**4. Discussion**

In recent years, several approaches have been proposed to employ "like-a-brush" LOCs as useful tools in the field of NA-based formulations [10,14,18,22,33,35,53–56]. Most of them rely on the conjugates bearing the diacyl lipid group. A few data have been reported on the features of triple lipophilic chains-tethered oligonucleotides [14,33]. Their investigations are generally limited by high hydrophobicity, fast self-aggregation, and salting out in aqueous solution. So far, only one paper reports two 15-mer oligoadenylate and oligothymidylate LOCs containing three "like-a-brush" hydrophobic chains [14]. The particle size and shape for the latter conjugate were characterized by DLS, TEM, and scanning electron microscopy, while highly hydrophobic oligoadenylate LOC irreversibly self-aggregated immediately after the deblocking procedure [14].

The present research aimed to investigate 13-, 17-, and 22-mer "like-a-brush" triple chains- contained DOCs for their self-assembly features and the ability to enter the cells. We have chosen heteronucleotide sequences for these oligomers and inserted two phosphoryl guanidine modifications at 3- -end of some conjugates during standard phosphoramidite solid-phase synthesis to provide additional nuclease stability [37,39]. Due to high hydrophobicity, DOCs form micellar particles in micromolar concentrations and require extreme caution to avoid their salting out during the work.

We observed a larger size of D-13 and D-17 DOC particles as compared with another 15-mer oligothymidylate LOC, also containing "like-a-brush" hydrophobic moiety with three aliphatic tails [14]. In turn, the conjugates containing phosphoryl guanidine modifications D-13PG, D-17PG, D-22PG formed larger micellar assemblies than their DNA counterparts. There is a little discussion in the literature that tends to focus on the oligonucleotide length, sequence, and modified sugar-phosphate backbone of the LOC regarding the size of a micelle formed. The research also reported various sizes of LOC self-assemblies, which demonstrated the required biological effect. The impact of nucleotide sequence on LOC micellar assemblies' size was described for two 19-mer PS conjugates bearing identical diacyl lipid groups at their 5- -ends. While the oligomer 5- -AACTTGTTTCCTGCAGGTGA-3 formed small particles of ~11 nm, 5- -CGTGTAGGTACGGCAGATC-3 yielded micellar structures with the size of more than 100 nm [10]. Recently it was shown [11] that BODIPY-conjugated 10- and 25-mer oligodeoxythymidylates self-aggregate with a formation of the same assemblies of 94.1 ± 20.4 and 75.5 ± 4.4 nm, correspondingly. Self-association of siRNA with one BODIPY-attached strand into nanosized aggregates of ~140 nm in aqueous solution was shown to be indispensable for the high cellular uptake of these duplexes and efficient gene regulation by RNA interference [11]. The siRNA-squalene conjugates also demonstrated self-organizing in water with the formation of ~165 nm particles, and after intravenous injections, inhibited tumor growth in a mice xenograft model of papillary thyroid carcinoma [21]. Most reports deal with the development of LOC-based constructions for anticancer therapy [10,13,21,23,24,29,35]. Passive targeting via enhanced permeability and retention (EPR) effect requires a 5–200 nm hydrodynamic size range for the formulations [57,58]. Therefore, the ability of DOCs to self-assembly into micellar particles and their aggregates of 30–170 nm in size seems to open the way for further in vitro and in vivo studies to examine their cell penetration efficacy.

Given the high plasma concentration of albumin in vivo, we evaluated the affinity of DOCs for association with BSA. Other findings confirm our BSA binding experiments. It was reported that approximately 93% of LOC micelles dissociated into a non-micellar state when incubated in vitro with 0.1 mM BSA [22]. Size exclusion chromatography method has shown that diacyl lipid conjugated PS oligonucleotide in aqueous solution elutes as micelles, but after the following incubation with FBS, nearly 50% of this conjugate co-migrate with the albumin fraction [54]. In another work, siRNA bearing short-chain fatty acid residues such as lauroyl did not bind to lipoproteins in vivo, either associated with serum albumin or remained unbound [26]. Interestingly, monododecyl-containing siRNA bound

to albumin with the highest affinity (Kd ~200 μM) among other investigated lipophilic residues, and the protein was saturated with these conjugates at 1:3.6 molar ratio [26].

The readily occurred interaction of DOCs with albumin can increase their overall in vivo lifetime [5]. An analogy can be drawn with recent reviews regarding more extensively studied interaction of PS modified therapeutics with proteins [59,60]. Oligonucleotides modified in PS backbone bind to a number of plasma proteins, including albumin. Plasma proteins binding increase the circulation half-life of PS oligomers which is crucial to maintain their distribution to peripheral tissues [60].

On the other hand, a well-known criticism of increased affinity of LOCs for binding by albumin refers to observations of subsequent conjugates accumulation in the liver and lymph nodes than in other organs [54,61]. To find a possible solution to this challenge, an interesting experiment was carried out with stable G-quadruplex-locked DNA micelles, which could not associate with serum albumin [22]. Cellular uptake of these conjugates was negligible in comparison with DNA micelles without intermolecular G-quadruplexes, which retain the ability to albumin bind [22].

In contrast to this limitation, other studies established albumin as a drug carrier [62]. Of particular interest are several features of albumin, which are responsible for the accumulation of this plasma protein in solid tumors [34,63]. The Paclitaxel albumin-bound particles known as Abraxane® are used in cancer therapy [62]. Recent research [35] suggested using in situ albumin targeting for development of carrier-free RNAi-based cancer therapies. The synthesized siRNA conjugated to a diacyl lipid moiety, which rapidly binds albumin in situ, was shown to achieve 19-fold greater tumor accumulation and 46-fold increase in per-tumor-cell uptake in a mouse orthotopic model of human triple-negative breast cancer as well as elicits sustained silencing in an in vivo tumor model [35]. The tumor:liver accumulation ratio of more than 40:1 achieved by this diacyl lipid-tethered siRNA is a promising result for LOC-based formulations. Future studies in this area are therefore required more attention to design of the research. Although most LOCs bind to albumin with high affinity, studies reporting the effect on intracellular uptake during the transfection are scarce. Recently it was found that BSA treatment reduces cell penetration of PS ASOs and their lipid conjugates in a protein concentration-dependent manner [25]. Interestingly, the cellular uptake efficacy of the lipid-conjugated PS ASO was reduced much stronger than that of the parental unconjugated oligonucleotide. Almost all other articles describe cells in vitro transfection in the absence of serum and/or albumin in the media [10,11,21,22,25], although understanding the features of interactions between LOCs and proteins is crucial for their use in vivo.

Taken together, in this study, we obtained key results proving that DOCs represent the attractive objects for further design of transport systems in oligonucleotide-based therapeutics. For future in vivo research of the DOCs, a principal issue is to evaluate the importance of the ability to form spherical micellar particles in aqueous media in front of their high protein binding affinity.
