A comprehensive evaluation of a polymeric zwitterionic hydrophilic monolith for nucleotide separation.

Rapid and effective separation of nucleotides (NTs) and their derivatives is crucial for studying their physiological functions. In this work, we comprehensively evaluated the separation ability of a zwitterionic hydrophilic monolith, i.e., poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammonium betaine-co-N,N'-methylenebisacrylamide) (poly(SPP-co-MBA)) for NTs analysis, including its selectivity, chemical stability under extremely basic condition and compatibility with hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (HILIC-MS). The poly(SPP-co-MBA) monolith exhibited excellent chemical stability, as evidenced by the low relative standard deviation of retention time (0.16-1.05%) after 4000 consecutive injections over one month under strong alkaline elution condition (pH 10). After optimizing the separation conditions, including buffer pH and concentration, organic solvent content and column temperature, four nucleoside triphosphates, five nucleoside diphosphates and five nucleoside monophosphates were baseline separated within 7 min. Additionally, the mixtures containing one nucleoside and its corresponding mono-, di-, and triphosphates were baseline separated within only 3 min, respectively. It is good HILIC-MS compatibility was also confirmed by the satisfactory peak shape and high response of nine NTs. Overall, the proposed poly(SPP-co-MBA) monolith exhibited good mechanical stability and compatibility of HILIC-MS, making it a promising technique for NTs analysis.

Simultaneous chromatographic separation of the anomers of saccharides on a polymer sulfobetaine zwitterionic stationary phase.

Simultaneous chromatographic separation of the anomers of saccharides was achieved by using a polymer zwitterionic stationary phase functionalized by acrylamide-type sulfobetaine. By optimization of separation parameters including column temperature, pH, and flow rate, the column operated in hydrophilic interaction chromatography mode exhibited excellent separation selectivity toward five monosaccharides and their anomers (including ribose, xylose, galactose, glucose, and arabinose) and two disaccharides (lactose and maltose). Baseline separation could be achieved at mild operation conditions such as 20-30°C of column temperature or typical mobile phase composition (85% acetrontrile-15% 20 mM ammonium formate [NH4 FA]) with wide pH tolerance range of 2-8. This offers a rapid way to determine the configuration of α or ß anomer of the saccharides.

High-resolution ion mobility spectrometry-mass spectrometry for isomeric separation of prostanoids after Girard's reagent T derivatization.

RATIONALE: Isomeric separation of prostanoids is often a challenge and requires chromatography and time-consuming sample preparation. Multiple prostanoid isomers have distinct in vivo functions crucial for understanding the inflammation process, including prostaglandins E2 (PGE2 ) and D2 (PGD2 ). High-resolution ion mobility spectrometry (IMS) based on linear ion transport in low-to-moderate electric fields and nonlinear ion transport in strong electric fields emerges as a broad approach for rapid separations prior to mass spectrometry. METHODS: Derivatization with Girard's reagent T (GT) was used to overcome inefficient ionization of prostanoids in negative ionization mode due to poor deprotonation of the carboxylic acid group. Three high-resolution IMS techniques, namely linear cyclic IMS, linear trapped IMS, and nonlinear high-field asymmetric waveform IMS, were compared for the isomeric separation and endogenous detection of prostanoids present in intestinal tissue. RESULTS: Direct infusion of GT-derivatized prostanoids proved to increase the ionization efficiency in positive ionization mode by a factor of >10, which enabled detection of these molecules in endogenous concentration levels. The high-resolution IMS comparison revealed its potential for rapid isomeric analysis of biologically relevant prostanoids. Strengths and weaknesses of both linear and nonlinear IMS are discussed. Endogenous prostanoid detection in intestinal tissue extracts demonstrated the applicability of our approach in biomedical research. CONCLUSIONS: The applied derivatization strategy offers high sensitivity and improved stereoisomeric separation for screening of complex biological systems. The high-resolution IMS comparison indicated that the best sensitivity and resolution are achieved by linear and nonlinear IMS, respectively.

Osmolyte effect on enzymatic stability and reaction equilibrium of formate dehydrogenase.

