Galactose Conjugates Research Articles

Surface-Conjugated Galactose on Electrospun Polycaprolactone Nanofibers: An Innovative Scaffold for Uterine Tissue Engineering.

The uterus, a vital organ in the female reproductive system, nurtures and supports developing embryos until maturity. This study focuses on addressing uterine related problems by creating a nanofibrous scaffold to regenerate uterine myometrial tissue, closely resembling the native extracellular matrix (ECM) for enhanced efficacy. To achieve this, we utilized polycaprolactone (PCL) as a biomaterial and employed an electrospinning technique to generate PCL nanofibers in both random and aligned orientations. Due to the inherent hydrophobic nature of PCL nanofibers, a two-step wet chemistry surface modification technique is used, involving the conjugation of galactose onto them. Galactose, a lectin-binding sugar, was chosen to enhance the scaffold's hydrophilicity, thereby improving cell adhesion and fostering l-selectin-based interactions between the scaffold and uterine cells. These interactions, in turn, activated uterine fibroblasts, leading to ECM remodeling. The optimized electrospinning process successfully generated random and aligned nanofibers. Subsequent surface modification was carried out, and the modified scaffold was subjected to various physicochemical characterization, such as the ninhydrin assay, enzyme-linked lectin assay techniques that revealed successful galactose conjugation, and mechanical characterization to assess any changes in material bulk properties resulting from the modification. The tensile strength of random galactose-modified PCL fibers reached 0.041 ± 0.01 MPa, outperforming random unmodified PCL fibers (0.026 ± 0.01 MPa), aligned unmodified PCL fibers (0.011 ± 0.001 MPa), and aligned modified PCL fibers (0.016 ± 0.002 MPa). Cytocompatibility studies with human uterine fibroblast cells showed enhanced viability and proliferation on the modified scaffolds. Initial pilot studies were attempted in the current study involving subcutaneous implantation in the dorsal area of Wistar rats to assess biocompatibility and tissue response before proceeding to intrauterine implantation indicated that the modification did not induce adverse inflammation in vivo. In conclusion, our study introduces a surface-modified PCL nanofibrous material for myometrial tissue engineering, offering promise in addressing myometrial damage and advancing uterine health and reproductive well-being.

Design, synthesis and biological evaluation of the positional isomers of the galactose conjugates able to target hepatocellular carcinoma cells via ASGPR-mediated cellular uptake and cytotoxicity

Galactose as a recognizing motif for asialoglycoprotein receptor (ASGPR) is a widely accepted vector to deliver cytotoxic agents in the therapy of hepatocellular carcinoma (HCC), however, the individual hydroxyl group of galactose (Gal) contributed to recognizing ASGPR is obscure and remains largely unanswered in the design of glycoconjugates. Herein, we designed and synthesized five positional isomers of Gal-anthocyanin Cy5.0 conjugates and three Gal-doxorubicin (Dox) isomers, respectively. The fluorescence intensity of Gal-Cy5.0 conjugates accumulated in cancer cells hinted the optimal modification sites of positions C2 and C6. Comparing to the cytotoxicity of other conjugates, C2-Gal-Dox (11) was the most potent. Moreover, Gal-Dox conjugates significantly the toxicity of Dox. A progressively lower internalization capacity and siRNA technology implied the cellular uptake and cytotoxicity directly related to the ASGPR expression level. Accordingly, position C2 of galactose may be the best substitution site via ASGPR mediation in the design of anti-HCC glycoconjugates.

Synthesis and In Vitro Anti-tumor Evaluation of Novel Glycoconjugated SN38 Derivatives

Abstract Five types of novel glycoconjugated 7-ethyl-10-hydroxycamptothecin (SN38) derivatives were synthesized to evaluate their anti-tumor activity against HCT116 human colon cancer cells, and the results showed that all of them demonstrated potent anti-tumor activity. Notably, glucose and galactose conjugates exhibited the better anti-tumor activity, while the mannose and allose conjugates had slightly lower activity. Our findings suggested that the anti-tumor activity of glycoconjugated SN38 derivatives was affected by the type of modifying molecule used and the position of the modification. These findings could have implications for the development of novel anti-tumor agents targeting glucose transporters.

