nanomaterials Article

Human Serum Albumin Nanoparticles for Use in Cancer Drug Delivery: Process Optimization and In Vitro Characterization Nikita Lomis 1,2 , Susan Westfall 1 , Leila Farahdel 1 , Meenakshi Malhotra 3 , Dominique Shum-Tim 4 and Satya Prakash 1, * 1

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Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, 3775 University Street, Montreal, QC H3A 2B4, Canada; [email protected] (N.L.); [email protected] (S.W.); [email protected] (L.F.) Division of Experimental Medicine, 1110 Pins Avenue, Montreal, QC H3A 1A3, Canada Department of Microbiology, Immunology and Infectious Diseases, CHU St. Justine Research Center, University of Montreal, 3175 Cote-Ste-Catherine, Montréal, QC H3T 1C5, Canada; [email protected] Division of Cardiac Surgery and Surgical Research, Royal Victoria Hospital, 1001 Boulevard Décarie, Montréal, QC H4A 3J1, Canada; [email protected] Correspondence: [email protected]; Tel.: +1-514-398-3676; Fax: +1-514-398-7461

Academic Editor: Luigi Pasqua Received: 12 April 2016; Accepted: 3 June 2016; Published: 15 June 2016

Abstract: Human serum albumin nanoparticles (HSA-NPs) are widely-used drug delivery systems with applications in various diseases, like cancer. For intravenous administration of HSA-NPs, the particle size, surface charge, drug loading and in vitro release kinetics are important parameters for consideration. This study focuses on the development of stable HSA-NPs containing the anti-cancer drug paclitaxel (PTX) via the emulsion-solvent evaporation method using a high-pressure homogenizer. The key parameters for the preparation of PTX-HSA-NPs are: the starting concentrations of HSA, PTX and the organic solvent, including the homogenization pressure and its number cycles, were optimized. Results indicate a size of 143.4 ˘ 0.7 nm and 170.2 ˘ 1.4 nm with a surface charge of ´5.6 ˘ 0.8 mV and ´17.4 ˘ 0.5 mV for HSA-NPs and PTX-HSA-NPs (0.5 mg/mL of PTX), respectively. The yield of the PTX-HSA-NPs was ~93% with an encapsulation efficiency of ~82%. To investigate the safety and effectiveness of the PTX-HSA-NPs, an in vitro drug release and cytotoxicity assay was performed on human breast cancer cell line (MCF-7). The PTX-HSA-NPs showed dose-dependent toxicity on cells of 52%, 39.3% and 22.6% with increasing concentrations of PTX at 8, 20.2 and 31.4 µg/mL, respectively. In summary, all parameters involved in HSA-NPs’ preparation, its anticancer efficacy and scale-up are outlined in this research article. Keywords: human serum albumin; nanoparticles; paclitaxel; cancer; MCF-7

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 [3–5]. 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

Nanomaterials 2016, 6, 116; doi:10.3390/nano6060116

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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).

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2. Results Nanomaterials 2016, 6, 116

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2.1. Nanoparticles Optimization and Preparation

