Dmso d6

Dry solvents
in septum bottles were purchased from Sigma-Aldrich Ltd. (Prague,
Czech Republic). All other solvents as well as sulfuric acid, HCl
(aq), acetic acid, acetic anhydride, NaOH, KOH, and Na₂SO₄ were purchased from Lachner Ltd. (Neratovice, Czech
Republic) and were of analytical grade. d(+)-glucose and
paraformaldehyde were purchased from Carl Roth GmbH + Co. KG (Karlsruhe,
Germany), and EDC hydrochloride was purchased from Carbolution Chemicals
GmbH (St. Ingbert, Germany). The origin of chemicals used in bioexperiments
is specified in Section 2.3. All other chemicals were purchased from Sigma-Aldrich Ltd.
(Prague, Czech Republic). Chlorobenzene, 2-ethyl-2-oxazoline, and tert-butyl bromoacetate used in the synthesis of the polymers
were dried over CaH₂ or P₂O₅ under
argon, distilled and stored over 4 Å molecular sieves prior to
use. All other chemicals were used as received.
Sephadex-LH20
was purchased from Cytiva via Sigma-Aldrich Ltd.
(Prague, Czech Republic), and equilibrated in methanol (MeOH) for
3 h before packing in a gravity-driven separation column.
Chloroform-d,
MeOD, and DMSO-d₆ were
purchased from Eurisotop (Cambridge, U.K.).
Proton nuclear magnetic
resonance (¹H NMR) measurements
were performed on a 400 MHz Bruker Avance Neo spectrometer using CDCl₃ MeOD, or DMSO-d₆ as a deuterated
solvent. For calibration, the specific signals of the nondeuterated
species were used.
Electron spray ionization mass spectrometry
(ESI-MS) was carried
out on a LCQ Fleet hybrid mass spectrometer (Thermo Fisher Scientific,
Waltham, USA) equipped with an LTQ Orbitrap XL using methanol as mobile
phase (flow rate 10 μL min^–1) in positive
mode. The data was processed with the Xcalibur Software (Thermo Fisher
Scientific).
Matrix-assisted laser desorption ionization–time-of-flight
mass spectrometry (MALDI-TOF MS) mass spectra were acquired with the
UltrafleXtreme TOF – TOF mass spectrometer (Bruker Daltonics,
Bremen, Germany) equipped with a 2000 Hz smartbeam-II laser (355 nm)
using the positive ion linear mode. Panoramic pulsed ion extraction
and external calibration were used for molecular weight assignment.
The dried droplet method was used in which solutions of the sample
(20 mg mL^–1), the matrix (DHB, 2,5-dihydroxybenzoic
acid, 20 mg mL^–1), and the ionizing agent sodium
trifluoroacetate (10 mg mL^–1) in methanol are mixed
in the volume ratio 4:20:1. One μL of the mixture was deposited
on the ground-steel target.
Gel permeation chromatography (GPC)
measurements in MeOH/acetate
buffer were carried out on a Dionex UltiMate 3000 UHPLC chromatograph
(ThermoFisher Sci, USA) equipped with an autosampler, an UV–VIS
detector (323 nm), an Optilab rEX differential refractometer and a
DAWN 8+ multiangle light scattering (MALS) detector (Wyatt; Santa
Barbara, CA, USA). A TSK SuperAW3000 column with methanol and sodium
acetate buffer (pH = 6, 8:2 v/v) as an eluent at a flow rate of 0.5
mL min^–1 was used.
Gel permeation chromatography
(GPC) measurements in DMSO were performed
using a DeltaChrom SDS 030 pump (Watrex Ltd., Czech Republic) with
a flow rate of 0.5 mL min^–1. The two PLgel 10 μm
mixed B LS columns (Polymer Laboratories, UK, separation range of
approximately 5 × 102 ≤ M ≤ 1 × 107 as determined
using PS standards) were used in a series. A DAWN HELEOS II MALS detector
(Wyatt Technology Corp., Germany) with a laser operating at a wavelength
λ = 658 nm, and an Optilab T-rEX RI detector (Wyatt Technology
Corp., Germany) were used. Dimethyl sulfoxide (≥99%, HPLC grade,
Fisher Scientific, Czech Republic) with 0.05 M LiBr (≥99%,
Merck, Czech Republic) as an additive was used as the mobile phase
at ambient temperature. The sample injection volume was 100 μL.