Galactosyl, alkyl, and acidic groups modify uptake and subcellular deposition of pyropheophorbide-a by epithelial tumor cells and determine photosensitizing efficacy

Photosensitizers currently used for photodynamic therapy of cancer show enhanced accumulation in tumor tissue but lack cancer cell specificity. To augment cellular uptake, the targeting of pyropheophorbide-a to carbohydrate-binding components of cancer cells was explored. Galactose was attached to pyropheophorbide-a at positions 172 and 20. Since the modification at position 172 removed a carboxylic group, which is relevant for cell specificity, this study evaluated the relative contribution of galactosyl and carboxyl groups at either position 172 or 20, with or without a (hexyloxy)ethyl at position 3, to cellular uptake by human epithelial cancer cells. The subcellular deposition was monitored using fluorescence microscopy and the photoreaction was quantified using biomarkers. The results demonstrated that any galactose addition suppresses transmembrane diffusion and promotes endocytosis and lysosomal accumulation. An anionic group at position 172 or 20 enhances lysosomal retention. Neutralization of the carboxylic group at position 172 facilitates transfer to mitochondria/endoplasmatic reticulum and promotes tumor cell-specific retention. Replacing (hexyloxy)ethyl with an ethyl group at position 3 increased both cellular uptake and egress but did not alter subcellular localization. These findings suggest that specific neutral galactosylated pheophorbides may provide an enhanced therapeutic effect for those tumor types that do not retain unmodified pyropheophorbide. However, the galactose conjugates also serve as substrates for preferential uptake by liver cells resulting in hepatic sequestration, reduced systemic distribution, and lower accumulation in tumor tissue.

Shell-Sheddable Micelles Based on Poly(ethylene glycol)-hydrazone-poly[R,S]-3-hydroxybutyrate Copolymer Loaded with 8-Hydroxyquinoline Glycoconjugates as a Dual Tumor-Targeting Drug Delivery System.

The development of selective delivery of anticancer drugs into tumor tissues to avoid systemic toxicity is a crucial challenge in cancer therapy. In this context, we evaluated the efficacy of a combination of nanocarrier pH-sensitivity and glycoconjugation of encapsulated drugs, since both vectors take advantage of the tumor-specific Warburg effect. Herein, we synthesized biodegradable diblock copolymer, a poly(ethylene glycol)-hydrazone linkage-poly[R,S]-3-hydroxybutyrate, which could further self-assemble into micelles with a diameter of ~55 nm. The hydrazone bond was incorporated between two copolymer blocks under an acidic pH, causing the shell-shedding of micelles which results in the drug’s release. The micelles were stable at pH 7.4, but decompose in acidic pH, as stated by DLS studies. The copolymer was used as a nanocarrier for 8-hydroxyquinoline glucose and galactose conjugates as well as doxorubicin, and exhibited pH-dependent drug release behavior. In vitro cytotoxicity, apoptosis, and life cycle assays studies of blank and drug-loaded micelles were performed on Normal Human Dermal Fibroblasts-Neonatal (NHDF-Neo), colon carcinoma (HCT-116), and breast cancer (MCF-7) for 24, 48, and 72 h. A lack of toxicity of blank micelles was demonstrated, whereas the glycoconjugates-loaded micelles revealed enhanced selectivity to inhibit the proliferation of cancer cells. The strategy of combining pH-responsive nanocarriers with glycoconjugation of the drug molecule provides an alternative to the modus operandi of designing multi-stimuli nanocarriers to increase the selectivity of anticancer therapy.

