Self-Dividing Micelles: A Mechanistic Look with Evolutionary and Clinical Implications

Micellar therapy has become a usefully viable treatment arm in various fields, ranging from oncology to bioimaging. As such, research leading to any improvements or adaptations in administration and techniques can have far-reaching consequences. Potential aspects of prebiotic chemistry may also be explored in such research as well. To that end, proof-of-concept experiments were performed to elucidate a possible mechanism of action for prebiotic protocell division. Representative potentially prebiotically plausible biomolecules, i.e., a fatty acid, amino acid, and nucleotide were mixed and heated in water and subjected to microscopic examination for observation of possible self-division and laboratory testing for the presence of polypeptides and polynucleotides (Biuret, MALDI mass-spec, etc.) with and without the presence of nucleotide. The results are presented for the first time here and a mechanism is proposed that best fits the data obtained. The evolutionary, e.g., prebiotic biomolecular cooperativity, and clinical, e.g., potential antineoplastic micellar/vesicular therapy, ramifications are discussed as well.


Introduction
Micellar therapy has become a usefully viable treatment arm in various fields, ranging from oncology to bioimaging [1]. As such, research leading to any improvements or adaptations in administration and techniques can have farreaching consequences. Potential aspects of prebiotic chemistry may also be explored in such research as well. To that end, proof-ofconcept experiments were performed to elucidate a possible mechanism of action for prebiotic protocell division. Although successful pioneering research in this area has been previously performed by Dreamer [2] and Szostak [3] among others, to this author's knowledge none has satisfactorily addressed 1) the dependence upon nucleic acid for protocell self-division, 2) the resultant production of other prebiotically relevant biomolecules, i.e., ATP, that further promulgate the self-division process, and 3) the recycling of the process to promulgate further self-divisions. This paper addresses all three. The results are presented for the first time here and a mechanism is proposed that best fits the data obtained. The evolutionary, e.g., prebiotic biomolecular cooperativity, and clinical, e.g., potential Page: 9 www.raftpubs.com antineoplastic micellar/vesicular therapy, ramifications are discussed as well.

Experimental
All research was conducted at Blue Ridge Community and Technical College in Martinsburg, WV, USA from April of 2018 through January of 2019. All pH measurements were made using a Horiba TwinpH waterproof B-213 Compact pH meter. Glass microscope well slides were procured through American Science and Surplus. Zeiss Primo Star binocular microscope and corresponding software equipment was used for imaging. All chemical supplies were purchased from Fisher Scientific. All Pyrex glassware was sterilized at 130° C for one hour [4] via autoclave using a Quincy Lab Inc. Model 30 GC Lab Oven. All measurements of chemicals were standardized to 0.1 Molarity (M) ± 5% using an Ohaus Analytical Plus electronic balance accurate to within ± 0.0001 gram (g). Three trials per step were performed and recorded with the data presented here representing the average of that total data. All data collected fell within a statistically acceptable ± 5% (p=0.05) internal margin of variance [5] with no outliers using Microsoft Excel Analyze-it add-in software. Concentrations of chemicals used were sufficient to achieve critical micelle concentration (CMC) as per Fisher Scientific product information. To 30 milliliters (mL) of sterile deionized water adjusted to a pH of 7.0 via dropwise titration with 0.1 M hydrochloric acid (HCl) and 0.1 M sodium hydroxide (NaOH) to facilitate imidazolium ion formation in L-histidine (His) [6] was added 0.3 gram (g) of n-Decyl-β-D-malt side and 0.1 g of His and heated to 100° Celsius (C) for 10 minutes (min) while stirred with a magnetic stirring rod. Samples were procured via dropper and subjected to a Biuret Test for polypeptides, with additional samples added to microscope well slides with methylene blue dye added to aid microscopic visualization. Observations were made at 100x oil immersion magnification and recorded. 0.1 g of adenosine 5ʹ-monophosphate (AMP) sodium ( Figure 1) was then added to the original mixture and heated to 100° C for 10 min while stirred with a magnetic stirring rod. Samples were procured via dropper and added to microscope well slides with methylene blue dye added. Observations were then made at 100x oil immersion magnification and recorded.