The data was collected using the Astra software (Wyatt Technology
Corp.). M_w and M_n were calculated with a dn/dc = 0.15.
Dynamic light scattering (DLS) measurements of the
polymers and
SLNP in water were performed on an ALV-6010 SLS/DLS instrument (ALV-GmbH,
Germany) equipped with a 22 mW He–Ne laser (λ = 632.8
nm) at a detection angle of 90°. Measurements were carried out
at 25 °C. Solvent viscosity and refractive index were automatically
adjusted to the temperature of the thermostat. The CONTIN algorithm
was applied to analyze the obtained correlation functions. Apparent
hydrodynamic radii were calculated according to the Stokes–Einstein
equation where R_H is the
hydrodynamic radius, k_B is the Boltzmann
constant, T is the absolute temperature, η
is the dynamic viscosity of the solvent, and D_T is the translational diffusion coefficient.
DLS measurements
of the polymers and SLNP in DMEM at 37 °C
were carried out on a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments,
UK). DMEM was filtered with a 0.22 μM filter prior to use.
Fluorescence measurements for the determination of the critical
micelle concentration were carried out on a Multimode Microplate Reader
(BioTek Synergy H1, Agilent, US). A stock solution of Nile Red in
DMEM (0.12 mmol) was prepared by mixing 4 μL of a Nile Red solution
(3.1 mmol in THF) with 100 mL of DMEM. The 2 mg mL^–1 stock solutions of the polymers in DMEM were diluted with pure DMEM
and DMEM containing Nile Red to obtain solutions with a concentration
of 1, 0.5, 0.25, 0.05, and 0.01 mg mL^–1, respectively.
The fluorescence of the Nile Red-containing solutions was measured
on a 96-well plate with an excitation wavelength of 515 nm and an
emission wavelength of 585 nm.
High-resolution scanning transmission
electron microscopy (STEM)
imaging of stabilized solid lipid nanoparticles was performed on a
custom-modified Quanta 650 FEG environmental scanning electron microscope
(ESEM) (Thermo Fisher Scientific, MA, USA) equipped with a scanning
transmission electron microscopy (STEM) detector.^{61 (link)} The samples, dissolved in distilled water, were applied
to a lacey carbon film on a copper TEM grid.^{62 (link)} Then, the samples were in situ freeze-dried at −20 °C
and 10 Pa in the ESEM specimen chamber (operated under environmental
mode). Observation was performed at a beam energy of 30 keV, a beam
current of 5 pA, and a working distance of 5.3 mm in high vacuum mode
using a dark field STEM detector. The micrographs were postprocessed
using MountainsSEM software (Digital Surf, France). Particle sizes
were measured using ImageJ software.
Fourier transform infrared
(FTIR) spectra were measured on a Spectrum
100T FT-IR spectrometer (PerkinElmer, USA) equipped with a deuterated
triglycine sulfate detector using the attenuated total reflectance
(ATR) technique. Four scans per spectrum (650–4000 cm^–1) at the resolution of 4 cm^–1 were measured.
UV/vis spectra of the cyanine 3/cyanine 5 (cy3/cy5)-labeled compounds
were recorded on an Evolution 220 UV/vis spectrometer (Thermo Scientific,
USA) using solutions of the compounds in micropure water (10 μg
mL^–1).
Fluorescence correlation spectroscopy
(FCS) uses time change in
fluctuations of fluorescence intensity to obtain separate FCS autocorrelation
functions (ACFs) of individual fluorophore populations in a mixture.
