**8. Summary and conclusions**

In the present study, new proteinoids were prepared using L-glutamic acid, L-aspartic acid, L-phenylalanine, L-lysine and low molecular weight poly(L-lactic acid). The polymerization was carried out by a simple straight-forward condensation polymerization in heat. The optimal conditions for the polymerization were tested, including changing the temperature and using microwave radiation. The proteinoids made are of high molecular weights with narrow size distributions and possess optical activity, which can be later used in specific drug delivery using chirality. The proteinoids are thermally stable and each proteinoid has enough carbox‐ ylic acid and/or amine functional groups, which can be later used to bind covalently desired molecules, such as drugs and dyes. The incorporation of 2000 Da PLLA into the proteinoid backbone presented a stable proteinoid as well. Proteinoids were manipulated in several ways to give proteinoid sphere-shaped nano/micro-particles, and to optimize this process. Specific conditions were found for the production of hollow particles of narrow size distribution. The incorporation of PLLA segments into the proteinoids increased the hydrophobic interior part and resulted in smaller size hollow particles. The particles were found to be non-toxic and stable over time. Also, encapsulation of different materials was carried out, giving organicfilled particles and fluorescent particles. In summary, proteinoid formation and cytotoxicity tests indicate that these particles are suitable for further in vivo testing.

The copolymer Prot8 containing 10% PLLA, was chosen to be used for the biomedical study, since it provided the smallest particles with the biodegradability derived from the addition of PLLA to the proteinoid backbone. The proteinoid-PLLA copolymer was self-assembled in the presence of the NIR fluorescent dye ICG to yield NIR fluorescent Prot8 particles of 70 ± 15 nm dry diameter. These new NIR fluorescent particles were found to be stable, avoiding leakage and photobleaching of the NIR dye over time. Furthermore, these particles are non-toxic, as shown by LDH and XTT assays for cytotoxicity and cell viability on human colorectal adenocarcinoma cell lines. The particles biodistribution in a mouse model was tested, follow‐ ing an i.v. injection, and the results showed that the particles penetrate a variety of organs, including the brain and bones. Nevertheless, 1 h post injection, the particles concentrate at specific sites of the body and are evacuated almost completely over 24 h. It was established, using the chicken embryo CAM model and the tumor-implanted mouse model, that the NIR fluorescent particles may be very useful for tumor diagnosis in vivo due to their low auto‐ fluoresence of the background and the deep penetration into biomatrices. In the CAM model, when the NIR fluorescent proteinoid-PLLA particles are conjugated to a bioactive ligand (PNA or anti-CEA), they preserve their activity and specifically detect the cancer cells with upregu‐ lated receptors (LS174t vs. SW480). Non-conjugated and anti-rabbit IgG-conjugated NIR fluorescent particles (control particles) marked the cells without specific recognition at a much lower fluorescence intensity. In the mouse model, human tumor cells were implanted in mice colons, and the mice were treated with the NIR fluorescent particles. The results showed that the anti-CEA-conjugated particles specifically label the colon tumors, as opposed to those conjugated with anti-rabbit IgG antibodies.

In summary, the chapter introduced a new series of newly-made proteinoids of a narrow selection of amino acid monomers, with high molecular weight and very narrow polydisper‐ sity index. Furthermore, this chapter has described proteinoid fluorescent particles as potential probes for precancerous colorectal adenomatous polyps or colon cancer tumors. Selective and specific fluorescent labelling of tumors is possible, with very low background signal, however improvements are still necessary for optimizing the targeting efficiency of the particles. Recent and ongoing developments in fluorescence imaging systems will open up the scope for effective detection of fluorescently labelled neoplasm. The proteinoid particles may further be developed to serve for both diagnostics and therapy combined.