Ketogal Safety Profile in Human Primary Colonic Epithelial Cells and in Mice

In our previous studies, a ketorolac–galactose conjugate (ketogal) showed prolonged anti-inflammatory and analgesic activity, causing less gastric ulcerogenic effect and renal toxicity than its parent drug ketorolac. In order to demonstrate the safer profile of ketogal compared to ketorolac, histopathological changes in the small intestine and liver using three staining techniques before and after repeated oral administration in mice with ketorolac or an equimolecular dose of its galactosylated prodrug ketogal were assessed. Cytotoxicity and oxidative stress parameters were evaluated and compared in ketorolac- and ketogal-treated Human Primary Colonic Epithelial cells at different concentrations and incubation times. Evidence of mitochondrial oxidative stress was found after ketorolac treatment; this was attributable to altered mitochondrial membrane depolarization and oxidative stress parameters. No mitochondrial damage was observed after ketogal treatment. In ketorolac-treated mice, severe subepithelial vacuolation and erosion with inflammatory infiltrates and edematous area in the intestinal tissues were noted, as well as alterations in sinusoidal spaces and hepatocytes with foamy cytoplasm. In contrast, treatment with ketogal provided a significant improvement in the morphology of both organs. The prodrug clearly demonstrated a safer profile than its parent drug both in vitro and ex vivo, confirming that ketogal is a strategic alternative to ketorolac.

Polysaccharide conjugates surpass monosaccharide ligands in hepatospecific targeting – Synthesis and comparative in silico and in vitro assessment

Ligands with the polysaccharide headgroups have been recently reported by our group to possess enhanced interaction with asialoglycoprotein receptor (ASGPR) in silico as compared to ligands having galactose moieties. This enhanced interaction is a result of the polymer's backbone support in anchoring the ligand in a specific orientation within the bilayer. In this paper, we have attempted to provide an in vitro proof of concept by performing a comparative evaluation of polysaccharide and monosaccharide-based ligands. Docking was performed to understand interaction with ASGPR in silico. Agarose and galactose conjugates with behenic acid were synthesized, purified, and characterized to yield biocompatible hepatospecific ligands which were incorporated into nanoliposomes. Cellular internalization of these targeted liposomes was studied using confocal microscopy and flow cytometry. The toxicity potential was assessed in vivo. Results indicated that the polysaccharide-based ligand increased cellular uptake due to better interaction with the receptor as compared to ligand bearing a single galactose group. In addition to developing novel liver targeting ligands, the study also established proof of concept that has been suggested by earlier in silico investigations. The approach can be used to design targeting ligands and develop formulations with improved targeting efficacy.

Biodegradable pH-responsive micelles loaded with 8-hydroxyquinoline glycoconjugates for Warburg effect based tumor targeting

Biodegradable triblock copolymer poly(ethylene glycol)-b-polycarbonate-b-oligo([R]-3-hydroxybutyrate) was prepared via metal-free ring-opening polymerization of ketal protected six-membered cyclic carbonate followed by esterification with bacterial oligo([R]-3-hydroxybutyrate) (oPHB). Amphiphilic triblock copolymer self-organizes into micelles with a diameter of ~25 nm. Acid-triggered hydrolysis of ketal groups to two hydroxyl groups causes an increase in hydrophilicity of the hydrophobic micelle core, resulting in the micelles swell and drug release. oPHB was added as core-forming block to increase the stability of prepared micelles in all pH (7.4, 6.4, 5.5) studied. Doxorubicin and 8-hydroxyquinoline glucose- and galactose conjugates were loaded in the micelles. In vitro drug release profiles in PBS buffers with different pH showed that a small amount of loaded drug was released in PBS at pH 7.4, while the drug was released much faster at pH 5.5. MTT assay showed that the blank micelles were non-toxic to different cell lines, while glycoconjugates-loaded micelles, showed significantly increased ability to inhibit the proliferation of MCF-7 and HCT-116 cells compared to free glycoconjugates. The glycoconjugation of anti-cancer drugs and pH-responsive nanocarriers have separately shown great potential to increase the tumor-targeted drug delivery efficiency. The combination of drug glycoconjugation and the use of pH-responsive nanocarrier opens up new possibilities to develop novel strategies for efficient tumor therapy.

