Molecular Conjugates Research Articles

Optimization of cold-induced aerated gels formed by Maillard-driven conjugates of SPI-gellan gum as an oil substitute in mayonnaise sauce

This research aimed at chracaterization of composite cold-set aerated gels composed of SPI-gellan gum Maillard conjugates. The optimized gel was eventually incorporated in mayonnaise sauce as an oil substitute. The optimum conditions were statistically determined as 1.5% SPI, 300 mM CaCl2, and 90 min heating time. All of which resulted 35% glycation degree and high molecular weight conjugates on top of SDS-PAGE injection wells. Increasing CaCl2 concentration enhanced the adsorption of conjugates at air-water interface, decreasing the density but increasing the WHC and hardness. Increasing heating time facilitated gelation which improved gel hardness. The optimized gel was microstructurally homogeneous with increased overrun (20.8%) and H-bonds. The rheological measurements showed viscoelastic gel network which was thermally stable up to 90°C, besides increasing G’, G” and η* at 85°C. Substitution of optimized gel in mayonnaise sauce improved the nutritional value and thermal stability (77.13%), but declined calorie. The substituted mayonnaise sauce was greatly accepted by panelists. Thus, the aerated gel formed at optimum conditions had great structural and mechanical characteristics and its usage as an oil analogue induced a low-calorie mayonnaise sauce with acceptable sensory properties.

Fully Conjugated Covalent Triazine Framework Integrating Hexaazatrinaphthylene Unit as Anode Material for High-Performance Hybrid Lithium-Ion Capacitors.

As a high-performance energy storage device consisting of a battery-type anode and a capacitor-type cathode, hybrid lithium-ion capacitors (HLICs) combine the advantages of high energy density of batteries and high power density of capacitors. However, the imbalance in electrochemical kinetics between the battery-type anode and the capacitor-type cathode hinders the further development of HLICs. Fully conjugated covalent organic frameworks have great potential as electrode materials for HLICs due to the designability of their structure. Herein, a fully conjugated covalent triazine framework (PT-CTF) integrating the hexaazatrinaphthylene unit was constructed, which provides abundant active sites (C═N and C═C groups) as the pseudocapacitive anode material for HLICs. And the connection of the triazine unit of PT-CTF improves the molecular conjugate degree, facilitating the transport of electrons. The fabricated PT-CTF||AC HLICs exhibit a high energy density (164.9 Wh kg-1 at 100 mA g-1), large power density (13.1 kW kg-1 at 4 A g-1), and excellent cycling capability (72% after 10 000 cycles at 2 A g-1).

Unveiling the anti-cancer potentiality of phthalimide-based Analogues targeting tubulin polymerization in MCF-7 cancerous Cells: Rational design, chemical Synthesis, and Biological-coupled Computational investigation

The present study deals with an anti-cancer investigation of an array of phthalimide-1,2,3-triazole molecular conjugates with various sulfonamide fragments against human breast MCF-7 and prostate PC3 cancer cell lines. The targeted 1,2,3-triazole derivatives 4a-l and 6a-c were synthesized from focused phthalimide-based alkyne precursors using a facile click synthesis approach and were thoroughly characterized using several spectroscopic techniques (IR, 1H, 13C NMR, and elemental analysis). The hybrid click adducts 4b, 4 h, and 6c displayed cytotoxic potency (IC50 values of 1.49, 1.07, and 0.56 μM, respectively) against MCF-7 cells. On the contrary, none of the synthesized compounds showed apparent cytotoxic efficacy for PC3 cells (IC50 ranging from 9.87- >100 μM). As a part of the mechanism analysis, compound 6c demonstrated a potent inhibitory effect (78.3 % inhibition) of tubulin polymerization in vitro with an IC50 value of 6.53 µM. In addition, biological assays showed that compound 6c could prompt apoptotic cell death and induce G2/M cell cycle arrest in MCF-7 cells. Accordingly, compound 6c can be further developed as an anti-breast cancer agent through apoptosis-induction.