Osmolytes are well-known biocatalyst stabilisers as they promote the folded state of proteins, and a stabilised biocatalyst might also improve reaction kinetics. In this work, the influence of four osmolytes (betaine, glycerol, trehalose, and trimethylamine N-oxide) on the activity and stability of Candida bondinii formate dehydrogenase cbFDH was studied experimentally and theoretically. Scanning differential fluorimetric studies were performed to assess the thermal stability of cbFDH, while UV detection was used to reveal changes in cbFDH activity and reaction equilibrium at osmolyte concentrations between 0.25 and 1 mol kg-1. The thermodynamic model ePC-SAFT advanced allowed predicting the effects of osmolyte on the reaction equilibrium by accounting for interactions involving osmolyte, products, substrates, and water. The results show that osmolytes at low concentrations were beneficial for both, thermal stability and cbFDH activity, while keeping the equilibrium yield at high level. Molecular dynamics simulations were used to describe the solvation around the cbFDH surface and the volume exclusion effect, proofing the beneficial effect of the osmolytes on cbFDH activity, especially at low concentrations of trimethylamine N-oxide and betaine. Different mechanisms of stabilisation (dependent on the osmolyte) show the importance of studying solvent-protein dynamics towards the design of optimised biocatalytic processes.

Zwitterionic surface chemistry enhances detachment of bacteria under shear.

The ubiquitous nature of microorganisms, especially of biofilm-forming bacteria, makes biofouling a prevalent challenge in many settings, including medical and industrial environments immersed in liquid and subjected to shear forces. Recent studies have shown that zwitterionic groups are effective in suppressing bacteria and protein adhesion as well as biofilm growth. However, the effect of zwitterionic groups on the removal of surface-bound bacteria has not been extensively studied. Here we present a microfluidic approach to evaluate the effectiveness in facilitating bacteria detachment by shear of an antifouling surface treatment using (3-(dimethyl;(3-trimethoxysilyl)propyl)ammonia propane-1-sulfonate), a sulfobetaine silane (SBS). Control studies show that SBS-functionalized surfaces greatly increase protein (bovine serum albumin) removal upon rinsing. On the same surfaces, enhanced bacteria (Pseudomonas aeruginosa) removal is observed under shear. To quantify this enhancement a microfluidic shear device is employed to investigate how SBS-functionalized surfaces promote bacteria detachment under shear. By using a microfluidic channel with five shear zones, we compare the removal of bacteria from zwitterionic and glass surfaces under different shear rates. At times of 15 min, 30 min, and 60 min, bacteria adhesion on SBS-functionalized surfaces is reduced relative to the control surface (glass) under quiescent conditions. However, surface-associated bacteria on the SBS-functionalized glass and control show similar percentages of live cells, suggesting minimal intrinsic biocidal effect from the SBS-functionalized surface. Notably, when exposed to shear rates ranging from 104 to 105 s-1, significantly fewer bacteria remain on the SBS-functionalized surfaces. These results demonstrate the potential of zwitterionic sulfobetaine as effective antifouling coatings that facilitate the removal of bacteria under shear.

Hyperosmotic Stress Allosterically Reconfigures Betaine Binding Pocket in BetP.

The transporter BetP in C. glutamicum is essential in maintaining bacterial cell viability during hyperosmotic stress and functions by co-transporting betaine and Na+ into bacterial cells. Hyperosmotic stress leads to increased intracellular K+ concentrations which in turn promotes betaine binding. While structural details of multiple end state conformations of BetP have provided high resolution snapshots, how K+ sensing by the C-terminal domain is allosterically relayed to the betaine binding site is not well understood. In this study, we describe conformational dynamics in solution of BetP using amide hydrogen/deuterium exchange mass spectrometry. These reveal how K+ alters conformation of the disordered C- and N-terminal domains to allosterically reconfigure transmembrane helices 3, 8, and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, reveals a critical extrahelical H-bond mediated by TM3 with betaine.

Effect of Molecular Weights on Metal-Mediated Grafting of Sulfobetaine Polymers onto Solid Surfaces for Non-Biofouling Applications.

The grafting of zwitterionic molecules onto solid surfaces is an important tool for decreasing the unwanted adsorption of biomolecules, such as proteins, bacteria, and cells. This has been achieved through various approaches, such as zwitterionic monolayer/multilayer formation, surface-initiated polymerization of zwitterionic monomers, and grafting of presynthesized zwitterionic polymers. Recently, a coordination-driven approach to grafting zwitterionic polymers onto solid surfaces has been discovered to be an effective method because of its versatility and robustness. However, the bacterial adhesion resistance of zwitterionic polymer grafting has been explored using only one molecular weight, and the non-biofouling performance against other fouling organisms has remained unexamined. In this study, the characteristics of coordination-driven surface zwitteration are systematically investigated. Sulfobetaine (SB) polymers with three different molecular weights are synthesized and employed for surface grafting. Polydopamine is used as a surface primer, and SB polymers are grafted onto the surfaces via the formation of metal-mediated coordinate bonds. The effect of molecular weight on the grafting efficiency and non-biofouling performance is investigated via protein adsorption and marine diatom adhesion assays. The SB polymer with a high molecular weight is found to be crucial for achieving strong resistance to protein adsorption and marine fouling.

