molecules Article Incorporation of Magnetic Nanoparticles into Protoplasts of Microalgae Haematococcus pluvialis: A Tool for Biotechnological Applications Maria G. Savvidou 1 , Angelo Ferraro 1 , Evangelos Hristoforou 2 , Diomi Mamma 1 , Dimitris Kekos 1 and Fragiskos N. Kolisis 1, * 1 Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str, Zografou Campus, 15780 Athens, Greece; msavvid@central.ntua.gr (M.G.S.); an.ferraro2@gmail.com (A.F.); dmamma@chemeng.ntua.gr (D.M.); kekos@chemeng.ntua.gr (D.K.) 2 Laboratory of Electronic Sensors, School of Electrical and Computer Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str, Zografou Campus, 15780 Athens, Greece; hristoforou@ece.ntua.gr * Correspondence: kolisis@chemeng.ntua.gr; Tel.: +30-210-772-3156 Academic Editor: Derek J. McPhee Received: 27 September 2020; Accepted: 30 October 2020; Published: 1 November 2020   Abstract: Intensive research on the use of magnetic nanoparticles for biotechnological applications of microalgae biomass guided the development of proper treatment to successfully incorporate them into these single-cell microorganisms. Protoplasts, as cells lacking a cell wall, are extensively used in plant/microalgae genetic manipulation as well as various biotechnological applications. In this work, a detailed study on the formation of protoplasts from Haematococcus pluvialis with the use of enzymatic and mechanical procedures was performed. The optimization of several parameters affecting the formation of protoplasmic cells and cell recovery was investigated. In the enzymatic treatment, a solution of cellulase was studied at different time points of incubation, whereas in the mechanical treatment, glass beads vortexing was used. Mechanical treatment gave better results in comparison to the enzymatic one. Concerning the cell recovery, after the protoplast formation, it was found to be similar in both methods used; cell viability was not investigated. To enhance the protoplast cell wall reconstruction, different “recovery media” with an organic source of carbon or nitrogen were used. Cell morphology during all treatments was evaluated by electron microscopy. The optimal conditions found for protoplast formation and cell reconstruction were successfully used to produce Haematococcus pluvialis cells with magnetic properties. Keywords: magnetic nanoparticles; microalgae; Haematococcus pluvialis; protoplasts transformation; protoplasts regeneration; cellulose treatment 1. Introduction Magnetic nanoparticles are a new trend in various scientific fields, such as drug delivery, DNA/RNA purification, improved magnetic resonance imaging (MRI), immobilization, food industry, medical diagnostics, cell harvesting, bioremediation, and others [1–4]. They are a class of particles consisting of a magnetic and a functional chemical component with a diameter varying from 1 to 100 nm and can be controlled by magnetic fields displaying superparamagnetism. Haematococcus pluvialis (H. pluvialis) is a microalgae strain rich in astaxanthin as well as lipids (20–25% per dry weight with 10% of it being polyunsaturated fatty acids), proteins (29–45% per dry weight), and carotenoids (2–5% per dry weight). Its ability to grow even in extreme conditions makes it a strategic tool for human diet and animal feeding and can be used even for anticancer and anti-inflammatory purposes [5]. Skin, heart and eye health, and photoprotection are also fields where H. pluvialis is an important implement [6]. Molecules 2020, 25, 5068; doi:10.3390/molecules25215068 www.mdpi.com/journal/molecules