Encapsulation of probiotic Lacticaseibacillus casei with whey protein isolate/soybean hull polysaccharide enhances cell viability in harsh conditions

Whey protein isolate (WPI)-soybean hull polysaccharide (SHP) Maillard reaction conjugates were used to encapsulate Lacticaseibacillus casei and determine the effect of encapsulation on bacterial viability during freeze drying, simulated pasteurization simulated gastrointestinal digestion and storage. The WPI-SHP conjugates were characterized by degree of conjugation, size-exclusion chromatography, infrared and fluorescence spectroscopy and other analyses. The degree of conjugation, the formation of Maillard reaction products and the content of high molecular weight conjugates increased with reaction time. The emulsification capacity and glass transition temperature (Tg) of the WPI-SHP conjugates peaked after 4 days of reaction, at a Tg of 140.51 ± 2.76 °C, an emulsification activity index of 9.12 m2/g and emulsion stability index of 87.01%. WPI-SHP4d had the best protection effect of probiotics under freeze drying, simulated pasteurization and gastrointestinal digestion. These findings provide valuable insights into the selection of encapsulating agents to improve probiotic viability in functional foods.

Scalable Approach to Molecular Motor-Polymer Conjugates for Light-Driven Artificial Muscles.

The integration of molecular machines and motors into materials represents a promising avenue for creating dynamic and functional molecular systems, with potential applications in soft robotics or reconfigurable biomaterials. However, the development of truly scalable and controllable approaches for incorporating molecular motors into polymeric matrices has remained a challenge. Here, it is shown that light-driven molecular motors with sensitive photo-isomerizable double bonds can be converted into initiators for Cu-mediated controlled/living radical polymerization enabling the synthesis of star-shaped motor-polymer conjugates. This approach enables scalability, precise control over the molecular structure, block copolymer structures, and high-end group fidelity. Moreover, it is demonstrated that these materials can be crosslinked to form gels with quasi-ideal network topology, exhibiting light-triggered contraction. The influence of arm length and polymer structure is investigated, and the first molecular dynamics simulation framework to gain deeper insights into the contraction processes is developed. Leveraging this scalable methodology, the creation of bilayer soft robotic devices and cargo-lifting artificial muscles is showcased, highlighting the versatility and potential applications of this advanced polymer chemistry approach. It is anticipated that the integrated experimental and simulation framework will accelerate scalable approaches for active polymer materials based on molecular machines, opening up new horizons in materials science and bioscience.

Sulfadiazine analogs: anti-Toxoplasma in vitro study of sulfonamide triazoles.

Toxoplasmosis is an infection that prevails all over the world and is caused by the obligate intracellular protozoan parasite Toxoplasma gondii (T. gondii). Promising novel compounds for the treatment of T. gondii are introduced in the current investigation. In order to test their in vitro potency against T. gondii tachyzoites, six 1,2,3-triazoles-based sulfonamide scaffolds with terminal NH2 or OH group were prepared and investigated as sulfadiazine equivalents. When compared to sulfadiazine, which served as a positive control, hybrid molecules showed much more anti-Toxoplasma activity. The results showed that the IC50 of the examined compounds 3(a-f) were recoded as 0.07492 μM, 0.07455 μM, 0.0392 μM, 0.03124 μM, 0.0533 μM, and 0.01835 μM, respectively, while the sulfadiazine exhibited 0.1852 μM. The studied 1,2,3-triazole-sulfadrug molecular conjugates 3(a-f) revealed selectivity index of 10.4, 8.9, 25.4, 21, 8.3, and 29; respectively. The current study focused on the newly synthesized amino derivatives 3(d-f), as they contain the more potent amino groups which are recognized to be essential elements and promote better biological activity. Extracellular tachyzoites underwent striking morphological alterations after 2 h of treatment as seen by scanning electron microscopy (SEM). Additionally, the intracellular tachyzoite exposed to the newly synthesized amino derivatives 3(d-f) for a 24-h period of treatment revealed damaged and altered morphology by transmission electron microscopic (TEM) indicating cytopathic effects. Moreover, compound 3f underwent the most pronounced changes, indicating that it had the strongest activity against T. gondii.

