**3. Proteinoid microspheres**

biodegradable polymers by synthesizing new proteinoids carrying various stereogenic centers. The main goal is to provide a large choice of biodegradable proteinoids with predetermined characteristics, while using amino acids as building blocks. Hence, the new selection is based on chiral building blocks carrying positive/negative charge, hydrophilic/ hydrophobic nature or any desired combination of the above [16]. Homopolymers, random copolymers and block-copolymers of two amino acids or more can be designed and synthesized, presenting thereby new proteinoid materials with specific, desired nature. The large variety of amino acids, either natural or synthetic, makes it possible to obtain a large library of different proteinoids, by simply changing the amino acids ratios. One protei‐ noid is completely different from another, by the fact that they are made of different amino acid monomers. This fact provides each proteinoid special features, and possibly influen‐

**Figure 1.** Thermal polymerization of amino acids through pyroglutamic acid catalysis.

Up until now, most, if not all, of the reported proteinoids in the literature were synthesized from at least four amino acids and possessed relatively low molecular weights [21,22]. Thus, high molecular weight proteinoids of narrow size distribution made of two to three natural amino acids, along with proteinoids containing poly(L-lactic acid) (PLLA) segments are presented in this study. Furthermore, the study presents NIR fluorescent proteinoid-based particles, which can be used for colon cancer detection. Some of the methods used have been

**2. Incorporation of poly(L-lactic acid) into the proteinoid backbone**

Poly(lactic acid) or PLA is the thermoplastic aliphatic polyester of lactic acid, derived from renewable sources. It is the one of the most important bioplastics in the world in terms of

ces the character of particles made from it [17-20].

published in a previous work [23].

50 Advances in Bioengineering

After preparation, the crude proteinoids can self-assemble to form micro- and nano-sized particles [33, 34]. As opposed to polystyrene microparticles, for example, which are formed during the polymerization process of styrene in the presence of a surfactant [35], the proteinoid particles are formed through a self-assembly process. This process is complet‐ ed only after the polymerization itself. The procedure involves either dissolving the dried crude proteinoid in water by heating and then cooling slowly,[36] or by pH changes of the proteinoid solution in water [37]. This way, particles of the size range of several nanome‐ ters to 10 μm may be formed [38].

The idea standing behind the unique self-assembly process lies in the existence of many functional groups as a part of the random polymer backbone. The preparation of proteinoid microspheres does not require the addition of any toxic cross-linking agents or surfactants. The conversion of the polypeptidic proteinoid solution into particles with a slight change of the environment involves a complex process of functional group changes occurring in the polymer. The great amount of carboxylates in the backbone, or aminium ions when dealing with a lysine-rich proteinoid, cause the solubility of the proteinoid in certain conditions and insolubility in the reverse conditions [39]. The hydrophobic portions of the proteinoid are assembled within the particle matrix, and the carboxylic acid moieties are exposed, as illustrated in Figure 2 [40].

When the self-assembly procedure is done in the presence of a suitable molecule such as drug or dye, a proteinoid particle containing the molecule is formed [41]. The proteinoids may be used to encapsulate materials such as drugs for drug delivery purposes, e.g. for the oral delivery of methotrexate [40], Hydroxyapetite [42], Cholesterol [37] and for diagnostics [21,43-48].

**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 amine groups.