In this this study, study, various various parameters parameters crucial crucial in in the the preparation preparation of of PTX-HSA-NPs PTX-HSA-NPs of of sizes sizes 100–200 100–200 nm nm In wereoptimized optimizedusing usingaahigh highpressure pressurehomogenizer. homogenizer.The Theworking working the high pressure homogenizer were ofof the high pressure homogenizer is is based principal that applyinga avery veryhigh highpressure pressure(5,000–60,000 (5,000–60,000psi) psi)to tothe theemulsion emulsion passed passed based onon thethe principal that applying through aa homogenization homogenization valve breaks the emulsion into nano-sized emulsion droplets [14,23,24]. through Evaporating the the organic organic solvent solvent from from the theemulsion emulsionleads leadsto tothe theformation formationof ofnanoparticles. nanoparticles. Evaporating 2.1.1. 2.1.1. Effect of Homogenization Homogenization Pressure Pressure on on Nanoparticles Nanoparticles To pressure was optimized by by applying 10,000 psi, psi, 15,000 psi Toform formHSA-NPs, HSA-NPs,the thehomogenization homogenization pressure was optimized applying 10,000 15,000 and 20,000 psi,psi, to the starting emulsion. 20 mg/mL mg/mLwith witha psi and 20,000 to the starting emulsion.The Thestarting starting HSA HSA concentration concentration was 20 achloroform chloroformconcentration concentrationofof3% 3%v/v, v/v,and and12 12homogenization homogenizationcycles cycles were were applied. Results suggested suggested that on increasing increasingthe thepressure pressure above 10,000 pounds per square inch the (psi), the average size± that on above 10,000 pounds per square inch (psi), average size (mean (mean ˘ standard deviation (SD)) of the HSA-NPs was reduced from around 337.7 ˘ 14.8 nm down standard deviation (SD)) of the HSA-NPs was reduced from around 337.7 ± 14.8 nm down to 248.2 ± to 6.6 nmpsi atand 15,000 psi± and 15.5 nm 20,0001a). psiThe (Figure 1a). sizes The HSA-NP at 6.6248.2 nm at˘15,000 254.7 15.5 254.7 nm at˘20,000 psi at (Figure HSA-NP at 15,000 sizes psi and 15,000 20,000 psi were not different. significantly The zeta of the nanoparticles 20,000 psi psiand were not significantly Thedifferent. zeta potentials of potentials the nanoparticles were aroundwere 5–8 around 5–8 mV 1b). The polydispersity index all of was the samples 0.3. mV (Figure 1b). (Figure The polydispersity index (PDI) for all (PDI) of the for samples less thanwas 0.3.less For than the next For the next experiments, the homogenization waspsi set to toobtain 20,000smaller-sized psi to obtain smaller-sized experiments, the homogenization pressure waspressure set to 20,000 nanoparticles nanoparticles [24]. [24].

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(b)

Figure 1. 1. Effect of homogenization homogenization pressure pressure (psi) (psi) on on the the (a) (a) human human serum serum albumin-nanoparticle albumin-nanoparticle Figure Effect of (HAS-NP) size (mean ± SD, n = 10), represented as columns, and polydispersity index, represented as (HAS-NP) size (mean ˘ SD, n = 10), represented as columns, and polydispersity index, represented line; (b) zeta potential of HSA-NPs (mean ± SD, n = 10), represented as columns, prepared from 20 as line; (b) zeta potential of HSA-NPs (mean ˘ SD, n = 10), represented as columns, prepared from mg/mL starting HSA concentration, 12 homogenization cycles andand a chloroform concentration of 3% 20 mg/mL starting HSA concentration, 12 homogenization cycles a chloroform concentration of v/v v/v of starting HSAHSA solution (*** (*** p < 0.0001; ns =ns not= significant). 3% of starting solution p < 0.0001; not significant).

2.1.2. Effect Effect of of Homogenization Homogenization Cycles Cycles on on Nanoparticles Nanoparticles 2.1.2. The effect effectof ofthe thenumber numberofofhomogenization homogenizationcycles cycleson onHSA-NP HSA-NPsize sizewas was evaluated applying The evaluated byby applying 6, 6, 9, 12 and 15 cycles. Starting with a 20 mg/mL HSA concentration, 3% v/v chloroform and 20,000 psi 9, 12 and 15 cycles. Starting with a 20 mg/mL HSA concentration, 3% v/v chloroform and 20,000 psi pressure, results results indicated indicated that that on on applying applying 12 12 homogenization homogenization cycles, cycles, HSA-NPs HSA-NPs of of an an average average size size pressure, 250 ± 11.7 nm were formed, which was significantly lower than the other samples (Figure 2a). The 250 ˘ 11.7 nm were formed, which was significantly lower than the other samples (Figure 2a). The zeta zeta potential of the nanoparticles was −18 ± 2.9 mV. Comparing the particle size obtained potential of the nanoparticles was ´18 ˘ 2.9 mV. Comparing the particle size obtained by applyingby 6, applying 6, 9 and 15 homogenization cycles did not show any significant difference. Furthermore, 9 and 15 homogenization cycles did not show any significant difference. Furthermore, there was no there was no significant difference in thevalues zeta potential values 2b).particles The PDIwas of the significant difference in the zeta potential (Figure 2b). The (Figure PDI of the less particles than 0.4. was less than 0.4. Increasing the number of homogenization cycles combined with Increasing the number of homogenization cycles combined with the applied pressure led the to a applied greater pressure led to a greater reduction in particle size [25]. reduction in particle size [25].