Redox-sensitive self-assembled micelles based on low molecular weight chitosan-lipoic acid conjugates for the delivery of doxorubicin: Effect of substitution degree of lipoic acid

Amphiphilic low molecular weight chitosan-lipoic acid (LC-LA) conjugates with different degrees of substitution (DS) of LA were synthesized by N, N′‑carbonyldiimidazole (CDI) catalysis to self-assemble into redox-sensitive micelles. Critical micelle concentration (CMC), size, zeta potential, biocompatibility and redox-sensitive behavior of blank micelles were investigated. The results indicated that blank micelles with low CMC, nanoscale size and positive zeta potential showed excellent biocompatibility and redox-sensitive behavior. Doxorubicin (Dox) loaded micelles were prepared by encapsulating Dox into blank micelles. The loading ability, trigger-release behavior, antitumor activity and cellular uptake of Dox loaded micelles were studied. The results demonstrated that Dox loaded micelles with superior loading ability exhibited redox-trigger behavior, strong antitumor activity and increased cellular uptake efficiency against A549 cell. Besides, the effect of DS of LA on above properties was estimated. An increase in DS of LA reduced the CMC and cumulative release amount of Dox, but improved the loading efficiency, antitumor activity, and cellular uptake of Dox loaded micelles, which resulted from stronger interaction of hydrophobic groups in micelles with the DS of LA increased. Overall, self-assembled LC-LA micelles with good biosecurity and redox-sensitive behavior hold promising application prospects in Dox delivery and improving cancer therapeutic effect of Dox.

Formation and Structure Research of a Molecular Conjugate in the L-Ascorbic Acid – Isomaltite System

The formation of L-ascorbic acid molecular complexes with various biologically active substances opens the way to the production of a new drug type promising for the pharmaceutical, food and cosmetic industries use. The research concerns the possibility study of obtaining a conjugate of L-ascorbic acid with isomaltite in an aqueous ethanol (40 %) medium. The method of the target product isolating involves thermostating a solution containing equimolar amounts (0.001 mmol) of components (50 °C, 1 hour), followed by slow solvent removal for 24 hours (25 °C). The authors examined formation processes dynamics by molecular spectroscopy methods through sequential sampling. The optical density values at 350 Nm in the electronic spectra and 1,674 and 1,754 cm−1 bands position in the IR-Fourier solution spectra indicate the components destruction absence during thermostate control. When treated with carbon tetrachloride or diethyl ether, the syrupy mass obtained after solvent removal containing significant solvent amounts crystallizes a yellowish powder (0.32 g, ice point at 146.6 °C). A man studied its structure by the IR-Fourier spectroscopy. The authors recorded the spectra in the transmission mode (in the KBr tablets) and using the diffusion reflection method enabling to record changes in the absorption bands characteristic positions of the molecular complex formation. The transmission spectra of the high-frequency area indicate the 1-O-glycosylated isomaltite component involvement in the conjugation with ascorbic acid. The shifts of the C=C, C−O and C−O−H bond oscillation bands in the diffusion reflection spectrum of the isolated product relative to their position in the initial component spectra in the low-frequency area indicate the endiol ascorbic acid fragment interaction with the secondary OH groups of the acyclic isomaltite component. Structural analogues of the studied molecular conjugate are promising antioxidant preparations. This determines the further study relevance of its properties.

Deep learning for the encounter of inorganic nanomaterial for efficient photochemical hydrogen production

A strong photosynthetic multifaceted called photosystem I (PS I) successfully catches photons to produce a charge detached phase through a quantum yield. The low latent reductant that is created is dignified at a redox potential that is conducive to H2 development, and this charge detached phase is constant for around 100 ms. By using dithiol as a molecular wire to link Photo System I to the particles and transport electrons as of the incurable electron transport cofactor of Photo System I, towards the nanoparticle, PS I consumes been covalently connected to the surfaces of like as Pt and also Au nanoparticles. The Photosystem I or molecular wire or nanoparticle bio conjugates are capable of catalysing the reaction 2H+ + 2e H2 when illuminated. H2 development is not slowed down by the transference of electrons from PS I in the direction of the nanoparticle via the molecular wire. The speed of H2 evolution is increased fivefold when the system is supplied by means of more effective donor sideways electron-donating kinds. When evaluating hydrogen production based on predicted energy from the sun with observed values falling on unstable slopes, the root mean square error called as RMSE as well as coefficient of determination called R2 are utilised as performance metrics. The most effective model among those taken into consideration, the Deep Learning model performs well and is quite compatible with the observational data.

