Amine Protons Research Articles

Mechanistic Insights into Curvature Formation in Synthetic Vesicles.

The mimicking of natural lipid bilayers with synthetic amphiphilic systems is of great interest to researchers, as insights could lead to better understanding of the complexities of cell membranes, as well as new materials and healthcare technologies. Recapitulating natural lipid asymmetry across bilayer membranes has important implications for curvature in cell, vesicle, and organelle morphologies, but has been challenging to achieve with synthetic lipid combinations or standard amphiphilic block copolymers. In a recent article, Elizebath et al. report the synthesis of a new type of synthetic amphiphile able to induce asymmetry in an artificial bilayer membrane dynamically. The molecules were designed around an extended π-conjugated hydrophobic core with tertiary amine-terminated oxyalkylene side chains. Protonation of the tertiary amines on the bilayer exterior leads to curvature induction, bilayer fission, and vesicle formation as monitored by time-resolved spectroscopy techniques and microscopy. The results were further validated with density functional theory (DFT) calculations. The delicate balance between different molecular scale interactions in the supramolecular structures led to the dynamic transformation of the bilayer membranes. Insights described could be used to advance the assembly of hierarchical life-like materials.

Mechanistic Insights into Curvature Formation in Synthetic Vesicles

AbstractThe mimicking of natural lipid bilayers with synthetic amphiphilic systems is of great interest to researchers, as insights could lead to better understanding of the complexities of cell membranes, as well as new materials and healthcare technologies. Recapitulating natural lipid asymmetry across bilayer membranes has important implications for curvature in cell, vesicle, and organelle morphologies, but has been challenging to achieve with synthetic lipid combinations or standard amphiphilic block copolymers. In a recent article, Elizebath et al. report the synthesis of a new type of synthetic amphiphile able to induce asymmetry in an artificial bilayer membrane dynamically. The molecules were designed around an extended π‐conjugated hydrophobic core with tertiary amine‐terminated oxyalkylene side chains. Protonation of the tertiary amines on the bilayer exterior leads to curvature induction, bilayer fission, and vesicle formation as monitored by time‐resolved spectroscopy techniques and microscopy. The results were further validated with density functional theory (DFT) calculations. The delicate balance between different molecular scale interactions in the supramolecular structures led to the dynamic transformation of the bilayer membranes. Insights described could be used to advance the assembly of hierarchical life‐like materials.

Molecular Necklaces Formed Through Zn2+-Templated Binding of Macrocycles with Dynamically Formed Imines as Guest Recognition Sites.

Herein we demonstrate an "in-ring establishing" strategy for assembling interlocked molecules through dynamic imine formation, "establishing" the host recognition sites in situ. Using Zn2+ ions to template the assembly of a pyridine-containing macrocycle with semidumbbell-shaped triazole-containing aldehyde and amine derivatives, we obtained the corresponding [2]rotaxane in high yield (85 %) after subsequent imine reduction (NaBH4) and amine protonation (NH4PF6). We performed the same three steps (assembly, reduction, protonation) to prepare a stable and highly symmetrical [5]molecular necklace ([5]MN) from 12 components (two almost-90°-oriented dialdehydes, two almost-90°-oriented diamines, four macrocycles, four Zn2+ ions) in an overall yield of 69 %.

Sonochemical synthesis, characterization, and ADMET studiesof Fe (II) and Cu (II) nano-sized complexes of trimethoprim