Paracetamol-Galactose Conjugate: A Novel Prodrug for an Old Analgesic Drug.

Paracetamol has been one of the most commonly used and prescribed analgesic drugs for more than a hundred years. Despite being generally well tolerated, it can result in high liver toxicity when administered in specific conditions, such as overdose, or in vulnerable individuals. We have synthesized and characterized a paracetamol galactosylated prodrug (PARgal) with the aim of improving both the pharmacodynamic and pharmacological profile of paracetamol. PARgal shows a range of physicochemical properties, solubility, lipophilicity, and chemical stability at differing physiological pH values and in human serum. PARgal could still be preclinically detected 2 h after administration, meaning that it displays reduced hepatic metabolism compared to paracetamol. In overdose conditions, PARgal has not shown any cytotoxic effect in in vitro analyses performed on human liver cells. Furthermore, when tested in an animal pain model, PARgal demonstrated a sustained analgesic effect up to the 12th hour after oral administration. These findings support the use of galactose as a suitable carrier in the development of prodrugs for analgesic treatment.

Aceclofenac–Galactose Conjugate: Design, Synthesis, Characterization, and Pharmacological and Toxicological Evaluations

Aceclofenac is a popular analgesic, antipyretic, and nonsteroidal anti-inflammatory drug (NSAID) used for prolonged treatment (at least three months) in musculoskeletal disorders. It is characterized by several limitations such as poor water solubility and low oral bioavailability. The main side-effect of aceclofenac, as well as all NSAIDs, is the gastrotoxicity; among other adverse effects, there is the risk of bleeding since aceclofenac reversibly inhibits platelet aggregation. With the aim to reduce these drawbacks, we have designed, synthesized, and characterized, both in vitro and in vivo, an orally administrable pro-drug of aceclofenac (ACEgal). ACEgal was obtained by conjugating carboxyl group with the 6-OH group of d-galactose; its structure was confirmed by X-ray powder diffractometry. The pro-drug was shown to be stable at 37 °C in simulated gastric fluid (SGF-without pepsin, pH = 1.2) and moderately stable in phosphate buffered saline (PBS, pH = 7.4). However, it hydrolyzed in human serum with a half-life ( t1/2) of 36 min, producing aceclofenac. Furthermore, if compared to its parent drug, ACEgal was four-times more soluble in SGF. To predict human intestinal absorption, cell permeability in a Caco-2 model of aceclofenac and ACEgal was determined. Anti-inflammatory, analgesic, and ulcerogenic activities have been investigated in vivo. In addition, oxidative stress parameters (thiobarbituric acid reactive substances, TBARS, and glutathione, GSH) and platelet antiaggregatory activity both of parent drug and pro-drug were evaluated. Results clearly showed that the conjugation of aceclofenac to a galactose molecule improves physicochemical, toxicological (at gastric and blood level), and pharmacological profile of aceclofenac itself without changing intestinal permeability and antiplatelet activity (in spite the new sugar moiety).

Comparative Studies on Physicochemical Properties of Bovine Serum Albumin–Glucose and Galactose Conjugates Formed by Glycation Combined with Ultrasonic Pretreatment

Abstract The effects of ultrasonication on the physicochemical properties of bovine serum albumin (BSA)–glucose and the galactose conjugates formed by glycation was investigated. Fourier transform ion cyclotron resonance mass spectra analysis showed that the conjugates had a higher molecular weight than the native and ultrasonicated BSA. Ultrasonicated BSA had significantly higher emulsifying and foaming properties than native BSA. The browning intensity, surface hydrophobicity, emulsifying property and foaming capacity of the conjugates were substantially improved while the free amino groups, intrinsic fluorescence emission and foaming stability were decreased compared to native and ultrasonicated BSA. The results of this study indicate that ultrasonication is an efficient technique to improve the physicochemical properties of proteins. The glycation between ultrasonicated BSA and monosaccharide can effectively improve the physicochemical properties of BSA, and the glycation rate order is galactose \gt glucose. It also shows the critical role of monosaccharide conformational changes in improving the glycation and physicochemical properties of proteins.

