and DMSO (less than a 0.5% final concentration of DMSO in solution) to obtain dilutions of 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM and 0.001 μM.
The IC50 was calculated using the GraphPad Prism software, version 5.01 (GraphPad Software Inc., La Jolla, CA, USA), following nonlinear regression analysis.