Theoretical, Semiempirical, and Experimental Solvatochromic Comparison Methods for the Construction of the α1 Scale of Hydrogen-Bond Donation of Solvents.

Today, the hydrogen bonding donation (HBD) ability parameter of new solvents, α, is generally determined either by the Kamlet-Taft solvatochromic comparison of two probes, Reichardt betaine dye B(30) and 4-nitroanisole, or by the measurement of a single probe (e.g., solvatochromism of an iron coordination complex). This work highlights the shortcomings of these probes and recommends three replacement methods: (a) the theoretical comparison of the experimental and PCM-TD-DFT calculated transition energies ET(30) of B(30), (b) the semiempirical comparison of the experimental and McRae calculated ET(30), and, (c) for ionic liquids, the experimental comparison of ET(30) and ET(33) lying on the lower basicity of the betaine dye B(33) compared to B(30). These methods yield a new HBD parameter, α1, for 101 molecular solvents and 30 ionic liquids. The novelty is emblematic for water, with α1 = 1.54 instead of α (Kamlet-Taft) = 1.17. The solvent parameter α1 is not equivalent to the solute hydrogen-bond acidity parameter α2H, partly because of the self-association of HBD solvents.

Mechanism of Osmolyte Stabilization-Destabilization of Proteins: Experimental Evidence.

In this work, we investigated the influence of stabilizing (N,N,N-trimethylglycine) and destabilizing (urea) osmolytes on the hydration spheres of biomacromolecules in folded forms (trpzip-1 peptide and hen egg white lysozyme─hewl) and unfolded protein models (glycine─GLY and N-methylglycine─NMG) by means of infrared spectroscopy. GLY and NMG were clearly limited as minimal models for unfolded proteins and should be treated with caution. We isolated the spectral share of water changed simultaneously by the biomacromolecule/model molecule and the osmolyte, which allowed us to provide unambiguous experimental arguments for the mechanism of stabilization/destabilization of proteins by osmolytes. In the case of both types of osmolytes, the decisive factor determining the equilibrium folded/unfolded state of protein was the enthalpy effect exerted on the hydration spheres of proteins in both forms. In the case of stabilizing osmolytes, enthalpy was also favored by entropy, as the unfolded state of a protein was more entropically destabilized than the folded state.

Structure-Activity Relationship of the Thiacalix[4]arenes Family with Sulfobetaine Fragments: Self-Assembly and Cytotoxic Effect against Cancer Cell Lines.

Regulating the structure of macrocyclic host molecules and supramolecular assemblies is crucial because the structure-activity relationship often plays a role in governing the properties of these systems. Herein, we propose and develop an approach to the synthesis of the family of sulfobetaine functionalized thiacalix[4]arenes with regulation of the self-assembly and cytotoxic effect against cancer cell lines. The dynamic light scattering method showed that the synthesized macrocycles in cone, partial cone and 1,3-alternate conformations form submicron-sized particles with Ag+ in water, but the particle size and polydispersity of the systems studied depend on the macrocycle conformation. Based on the results obtained by 1H and 1H-1H NOESY NMR spectroscopy and transmission electron microscopy for the macrocycles and their aggregates with Ag+, a coordination scheme for the Ag+ and different conformations of p-tert-butylthiacalix[4]arene functionalized with sulfobetaine fragments was proposed. The type of coordination determines the different shapes of the associates. Cytotoxic properties are shown to be controlled by the shape of associates, with the highest activity demonstrated by thiacalix[4]arenes in partial cone conformation. This complex partial cone/Ag+ is two times higher than the reference drug imatinib mesylate. High selectivity against cervical carcinoma cell line indicates the prospect of their using as components of new anticancer system.

Unraveling the Structured Solvation Shell of Zwitterion Nanoparticles for Controlled Release of Nitric Oxide.

