Article Cite This: Langmuir 2018, 34, 13244−13251
pubs.acs.org/Langmuir
Covalent Surface Modification of Lipid Nanoparticles by Rapid Potassium Acyltrifluoroborate Amide Ligation Sean Oriana,†,‡,§ Alessandro Fracassi,†,‡,§ Corey Archer,†,‡ and Yoko Yamakoshi*,†,‡ †
Laboratorium für Organische Chemie, ETH Zürich, Vladimir-Prelog-Weg 3, CH8093 Zürich, Switzerland Institut für Geochemie und Petrologie, ETH Zürich, Clausiusstrasse 25, CH8092 Zürich, Switzerland
‡
Downloaded via UNIV FRANKFURT on December 2, 2018 at 20:11:16 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: Because of the recent increasing demand for the synthetic biomimetic nanoparticles as in vivo carriers of drugs and imaging probes, it is very important to develop reliable, stable, and orthogonal methods for surface functionalization of the particles. To address these issues, in this study, a recently reported chemoselective amide-forming ligation reaction [potassium acyltrifluoroborate (KAT) ligation] was employed for the first time, as a mean to provide the surface functionalization of particles for creating covalent attachments of bioactive molecules. A KAT derivative of oleic acid (OAKAT, 1) was added to a mixture of three lipid components (triolein, phosphatidyl choline, and cholesteryl oleate), which have been commonly used as substrates for lipid nanoparticles. After sonication and extrusion in a buffer, successfully obtained lipid nanoparticles containing OA-KAT (NP-KAT) resulted to be well-dispersed with mean diameters of about 40−70 nm by dynamic light scattering. After preliminary confirmation of the fast and efficient KAT ligation in a solution phase using the identical reaction substrates, the “on-surface (on-particle)” KAT ligation on the NP-KAT was tested with an N-hydroxylamine derivative of fluorescein 2. The ligation was carried out in a phosphate buffer (10 mM, pH 5.2) at room temperature with reactant concentration ranges of 250 μM. Reaction efficiency was evaluated based on the amount of boron (determined by inductively coupled plasma mass spectrometry) and fluorescein (determined by fluorescence emission) in the particles before and after the reaction. As a result, the reaction proceeded in a significantly efficient way with ca. 40−50% conversion of the OA-KAT incorporated in the particles. Taken together with the fact that KAT ligation does not require any additional coupling reagents, these results indicated that the “on-surface” chemical functionalization of nanoparticles by KAT ligation is a useful method and represents a powerful and potentially versatile tool for the production of nanoparticles with a variety of covalently functionalized biomolecules and probes.
■
INTRODUCTION Nanoparticle-based vehicles have been recognized as promising tools for the efficient and selective delivery of medicines and imaging probes in vivo.1,2 Interestingly, recent emerging developments in nanotechnology analytical methods such as electron and scanning microscopies [transmission electron microscopy (TEM), scanning electron microscopy, atomic force microscopy, scanning tunneling microscopy, etc.] enabled detailed characterization of nanomaterials promoting the development of a variety of nanoparticles with precisely designed structures.3 Supported by such analytical techniques, it is important to develop new and efficient production methods of the nanoparticles by covalent functionalization with a variety of bioactive molecules such as (1) ligands for the receptors expressed in diseased tissues, (2) bioactive proteins and peptides, (3) water-soluble polymers to increase the solubility and biocompatibility of the particles, and (4) imaging © 2018 American Chemical Society