Nanoparticles exhibit distinct physical and chemical characteristics and are becoming increasingly significant in the production of innovative nanodevices for many applications in physics, biology, biomedicine, and pharmaceuticals. The aim of this research work is to synthesize Fe (II) and Cu (II) nano-sized complexes of trimethoprim (TMP) using the sonication method, characterize them using physical and spectroscopic methods, and carry out ADMET studies on the synthesized complexes. The spectroscopic and physical studies showed a change in colour and an increase in melting point due to coordination. The novel compounds were slightly soluble in water. The XRD tests revealed that the new nanocomplexes were crystalline. The Fe (II) nanocomplexes had a size of 57.56 nm and the Cu (II) nanocomplexes had a size of 69.88 nm. These values were found using Debye-Scherrer’s equation. The FTIR results of the TMP, Fe (II), and Cu (II) nanometal complexes showed a shift of the amino group band from 3317 to 3295 and 3202 cm?1 and the azomethine band from 1633 to 1625 and 1592 cm?1 in the complexes. In the complexes, the proton NMR spectra revealed an upfield shift of the amine proton. The carbon-13 NMR spectra showed that CH2 was involved in coordination with the metal ions. The spectra studies indicated that TMP coordinated with the metal ions through the methylene and amino groups. A trigonal bipyramid structure was proposed for the complexes. The results of the Rule of 5 studies indicated that the test compounds had a good drug-likeness prediction, with only one violation. The ADMET prediction showed that all of the compounds demonstrated improved pharmacokinetic characteristics and adhered to the RO5 requirement. These findings highlight the therapeutic potential of Fe (II) and Cu (II) nano-sized TMP complexes as bioactive compounds that warrant further investigation for pharmaceutical applications.

Electrochemistry of Diquinonyl Amines with an Internal Proton Source

It is well established that quinones play an important role in various biological systems. Studying electrochemical properties of quinones affords fundamental knowledge of semi-quinone radicals formation in vivo and in vitro in different media.Following our previous work on electrochemical properties of various quinonyl amines [1], Scheme 1 below describes seven diquinonyl amines. Noteworthy that six of them (1-6) contain an internal proton donor (‘NH’) except for the seventh one (7), in which both quinone moieties are attached to ‘NMe’ group. Their redox potentials were measured by cyclic voltammetry in dichloromethane [2].The results show a strong dependence of the nature of substituent on the first reduction potentials, and that protonation of diquinonyl amines is feasible by internal proton source even in a non-polar medium.

Modifying Specific Ion Effects: Studies of Monovalent Ion Interactions with Amines.

Specific ion effects in the interactions of monovalent anions with amine groups─one of the hydrophilic moieties found in proteins─were investigated using octadecylamine monolayers floating at air-aqueous solution interfaces. We find that at solution pH 5.7, larger monovalent anions induce a nonzero pressure starting at higher areas/molecules, i.e., a wider "liquid expanded" region in the monolayer isotherms. Using X-ray fluorescence at near total reflection (XFNTR), an element- and surface-specific technique, ion adsorption to the amines at pH 5.7 is confirmed to be ion-specific and to follow the conventional Hofmeister series. However, at pH 4, this ion specificity is no longer observed. We propose that at the higher pH, the amine headgroups are only partially protonated, and large polarizable ions such as iodine are better able to boost amine protonation. At the lower pH, on the other hand, the monolayer is fully protonated, and electrostatic interactions dominate over ion specificity. These results demonstrate that ion specificity can be modified by changing the experimental conditions.

Sulfato‐β‐cyclodextrin induced multivalent supramolecular directional aggregation of cyanovinylene derivatives for achieving reversible near‐infrared fluorescence

AbstractMolecular aggregation or supramolecular aggregation‐induced emission is one of the research hotspots in chemistry, biology, and materials. Herein, we report negatively charged sulfato‐β‐cyclodextrin (SCD) induced cyanovinylene derivatives (DPy‐6C) directional aggregation to form regular nanorods (DPy‐6C@SCD) through supramolecular multivalent interactions, not only achieves ultraviolet‐visible absorption redshifted from 453 to 521 nm but also displays near‐infrared (NIR) aggregation‐induced emission with a large spectral redshift of 135 nm. The DPy‐6C monomer presents random nanosheets with weak fluorescence but obtains regular aggregates after assembly with SCD through electrostatic interactions. In the presence of H+, the DPy‐6C@SCD can further aggregate into elliptical nanosheets without fluorescence changes due to the protonation of secondary amines. In contrast, the morphology of DPy‐6C@SCD becomes flexible and sticks together upon the addition of OH− with an emission blue shift of 72 nm and a 90‐fold intensity increase because of disrupting the stacking mode of aggregates, thereby achieving acid‐base regulated reversible fluorescence behaviors that cannot be realized by DPy‐6C monomer. The DPy‐6C@SCD can efficiently select the detection of volatile organic amines both in liquid and gas phases within 5 s at the nanomolar level. Taking advantage of RGB analysis and calculation formula application, the DPy‐6C@SCD has been successfully used to monitor various organic amines on a smartphone, accompanied by naked‐eye visible photoluminescence. Therefore, the present research provides an efficient directional aggregation method through supramolecular multivalent interactions, which not only realizes topological morphology transformation but also achieves reversible NIR luminescent molecular switch and high sensitivity organic amines fluorescent sensing devices.