Zwitterions have been attracting emerging attention as an anti-fouling polymer. However, the relationship between structured solvation shells and controlled drug release induced by deceleration of water molecule's translational and vibrational motions of zwitterions is an uncharted territory. Herein, sulfobetaine zwitterion nanoparticles (ZWNPs) were designed as a stable nitric oxide (NO)-delivering carrier. The condensed water structure of the solvation shell at its isoelectric point (PI) and the loose structure of water under different pH conditions were investigated through rheological and thermodynamical analyses. The ZWNPs showed a sustained-release profile at the PI, which presented a structured solvation barrier. On the other hand, NO-loaded ZWNPs showed different release profiles with the burst release at pH 5.5. Notably, an increased cell proliferation rate and a decreased antibacterial effect were observed at the same concentration depending on solvation shell's characteristics.

Influence of Zwitterionic CAPB on Flocculation of the Aqueous Cationic Guar Gum/Glauconite Suspensions at Various pH.

The influence of the pseudoamphoteric zwitterionic surfactant cocamidopropylbetaine (CAPB) on the stabilizing flocculating properties of the aqueous suspensions of glauconite (GT) with cationic guar gum (CGG) at various pH values was investigated. The following techniques were used: turbidimetry, UV-VIS spectrophotometry, tensiometry, electrophoretic mobility measurements, SEM, CHN, XRD, and FT-IR. It was established that CGG is an effective glauconite flocculant. Moreover, the most probable mechanism that is responsible for flocculation is bridge flocculation resulting from polymer adsorption on the glauconite surface. The adsorption process is caused by electrostatic interactions between the negatively charged glauconite surface and the positively charged polymer. The amount of CGG adsorption increases with the increase of the pH, which was confirmed by the adsorption and zeta potential measurements. The addition of CAPB increases the amount of the polymer adsorption due to the formation of intermolecular polymer-surfactant complexes; however, it reduces flocculation effectiveness.

A green approach to starch modification by solvent-free method with betaine hydrochloride.

In this study, a novel simple and eco-efficient, semi-dry method with a spray system for starch modification has been developed. Compared to conventional semi-dry methods, this method does not use solvents so that no slurry or semi-liquid mixture is obtained, the material is in a moisted/semi-moisted state. The modification of starch was performed using betaine hydrochloride (BHC) as the cationic reagent, and the characteristics of such starch derivates were compared with cationic starches obtained using glycidyltrimethylammonium chloride (GTMAC). Due to the instability, toxicity, and high cost of the most commonly used GTMAC, it should be replaced with more eco-friendly reagents, such as BHC, which is derived from betaine found in most green plants (e.g., spinach - Spinacia oleracea, beets - Beta vulgaris). The influence of processing conditions such as temperature, concentration of cationic reagents, presence and concentration of natural plasticizers/catalyst on physico-chemical and structural properties of cationic starches have also been studied. The cationic degree varied from 0.045-0.204 for the starch-BHC samples and within the range of 0.066-0.245 for the starch-GTMAC samples. The modification of starch with cationic reagents resulted in an increased solubility and swelling capacity, followed by decreased viscosity of the modified starches.

Inhibitors of Mycobacterium tuberculosis EgtD target both substrate binding sites to limit hercynine production.

Ergothioneine (EGT) is a low molecular weight histidine betaine essential in all domains of life but only synthesized by selected few organisms. Synthesis of EGT by Mycobacterium tuberculosis (M. tb) is critical for maintaining bioenergetic homeostasis and protecting the bacterium from alkylating agents, oxidative stress, and anti-tubercular drugs. EgtD, an S-adenosylmethionine-dependent methyltransferase (AdoMet), catalyzes the trimethylation of L-Histidine to initiate EGT biosynthesis and this reaction has been shown to be essential for EGT production in mycobacteria and for long-term infection of murine macrophages by M. tb. In this work, library screening and structure-guided strategies identified multiple classes of M. tb EgtD inhibitors that bind in various regions of the enzyme active site. X-ray crystal structures of EgtD-inhibitor complexes confirm that L-Histidine analogs bind solely to the L-Histidine binding site while drug-like inhibitors, such as TGX-221, and S-Glycyl-H-1152 span both the L-Histidine and AdoMet binding sites. These enzyme-inhibitor complexes provide detailed structural information of compound scaffolds useful for developing more potent inhibitors that could shorten Tuberculosis treatment regimens by weakening important bacterial defenses.

