Synthesis and Characterization of New Biocompatible Amino Amphiphilic Compounds Derived from Oleic Acid as Nanovectors for Drug Delivery

Amphiphilic molecules have been actively explored as promising materials in the field of bio and nanotechnology. These molecules are constituted by a polar head and a lipophilic tail and in an aqueous medium are self-assemble to form different types of macromolecular structures such as micelles, monolayer vesicles, bars, sheets and tubes. In this work, a convergent synthetic approach for the synthesis of two new amphiphilic compounds based on a versatile amino polar head, a tetraethylene glycol spacer and a lipophilic tail derived from oleic acid has been developed. Subsequently, after a self-assembly process in aqueous medium, nanostructures as micelles have been obtained and characterized. Finally, a procedure for the inclusion of the highly lipophilic drug Dexamethasone has been carried out in order to study the ability of these micelles to act as nanovectors for drug delivery.


Introduction
In the last years, amphiphilic molecules have been highly used in the development of nanostructures, representing a great promise for targeted delivery, improved bioavailability, and drugs controlled release 1-7]. These molecules consist of a polar head and a lipophilic tail that are distributed in an aqueous medium to form different types of structures such as micelles, monolayer vesicles (also known as liposomes), bars, sheets and tubes 8]. The cell membrane of living cells, formed by a bilayer self-organization is the most illustrative example of a complex nanosystem formed from units of phospholipid. Among the different types of structures formed by amphiphilic compounds, micelles have received growing scientific attention 9]. Micelles, in general, are selfassembled particles composed of amphiphilic compounds 10]. In an aqueous environment, these compounds are distributed with the hydrophobic tails in the centre of the micelle, and the polar heads towards the aqueous medium. In this way, they auto-assemble to form spheroidal structures with a hydrophilic shell and a hydrophobic core to minimize the contact of the hydrophobic segments with the aqueous environment by allowing a good grade of stability 11]. CMC, critical micelle concentration, is a relatively small range of concentrations separating the limit below which virtually no micelles are detected and the limit above which virtually all additional amphiphilic molecules form micelles.
Micelles have a particle size between 10 and 100 nm that is important to allow a high stability and a high bioavailability. This size makes it possible to inject these micelles into systemic circulation without risk of blood vessel blockage. The fate in vivo of micelles depends on their sizes, particles under 200 nm are less phagocytosed by macrophages after the opsonization, compared to those with larger dimensions 12].
One of the advantages of using micelles in drug delivery is their ability to transport drugs with different degrees of polarity thanks to their structure consisting in a hydrophilic shell and a hydrophobic core.
Drugs will be distributed differently by chemical conjugation, physical entrapment or ionic interactions depending on the nature of the drug and the amphiphilic compound properties: Hydrophilic drugs will bind to the surface (case 1), those with different hydrophilicity and lipophilicity ratios will be between the polar part and the lipophilic part of the nanosystem (case 2), and finally very lipophilic drugs will be distributed inside the micelle core (case 3) (Figure 1) 9]. It is known that about 90% of drugs are lipophilic and are characterized by a low solubility in water, this causing a difficult distribution and accumulation in fatty tissues leading to a delayed release of the drug and an increase of side effects. Micelles are therefore used for the transport of highly lipophilic drugs, increasing the solubility of drugs from 10 to 8400 times and consequently their bioavailability 13,14].
One of the most successful examples of micellar formulation as alternative solubilizing agents is the formulation of paclitaxel (PTX) in a poly (D, L-lactide) MePEG diblock copolymer which increases the solubilized PTX levels in water around 5000 times 15].
In summary, the main advantages of micelles in drug delivery are the following: i) high dynamic stability that permits their application in vivo ii) the hydrophobic core of micelles confers them the ability to transport highly lipophilic drugs iii) the hydrophilic shell of micelles increases their solubility in water, resulting in greater bioavailability and lower toxicity for poorly water-soluble drugs. iv) the possibility of modifying their surface with specific ligands confers the ability to direct drugs to specific targets.
This work is placed in the field of nanotechnologies applied to drug delivery and specifically focused on the synthesis of amphiphilic compounds in order to obtain a new family of micelles as drug nanovectors within the organism.
Both of these amphiphilic compounds synthesized present a versatile amino polar head, a spacer and a lipophilic tail. The spacer in all cases is tetraethylene glycol, a polymer derived from polyethylene glycol which presents two important advantages: (i) an adequate hydrophilichydrophobic balance for the optimal formation of the micelle and (ii) the ability to avoid the activation of the immune system. It has been discovered that PEG derivatives are biocompatible and they are not attacked by macrophages escaping the opsonization. Furthermore, the amphiphilic compounds synthesized present oleic acid as the lipophilic tail (Scheme 1). Scheme 1. General structure of the amphiphilic compounds.
Micelles have been obtained in water by a self-assembly process of the amphiphilic compounds synthesized. After the characterization of micelles, the internalization of Dexamethasone, a synthetic highly lipophilic anti-inflammatory corticosteroid drug 23], has been studied in order to verify the advantages of the use of our micelles in drug delivery (Scheme 2). Scheme 2. Formation of micelles by supramolecular self-assembly of amphiphilic compounds and internalization of Dexamethasone.

