ISSN 0012-5016, Doklady Physical Chemistry, 2018, Vol. 478, Part 1, pp. 1–5. © Pleiades Publishing, Ltd., 2018. Original Russian Text © E.Yu. Ladilina, S.A. Lermontova, L.G. Klapshina, N.S. Zakharycheva, Yu.P. Klapshin, G.A. Domrachev, 2018, published in Doklady Akademii Nauk, 2018, Vol. 478, No. 1, pp. 48–53.
PHYSICAL CHEMISTRY
Polyfunctional Siloxane Water-Soluble Nanoparticles for Biomedical Applications E. Yu. Ladilinaa, *, S. A. Lermontovaa, b, L. G. Klapshina a, N. S. Zakharychevab, Yu. P. Klapshinb, and Corresponding Member of the RAS G. A. Domracheva Received July 14, 2017
Abstract—Hydrolysis of N-methyl-N-(2,3,4,5,6-pentahydroxyhexyl)-N'-(3-triethoxysilylpropyl)urea gave water-soluble polysiloxane nanoparticles. They can be used for the preparation of intensely luminescent stable aqueous suspensions of water-insoluble or poorly soluble compounds (Eu(BTFA)3 ⋅ 6H2O complex and tetracyano-tetraaryl-porphyrazines) for biomedical applications, in particular, for bioimaging. DOI: 10.1134/S0012501618010013
Most of antibacterial and antiviral agents and drugs for the treatment of central nervous system and cancer are water-insoluble, which reduces their bioavailability [1]. The bioavailability of substances can be increased by using a dosage form such as nanosuspension [2]. Multifuctional linear polymers are currently most popular and widely used for biomedical applications, in particular, for the preparation of nanosuspensions [3]. Dendrites are intensely studied as regards the use for increasing the solubility of various drugs [4, 5] and hyperbranched polymers [6]. Nanoparticles based on mesoporous silica can also be used as delivery systems, since silicon dioxide is chemically inert, thermally stable, and biocompatible [7]. However, organic-inorganic hybrid materials seem to be the most attractive delivery agents [8]. Hydrolysis of tet-
(EtO)3Si
raethoxysilane (TEOS) or trialkoxysilanes containing various organic substituents or co-hydrolysis of these compounds [9] can give siloxane-based nanoparticles with a functionalized surface. Apart from water solubility and the lack of toxicity, an important characteristic of these drug delivery systems is the presence of numerous readily accessible functional groups for the interaction with the drug being delivered [10]. This communication describes the synthesis of a new organosilicon sol-gel monomer as well as solvating properties of water-soluble polyfunctional nanoparticles obtained by its hydrolysis. The monomer, N-methyl-N-(2,3,4,5,6-pentahydroxyhexyl)-N'-3-triethoxysilylpropylurea, was synthesized from isocyanatopropyltriethoxysilane and Nmethylglucamine according to the following scheme:
Me N C O + HN
OH OH OH OH OH
(EtO)3Si
Me N
NH O
OH OH OH OH OH
1
Compound 1 contains the easily hydrolyzable triethoxysilyl moiety and the carbamide group, which can efficiently solvate organic compounds and metal complexes. The linear substituent at the nitrogen atom with five hydroxyl groups can simultaneously interact
a Razuvaev
Institute of Organometallic Chemistry, Russian Academy of Sciences, Nizhny Novgorod, 603950 Russia b Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, 603022 Russia *e-mail:
[email protected] 1
2
LADILINA et al.