We used this technique to probe the presence of different fluorescently
labeled species in our polymer and LNP solutions. The samples were
diluted to obtain reasonable concentrations of the fluorophore within
the observation confocal volume (200× in case of the LNP, and
600× in case of the polymer solution, starting from solutions
based on 1 mg mL^–1 polymer) and subsequently excited
by an LDH-D-C-640 laser diode emitting 640 nm light, driven by a PDL
828 Sepia II driver in picosecond pulsed mode at a 20 MHz repetition
rate (both devices: PicoQuant) through the 635 nm dichroic mirror
built into the IX83 scan head. An Olympus UPlanSApo water immersion
objective (60×, 1.2 NA) delivered the excitation light into a
diffraction-limited spot and collected the emitted fluorescence. The
laser intensity was maintained at approximately 10 μW average
power at the objective entrance pupil to avoid photobleaching and/or
saturation. The collected fluorescence light passed through a Semrock
690/70 nm BrightLine emission filter and was detected by a hybrid
photomultiplier (PMA Hybrid-40 from PicoQuant) operated in photon
counting mode. Photon counts were recorded using a PicoHarp300 TCSPC
module in a T3 time tagging mode. The SymPhoTime64, ver. 2.1 software
from PicoQuant was used for data acquisition and FCS data analysis.
Each acquisition took 1 min, and the measurements were performed at
23 ± 1 °C. The FCS autocorrelation function (ACF) for the
simplest case of one diffusing component is mathematically given by
equation wherein N_p is
the average number of diffusing fluorescent particles in the confocal
volume, t is the correlation time, the diffusion
time τ_D refers to the residence time of fluorescent
objects in focus and k is the ratio of axial to radial
radii of the confocal volume, k = w_z/w_xy with w_xy and w_z being the dimensions of the
focal spot in the x–y plane
(perpendicular to the optical axis) and along the z-axis. Then, the diffusion time can be expressed as τ_D= w²_xy/4D_T, where D_T is the coefficient of translational diffusion of particles.
Diffusion coefficients were obtained by fitting of measured ACFs with
appropriate model functions and hydrodynamic radii of the polymers
and LNP in aqueous solution were subsequently obtained using the Stokes–Einstein
equation where R_H is the
hydrodynamic radius, k_B is the Boltzmann
constant, T is the absolute temperature, η
is the dynamic viscosity of the solvent, and D_T is the translational diffusion coefficient.

All solvents were purchased from commercial sources and were dried according to standard methods. All chemicals and starting materials (2-oxoindoline-5-carboxylic acid, (S)-1-phenylethan-1-amine, HATU, 3,5-dimethyl-4-nitro-1H-pyrrole-2-carbaldehyde, bromoacetic acid, DIPEA, piperidine and zinc powder) were purchased from Fluorochem and Sigma-Aldrich. Reactions were monitored by TLC using 0.25 mm silica gel 60 F₂₅₄ TLC plates purchased from Merck. Spots were visualized under ultraviolet light (254 nm).
¹H, ¹³C APT NMR spectra were acquired on a Bruker Avance 400 spectrometer equipped with a 5 mm QNP probe operating at 400.16 MHz and 100.61 MHz for ¹H and ¹³C respectively, at 293K. Spectra of all compounds were recorded using DMSO-d₆ (99.9%; Eurisotop, UK). Spectroscopic data of all compounds was matched with the one previously reported.⁴⁵ (link) High-Resolution-Mass Spectrometry (HR-MS) and low-resolution Mass Spectrometry (MS) spectra were performed on an Impact II QTOF (Bruker, Bremen, Germany) mass spectrometer with an electrospray ionization source (ESI). The method consisted of direct infusions with MS/MS scans, in the positive and negative modes.

The identity of the synthesized glycosides was confirmed by NMR. The acquisitions were carried out in deuterium oxide (D₂O, 99.96% 2H, Eurisotop, Saint-Aubin Cedex, France), dimethylsulfoxide (DMSO-D6, 99.80% ²H, Eurisotop, Saint-Aubin Cedex, France) or mixtures thereof. The spectra of flavonoid glucosides were acquired on a Jeol JNM-ECZL 500 MHz NMR spectrometer with Royal HFX-probe (automatic tuning and matching). Frequency ¹H: 499.7189 MHz; ¹³C: 125.6544 MHz. The Jeol Delta 6.1 software was used for the measurements. The acquisitions of phenylpropanoid glycosides were carried out on a Bruker AVANCE III 300-MHz spectrometer (Bruker, Rheinstetten, Germany) with an autosampler. The Bruker Topspin 3.5 software was used for the measurements. The spectra were analyzed using MestReNova 16.0 (Mestrelab Research, S.L.) and JASON (Jeol).