Materials and Techniques
Unless otherwise stated, the starting materials, reagents, and solvents were purchased as highgrade commercial products from Sigma-Aldrich. THF, CH2Cl2, DMF and Toluene were dried using molecular sieves, and highest quality solvents were used. All non-aqueous reactions were performed under an argon atmosphere in oven-dried glassware.
Analytical TLC was run on silica gel plates supported by alluminio Alufram® Sil.G / V245 Merck di 0.25 nm. Plates eluted and dried with 5% of phosphomolybdic acid in ethanol.
Flash chromatography was performed on glass column using silica gel type 60 (particle size 230-400 mesh, Merck). The composition of the eluent used is different for each compound, as indicated.
High resolution mass spectrometry (HRMS) was carried out on a Kratos MS-80-RFA spectrometer and in a AutoSpec micro-mass spectrometer at the centre of Research, Technology and Innovation of the University of Seville.

Synthesis of the Amphiphilic Derivatives
Synthesis of the First Amphiphilic Derivative

1,11-diazido-3,6,9-trioxaundecane (2)
To a solution of 1 (18.80 g, 53.66 mmol) in dry EtOH (35.8 mL) was added sodium azide (8.72 g, 134.15 mmol). The mixture was allowed to reflux during 24 h, under argon atmosphere and after this time, was added NaCl (50 mL) to deactivate the sodium azide. Then the solvent (EtOH) was removed by rotary evaporation. Successively the mixture was extracted with CH2Cl2 in order to obtain the product in the organic phase, and after all, was evaporated the solvent. Then the crude product was purified by flash chromatography column on silica gel with AcOEt: Hexane (1:2), to yield 2 as a yellowish oil (11.9 g, 48.71 mmol, 91%).

11-azido-3,6,9-trioxaundecan-1-amine (3)
To a solution of 2 (7.83 g, 32.04 mmol) in HCl 1M (96.10 mL) and ethyl acetate (56.91 mL) at 0 °C, was added dropwise a solution of triphenylphosphine (9.24 g, 35.24 mmol) in ethyl acetate (85.36 mL). Afterwards, the temperature was allowed to reach room temperature and stirred over 7 h. The mixture was separated in a separatory funnel, and in the aqueous phase was added NaOH until PH >14 (5 mL). Successively was added CH2Cl2 (30 mL) and separated the organic phase which was dried over anhydrous Na2SO4. The solvent was removed by rotary evaporation and the residue was purified by a flash column chromatography on silica gel eluting with AcOEt: Hexane (1:1), to obtain product 3 (4.70 g, 21.52 mmol, 72%), as a colorless oil.  N-(2-propin-1-yl) oleamide (4) A solution of propargylamine (0.15 mL, 2.34 mmol) and dimethylaminopyridine (0.066 g, 0.54 mmol) in dry CH2Cl2 (4 mL) was added to a solution of oleoyl chloride (0.50 g, 1.8 mmol) and diisopropylcarbodiimide (0.4 mL, 2.7 mmol) in dry CH2Cl2 (4 mL), under argon atmosphere and was stirred overnight. Afterwards the reaction was dissolved in CH2Cl2 then was treated with HCl 4N (2 × 7 mL), neutralized with NaHCO3 (7 mL) and finally washed with brine (7 mL). The organic phase was dried over anhydrous Na2SO4, and the solvent evaporated to afford the crude product. To obtain the pure product 5 (0.505 g, 1.34 mmol, 57%), as a white solid, the crude was purified with column chromatography on silica gel using AcOEt: Hexane (1:5).  (6 mL), was added to a solution of CuSO4 (0.018 g, 0.11 mmol) and sodium ascorbate (0,059 g, 0.30 mmol) in water (8 mL). Afterwards, the reaction mixture was stirred vigorously over three days. Then the mixture was separated and the organic phase was dried over anhydrous Na2SO4. The CH2Cl2 was removed by rotary evaporation to yield the crude product. The triazole derivative, was isolated by flash column chromatography on silica gel, eluting with a CH2Cl2:MeOH (9:1) mixture. Thus, product 5 was obtained as a white solid (0.34 g, 0.51 mmol, 30%).