Sonochemical synthesis and characterization of Fe(II) and Cu(II) nano-sized complexes of sulfamethoxazole

Nanoparticle drug delivery systems are precisely designed technologies that utilize nanoparticles to deliver therapeutic drugs to specific targets and regulate their release. In recent times, nanoparticles have garnered significant interest owing to their potential for efficient drug delivery. This work is aimed at synthesizing Fe(II) and Cu(II) nano-sized complexes of sulfamethoxazole (SMX) using the sonication method. The physical and spectroscopic studies showed a colour change from white to grey, and a decrease in melting points suggested the formation of the metal complexes. The nanometal complexes were insoluble in water. XRD analysis showed that the crystallite sizes of Fe(II) and Cu(II) nanometal complexes were determined to be 76.08 nm and 37.13 nm, respectively, using the Debye-Scherrer equation. The FTIR results of the SMX, Fe(II), and Cu(II) nanometal complexes showed a shift of the amine band from 3243 to 3191 cm^-1 and the sulfone band from 1154 to 1092 cm^-1 in both complexes. The proton NMR showed a shift of the amine proton from 6.100 ppm to 6.035 ppm in the spectra of the Cu(II) complex. The amine chemical shift was absent in the spectra of the Fe(II) complex, showing deprotonation. The carbon-13 NMR spectra showed a similar chemical shift. The spectra studies indicated that SMX coordinated with the metal ions through the amino and sulfone groups. A tetrahedral structure was proposed for the complexes. SMX coordinated as a bidentate ligand to Fe(II) and Cu(II) ions.

PH- and Photoresponsive Liquid Plasticine.

Liquid marbles (LMs) can be prepared by adsorption of hydrophobic particles at the air-liquid interface of a water droplet. LMs have been studied for their application as microreaction vessels. However, their opaqueness poses challenges for internal observation. Liquid plasticines (LPs), akin to LMs, can be prepared by the adsorption of hydrophobic particles with a diameter of 50 nm or less, at the air-liquid interface of a water droplet. Unlike LMs, LPs are transparent, allowing for internal observation, thus presenting promising applications as reactors and culture vessels on a microliter scale. In this study, the surface of silica particles, approximately 20 nm in diameter, was rendered hydrophobic to prepare hydrophobic silica particles (SD0). A small amount of poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) was then grafted onto the surface of SD0, yielding SD1. SD0 particles exhibited consistent hydrophobicity irrespective of the environmental pH atmosphere. Under acidic conditions, SD1 became hydrophilic due to the protonation of pendant tertiary amines in the grafted PDPA chains. However, SD1 alone was unsuitable for LP preparation due to its high surface wettability regardless of atmospheric pH, attributable to the presence of PDPA-grafted chains. Therefore, to prepare pH-responsive LP, SD1 and SD0 were mixed (SD1/SD0 = 3/7). Upon exposure to HCl gas, these LPs ruptured, with the leaked water from the LPs being absorbed by adjacent paper. Moreover, clear LPs, prepared using an aqueous solution containing a water-soluble photoacid generator (PAG), disintegrated upon exposure to light as PAG generated acid, leading to LP breakdown. In summary, pH-responsive LPs, capable of disintegration under acidic conditions and upon light irradiation, were successfully prepared in this study.