Results and Discussion
Samples sans dye without AMP pre-and postheating were subjected to a Biuret Test [7] in sterile test tubes and revealed the following:       Page: 11 www.raftpubs.com as a representative fatty acid-like nonionic detergent amenable to MALDI spectroscopy (Fisher Scientific product information); His was chosen as a representative prebiotically plausible amino acid [8] and due to its dual polymerizing and cleaving capacity as a polyimide [9]; AMP was chosen as a representative prebiotically plausible nucleotide [10] and due to its being the direct precursor of adenosine 5ʹ-triphosphate (ATP) [11] (Figure 8). Heating was applied to induce amino acid and nucleotide polymerization via condensation dehydration reactions and appears to have been successful as evidenced by positive Biuret tests and MALDI spectroscopy peaks in the blue 1/20 dilution middle line of Figure 7 at 1380 and 990 m/z, respectively indicating a 10-mer polymer decapeptide of protonated poly-L-histidine imidazolium ions [12] and a 3mer polymer trinucleotide of protonated polyadenylate [13]. Interestingly, the peak at 507 m/z in the green 1/200 dilution top line indicates the presence of unhydrolyzed ATP [14] and suggests a possible mechanism for not only the robust polymerization of short nucleotides that mimics nature's own, but Page: 12 www.raftpubs.com for micelle self-division as well. The mechanism proposed here begins with the heat-induced condensation dehydration polymerization of individual His molecules into 10-mer protonated imidazolium ion (Figure 9) poly-L-histidine decapeptides (Figure 10), present in the micelles pictured in Figure 3, with the partially negative protonated hydroxy dipole moiety of malt side on the surface of the micelle forming an electrostatic or possibly even hydrogen bond with the partially positive protonated amino dipole moiety adjacent to the α-carbon of the poly-L-histidine ( Figure 11).   Page: 13 www.raftpubs.com With the addition of AMP, the negatively charged oxygens of the molecule's phosphate moieties align with the positively charged imidazolium ions of the poly-L-histidine decapeptide ( Figure 12) to accommodate at least nine AMPs per poly-L-histidine decapeptide which would facilitate, accompanied by the hydrolysis of six adenosine moieties out of every nine AMPs, the formation of three ATP molecules. Such accommodation of at least nine AMPs and subsequently three ATPs would mimic nature and facilitate the formation of polyadenylate trinucleotide accompanied by the hydrolysis of three inorganic pyrophosphate (PPi) molecules, inducing the momentary inversion of the micelle that would facilitate both release of the ATP and polyadenylate trinucleotide respectively, and ultimately self-division of the micelle (Figure 13). The detection of any adenosine moieties and PPi molecules fell below the limits of the MALDI analysis performed here.   Figure 11) that, with the addition of at least 9 AMP molecules, inverts the micelle (B) (inverted surface proposed structure pictured in Figure 14), polymerizing the AMP and hydrolyzing 6 adenosine moieties, forming 3 ATP molecules which subsequently hydrolyze 3 PPi, synthesizing the 3-mer trinucleotide triadenylate, and forms two "daughter" micelles (C), both now with the same outer surface structure as A. Page: 14 www.raftpubs.com

Evolutionary Implications
As previouosly discussed, all materials used except for the fatty acid-like n-Decyl-β-Dmaltoside are prebiotically plausible molecules. Fatty acids themselves, however, are indeed prebiotically plausible molecules [15], and to that end were also employed in these experiments using mono-and polyunsaturated fatty acids, L-lysine, and AMP (see Supplemental Figures S1-S4), as well as n-Decyl-β-D-maltoside, L-lysine, and AMP (see Supplemental Figures S5-S9) with mixed results. Interestingly in all cases though, similar self-dividing protocell vesicle/micelle interactions were observed microscopically with both monolayer and bilayer surfaces, pointing to the reasonable conclusion that life likely arose via inclusive cooperation among prebiotically present biomolecules such as fatty acids, amino acids, and nucleotides under condensing dehydrating conditions rather than the more exclusive prevailing theories like the RNA World Hypothesis. Also interesting is the preferential synthesis of triplet trinucleotides, implicated in everything from the possible origins of metabolism [16], through triplet nucleotide sequences having been shown experimentally to act as templates for RNAcatalyzed RNA ribozymes when nucleotide triphosphates were used as substrates [17], to the highly likely 93-nucleotide primordial Page: 15 www.raftpubs.com precursor transfer RNA (tRNA) molecule proposed by Burton et al [18].

Clinical Implications and Prospectus
One of the clinical implications of either selfdividing monolayer micelles or bilayer liposomes is arguably obvious, with the former delivering lipophilic chemotherapeutic antineoplastic agents and the latter delivering hydrophilic ones, offering a distinct advantage over their current non-dividing counterparts presently in use, providing significantly improved coverage over a much larger surface area, particularly in treating solid tumors. The choice of polypeptide employed may also not need be limited to poly-L-histidine but can also be expanded to include any of the basic sidechain amino acids, such as the nowpredominant poly-L-lysine, as well as possibly poly-L-arginine. Another not as readily apparent implication involves the potential clinical application of the triplet trinucleotides themselves, specifically the possible use of messenger RNA (mRNA) stop codons. With the highly touted mRNA technology currently being successfully incorporated into several of the SARS-CoV-2 (Covid-19) vaccines now in production, the prospect of the use of this technology in potential cancer vaccines is presently in various stages of implementation [19]. The proposition is therefore thus put forth here for the possible development of an mRNA cancer vaccine or vaccines utilizing self-dividing micelles and/or liposomes using only stop codons for the treatment and destruction of rapidly-dividing solid tumors, after which time clinical trials assessing safety and efficacy would be justified.    Page: 17 www.raftpubs.com Figure S3: Zoom in to show poly-asparagine standardpeaks separated by mass ~114 which is the residue mass of asparagine (asparagine can also deaminate to aspartate which has a residue mass of 115). Poly-lysine should have a repeating unit of mass 128. There is not a clear indication of this in the 'A' sample dilutions that were tried. Figure S4: Prepared as last time, using a ZipTipC18 pipette tip to desalt the samples. Could detect the oligonucleotide standard but could not see any obvious signal for sample B in negative ion mode.  Page: 18 www.raftpubs.com Figure S6: From left to right: negative Biuret tests for n-Decyl-β-D-maltoside in sterile deionized water at room temperature and heated to 100° C; minimally positive Biuret test for poly-L-lysine in n-Decylβ-D-maltoside/Lys, highly positive Biuret test for poly-L-lysine in n-Decyl-β-D-maltoside heated to 100° C, and another minimally positive Biuret test after addition of AMP.   Page: 19 www.raftpubs.com Figure S9: Diluted sample A and analyzed by LCMS using a reversed phase C18 column with an ionpairing (perfluoroheptanoic acid) containing mobile phase to get lysine retained. The MS was set up to look for lysine (m/z 147) and several polylysine masses (275, 403, 531, and 659). Only a lysine peak was seen. Scanning up to higher mass (m/z 1500) did not reveal anything either.