Synthesis of the Amphiphilic Compounds
The first synthesized amphiphilic compound, 5, has a tetraethylene glycol chain as a spacer, the versatile amine group as a polar head and the oleic acid fragment as a lipophilic tail. It was obtained through a five steps sequence (Scheme 3). Mesylation of tetraethylene glycol with mesyl chloride gave the dimesylated derivative 1, which was transformed into diazide 2 by substitution with sodium azide, and then reduced with triphenylphosphine to yield compound 3. The linking of 3 to the lipidic part of the amphiphilic compound was carried out through a Cu (I) catalyzed Huisgen reaction. Whith this purpous, the corresponding alkynyl derivative of oleic acid 4 was previously prepared by amidation of oleic acid with propargylamine, and the Huisgen reaction was carried out with the corresponding azide 3, obtaining the final compound 5. The presence of the amine group in 5, as a versatil polar head, may constitute a binding site to other specific functional groups as a drug, a recognition ligand or an anionic group like phosphonate.
In the case of the second synthesized amphiphilic compound 7, with a structure similar to that of compound 5, the connection between the polar head and the lipophilic chain is an amide group instead of a triazole one (Scheme 4). The reaction between compound 3 and oleoyl chloride yielded the amide 6, and the subsequent regioselective reduction of the azide group with LAH gave compound 7, without reducing the amide function.

Preparation and Characterization Of Micelles
In all cases, the range of CMC obtained was [0.01 mM-0.08 mM] using the pyrene method 24], very similar to the CMC values of polymeric micelles 25-27].
Micelles were formed in water solutions at a concentration of 1.25 mg/mL (0.02 M in the case of compound 7) much greater than CMC. Micelle formation process was previously optimized and consists on the dispersion of the amphiphilic compound in MilliQ water. Then, the sample was ultrasonicated by a sonic tip (Digital ultrasonic sonicator Q500 of 500 watts), for 30 min. After sonication, a microfiltration process was carried out with a 30 mm membrane filter (Interlab Ltd. Customables syringe filters) in order to eliminate suspended particles.
Micelles were then characterised by DLS and TEM. Figure 2 represents two electronic transmission microscope pictures with different magnification of micelles obtained from amphiphilic compound 5 (M5). As it can be seen, micelles are monodisperse, therefore aggregates are not present, and they have sizes in a range of 50-89 nm. In addition to microscopic analysis, the sample was analysed by DLS (Dynamic light scattering) in order to determine their hydrodynamic size. In the case of M5, it was of 99.80 nm (Figure 3). As can be seen, both techniques determined similar micelles size. Figure 4 represents a TEM photograph of micelles from compound 7 (M7) with diameters between 70-120 nm and DLS results with hydrodynamic size of 40 nm. In this case, one more time, both techniques confirm the presence of micelles.

Inclusion of Dexamethasone in Micelles
Dexamethasone (Dexa) was introduced in the synthesized micelles, in order to verify the ability of these nanocarriers to contain a highly insoluble drug. This test was performed using synthesized micelles of compound 5 (M5) and 7 (M7) ( Table 1).
A procedure in 3 steps has been carried out: (i) Addition of the solid drug (5.9 mg) directly to the previously prepared water solution of micelles and stirring 72 h at 50 °C. During this time, the sample was covered with an aluminum foil to prevent the degradation of the photosensitive drug Dexamethasone. (ii) Centrifugation at 2000 rpm during 15 min, obtaining a precipitate, which represents the drug not included, and a solution containing the micelles with the drug inside. (iii) Lyophilization for the elimination of water from the samples. To confirm the inclusion of the drug in the micelle, the analysis of the samples by 1 H-NMR using different deuterated solvents has been carried out and the results obtained are shown in Figure 5. 1 H-RMN of micelles + Dexa in D2O highlights only the signals corresponding to the protons of the amphiphilic compound, whereas 1 H-NMR of micelles + Dexa in DMSO which is an organic solvent and causes the leakage of the drug, shows the proton signals corresponding to both, the amphiphilic compound and Dexamethasone. This analysis represents a further indication of the internalization of the drug (Scheme 5).
In conclusion, it is possible to considerate that the drug is located inside the hydrophobic cavity of the micelles. This is evidenced not only by a gravimetric method, but also by NMR which shows all the signals corresponding to the drug.

Conclusions
The goals of this experimental work were to synthesize a new family of amphiphilic compounds in order to obtain micelles as drug nanocarriers. For this purpose, we prepared two amphiphilic compounds 5 and 7, with an amine as a versatile polar head, which lead to the corresponding micelles, M5 and M7 respectively, characterized by DLS and TEM, which have different sizes and distribution in water.
In order to verify the ability of these micelles, characterized by an average size of about 100 nm, as drug transport agents, the inclusion of the highly lipophilic drug Dexamethasone into both micelles M5 and M7 was performed, containing about 54 % and 74% of the previously added drug, respectively. This represents a good percentage and demonstrates their ability to encapsulate a highly lipophilic drug in their core.
As a project in the near future, in vitro release of encapsulated Dexa studies will be performed and M5 and M7 micelles will be functionalized by exploiting the free amine groups of their amphiphilic monomers, in order to address them to a specific target.