Anion-Binding Properties of Short Linear Homopeptides.

A comprehensive thermodynamic and structural study of the complexation affinities of tetra (L1), penta (L2), and hexaphenylalanine (L3) linear peptides towards several inorganic anions in acetonitrile (MeCN) and N,N-dimethylformamide (DMF) was carried out. The influence of the chain length on the complexation thermodynamics and structural changes upon anion binding are particularly addressed here. The complexation processes were characterized by means of spectrofluorimetric, 1H NMR, microcalorimetric, and circular dichroism spectroscopy titrations. The results indicate that all three peptides formed complexes of 1:1 stoichiometry with chloride, bromide, hydrogen sulfate, dihydrogen phosphate (DHP), and nitrate anions in acetonitrile and DMF. In the case of hydrogen sulfate and DHP, anion complexes of higher stoichiometries were observed as well, namely those with 1:2 and 2:1 (peptide:anion) complexes. Anion-induced peptide backbone structural changes were studied by molecular dynamic simulations. The anions interacted with backbone amide protons and one of the N-terminal amine protons through hydrogen bonding. Due to the anion binding, the main chain of the studied peptides changed its conformation from elongated to quasi-cyclic in all 1:1 complexes. The accomplishment of such a conformation is especially important for cyclopeptide synthesis in the head-to-tail macrocyclization step, since it is most suitable for ring closure. In addition, the studied peptides can act as versatile ionophores, facilitating transmembrane anion transport.

Dual pH/redox-responsive hyperbranched polymeric nanocarriers with TME-trigger size shrinkage and charge reversible ability for amplified chemotherapy of breast cancer

A novel pH/redox-responsive hyperbranched MeO-PEG-b-(NIPAAm-co-PBAE) nanoparticles (NPs) were designed with size shrinkage and charge-reversible potential for targeted delivery of docetaxel (DTX) to MDA-MB-231 cell lines. In the tumor microenvironment (TME), amine protonation induces charge reversal and disulfide bond cleavage under high TME GSH concentration causing size shrinkage, improved deep tumor penetration, and active targeting of the therapeutic agents. These nano drug delivery systems (NDDSs) significantly promoted cancer cell uptake (~ 100% at 0.5 h), facilitating site-specific delivery and deep tumor penetration. The MTT assay revealed significantly higher cytotoxicity (P value < 0.0001) for DTX-loaded NPs compared to free DTX. Cell cycle analysis revealed G2/M (58.3 ± 2.1%) and S (21.5 ± 1.3%) arrest for DTX-loaded NPs, while free DTX caused G2/M (67.9 ± 1.1%) and sub-G1 (10.3 ± 0.8%) arrest. DTX-loaded NPs induced higher apoptosis (P value < 0.001) in MDA-MB-231 cells (71.5 ± 2.8%) compared to free DTX (42.3 ± 3.1%). Western blotting and RT-PCR assays confirmed significant up-regulation of protein levels and apoptotic genes by DTX-loaded NPs compared to free DTX. In conclusion, TME-responsive charge reversal and size-shrinkable smart NDDSs designed based on low pH, and high glutathione (GSH), offer more effective site-specific delivery of therapeutic agents to tumors.

Spontaneous Curvature Induction in an Artificial Bilayer Membrane.

Maintaining lipid asymmetry across membrane leaflets is critical for functions like vesicular traffic and organelle homeostasis. However, a lack of molecular-level understanding of the mechanisms underlying membrane fission and fusion processes in synthetic systems precludes their development as artificial analogs. Here, we report asymmetry induction of a bilayer membrane formed by an extended π-conjugated molecule with oxyalkylene side chains bearing terminal tertiary amine moieties (BA1) in water. Autogenous protonation of the tertiary amines in the periphery of the bilayer by water induces anisotropic curvature, resulting in membrane fission to form vesicles and can be monitored using time-dependent spectroscopy and microscopy. Interestingly, upon loss of the induced asymmetry by extensive protonation using an organic acid restored bilayer membrane. The mechanism leading to the compositional asymmetry in the leaflet and curvature induction in the membrane is validated by density functional theory (DFT) calculations. Studies extended to control molecules having changes in hydrophilic (BA2) and hydrophobic (BA3) segments provide insight into the delicate nature of molecular scale interactions in the dynamic transformation of supramolecular structures. The synergic effect of hydrophobic interaction and the hydrated state of BA1 aggregates provide dynamicity and unusual stability. Our study unveils mechanistic insight into the dynamic transformation of bilayer membranes into vesicles.