Chitosan/Polyvinyl Alcohol/ Lauramidopropyl Betaine/2D-HOF Mixed Film with Abundant Hydrogen Bonds Acts as High Mechanical Strength Artificial Skin.

The mechanical properties of artificial skins are complicated to maintain under ensuring air permeability and antimicrobial. Thus, a series of hydrophilic antimicrobial polymer networks are prepared by crosslinking chitosan and polyvinyl alcohol with the lauramidopropyl betaine and hydrogen bond organic framework (CS/PVA/LPB/2D-HOF). The mechanical performance of the control groups and the complex are systematically evaluated to attain an artificial strength skin. The CS/PVA/LPB/2D-HOF complex exhibits strong mechanical abilities than other control groups. By analyzing the IR spectra and the morphology, the synergistic effect of hydrogen bonds between molecules and cracks significantly improves the mechanical properties of the complex. Its maximum tensile strength can reach 29 MPa, and its maximum load capacity can reach 3700 g. Notably, the composite membrane also performs an excellent antimicrobial activity. In vivo and in vitro experiments show that the hybrid membrane can promote tissue regeneration and wound healing (95％). These results may open up the opportunity for future composite material investigations in the artificial skin and tissue engineering field.

The MttB superfamily member MtyB from the human gut symbiont Eubacterium limosum is a cobalamin-dependent γ-butyrobetaine methyltransferase.

The production of trimethylamine (TMA) from quaternary amines such as l-carnitine or γ-butyrobetaine (4-(trimethylammonio)butanoate) by gut microbial enzymes has been linked to heart disease. This has led to interest in enzymes of the gut microbiome that might ameliorate net TMA production, such as members of the MttB superfamily of proteins, which can demethylate TMA (e.g., MttB) or l-carnitine (e.g., MtcB). Here, we show that the human gut acetogen Eubacterium limosum demethylates γ-butyrobetaine and produces MtyB, a previously uncharacterized MttB superfamily member catalyzing the demethylation of γ-butyrobetaine. Proteomic analyses of E. limosum grown on either γ-butyrobetaine or dl-lactate were employed to identify candidate proteins underlying catabolic demethylation of the growth substrate. Three proteins were significantly elevated in abundance in γ-butyrobetaine-grown cells: MtyB, MtqC (a corrinoid-binding protein), and MtqA (a corrinoid:tetrahydrofolate methyltransferase). Together, these proteins act as a γ-butyrobetaine:tetrahydrofolate methyltransferase system, forming a key intermediate of acetogenesis. Recombinant MtyB acts as a γ-butyrobetaine:MtqC methyltransferase but cannot methylate free cobalamin cofactor. MtyB is very similar to MtcB, the carnitine methyltransferase, but neither was detectable in cells grown on carnitine nor was detectable in cells grown with γ-butyrobetaine. Both quaternary amines are substrates for either enzyme, but kinetic analysis revealed that, in comparison to MtcB, MtyB has a lower apparent Km for γ-butyrobetaine and higher apparent Vmax, providing a rationale for MtyB abundance in γ-butyrobetaine-grown cells. As TMA is readily produced from γ-butyrobetaine, organisms with MtyB-like proteins may provide a means to lower levels of TMA and proatherogenic TMA-N-oxide via precursor competition.

Four Novel Pharmaceutical Cocrystals of Oxyresveratrol, Including a 2 : 3 Cocrystal with Betaine.

Cocrystal engineering can alter the physicochemical properties of a drug and generate a superior drug candidate for formulation design. Oxyresveratrol (ORV) exhibits a poor solubility in aqueous environments, thereby resulting in a poor bioavailability. Extensive cocrystal screening of ORV with 67 cocrystal formers (coformers) bearing various functional groups was therefore conducted using grinding, liquid-assisted grinding, solvent evaporation, and slurry methods. Six cocrystals (ORV with betaine (BTN), L-proline (PRL), isonicotinamide, nicotinamide, urea, and ethyl maltol) were found, including four novel cocrystals. Powder X-ray diffraction, low frequency Raman spectroscopy, and thermal analysis revealed unique crystal forms in all obtained samples. Conventional Raman and infrared data differentiated the cocrystals by the presence or absence of a hydrogen bond interacting with the aromatic ring of ORV. The crystal structures were then elucidated by single-crystal X-ray diffraction. Two new cocrystals consisting of ORV : BTN (2 : 3) and ORV : PRL : H2O (1 : 2 : 1) were identified, and their crystal structures were solved. We report novel cocrystalline solids of ORV with improved aqueous solubilities and the unique cage-like crystal structures.