Tuning Electrochemical Properties of Nitroaromatic Cathodes by Function‐Oriented Design for Rechargeable Lithium‐Ion Batteries

AbstractRechargeable lithium‐ion batteries (LIBs) based on organic cathodes are an attractive alternative energy storage technology owing to the low cost and sustainability. Recently, nitro functionality in dinitrobenzenes is successfully demonstrated as an electrochemically reversible high‐capacity redox group. In this study, a function‐oriented design is employed to further disclose the effects of substituting functional groups and molecular conjugate structures on electrochemical properties of a range of nitroaromatic derivatives as organic cathodes for rechargeable LIBs. In specific, it is revealed that the redox potential of nitroaromatic cathodes can be effectively adjusted by introducing distinct electronically inducible functional groups, while the cyclic life can be significantly prolonged with the introduction of the hydrophilic groups. When constructed with extended π‐conjugated structures, the electronic conductivity and electrochemical kinetics of nitroaromatics are increased significantly owing to their various long‐range π–π stacking. Moreover, density‐functional theory calculations further provide theoretical insights into the distinct electrochemical behaviors of the various nitroaromatics in the molecular level. This is the first study that reveals the influences of substituting groups and conjugated structures on the electrochemical performance of nitroaromatic cathode materials, which enables a function‐oriented molecular design of such organic materials and sheds light on their future development.

Ionic Liquid-Mediated Approach for the Synthesis of Site-Specific Thioether Conjugates.

Site-specific conjugation approaches are of great importance in drug discovery, notably for the synthesis of biochemical probes or molecular conjugates for targeted delivery. Herein, we report a mild ionic liquid (IL)-mediated thiolation technique that relies on the use of 1,3-ethyl-methyl imidazolium acetate, [C2 mim][OAc] as a solvent and precursor to generate activated IL, as well as a solvent for the conjugation reaction. First, a focused library of active ILs was prepared for functionalizing/conjugating cysteine-containing small molecules and unprotected peptides. Interestingly, a bifunctional active IL could also be successfully employed as a linker for the conjugation of peptides lacking Cys. This study sets the ground for further investigation of the use of active ILs for modifying, labeling or conjugating larger and more complex therapeutic modalities such as proteins and antibodies.

Inhibition of atherosclerosis progression by modular micelles.

Atherosclerosis is a chronic disease initiated by lipid-mediated vascular inflammation. From the perspective of conventional treatment, it is difficult to achieve good therapeutic effects via regulation of a single lipid or anti-inflammatory effects. Herein, we designed an amphiphilic low molecular weight heparin-unsaturated fatty acid conjugate (LMWH-uFA) that acted as both an antiatherosclerotic agent and a nanocarrier with self-delivery properties. Structurally, LMWH-uFA self-assembled to form micelles with LMWH as the shell and uFA as the core, without any additives, which guaranteed their biosafety. Functionally, the hydrophilic segment, LMWH, prevented monocyte adhesion to inhibit early vascular inflammation, and the hydrophobic segment, uFA, could participate in the regulation of blood lipids. The anti-inflammatory drug rapamycin (RAP) was encapsulated in the micellar core, which improved its water solubility, and cooperated with LMWH to achieve targeted blockade of the vascular inflammation cascade at P-selectin. The three treatment modules, LMWH, uFA and RAP, were integrated into one system for different therapeutic targets in anticipation of better efficacy. In an atherosclerosis mouse model, RAP-loaded NPs significantly reduced the plaque area and showed satisfactory curative effects, which were related to the targeting of lipid regulation and inflammation. Thus, these modular micellar nanoparticles offer a promising approach for the clinical treatment of atherosclerosis.

Nintedanib-αVβ6 Integrin Ligand Conjugates Reduce TGFβ-Induced EMT in Human Non-Small Cell Lung Cancer.

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGFβ is one of the most powerful inducers of this transition, as it induces overexpression of the fibronectin receptor, αvβ6 integrin, in cancer cells which, in turn, is strongly associated with EMT. Thus, αvβ6 integrin receptors may be exploited as a target for the selective delivery of anti-tumor agents. We introduce three novel synthesized conjugates, in which a selective αvβ6 receptor ligand is linked to nintedanib, a potent kinase inhibitor used to treat advanced adenocarcinoma lung cancer in clinics. The αvβ6 integrin ligand directs nintedanib activity to the target cells of the tumor microenvironment, avoiding the onset of negative side effects in normal cells. We found that the three conjugates inhibit the adhesion of cancer cells to fibronectin in a concentration-dependent manner and that αvβ6-expressing cells internalized the conjugated compounds, thus permitting nintedanib to inhibit 2D and 3D cancer cell growth and suppress the clonogenic ability of the EMT phenotype as well as intervening in other aspects associated with the EMT transition. These results highlight αvβ6 receptors as privileged access points for dual-targeting molecular conjugates engaged in an efficient and precise strategy against non-small cell lung cancer.

