**5. Proteinoid analysis and characterization**

**4. Preparation of proteinoids by thermal condensation polymerization**

lyophilized to obtain a yellow-white proteinoid powder.

amine groups.

52 Advances in Bioengineering

**Polymer** Amino acid content (g)a

**4.1. Polymerization kinetics study at different temperatures**

**Prot1** 5 - - - - **Prot2** 2.5 2.5 - - - **Prot3** 2.5 - - 2.5 - **Prot4** 1.25 2.5 - 1.25 - **Prot5** 1.67 1.67 1.67 - - **Prot6** 1.67 - 1.67 1.67 - **Prot7** 1.25 - 2.5 1.25 - **Prot8** 2.25 - - 2.25 0.5

**Table 1. Amino acid content of the different proteinoids. a**In all proteinoids made by thermal condensation polymerization the total monomer content was 5-5.01 g; **<sup>b</sup>**made by microwave-assisted polymerization.

Polymerization kinetics was studied by collecting proteinoid samples from the reaction vessel at different time periods of the polymerization at 180, 190 and 200°C. The samples

L-glutamic acid was heated to the molten state (180°C) in an oil bath, under nitrogen atmos‐ phere. The molten mass was stirred at 180°C for 30 min. To this, different contents of additional L-amino acids were added to give a total monomer weight of 5-5.01 g, as specified in Table 1, and kept at 180°C under nitrogen. The mixture was mechanically stirred at 150 rpm for 3 h. The product is a highly viscous orange-brown paste, which hardens to give a glassy mass when cooled to room temperature. Then, water (10 mL) was added to the crude product, and the mixture was stirred for 20 min. The solution was then intensively dialyzed through a cellulose membrane (3500 Da MWCO) against distilled water. The content of the dialysis tube was then

**Figure 2.** Schematic representation of the self-assembled proteinoid particles. Hydrophobic moieties are represented by scribbled lines. When lysine is also a part of the proteinoid, as in Prot5-7, some carboxyl groups are exchanged with

L-Glu L-Asp L-Lys L-Phe PLLA

The molecular weights and polydispersity index of the dried crude proteinoids were deter‐ mined using Gel Permeation Chromatography (GPC) consisting of a Waters Spectra Series P100 isocratic HPLC pump with an ERMA ERC-7510 refractive index detector and a Rheodyne (Coatati, CA) injection valve with a 20 μL loop (Waters, MA). The samples were eluted with super-pure HPLC water through a linear BioSep SEC-s3000 column (Phenomenex) at a flow rate of 1 mL/min. The molecular weights were determined relative to poly(ethylene glycol) standards (Polymer Standards Service-USA, Silver Spring, MD) with a molecular weight range of 100-450000 Da, using Clarity chromatography software. The optical activities of the proteinoids were determined using a PE 343 polarimeter (PerkinElmer). All of the measure‐ ments were done in water, at 589 nm at 25°C. Table 2 shows the characteristic molecular weights, polydispersity and optical activity of the prepared proteinoids.


**Table 2. Mw, Mn, Mp, PDI and optical activity of the various proteinoids.a** The proteinoids were prepared at 180°C according to section 3.2.1; **<sup>b</sup>**molecular masses were measured by GPC, Mp is the molecular mass at the peak; **<sup>c</sup>** PDI is the polydispersity index, given by Mw/Mn;**<sup>d</sup>**specific optical rotation (c=1, in H2O, at 25°C); **<sup>e</sup>** made by microwave-assisted polymerization.

Table 2 indicates relatively low PDI values for the obtained proteinoids. This is unexpected since the polycondensation of the various amino acids is random and step-growth polymeri‐ zation processes, as in the present case, result usually in very broad size distribution polymers [50]. The highest PDI (2.32) was observed for Prot1, composed of the single amino acid Lglutamic acid, while the PDIs of the other proteinoids composed of at least 2 amino acids were ranging between 1.01 and 1.27. All of the thermally-made proteinoids have relatively high molecular masses of 26-195 kDa. This indicates that the polymerization procedure by thermal heating used here provides relatively long polymer chains. This fact may serve as an advantage for different uses later, since polymers with such high molecular weights are usually mechan‐ ically stronger and resemble natural proteins. Table 2 indicates that the lowest molecular weight was observed for the proteinoid composed of the single amino acid L-glutamic acid (Prot1) and the highest one for the proteinoid composed of L-glutamic acid, L-aspartic acid and L-lysine (Prot 5). Prot2, which was synthesized by microwave-assisted polymerization, reached an abundantly higher molecular weight. In this procedure, a 500 kDa proteinoid chain was prepared, about twice the size of the regular thermal proteinoid. This kind of procedure gives better yield over 60 min, compared to the 3 h needed usually. It can be used further for higher molecular weights and more rigid proteinoids. However, unfortunately, this kind of proteinoid does not self-assemble into spherically-shaped particles.

