A Method for Producing Highly Pure Magnetosomes in Large Quantity for Medical Applications Using Magnetospirillum gryphiswaldense MSR-1 Magnetotactic Bacteria Amplified in Minimal Growth Media

Abstract

We report the synthesis in large quantity of highly pure magnetosomes for medical applications. For that, magnetosomes are produced by MSR-1 Magnetospirillum gryphiswaldense magnetotactic bacteria using minimal growth media devoid of uncharacterized and toxic products prohibited by pharmaceutical regulation, i.e., yeast extract, heavy metals different from iron, and carcinogenic, mutagenic and reprotoxic agents. This method follows two steps, during which bacteria are first pre-amplified without producing magnetosomes and are then fed with an iron source to synthesize magnetosomes, yielding, after 50 h of growth, an equivalent OD₅₆₅ of ~8 and 10 mg of magnetosomes in iron per liter of growth media. Compared with magnetosomes produced in non-minimal growth media, those particles have lower concentrations in metals other than iron. Very significant reduction or disappearance in magnetosome composition of zinc, manganese, barium, and aluminum are observed. This new synthesis method paves the way towards the production of magnetosomes for medical applications.

Keywords: iron incorporation; magnetosome; magnetotactic bacteria; metals; minimal medium; yeast extract.

Figure 1

(A) Cell growth curve in a 70 L fermenter, represented by the variation of cell dry weight and OD₅₆₅ (measurement error < 5%) as a function of time during the growth step. (B) Consumed L lactic acid (L-LA) and ammonium (${\text{NH}}_4^{+}$) as a function of time during the growth step, estimated in gram of L-LA per gram of bacteria and gram of ${\text{NH}}_4^{+}$ per gram of bacteria. The dissolved O₂, dO₂, was maintained between 0.1 and 1% by the regulation of airflow, after which it decreased from 12% at 0 h (T = 0 h) to below 1% at 6 h (T = 6 h). The magnetic response is negative before 20 h (T = 20 h) and positive after. These analyses were performed in triplicate. The error bars represent standard deviations.

Figure 2

Transmission electron microscopy (TEM) images of a typical magnetotactic bacterium 0 h (T = 0 h), 23 h (T = 23 h), or 50 h (T = 50 h), showing a few individual magnetosomes at T = 0 h, an incompletely formed chain of magnetosomes at T = 23 h, and a fully formed chain of magnetosomes at T = 50 h. To carry out these observations, suspensions containing whole magnetotactic bacteria were deposited and dried on top of a carbon grid and then observed by TEM.

Figure 3

(A) Evolution of the repartition of iron during the 50 h of fermentation, which is divided between: (i) iron inside MTB in magnetosome chain form, (ii) iron outside MTB, and (iii) iron inside MTB both in magnetosome chains and other iron forms, where the quantities of the three different types of iron are estimated in μg of iron per mL of growth medium. The error bars represent standard deviations. Standard deviations values are, respectively: 0.005, 0.003, 0.006, 0.014, 0.222, and 0.066 at 20, 23, 26, 44, 47, and 50 h for iron inside MTB under magnetosome chains form; 0.001, 0.003, 0.002, 0.002, 0.010, 0.054, 0.040, 0.105 at 1, 3, 20, 23, 26, 44, 47, and 50 h for iron outside MTB; 1.10⁻⁴, 1.10⁻⁴, 1.10⁻⁴, 0.021, 0.012, 0.041, 0.383, 0.174, 0.179 at 0, 1, 3, 20, 23, 26, 44, 47, and 50 h for iron inside MTB. (B), Variation of the quantity of iron in magnetosome chain (MC) form, estimated in mg of magnetosome chains per gram of MTB, during the 50 h of the growth step. The error bars represent standard deviations. Standard deviations values are, respectively, 0.012, 0.004, 0.006, 0.007, 0.094, 0.029 at 20, 23, 26, 44, 47, and 50 h. T Extracellular iron is estimated in the supernate obtained after centrifugation of whole MTB. Total intracellular iron is obtained by estimating the quantity of iron in the bacterial pellet obtained after centrifugation. The intracellular iron, which is in the crystallized form (magnetosome chains) inside MTB, is obtained by estimating the quantity of iron in magnetosomes extracted from whole MTB, which are obtained following lysis of whole MTB. These analyses were performed in triplicates.

Figure 4

Evolution during the course of the growth step of the percentage in mass of iron in magnetosome chains (MC) relatively to the mass of all analyzed metals (Ag, Al, As, Ba, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sb, Se, Si, Ti, Tl, W, Zn) in magnetosome chains. These analyses were performed in triplicates. The error bars represent standard deviations. Standard deviations values are, respectively, 0.016, 0.009, 0.002, 7.10⁻⁴, 0.001, 4.10⁻⁴ at 20, 23, 26, 44, 47, and 50 h.