Observation of the triplet energy transfer in orthogonal photoexcited iodinated-BODIPY dimers.

Intramolecular charge and energy transfer processes initiated by light absorption can change the photosensitization properties of molecular conjugates. Transient optical and electron paramagnetic resonance (TREPR) spectroscopies are well suited for the study of these processes. In the TREPR spectra of the triplet state of an iodinated BODIPY dimer, we have observed the effect of the averaging of the zero-field (ZFS) parameter E, which becomes more efficient with increasing temperature. This property is associated with triplet energy transfer from one chromophore in the dimer to another, implying the presence of a dynamic equilibrium between the two chromophores of the dimer. From the comparison of the ZFS parameters of the monomer and the dimer, the rates of the reversible hopping are derived within the framework of a two-site model. The obtained data indicate that the triplet states are separated by an energy barrier of ca. 70 K (ca. 0.006 eV) and that below this temperature energy transfer occurs via tunneling.

Creation of single molecular conjugates of metal-organic cages and DNA.

Here we report the development of an equimolar conjugate of a metal-organic cage (MOC) and DNA (MOC-DNA). Several MOC-DNA conjugates were assembled into a programmed structure by coordinating with a template DNA having a complementary base sequence. Moreover, conjugation with the MOC drastically enhanced the permeability of DNA through the lipid bilayer, presenting great potential as a drug delivery system.

Photophysical characterization of a bisacridinium-diphenylporphyrin conjugate

A molecular conjugate made of a free-base diphenyl porphyrin chromophore (DPP) trans-linked to two N-acridinium units has been synthesized and photophysically characterized in acetonitrile. Interestingly, the emission of both fluorophores is quenched in the array at room temperature. Steady-state and time-resolved optical analysis proved that an ultrafast electron transfer from the porphyrin to the acridinium units occurs upon excitation of either the porphyrin or the acridinium moiety. On the other hand, at low temperature (77 K) the emission of the porphyrin is completely recovered, thanks to the inhibition of the electron transfer, and a photoinduced energy transfer from the acridinium to the porphyrin component is observed.

A pHe sensitive nanodrug for collaborative penetration and inhibition of metastatic tumors.

Current chemotherapies for metastatic tumors are seriously restricted by limited drug infiltration and deficient disturbance of metastasis-associated complex pathways involving tumor cell autocrine as well as paracrine loops in the microenvironment (TME). Of note, cancer-associated fibroblasts (CAFs) play a predominant role in shaping TME favoring drug resistance and metastasis. Herein, we constructed a tumor extracellular pH (pHe) sensitive methotrexate-chitosan conjugate (MTX-GC-DEAP) and co-assembled it with quercetin (QUE) to achieve co-delivered nanodrugs (MTX-GC-DEAP/QUE). The pHe sensitive protonation and disassembly enabled MTX-GC-DEAP/QUE for stroma-specific delivery of QUE and positive-charged MTX-GC-DEAP molecular conjugates, thereby achieving deep tumor penetration via the combination of QUE-mediated CAF inactivation and adsorption-mediated transcytosis. On the basis of significantly promoted drug availability, a strengthened "omnidirectional" inhibition of pre-metastatic initiation was generated both in vitro and in vivo from the CAF inactivation-mediated reversion of metastasis-promoting environments as well as the inhibition of epithelial-mesenchymal transition, local and blood vessel invasion via QUE-mediated direct regulation on tumor cells. Our tailor-designed versatile nanodrug provides a deep insight into potentiating multi-faceted penetration of multi-mechanism-based regulating agents for intensive metastasis inhibition.