with the solvated component and water molecules, thus providing efficient dissolution of both the monomer and the monomer-based functionalized siloxane species. Fast dissolution of the monomer in water is accompanied by slow hydrolysis of its triethoxysilyl group. Dynamic light scattering (DLS) examination of the resulting solution showed the presence of nanoparticles with a bimodal size distribution (91 and 268 nm peaks). The higher contribution to the total light scattering (85%) is made by the particles less than 170 nm in diameter, while larger particles are only aggregates of the smaller ones. The proneness of the particles to aggregation, although slight, is supported by the boundary value of the zeta-potential (–29 mV). The polymer that forms the nanoparticles is mainly a cross-linked polysiloxane, because its sol fraction is only 5% and is a mixture of di- and trisiloxanes. The 29Si NMR spectrum of the polymer exhibits two very broad signals, –68 and –58 ppm, corresponding to the Т 3 and Т 2 moieties, respectively [11] (Fig. 1), which is indicative of the formation of siloxane bonds within a variety of open-chain polyhedral and branched structural units. The integrated intensity of the signal for Т 3 is twice higher; thus, the polymer composition is as follows: OH
OH
OH
OH
HO
HO OH
OH
HO
HO
Me N
Me N
n:m=2:1 O
O
NH
HO
NH
SiO1.5
SiO n
OH m
OH 2
The polymer is amorphous and contains no distinct cyclic silsesquioxanes (its X-ray diffraction pattern shows one intense peak at 15°–25°). We studied the reaction of precursor 1 with waterinsoluble europium benzoyltrifluoroacetonate Eu(BTFA)3 ⋅ 6H2O (3). From a THF solution containing a mixture of compound 1 and complex 3 (37%), a smooth transparent film is formed. The IR spectrum of the film (Fig. 2) exhibits clear-cut changes in the absorption bands of the diketonate ligand of the complex: the band at 1532 cm–1 shifts to 1537 cm–1, that at 1576 cm–1 shifts to 1579 cm–1, while the band at 1614 cm–1 acquires a high-frequency shoulder at 1645 cm–1. Thus, solvation of the complex with monomer molecules induces considerable changes in
T3
T2
−50 −55 −60 −65 −70
−75 −80 δ, ppm
Fig. 1. 29Si NMR spectrum of polysiloxane 2.
the metal coordination sphere. The interaction with the metal involves the oxygen atoms of the amide groups. This is confirmed by a change in the position of the amide I band, corresponding to C=O stretching modes: in the monomer spectrum, this band occurs at 1606 cm–1, while in the spectrum of the film, it is at 1625 cm–1. Meanwhile, the amide II band is not shifted. Also, noticeable changes occur in the range of 3700–3000 cm–1, which shows the O–H and N–H vibrations. The IR spectrum of the monomer in this region (Fig. 2) had absorption bands at 3327 and 3278 cm–1, which is attributable to involvement of the O–H and N–H groups into the intermolecular interaction. The spectrum of complex 3 shows broad and fairly intense absorption of coordinated water (3656 and 3400 cm–1). However, the spectrum of the film shows only one intense broadened band at 3374 cm–1, which attests to the absence of a hydrogen bond between the hydroxyl groups of the organic substituent at the silicon atom and the amide group involved in the interaction with the complex. Further, on going from the monomer to the film, the 1083 cm–1 band disappears, but a band appears at 1073 cm–1; in addition, the intensities of the 950 and 770 cm–1 bands change. This indicates that the SiOEt groups of the monomer are hydrolyzed by water displaced from the metal coordination sphere, being converted to siloxane bonds within the film material. The initial monomer precipitates from a THF solution as a white powder, whereas the film is solid and transparent, that is, this is polysiloxane doped with the europium complex. An IR-spectroscopic study of a film formed from an aqueous suspension of nanoparticles of the polymer containing the same complex (Fig. 2) has shown that the band positions and intensities in the 1700– 1500 cm–1 range are identical to those in the spectrum
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3
Intensity, rel. units 200
4 3 Transmittance
2 1 100
4000
3000
1000 ν, cm−1
2000
0
Fig. 2. IR spectra of (1) monomer 1 (Nujol), (2) Eu(BTFA)3 ⋅ 6H2O (Nujol), (3) compound 1 with complex 3 (35%) (solid film), and (4) film of the aqueous suspension of nanoparticles doped with complex 3.
400
500
600 λ, nm
Fig. 3. Luminescence spectra of a suspension of polymer particles doped with europium complex (C3 = 1.47 × 10–5 mol/L, λex = 330 nm).
tion is 5D0 → 7F2 at 616 nm. However, the nanoparticles that form such suspensions are much more prone to aggregation than the undoped ones (the zeta-potential is –10 mV). The solution obtained by mixing of a 1% aqueous suspension of the polymer with an ethanol solution of the complex also exhibits a bimodal distribution, with peaks at 98 and 337 nm, with association and precipitation occurring for several days.