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(a)

(b)

Figure 2. Effect of varying the number of homogenization cycles on the (a)(b) HSA-NP size (mean ± SD), Figure 2. Effect of varying(a) the number of homogenization cycles on the (a) HSA-NP size (mean ˘ SD), n = 10), represented as columns, and polydispersity index, represented as line; (b) zeta potential of n = 10), represented columns, and polydispersity index, represented as line; size (b) zeta potential Figure 2. Effect ofas varying the number of homogenization cycles on the (a) HSA-NP (mean ± SD), of HSA-NPs (mean ± SD, n = 10), represented as columns, prepared from a 20 mg/mL starting HSA HSA-NPs (mean ˘ SD, as n= 10), represented as columns, from a 20(b) mg/mL startingofHSA n = 10), represented columns, and polydispersity index,prepared represented as line; zeta potential concentration, 20,000 psi pressure with a chloroform concentration of 3% v/v of starting HSA solution HSA-NPs 20,000 (mean ± SD, n = 10),with represented as columns, prepared of from 20 mg/mL starting concentration, psi pressure a chloroform concentration 3%av/v of starting HSAHSA solution (*** p < 0.0001; ns = not significant). concentration, 20,000 psi pressure with a chloroform concentration of 3% v/v of starting HSA solution (*** p < 0.0001; ns = not significant). (*** p < 0.0001; ns = not significant).

2.1.3. Effect of HSA Concentration on Nanoparticle Size 2.1.3. Effect of HSA Concentration on Nanoparticle Size 2.1.3. of HSA Concentration Nanoparticle TheEffect nanoparticle size was alsooninfluenced by Size varying the starting HSA concentration. As the The nanoparticle size was also influenced by varying the starting HSA concentration. As the HSA concentration was size increased from 10 mg/mL to 40 mg/mL, the HSA-NP size increased from The nanoparticle was also influenced by varying the starting HSA concentration. As the HSA concentration was increased from 10 mg/mL to 40 mg/mL, the HSA-NP size increased from 216.6 ± 9.8 nm to 343.6was ± 14.6 nm, respectively, as represented in (Figure 3a). Consequently, zeta HSA concentration increased from 10 mg/mL to 40 mg/mL, the HSA-NP size increased the from 216.6 ˘ 9.8 nm to 343.6 ˘ 14.6 nm, respectively, as represented in (Figure 3a). Consequently, potential from −14.7 ± 13.7 to −18.3 ± mV (Figure 3b). For the the 10 mg/mL 216.6 ± of 9.8the nmHSA-NPs to 343.6 ± varied 14.6 nm, respectively, as mV represented in6.5 (Figure 3a). Consequently, zeta the zeta potential ofthe theHSA-NPs HSA-NPs varied ˘which 13.7 mV ˘ that 6.5 3b). mV (Figure For the HSA concentration, the PDI varied was approximately 0.26, was±to lower of other concentrations. potential of fromfrom −14.7´14.7 ± 13.7 mV to −18.3 6.5´18.3 mVthan (Figure For the 103b). mg/mL 10 mg/mL HSA concentration, theapproximately PDI approximately which wasof lower than that ofalso other HSAalso concentration, was 0.26, which was0.26, than that other concentrations. It was noted thatthe onPDI increasing thewas HSA concentration inlower solution, the PDI of the solution It was also noted that on increasing the HSA concentration in solution, the PDI of the solution also concentrations. It was also noted that on increasing the HSA concentration in solution, the PDI of the increased. increased. solution also increased.

(a) (a)

(b) (b)

Figure 3. 3. Effect ofofthe (mg/mL) on onthe the(a) (a)HSA-NP HSA-NPsize size (mean ± SD, Figure Effect thestarting startingHSA HSAconcentration concentration (mg/mL) (mean ± SD, n =n = Figure 3. Effect of the starting HSA concentration (mg/mL) on the (a) HSA-NP size (mean ˘ SD, 10),10), represented asascolumns, representedasasline; line;(b) (b)zeta zeta potential HSArepresented columns,and andpolydispersity polydispersity index, index, represented potential of of HSAn = 10), represented as columns, and polydispersity index, represented as line; (b) zeta potential of NPs (mean ± SD, nn = 10), withaachloroform chloroformconcentration concentration NPs (mean ± SD, = 10),represented representedas ascolumns, columns, prepared prepared with of of 3%3% v/vv/v HSA-NPs (mean ˘ SD, n = 10), represented as columns, prepared with a chloroform concentration of starting HSA solution,1212homogenization homogenization cycles and pressure (***(*** p