**6. Preparation and characterization of the proteinoid nano/micro-particles by a self-assembly process**

Proteinoid particles were prepared by a self-assembly mechanism. Briefly, 100 mg of the dried proteinoid were added to 10 mL 10-5N NaCl solution. The mixture was then heated to 80°C until the crude proteinoid dissolves completely. Proteinoid particles were then formed by removal of the heating and leaving the mixture to cool to room temperature.

Hydrodynamic diameter and size distribution of the particles dispersed in double distilled (DD) water were measured at room temperature with a particle DLS analyzer model Nano‐ phox (SympatecGmbH, Germany).

Dried particle size and size distribution were measured with a Scanning Electron Microscope (SEM). SEM pictures were obtained with a JEOL, JSM-840 Model, Japan. For this purpose, a drop of dilute particle dispersion in distilled water was spread on a glass surface, and then dried at room temperature. The dried sample was coated with carbon in vacuum before viewing under SEM. The average particle size and distribution were determined by the measurement of the diameter of more than 200 particles with image analysis software (Analysis Auto, Soft Imaging System GmbH, Germany). Figure 5 exhibits the proteinoid particles made from self-assembly of Prot8. The procedure produced spherical proteinoid particles of 103 ± 11 nm hydrodynamic diameter and 70 ± 15 nm dry diameter. The dry diameter of the proteinoid particles is illustrated by the typical SEM photomicrograph shown in Figure 5A. The hydrodynamic diameter of these particles dispersed in water is illustrated by the typical light scattering measurement shown in Figure 5B. The difference in the particle size between the SEM and the light scattering measurements is probably due to the fact that SEM measurements determine the dry diameter, whereas light scattering measurements take into account the hydrated water layers adsorbed onto the particle's surface.

The density of the particles was determined by pycnometry [52]. Briefly, dry pre-weighed particles were put in a calibrated pycnometer, which was then filled with water. The density of the sample can then be calculated from the known density of the water, the weight of the pycnometer filled only with water, the weight of the pycnometer containing both the sample and water, and the weight of the sample, as described in the literature [52]. Density measure‐ ments indicated that all proteinoid particles possess a very low density, ranging from 0.001 to 0.014 g/mL indicating that the particles formed are probably hollow, as already indicated for the proteinoids prepared by Fox et al [6,38]. The hollow nature of the particles is significantly important for applications such as ultrasound imaging agents, drugs and dyes encapsulation, controlled released, etc.

As suggested in our and in previous studies, the proteinoid forms particles of different sizes according to the nature of its surrounding. The hydrophobic portions of the crude proteinoid

**Figure 5.** Hydrodynamic size histogram (A) and SEM image (B) of Prot8 particles.

are assembled within the particle matrix, while the polar hydrophilic groups (carboxyl and amines) are exposed to the aqueous environment, as illustrated before in Figure 2 [40].

#### **6.1. Particle stability in storage conditions**

Proteinoid particles aqueous dispersions (1 mg/mL) were put in a refrigerator at 4°C for 6 months. Samples were taken at different time periods, filtered through a centrifugation tube (Vivaspin 3000 Da MWCO) and the filtrate was checked by UV at 200-210 nm, to find aqueous soluble proteinoid. Also, the particle aqueous dispersions were checked by Nanophox for their size and size distribution. In order to check the particle stability after drying, the particles were lyophilized to dryness and then dispersed in an aqueous phase to their original concentration. The samples size and size distribution were then rechecked by Nanophox. Overall, the proteinoid particles remain in the same size after 6 months in storage at 4°C. Also, the degradation and/or dissolution of the proteinoid particles in the aqueous continuous phase was tested by the filtration centrifugation method and resulted in negative results in the filtrate, meaning no degradation or dissolution occurs at this temperature over 6 months. When lyophilized to dryness, the proteinoid particles can be redispersed in water completely while the particle size and size distribution remain the same. This means that the particles can be stored as a freeze-dried powder as well, and redispersed when needed, without the need to add cryoprotectants as mentioned in the literature [53].