Convergent charge interval spacing of zwitterionic 4-vinylpyridine carboxybetaine structures for superior blood-inert regulation in amphiphilic phases.

Antifouling materials are indispensable in the biomedical field, but their high hydrophilicity and surface free energy provoke contamination on surfaces under atmospheric conditions, thus limiting their applicability in medical devices. This study proposes a new zwitterionic structure, 4-vinylpyridine carboxybetaine (4VPCB), that results in lower surface free energy and increases biological inertness. In the design of 4VPCB, one to three carbon atoms are inserted between the positive charge and negative charge (carbon space length, CSL) of the pyridyl-containing side chain to adjust hydration with water molecules. The pyridine in the 4VPCB structure provides the hydrophobicity of the zwitterionic functional group, and thus it can have a lower free energy in the gas phase but maintain higher hydrophilicity in the liquid phase environment. Surface plasmon resonance and confocal microscopy were used to analyze the antiprotein adsorption and anti-blood cell adhesion properties of the P4VPCB brush surface. The results showed that the CSL in the P4VPCB structure affected the biological inertness of the surface. The protein adsorption on the surface of P4VPCB2 (CSL= 2) is lower than that on the surfaces of P4VPCB1 (CSL = 1) and P4VPCB3 (CSL = 3), and the optimal resistance to protein adsorption can be reduced to 7.5 ng cm-2. The surface of P4VPCB2 can also exhibit excellent blood-inert function in the adhesion test with various human blood cells, offering a potential possibility for the future design of a new generation of blood-inert medical materials.

Soapwort (Saponaria officinalis L.) Extract vs. Synthetic Surfactants-Effect on Skin-Mimetic Models.

Our skin is continuously exposed to different amphiphilic substances capable of interaction with its lipids and proteins. We describe the effect of a saponin-rich soapwort extract and of four commonly employed synthetic surfactants: sodium lauryl sulfate (SLS), sodium laureth sulfate (SLES), ammonium lauryl sulfate (ALS), cocamidopropyl betaine (CAPB) on different human skin models. Two human skin cell lines were employed: normal keratinocytes (HaCaT) and human melanoma cells (A375). The liposomes consisting of a dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3, mimicking the cell membrane of keratinocytes and melanoma cells were employed as the second model. Using dynamic light scattering (DLS), the particle size distribution of liposomes was analyzed before and after contact with the tested (bio)surfactants. The results, supplemented by the protein solubilization tests (albumin denaturation test, zein test) and oil emulsification capacity (using olive oil and engine oil), showed that the soapwort extract affects the skin models to a clearly different extent than any of the tested synthetic surfactants. Its protein and lipid solubilizing potential are much smaller than for the three anionic surfactants (SLS, ALS, SLES). In terms of protein solubilization potential, the soapwort extract is comparable to CAPB, which, however, is much harsher to lipids.

Dimethyl Sulfoxide-Free Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells Based on Zwitterionic Betaine and Electroporation.

The effective cryopreservation of mesenchymal stem cells (MSCs) is indispensable to the operation of basic research and clinical transplantation. The prevalent protocols for MSC cryopreservation utilize dimethyl sulfoxide (DMSO), which is easily permeable and able to protect MSCs from cryo-injuries, as a primary cryoprotectant (CPA). However, its intrinsic toxicity and adverse effects on cell function remain the bottleneck of MSC cryopreservation. In this work, we cryopreserved human umbilical cord mesenchymal stem cells (UCMSCs) using zwitterionic betaine combined with electroporation without any addition of DMSO. Betaine was characterized by excellent compatibility and cryoprotective properties to depress the freezing point of pure water and balance the cellular osmotic stress. Electroporation was introduced to achieve intracellular delivery of betaine, intending to further provide comprehensive cryoprotection on UCMSCs. Compared with DMSO cryopreservation, UCMSCs recovered from the protocol we developed maintained the normal viability and functions and reduced the level of reactive oxygen species (ROS) that are harmful to cell metabolism. Moreover, the in vivo distribution of thawed UCMSCs was consistent with that of fresh cells monitored by a bioluminescence imaging (BLI) system. This work opens a new window of opportunity for DMSO-free MSC cryopreservation using zwitterionic compounds like betaine combined with electroporation.