As further indicated from Table 2, all of the proteinoids exhibit optical activity, although the amino acid monomers are known to racemize during the thermal process [51]. This fact can become a benefit later in the design of a stereospecific drug carrier, for example.

Fourier Transform Infra-Red (FTIR) measurements of the crude proteinoids were done by the Attenuated Total Reflectance (ATR) technique, using Bruker ALPHA-FTIR QuickSnapTM sampling module equipped with Platinum ATR diamond module. All proteinoids showed characteristic peaks of NH stretching at 3360 and 2990 cm-l, amide CO stretching at 1565 cm-1, an amide NH bending band at 1450 cm-1 and CO bending at 500-700 cm-1. A representative spectrum of Prot3 is shown in Figure 4.

**Figure 4.** FTIR spectrum of Prot3.

**Proteinoida** Mw

54 Advances in Bioengineering

polymerization.

(Da) b

**Table 2. Mw, Mn, Mp, PDI and optical activity of the various proteinoids.a**

polydispersity index, given by Mw/Mn;**<sup>d</sup>**specific optical rotation (c=1, in H2O, at 25°C); **<sup>e</sup>**

proteinoid does not self-assemble into spherically-shaped particles.

Mn (Da) b

**Prot1** 26250 11300 11320 2.32 +6.5 **Prot2** 181540 144940 195300 1.25 -4.4 **Prot2e** 500240 497280 503070 1.01 +8.1 **Prot3** 164930 138250 158740 1.19 -9.0 **Prot4** 87660 84410 85250 1.04 -3.3 **Prot5** 195080 165870 191440 1.17 -7.4 **Prot6** 190390 163290 204050 1.16 -15.1 **Prot7** 72260 56880 42870 1.27 +2.8 **Prot8** 168300 156600 136800 1.07 -4.6

according to section 3.2.1; **<sup>b</sup>**molecular masses were measured by GPC, Mp is the molecular mass at the peak; **<sup>c</sup>**

Table 2 indicates relatively low PDI values for the obtained proteinoids. This is unexpected since the polycondensation of the various amino acids is random and step-growth polymeri‐ zation processes, as in the present case, result usually in very broad size distribution polymers [50]. The highest PDI (2.32) was observed for Prot1, composed of the single amino acid Lglutamic acid, while the PDIs of the other proteinoids composed of at least 2 amino acids were ranging between 1.01 and 1.27. All of the thermally-made proteinoids have relatively high molecular masses of 26-195 kDa. This indicates that the polymerization procedure by thermal heating used here provides relatively long polymer chains. This fact may serve as an advantage for different uses later, since polymers with such high molecular weights are usually mechan‐ ically stronger and resemble natural proteins. Table 2 indicates that the lowest molecular weight was observed for the proteinoid composed of the single amino acid L-glutamic acid (Prot1) and the highest one for the proteinoid composed of L-glutamic acid, L-aspartic acid and L-lysine (Prot 5). Prot2, which was synthesized by microwave-assisted polymerization, reached an abundantly higher molecular weight. In this procedure, a 500 kDa proteinoid chain was prepared, about twice the size of the regular thermal proteinoid. This kind of procedure gives better yield over 60 min, compared to the 3 h needed usually. It can be used further for higher molecular weights and more rigid proteinoids. However, unfortunately, this kind of

As further indicated from Table 2, all of the proteinoids exhibit optical activity, although the amino acid monomers are known to racemize during the thermal process [51]. This fact can

Fourier Transform Infra-Red (FTIR) measurements of the crude proteinoids were done by the Attenuated Total Reflectance (ATR) technique, using Bruker ALPHA-FTIR QuickSnapTM sampling module equipped with Platinum ATR diamond module. All proteinoids showed characteristic peaks of NH stretching at 3360 and 2990 cm-l, amide CO stretching at 1565 cm-1,

become a benefit later in the design of a stereospecific drug carrier, for example.

Mp (Da) b PDIc Optical Activity

The proteinoids were prepared at 180°C

made by microwave-assisted

[α]D25°C (°)d

PDI is the

The thermal behavior of the proteinoids was determined using Differential Scanning Calo‐ rimetry (DSC) and Thermo Gravimetric Analysis (TGA) with a TGA/DSC 1 STARe system (Mettler Toledo, Switzerland). The samples were heated between 25 - 400 °C at a rate of 10°C/ min under nitrogen atmosphere. The results are shown in Table 3.


**Table 3. Thermal properties of proteinoids produced by thermal polymerization.a** Tm and ∆Hm were measured by DSC; **<sup>b</sup>**Tdec (temperature of decomposition) was measured by TGA/DSC and refer to the exothermal peak in DSC, **c** commercial PLLA 2000 Da parameters were measured similar to the made proteinoids.