Spontaneous Curvature Induction in an Artificial Bilayer Membrane

AbstractMaintaining lipid asymmetry across membrane leaflets is critical for functions like vesicular traffic and organelle homeostasis. However, a lack of molecular‐level understanding of the mechanisms underlying membrane fission and fusion processes in synthetic systems precludes their development as artificial analogs. Here, we report asymmetry induction of a bilayer membrane formed by an extended π‐conjugated molecule with oxyalkylene side chains bearing terminal tertiary amine moieties (BA1) in water. Autogenous protonation of the tertiary amines in the periphery of the bilayer by water induces anisotropic curvature, resulting in membrane fission to form vesicles and can be monitored using time‐dependent spectroscopy and microscopy. Interestingly, upon loss of the induced asymmetry by extensive protonation using an organic acid restored bilayer membrane. The mechanism leading to the compositional asymmetry in the leaflet and curvature induction in the membrane is validated by density functional theory (DFT) calculations. Studies extended to control molecules having changes in hydrophilic (BA2) and hydrophobic (BA3) segments provide insight into the delicate nature of molecular scale interactions in the dynamic transformation of supramolecular structures. The synergic effect of hydrophobic interaction and the hydrated state of BA1 aggregates provide dynamicity and unusual stability. Our study unveils mechanistic insight into the dynamic transformation of bilayer membranes into vesicles.

Site-Selective Palladium-catalyzed Oxidation of Unprotected Aminoglycosides and Sugar Phosphates.

The site-selective modification of complex biomolecules by transition metal-catalysis is highly warranted, but often thwarted by the presence of Lewis basic functional groups. This study demonstrates that protonation of amines and phosphates in carbohydrates circumvents catalyst inhibition in palladium-catalyzed site-selective oxidation. Both aminoglycosides and sugar phosphates, compound classes that up till now largely escaped direct modification, are oxidized with good efficiency. Site-selective oxidation of kanamycin and amikacin was used to prepare a set of 3'-modified aminoglycoside derivatives of which two showed promising activity against antibiotic-resistant E. coli strains.

Exploring the effect of a pendent amine group poised over the secondary coordination sphere of a cobalt complex on the electrocatalytic hydrogen evolution reaction.

A CoIII complex (2) of a bispyridine-dioxime ligand (H2LNMe2) containing a tertiary amine group in the proximity of the Co center is synthesized and characterized. One of the oxime protons of the ligand is deprotonated, and the amine group remains protonated in the solid-state structure of the CoII complex (2a). The acid-base properties of 2 showed pKa values of 5.9, 8.4, and 9.6, which are assigned to the dissociation of two consecutive oxime protons and amine protons, respectively. The electrocatalytic proton reduction of 2 was investigated in an aqueous phosphate buffer solution (PBS), revealing a catalytic hydrogen evolution reaction (HER) at an Ecat/2 of -1.01 V vs. the SHE, with an overpotential of 673 mV and a kobs value of 2.6 × 103 s-1 at pH 7. For comparison, the HER of the Co complex (1) lacking the tert-amine group at the secondary sphere was investigated in PBS, which showed a kobs of 1.3 × 103 s-1 and an overpotential of 577 mV. At pH 4, however, 2 revealed a ∼3 times higher kobs value than 1, which suggests that the protonated amine group likely works as a proton relay site. Notably, no significant change in the reaction rate was observed at different pH values for 1, implying that oxime protons may not be involved in the intramolecular proton-coupled electron transfer reaction in the HER. The kobs values for Co complexes at pH 7.0 are significantly higher than those of the [Co(dmgH)2(pyridine)(Cl)] complex, implying that the primary coordination sphere around 1 or 2 enhances the HER and offers better catalyst stability in acidic buffer solutions.