probes to visualize the location of the particles in vivo. These modifications, ideally through covalent bonds, require chemical reactions which are chemoselective and efficient even under aqueous and often diluted conditions. Among many nanoparticles, lipoproteins have been recognized as a useful natural carrier for the selective in vivo delivery of medicines and imaging probes to diseased tissues. For example, the low-density lipoprotein (LDL) was used as a vehicle for the selective detection and drug delivery for tumors,4−12 and high-density lipoprotein (HDL)-based nanoparticles have been developed for the detection of atheromas.13−18 In our previous studies, we developed an LDL-based MRI contrast agent for the selective and efficient Received: June 9, 2018 Revised: September 8, 2018 Published: October 20, 2018 13244
DOI: 10.1021/acs.langmuir.8b01945 Langmuir 2018, 34, 13244−13251
Article
Langmuir detection of atheroplaques.13,19,20 The natural LDL (nLDL), which was isolated from the blood of healthy human donors, was modified with an oleic acid (OA) derivative of a Gd3+chelate (GdDO3A-OA) by intercalation of the oleyl moiety into the nLDL lipid monolayer. As a result, nLDL with GdDO3A-OA was successfully obtained in a high payload of Gd3+ (>200 Gd3+ atoms per nLDL) and strongly enhanced the contrast of the miniature atheroma of a mouse model in our previous in vivo tests. Although successful, there were some disadvantages in this previous system. The supply of nLDL was rather limited, and a risk of infection from the donor was a concern. To overcome these limitations, in this study, we used synthetic nanoparticles prepared from a mixture of relatively inexpensive and commercially available lipidic molecules [phosphatidyl choline (PC), triolein (TO), and cholesteryl oleate (CO)], which have been used in the preparation of synthetic LDL (sLDL) by several other groups.21−25 Taking advantage of these previous reports of sLDL, in this study, we synthesized a lipidic molecule [OA-potassium acyltrifluoroborate (KAT), 1 in Figure 1] possessing a chemically reactive
functionalization of the particle surface. In this study, we used a fluorescein-HA molecule (2 in Figure 1) to test the efficiency of KAT ligation, preliminarily in solution phase, and then on particle using NP-KAT. The in situ on-particle KAT ligation strategy would represent a general and versatile method to create a variety of lipid nanoparticles with functional moieties such as imaging probes (e.g., near infrared dyes, Gd-chelates) and targeting peptides for the selective in vivo delivery of particles to a specific diseased tissue.
■
EXPERIMENTAL SECTION
Synthesis of OA-KAT 1 and Fluorescein-HA 2. General. All of the solvents were purchased from Acros Organics (Thermo Fisher Sci. Inc.) or Sigma-Aldrich Inc. (Merck KGaA) and dried by a solvent system (Innovative Technology Inc.) or distilled if needed. All water used was from Millipore purification system. Column chromatography and analytical thin-layer chromatography (TLC) were performed on SILICYCLE SilicaFlash F60 (230−400 mesh) and Silica gel 60 F254 TLC (Merck KGaA), respectively. Details of the syntheses of all of the compounds and their intermediates are described in the Supporting Information with corresponding spectra. (Z)-1-(1-Phenoxynonadec-10-en-1-yl)-1H-benzo[d][1,2,3]triazole (5). To a solution of 3 (1.0 g, 4.4 mmol, 1 equiv) in dried and degassed tetrahydrofuran (THF) (40 mL), n-BuLi (1.6 M in hexane, 4.1 mL, 6.6 mmol, 1.5 equiv) was added slowly at −78 °C and stirred for 15 min. Subsequently, iodide 4 (2.51 g, 6.6 mmol, 1.5 equiv) was added, and the reaction mixture was slowly warmed to 0 °C. While stirring overnight under Ar atmosphere, the reaction mixture was gradually warmed up to room temperature. Subsequently, the reaction mixture was cooled to 0 °C in an ice bath and quenched with water and then extracted with EtOAc. Combined organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The crude mixture was purified by a silica gel flash