ИЗУЧЕНИЕ ЦИТОТОКСИЧЕСКОГО ДЕЙСТВИЯ МОЛЕКУЛЯРНОГО КОНЪЮГАТА НА ОСНОВЕ ХЛОРИНА Е6

Photodynamic therapy is a clinically approved method of cancer treatment that has shown great potential due to its non-invasiveness and selectivity for proliferating cells. However, the potential of this type of therapy has not been fully exploited, partly due to the lack of ideal photosensitizer drugs. One of the possible strategies for the molecular design of new generation drugs is the inclusion of heavy atoms into the structure of such compounds. The photodynamic and photocytotoxic properties of the newly synthesized molecular conjugate based on Chlorin e6 with europium were studied. Optimal irradiation conditions were selected and effective cell death of Ehrlich's ascitic carcinoma under the action of a molecular conjugate upon irradiation with red light at a wavelength of 645 nm for 20 min was shown. At this stage, the PEI/e6/DTPA/FA/Eu molecular conjugate exhibits confirmed photodynamic activity, which indicates the prospect of further research.

Effect of carrier molecular weight on physicochemical properties and the in vitro immune-stimulatory activity of the CpG-dextran conjugates

The effect of dextran molecular weight on the in vitro physicochemical and immune properties of cytosine-phosphate-guanine (CpG) oligodeoxynucleotide-amino-dextran conjugates is investigated. CpG-1668 was conjugated at the 3′-end to amino-dextran of differing molecular weight (20, 40, 70 or 110-kDa) via a stable bis-aryl hydrazone linkage. Conjugate formation was confirmed by agarose gel electrophoresis and dynamic light scattering measured the size and surface charge of conjugates. Uptake and immune-stimulatory activity of CpG-dextran by antigen-presenting cells was evaluated by flow cytometry and confocal microscopy. Degradation by DNase I was monitored by loss of the fluorescent signal from labelled CpG and changes in size and zeta potential. Hydrazone bond formation (UV 354 nm) showed on average four CpG molecules conjugated per polymer. CpG-dextran prepared from 20 or 40-kDa dextran had a size of 17 nm while 70 or 110-kDa was 30 nm. CpG-dextran was preferentially taken up by dendritic cells, followed by macrophages and then B-cells. Only the 20-kDa dextran conjugate significantly enhanced uptake by bone-marrow derived dendritic cells (BMDCs) compared to free CpG. Confocal microscopy showed that CpG and CpG-dextran accumulates in the endo-lysosomal compartment of BMDCs at 24 h. All conjugates upregulated activation markers (CD40, CD80 or CD86) of BMDCs to a similar level as for free CpG. CpG-dextran 40-kDa produced highest levels of cytokines (TNF-α, IL-6, and IL-12p70) secreted by BMDCs. Enzymatic protection assays showed that the conjugate made from dextran 20-kDa provided no protection for CpG while the higher molecular weight conjugates reduced degradation by DNase I. The 40-kDa dextran conjugate produced the greatest in vitro immune activity, this was due to the conjugate being relatively small in size for cell uptake while sufficiently large enough to protect CpG from nuclease attack. These in vitro studies identify the need to consider the molecular weight of the carrier in bioconjugate design.

Characterization of High Molecular Weight Pneumococcal Conjugate by SEC-MALS and AF4-MALS.

Infections by Streptococcus pneumoniae can cause serious pneumococcal diseases and other medical complications among patients. Polysaccharide-based vaccines have been successfully developed as prophylactic agents against such deadly bacterial infections. In the 1980s, PNEUMOVAX® 23 were introduced as the first pneumococcal polysaccharide vaccines (PPSV). Later, pneumococcal polysaccharides were conjugated to a carrier protein to improve immune responses. Pneumococcal conjugate vaccines (PCV) such as PREVNAR® and VAXNEUVANCE™ have been developed. Of the more than 90 pneumococcal bacteria serotypes, serotype 1 (ST-1) and serotype 4 (ST-4) are the two main types that cause invasive pneumococcal diseases (IPD) that could lead to morbidity and mortality. Development of a novel multi-valent PCV against these serotypes requires extensive biophysical and biochemical characterizations of each monovalent conjugate (MVC) in the vaccine. To understand and characterize these high molecular weight (Mw) polysaccharide protein conjugates, we employed the multi-angle light scattering (MALS) technique coupled with size-exclusion chromatography (SEC) separation and asymmetrical flow field flow fractionation (AF4). MALS analysis of MVCs from the two orthogonal separation mechanisms helps shed light on the heterogeneity in conformation and aggregation states of each conjugate.