The melting temperatures of the different proteinoids range between 78-246°C. The wide range of temperatures derives from the difference in the monomeric units used in each proteinoid. When using phenylalanine, as in Prot3, 4, 6 and 7, the resulted proteinoid gains significant rigidity in the overall structure, due to the aromatic rings which allow pi-stacking. Hence, these proteinoids melt at higher temperatures. When PLLA is incorporated into the proteinoid, as in Prot8 compared to Prot3, the Tm rises mildly (103°C and 117°C, respectively), due to the presence of 2000 Da rigid polymer chains in the overall proteinoid structure.

The TGA/DSC measurements of the proteinoids show decomposition temperatures of 268-385°C. Most proteinoids lose at this temperature range around 50% of their weight The decomposition measured at 400°C of most proteinoids is between 47-64%, except Prot4 (25%). Pure PLLA decomposes at 349°C almost completely (90% weight loss). Prot8, composed of PLLA segments (10% of the total monomer), has the lowest decomposition temperature of all proteinoids (268°C). This can be explained by the non-uniformity of the structure of the whole proteinoid due to the inserted segments of 2000 Da PLLA within the random segments of polymerized amino acids.

The content of free carboxyl groups in the synthesized proteinoids is an essential factor in determining their solubility in different media, thus helping to understand their stability at different sites in the human body with different pHs. In order to determine the free carboxyl groups in the synthesized proteinoids, a titrimetric method was carried out [40]. Briefly, to a known quantity of dry proteinoid, a known excess of 0.05 N NaOH was added, followed by the addition of 37% formaldehyde solution. The unreacted NaOH was back-titrated with standard 0.05 N HCl. A blank titration was also performed. In addition, human serum albumin (HSA) was titrated for comparison. Table 6 indicates the carboxyl group contents of the synthesized proteinoids, showing higher values of 80-155 mmol/g compared with albumin. This is true also in Prot5-7, where lysine is also a part of the polymer. Moreover, aspartic and glutamic acid moieties in the proteinoids, along with lysine, impart the hydrophilic nature of the whole proteinoid. The biodegradability rate of various amino acid polymers increases with their hydrophilicity [40]. Therefore, it is more appropriate to choose these proteinoids as ideal biomaterials for drug delivery applications.


**Table 4.** Carboxyl group content in the proteinoids and albumin.

#### **5.1. Incorporation of poly(L-lactic acid) into the proteinoids**

In order to effect the chemical and physical properties of the product, a thermal polymerization of L-glutamic acid and L-phenylalanine was carried out in the presence of low molecular weight poly(L-lactic acid) (PLLA, 2000 Da). The proteinoid-PLLA (Prot8) consists of 2.25 g of each amino acid and 0.5 g of PLLA. After polymerization, it was washed, dried and charac‐ terized as described earlier. The characterization of Prot8 is included in the tables above.

in Prot8 compared to Prot3, the Tm rises mildly (103°C and 117°C, respectively), due to the

The TGA/DSC measurements of the proteinoids show decomposition temperatures of 268-385°C. Most proteinoids lose at this temperature range around 50% of their weight The decomposition measured at 400°C of most proteinoids is between 47-64%, except Prot4 (25%). Pure PLLA decomposes at 349°C almost completely (90% weight loss). Prot8, composed of PLLA segments (10% of the total monomer), has the lowest decomposition temperature of all proteinoids (268°C). This can be explained by the non-uniformity of the structure of the whole proteinoid due to the inserted segments of 2000 Da PLLA within the random segments of

The content of free carboxyl groups in the synthesized proteinoids is an essential factor in determining their solubility in different media, thus helping to understand their stability at different sites in the human body with different pHs. In order to determine the free carboxyl groups in the synthesized proteinoids, a titrimetric method was carried out [40]. Briefly, to a known quantity of dry proteinoid, a known excess of 0.05 N NaOH was added, followed by the addition of 37% formaldehyde solution. The unreacted NaOH was back-titrated with standard 0.05 N HCl. A blank titration was also performed. In addition, human serum albumin (HSA) was titrated for comparison. Table 6 indicates the carboxyl group contents of the synthesized proteinoids, showing higher values of 80-155 mmol/g compared with albumin. This is true also in Prot5-7, where lysine is also a part of the polymer. Moreover, aspartic and glutamic acid moieties in the proteinoids, along with lysine, impart the hydrophilic nature of the whole proteinoid. The biodegradability rate of various amino acid polymers increases with their hydrophilicity [40]. Therefore, it is more appropriate to choose these proteinoids as ideal

presence of 2000 Da rigid polymer chains in the overall proteinoid structure.

polymerized amino acids.

56 Advances in Bioengineering

biomaterials for drug delivery applications.

**Polypeptide [Carboxyl groups] (mmol/g)**

Albumin 56 Prot1 150 Prot2 155 Prot3 90 Prot4 122 Prot5 88 Prot6 87 Prot7 80 Prot8 102

**Table 4.** Carboxyl group content in the proteinoids and albumin.

**5.1. Incorporation of poly(L-lactic acid) into the proteinoids**

In order to effect the chemical and physical properties of the product, a thermal polymerization of L-glutamic acid and L-phenylalanine was carried out in the presence of low molecular