Multi-parametric MRI assessment of melatonin regulating the polarization of microglia in rats after cerebral ischemia/reperfusion injury

ObjectivesMulti-parametric magnetic resonance imaging (MRI) can provide comprehensive and valuable information for precise diagnosis and treatment evaluation of a number of diseases. In this study, the neuroprotective effects of melatonin (Mel) on a rat model of cerebral ischemia/reperfusion injury (CIRI) were assessed by multi-parametric MRI combined with histopathological techniques for longitudinal monitoring of the lesion microenvironment. MethodsSixty Sprague Dawley (SD) rats were randomly divided into three groups: the Sham, CIRI and CIRI + Mel groups. At multiple time points after ischemia, MRI scanning was performed on a 7.0 Tesla MRI scanner. Multi-parametric MRI includes T2-weighted imaging (T2WI), diffusion weighted imaging (DWI), and chemical exchange saturation transfer (CEST)-MRI. CEST effects were calculated by the Lorentzian difference method, 3.5 ppm indicates amide protons of mobile proteins/peptide (Amide-CEST) and 2.0 ppm indicates amine protons (Guan-CEST). Multiple histopathological techniques were used to examine the histopathological changes and explore the therapeutic effects of Mel. ResultsT2WI and DWI-MRI could localize the infarct foci and areas in CIRI rats, which was further validated by staining, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin and eosin (H&E) staining, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining. After Mel treatment, T2WI and DWI-MRI showed smaller infarct volume, and neurons displayed improved morphology with less apoptosis rates. Notably, Amide-CEST and Guan-CEST signal decreased as early as 2 h after CIRI (all P <0.001), reflecting the change of pH after ischemia. After Mel treatment, both Amide-CEST and Guan-CEST signal increased in ischemic cortex and striatum compared with control group (all P < 0.001). The immunofluorescence staining and western blotting analysis suggested the expression of M2 microglia increased after Mel treatment; While，after Mel treatment the inflammatory factor interleukin-1β (IL-1β) decreased compared with control CIRI rats. ConclusionsMulti-parametric MRI was shown to be an effective method to monitor the brain damage in a rat model of CIRI and assess the therapeutic effects of Mel treatment. Amide-CEST and Guan-CEST were especially sensitive to the changes in brain microenvironment during the early stage after CIRI. Furthermore, the neuroprotective effect of Mel treatment is associated with its promotion of the microglia polarized to M2 type in CIRI rats.

In vivo lymph node CEST-Dixon MRI in breast cancer patients with metastatic lymph node involvement.

Axillary lymph nodes (LNs) often present a reservoir for metastatic breast cancer, yet metastatic LN involvement cannot be discerned definitively using diagnostic imaging. This study investigated whether in vivo CEST may discriminate LNs with versus without metastatic involvement. 3T MRI was performed in patients with breast cancer before clinically-indicated mastectomy or lumpectomy with LN removal, after which LN metastasic involvement was determined using histological evaluation. Non-contrast anatomical imaging, as well as B0 and B1 field maps, were acquired in sequence with three-point CEST-Dixon (3D turbo-gradient-echo; factor = 25; TR/TE1/ΔTE = 851/1.35/1.1 ms; spatial-resolution = 2.5 × 2.5 × 6 mm; slices = 10; four sinc-gauss pulses with duty-cycle = 0.5, total saturation duration = 701.7 ms; B1 = 1.5 μT; saturation offsets = -5.5 to +5.5 ppm; stepsize = 0.2 ppm; scan duration = 6 min 30 s). The mean z-spectrum from LNs with (n = 20) versus without (n = 22) metastatic involvement were analyzed and a Wilcoxon rank-sum test (significance: p < 0.05) was applied to evaluate differences in B0, B1 , and magnetization transfer ratio (MTR) in differing spectral regions of known proton exchange (nuclear Overhauser effect [NOE], amide, amine, and hydroxyl) between cohorts. No difference in axillary B1 (p = 0.634) or B0 (p = 0.689) was observed between cohorts. Elevated MTR was observed for the NOE (-1.7 ppm; MTR = 0.285 ± 0.075 vs. 0.248 ± 0.039; p = 0.048), amine (+2.5 ppm; MTR = 0.284 ± 0.067 vs. 0.234 ± 0.31; p = 0.005), and hydroxyl (+1 ppm; MTR = 0.394 ± 0.075 vs. 0.329 ± 0.055; p = 0.002) protons in LNs from participants with versus without metastatic involvement. Findings are consistent with a unique metastatic LN microenvironment detectable by CEST-Dixon and suggest that CEST MRI may have potential for mapping LN metastasis non-invasively in vivo.