#### **6.2. Cytotoxicity of the proteinoid particles**

In vitro cytotoxicity of the proteinoid particles was tested by using human colon adenocarci‐ noma LS174T cancer cell line. The tests were done on Prot2, Prot4, Prot5, Prot7 and Prot8. The cell line is adherent to the used culture dishes. LS174T cells were grown in MEM that was supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% glutamine and 1% penicillin/streptomycin. Cells were screened to ensure they remained mycoplasma-free using Mycoplasma Detection Kit [54]. Cell cytotoxicity was assessed by measuring the release of cytoplasmic lactate dehydrogenase (LDH) into cell culture supernatants. LDH activity was assayed using the Cytotoxicity Detection Kit according to the manufacturer's instructions [55]. Cells (3×105 cells per well) were seeded and grown to 90–95% confluency in 24 well plates before treatment with the proteinoid particles. Cell cultures that were not exposed to the particles were included in all assays as negative controls. Cell cultures that were treated with 1% Triton-x-100 were used as positive controls. To test if the particles can interact with LDH kit compounds, cell cultures were exposed to a mixture containing maximal nano/microparticles concentration dispersed in PBS and 1% Triton-x-100. The proteinoid particles were freshly dispersed in PBS (1.25 and 2.5 mg/mL) and then added to the 95% confluent cell culture in culture medium. The cell cultures were further incubated at 37°C in a humidified 5% CO2 incubator and then checked for cellular cytotoxicity at intervals of 24h. The percentage of cell cytotoxicity was calculated using the formula shown in the manufacturer's protocol [55]. All samples were tested in tetraplicates.

are assembled within the particle matrix, while the polar hydrophilic groups (carboxyl and amines) are exposed to the aqueous environment, as illustrated before in Figure 2 [40].

Proteinoid particles aqueous dispersions (1 mg/mL) were put in a refrigerator at 4°C for 6 months. Samples were taken at different time periods, filtered through a centrifugation tube (Vivaspin 3000 Da MWCO) and the filtrate was checked by UV at 200-210 nm, to find aqueous soluble proteinoid. Also, the particle aqueous dispersions were checked by Nanophox for their size and size distribution. In order to check the particle stability after drying, the particles were lyophilized to dryness and then dispersed in an aqueous phase to their original concentration. The samples size and size distribution were then rechecked by Nanophox. Overall, the proteinoid particles remain in the same size after 6 months in storage at 4°C. Also, the degradation and/or dissolution of the proteinoid particles in the aqueous continuous phase was tested by the filtration centrifugation method and resulted in negative results in the filtrate, meaning no degradation or dissolution occurs at this temperature over 6 months. When lyophilized to dryness, the proteinoid particles can be redispersed in water completely while the particle size and size distribution remain the same. This means that the particles can be stored as a freeze-dried powder as well, and redispersed when needed, without the need to

In vitro cytotoxicity of the proteinoid particles was tested by using human colon adenocarci‐ noma LS174T cancer cell line. The tests were done on Prot2, Prot4, Prot5, Prot7 and Prot8. The

**6.1. Particle stability in storage conditions**

58 Advances in Bioengineering

**Figure 5.** Hydrodynamic size histogram (A) and SEM image (B) of Prot8 particles.

add cryoprotectants as mentioned in the literature [53].

**6.2. Cytotoxicity of the proteinoid particles**

**Figure 6. Cytotoxic effect of the proteinoid particles on colon adenocarcinoma LS174T cells measured by the LDH as‐ say.** Cells (3×105 ) were incubated for 24 and 48 h with the proteinoid particles dispersed in PBS (1.25 mg/mL and 2.5 mg/mL) according to section 3.2.5.8. Cells were incubated with Triton-x-100 1% as positive control (100% toxicity). In ad‐ dition, cells were incubated with Triton-x-100 1% and each one of the proteinoids to revoke any interaction. Untreated cells (negative control) were similarly incubated. Each bar represents mean ± standard deviations of 4 separate samples.

When tested by the LDH quantitative assay, Prot2, 4, 5, 7 and 8 particles dispersed in PBS at concentrations of 1.25 and 2.5 mg/mL had none, or minor cytotoxic effect on the human colon adenocarcinoma cell line LS174T (Figure 6). Treatment of the cells with Prot2 and Prot5 particles at both concentrations produced the highest LDH levels (up to 13% toxicity), when compared to untreated (blank) cells, indicating minor toxicity of these proteinoids to this cell line. Prot8 had the lowest cytotoxic effect on the cells treated with both concentrations, almost zero toxicity. This proteinoid is therefore the most suitable for treating cells, considering its low toxicity.