of the film based on a monomer–complex mixture. Thus, the amide groups of polymer 2 also interact with complex 3 as aqueous suspensions are formed. Therefore, the use of aqueous solutions of polysiloxane 2 nanoparticles allows one to transfer the water-insoluble complex into the aqueous phase. As can be seen from Fig. 3, the spectrum of a suspension of polymer particles doped with the europium complex exhibits cation-centered luminescence as a series of bands for transitions from the excited 5D0 level of Eu(III) to the multiplet 7Fn ground level. The most intense transi-
The interaction of functionalized nanoparticles with metal-free luminophores was studied in relation to a series of porphyrazines: CN
R N
NC
NH
R N N
N HN
N R
CN
N R
NC H3C
H3CO
R=
OCH3 O 4
5
6
It is known [12, 13] that an efficient interaction of compounds of this type with polymers in water increases the intensity of their luminescence. Going DOKLADY PHYSICAL CHEMISTRY
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from aqueous solutions to suspensions of nanoparticles is accompanied by a considerable increase in the porphyrazine luminescence intensity (Table 1), but to 2018
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LADILINA et al.
Table 1. Change in the luminescence intensity of porphyrazines 4–7 in a suspension in comparison with that in aqueous solution (λex = 590 nm) Increase in the luminescence intensity (Isusp/ Iwater)
the preparation of intensely luminescent stable aqueous suspensions of water-insoluble and poorly soluble compounds. Despite the absence of covalent bond with the complex or dye being dissolved, the polymer can be used as a “container” for luminescent compounds in bioimaging.
Porphyrazine
C, mol/L
λmax, nm
4
7.7 × 10–6
637
3.6 (160/45)
5
7.1 × 10
–6
646
5.8 (69/12)
EXPERIMENTAL
6
7.9 × 10–6
648
2.4 (65/27)
7
–6
658
24.4 (122/5)
IR spectra were measured on an FSM FTIR spectrometer as Nujol mulls between KBr plates, or on ZnSe plates for polymer films. 1H NMR spectra were measured on a Bruker Avance DPX-200 instrument (200 MHz for 1H, 39.7 MHz for 29Si) at 25°C with Ме4Si as the internal reference. The hydrodynamic diameter of nanoparticles in aqueous suspensions was determined by DLC in the mode of polymodal analysis of the correlation function using a NanoBrook Omni spectrometer (Brookhaven Instruments). The particle size was measured at 25°C in polystyrene cells optical way length is 1 cm at a 90° angle for suspensions of pure and porphyrazine-loaded polymer nanoparticles and at a 173° angle for nanoparticles with the europium complex. The zeta-potential was determined by phase analysis electrophoretic light scattering (PALS). The electrophoretic mobility values were converted to zeta-potentials using the Hückel model. The luminescent properties of solutions and suspensions were measured on a Perkin-Elmer LS-55 spectrofluorimeter. Powder X-ray diffraction analysis was carried out with filtered CuKα radiation (λ = 1.54178 Å) on a Shimadzu XRD-6000 diffractometer. The polymer sol fraction was analyzed on a Knauer Smartline chromatograph with Phenogel Phenomenex 5u columns (300 × 7.8 mm) with a UV detector (λ = 254 nm) and calibration against polystyrene standards with molecular masses from 3420 to 2570000 Da. THF at a flow rate of 2 mL/min was used as the mobile phase. The compounds were analyzed at the analytical centers of the Razuvaev Institute of Organometallic Chemistry and the collective use center “New Materials and Resource-Saving Processes” at the Research Institute of Chemistry, Lobachevsky State University of Nizhny Novgorod.