Intramacromolecular Conformational Changes in Low Generation PAMAM Dendrimers Probed by Pyrene Excimer Formation.

Pyrene excimer formation (PEF) was used to probe the intramacromolecular conformational change experienced by low generation pyrene-labeled PAMAM dendrimers referred to as PyCX-PAMAM-GY, where X (=4, 8, or 12) and Y (=0, 1, or 2) represent the number of atoms in the pyrenyl linker and the dendrimer generation, respectively. Each sample was studied in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) with and without 5 mM HCl. Global analysis of the monomer and excimer time-resolved fluorescence decays using the model free analysis (MFA) yielded the average rate constant of excimer formation, ⟨k⟩, which was compared with the local pyrene concentration ([Py]loc) of the PyCX-PAMAM-GY samples calculated by assuming that the oligomeric segments constituting the dendrimer's interior obeyed Gaussian statistics. A notable decrease in ⟨k⟩ was observed upon the addition of 5 mM HCl to the PyCX-PAMAM-GY solutions and was attributed to swelling of the dendrimers resulting from the protonation of the internal tertiary amines. The reversibility of this conformational change could also be monitored via PEF. Solvent differences between DMF and DMSO were accounted for by dividing ⟨k⟩ by kdiff, the bimolecular rate constant for diffusive PEF of a n-hexyl-1-pyrenebutyramide model compound, to yield the ⟨k⟩/kdiff ratio. Comparison between the ⟨k⟩/kdiff ratios obtained for all the PyCX-PAMAM-GY samples with and without 5 mM HCl revealed a 13% increase in the radius of the PAMAM-GY dendrimers upon protonation of their internal tertiary amines in agreement with earlier reports. These experiments illustrate that PEF represents a powerful experimental means to quantitatively probe the intramacromolecular conformational changes of complex macromolecules in solution, in a manner that complements scattering techniques.

Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

Reliable Identification of Relevant Factors for the Reactive Extraction of Succinic Acid from Electrolyte Containing Solutions

ABSTRACT Reactive extraction of succinic acid by tertiary amines is an effective separation technique. Its potential application for the initial recovery of bio-based succinic acid from fermentation broth. However, the co-extraction of inorganic anions significantly impairs the extraction performance. In this study, we use a statistically backed design of experiments method (DoE) to screen and identify the factors with a significant effect and relevant impact on extraction yield and selectivity. Eight operational factors (Extractant, solvent, succinic acid concentration, amine concentration, initial pH, the type of anion, electrolyte concentration, and temperature) were screened by applying an orthogonal design plan for the experiments. The choice of the extractant and diluent as well as the type of anion had the most significant effect on the extraction yield. In contrast, the selectivity was mainly affected by the concentration ratios of succinic acid and electrolyte. The screening plan reduced the number of experiments from 1728 possible combinations to 72 experiments required to identify the factors while maintaining the significance of the identified effects. We suggest competing mass-action relationships between the pH-dependent protonation of amines and the complexation of succinic acid to explain the observed trends.