chromatography [hexane− EtOAc (3:1)] to provide brown oil 5 (1.95 g, 4.1 mmol, 93%); IR (ATR) νmax (cm−1): 2923.55 (s), 2853.29 (m), 1451.21 (w), 1493.31 (m), 1590.64 (w), 1278.75 (w), 1153.89 (w), 1079.53 (w), 1071.00 (w), 1001.18 (w), 888.42 (w), 746.68 (s), 690.97 (m), 666.68 (m), 628.30 (s), 557.57 (s); 1H NMR (400 MHz, CDCl3): 8.03 (d, J = 8.3 Hz, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.95 (m, 3H), 6.83 (t, J = 6.8 Hz, 1H), 5.34 (m, 2H), 2.49−2.32 (m, 2H), 2.00 (m, 4H), 1.27 (m, 24H), 0.88 (t, J = 5.9 Hz, 3H); 13C NMR (100 MHz, CDCl3): 156.2, 146.7, 131.1, 129.7, 127.7, 124.3, 122.9, 120.2, 116.2, 111.2, 88.3, 34.8, 34.0, 32.8, 32.6, 31.9, 29.7, 29.5, 29.3, 29.3, 29.2, 29.2, 28.9, 28.8, 28.2, 27.2, 27.2, 24.7, 22.7, 14.1; HRMS (MALDI) m/z: calcd for C31H46N3O, 476.3635; found, 476.3634 [M + H]+. Potassium Oleyltrifluoroborate (1). A solution of compound 5 (4.53 g, 9.6 mmol, 1.1 equiv) in dried and degassed THF (120 mL) was cooled down to −78 °C and stirred for 20 min in Ar atmosphere. Subsequently, n-BuLi (1.6 M in hexane, 5.45 mL, 8.73 mmol, 1.0 equiv) was added slowly and stirred for 5 min. Afterward, neat B(OMe)3 (1.81 g, 17.5 mmol, 2.0 equiv) was added dropwise, and the reaction mixture was stirred for 1 h at −78 °C and then at room temperature for 5 min in Ar atmosphere. To the reaction mixture, KHF2 aqueous solution was added slowly (9 mL × 4), and the mixture was warmed up to form a biphasic mixture. The reaction mixture was stirred additionally for 12 h, concentrated in vacuo, and dried under high vacuum. The resulting solid was stirred in acetone and then filtered (repeated for three times). The combined acetone filtrates were concentrated in vacuo, diluted in Et2O, and stirred until precipitates were formed. The precipitates were filtered, washed twice with Et2O, and dried under high vacuum to give colorless wax 1 (0.94 g, 2.43 mmol, 26%); IR (ATR) νmax (cm−1): 2922.33 (s), 2852.57 (m), 1661.38 (m), 1465.81 (w), 2403.71 (w), 1361.45 (w), 1155.23 (m), 1025.89 (s), 969.23 (s), 928.72 (s), 746.47 (m), 721.53 (m), 670.14 (m), 667.00 (m), 590.06 (w), 558.07 (w), 567.76 (w); 1H NMR (600 MHz, acetone-d6): 5.35 (m, 2H), 2.37 (t, J = 7.4 Hz, 2H), 1.44 (p, J = 7.4 Hz, 2H), 1.29 (m, 22H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (150 MHz, acetone d6): 130.6, 130.5, 32.6, 30.6, 30.5, 30.5,
Figure 1. Preparation of lipid nanoparticles with KAT (NP-KAT) from PC, TO, CO, and OA-KAT 1 and in situ on-particle functionalization by KAT ligation with fluorescein-hydroxylamine derivative 2.
KAT moiety, which was then added to the lipid components above. We expect that this OA-KAT will be incorporated into the nanoparticle through hydrophobic interaction of the OA moiety, and sufficient amount of chemically reactive KAT moiety will be exposed on the surface to produce nanoparticles with chemically reactive KAT surface (NP-KAT).26−28 We plan to use this NP-KAT as a “scaffold particle” for further development of many bioactive nanoparticles by attaching a variety of biomolecules of interest on demand via “on-particle KAT ligation” forming an amide bond with the hydroxylamine (HA) derivatives of the corresponding biomolecules. This relatively new ligation reaction between KAT and HA moieties is known to work efficiently in aqueous solvents and under diluted conditions. Importantly, this reaction can proceed in an orthogonal manner and can be used for the reaction of peptides with unprotected functional groups. Previously, this reaction has been studied intensively in the solution phase, but it has not yet been used for the direct 13245
DOI: 10.1021/acs.langmuir.8b01945 Langmuir 2018, 34, 13244−13251
Article
Langmuir
Figure 2. Scheme for KAT ligation of 2ab with OA-KAT 1 [reagents and conditions: (i) TFA, CH2Cl2, rt, 10 min; (ii) 10 mM phosphate buffer (pH 5.8), MeOH, acetone, 50 μM of 1, and 2ab, rt,