8.7 × 10
a different extent, depending on the nature and position of the substituent in the benzene ring. In the case of porphyrazine 5 with the para-methoxy substituent, the increase in the luminescence intensity is more pronounced than for compound 6, in which the same substituent in the ortho-position. Probably, this is caused by the hydrogen bonding of the polymer amide and hydroxyl groups not only with the nitrile groups and the central nitrogen atoms of the macrocycle, but also with oxygen-containing porphyrazine substituents, most efficiently, with those located in the paraposition. The non-covalent interaction also involves the π-system of the aryl substituent [14], since luminescence is considerably enhanced also for porphyrazine 4, devoid of oxygen-containing groups. The most pronounced luminescence enhancement is observed for porphyrazine 7 (Table 1), which attests to the most intense interaction with the functionalized nanoparticle surface in the suspension, because apart from ndonor oxygen atoms, this compound contains the largest number of aromatic rings. Aqueous suspensions of polysiloxane nanoparticles doped with porphyrazine 5 were studied by DLC. The particle diameter distribution is bimodal with the peaks at 71 and 176 nm. The larger particles, resulting from association of smaller particles, are not manifested in the numerical distribution, and the contribution of particles less than 90 nm in diameter to the total light scattering is 79%. Comparison of the Z-averaged particle diameter of pure (121 nm) and loaded (115 nm) polymers indicates that dye solvation is accompanied by some compression of particles. Previously, we have demonstrated that porphyrazine-containing polymer particles of this type can efficiently interact with proteins via the formation of strong hydrogen bonds between the proteins and the amide and hydroxyl groups of the polysiloxane side substituents. This gives rise to a considerable enhancement of their luminescence in the presence of albumin, which is the major blood serum protein [12]. Thus, we found that water-soluble monomer 1 and polymer 2 can efficiently solvate rare earth metal complexes with displacement of water from the metal coordination sphere. Polysiloxane 2 can be used for
Synthesis of N-methyl-N-(2,3,4,5,6-pentahydroxyhexyl)-N'-(3-triethoxysilylpropyl)urea (1). A solution of 3-triethoxysilylpropyl isocyanate (2.00 g, 8.09 mmol) in 25 mL of THF was added dropwise with stirring over a period of 30 min to a suspension of N-methylglucamine (1.59 g, 8.09 mmol) in 25 mL of THF. The reaction mixture was stirred for 2 h with heating (80– 90°C), and THF was evaporated in a vacuum to give 3.45 g (99%) of compound 1 as a white powder. For C17H38N2O9Si anal. calcd. (%): C, 46.14; H, 8.65; Si, 6.35. Found (%): C, 42.11; H, 8.15; Si, 5.14.
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IR, ν, cm–1: 3330, 3271 (N–H, O–H), 2977, 2933, 2926 (C–H), 1700, 1608, 1545 (C=O, N–H), 1104 (C–O–H), 1080 (Si–O–C), 953, 775, 723 (Si–O–Et). 1 H NMR (CD 3) 2CO, δ, ppm): 0.59 (m, 2H, –CH2–Si–), 1.18 (t, 9H, CH3–CH2–, 3JH,H = 7.0 Hz), 1.58 (m, 2H, –CH2–CH2–CH2), 2.96 (s, 3H, МеN), (3.14 (m, 2H, –NH–CH2–), 3.43 (m, 2H, МеN– CH2–), 3.60 (m, 2H, –CH2OH), 3.71 (m, 4H, –CH(OH)–), 3.80 (q, 6H, CH3–CH2, 3JH,H = 7.0 Hz), 3.91 (m, 2H, –CH2OH), 4.08 (m, 1H, –CH2OH), 4.19 (m, 1H, –CH2OH), 4.86 (br.d., 1H, –CH2OH), 5.98 (br.t, 1H, HN–CH2). Hydrolysis of compound 1. Compound 1 (0.81 g, 1.83 × 10–3 mol) was added with stirring to 81.84 g of water. The solution was kept for a week. Drying gave a transparent solid film of water- and ethanol-soluble polysiloxane. Upon mechanical removal of the film, the product was a white powder. IR: ν, cm−1: 3358 (N–H, O–H), 2937, 2878 (C– H), 1604, 1547 (C=O, N–H), 1398 (C–H), 1084 (C– O–H), 1057 (Si–O–Si). 1H NMR (D O, δ, ppm): 0.58 (m, 2H, –CH –Si–), 2 2 1.50 (m, 2H, –CH2–CH2–CH2), 2.85 (br.s, 4H, –CH(OH)–), 3.07 (m, 2H, –NH–CH2–), 3.65 (m, 5H, МеN–CH2–), 3.90 (m, 2H, –CH2OH), 4.57 (br.s, 1H, HN–CH2), 4.81 (br.s, 5H, –OH). 29Si NMR (D O, ppm): –58 (T 2), –68 (T 3). 2 Preparation of luminescent aqueous suspensions. A weighed portion (0.005–0.002 g) of porphyrazine 4–7 or complex 3 was dissolved in a minor amount of ethanol. A 1% aqueous suspension of nanoparticles of polymer 2 was added to the solution. The resulting suspensions were diluted with distilled water, if necessary, and used to measure the luminescence spectra. ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research (project nos. 16–34–60117 mol_a_dk, 15–02–05468-а) and the Ministry of Education and Science of the Russian Federation (4.5510.2017/BCh).
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Translated by Z. Svitanko
2018