Glycation of β-lactoglobulin under dynamic high pressure microfluidization treatment: Effects on IgE-binding capacity and conformation

The effects of dynamic high-pressure microfluidization (DHPM) (80, 120, and 160MPa) treatment and glycation with galactose on the IgE-binding capacity and conformation of β-lactoglobulin (β-Lg) were investigated. The binding capacity of immunoglobulin E (IgE) from patients' sera with cow's milk allergy on β-Lg glycated with galactose decreased after DHPM treatment. β-Lg treated after different DHPM methods and pressures yielded a significant discrepancy in IgE-binding capacity. When β-Lg was pretreated by DHPM, the IgE-binding capacity of β-Lg–galactose conjugates decreased with increasing pressure; however, the conjugates showed higher IgE-binding capacity at 120MPa than that at 80 and 160MPa when the β-Lg–galactose mixture was treated by DHPM. Results of thermal properties, intrinsic fluorescence spectroscopy, surface hydrophobicity, and circular dichroism (CD) spectra indicated the occurrence of protein unfolding, as well as the tertiary and secondary structural changes of β-Lg. The results suggested pretreatment by DHPM and glycation with galactose was a promising approach for eliminating the IgE-binding capacity of β-Lg.

A dual-functional supramolecular hydrogel based on a spiropyran-galactose conjugate for target-mediated and light-controlled delivery of microRNA into cells.

A supramolecular hydrogel formed by spiropyran conjugated galactose was developed for light-controlled release of miR-122 and target-mediated delivery of the miRNA into HepG2 cells cultured on top of the gel.

Cyclam glycoconjugates as lectin ligands and protective agents of metal-induced amyloid aggregation

Transition metal ion complexes of a number of chelators have been widely investigated due to their biological properties. The sugar conjugation of metal complexes has resulted in improved properties of the systems, such as solubility and lectin recognition. In this paper, we report the synthesis, the characterization of new glucose and galactose conjugates of 1,4,8,11-tetraazacyclotetradecane (cyclam) and their CuII complexes. The glycoconjugates were proved to be protective agents of metal-induced amyloid aggregation. The binding constants of the galactose conjugate KA1=(3.07±0.16)×107 and KA2=(2.13±0.04)×107M−1 with the lectin Ricinus communis agglutinin (RCA120) as a model of galactose-specific human lectin were obtained with surface plasmon resonance.

Photodynamic effect of glycochlorin conjugates in human cancer epithelial cells

The effect of glycochlorins in HeLa cells shows that the galactose conjugate is the most efficient photosensitizer.

Production and characterisation of potato patatin–galactose, galactooligosaccharides, and galactan conjugates of great potential as functional ingredients

Potato proteins are of high interest because of their high nutritional quality and multiple health benefits, but they are currently undervalued due to their limited solubility and stability. Glycated patatin (PTT) with galactose, galactooligosaccharides (GOSs) and galactan were produced through the Maillard reaction and characterised structurally and functionally. Fourier-transform infrared and fluorescence spectroscopy data revealed important changes in total secondary structures through glycation with GOSs (61.2%) and galactan (36.7%) and also significant tertiary structural changes leading to an exposure of tryptophan residues. These structural changes led to more heat stable forms of PTT with a higher unfolding temperature (70–90°C) than the unmodified protein (50–70°C) and with higher antioxidant activity. PTT:galactose conjugates exhibited similar thermal stability and pH-structural behaviour to native PTT. However, the high level of galactose conjugation to PTT and increased exposure of hydrophobic residues led to a significant increase in its emulsifying stability at pH 3.

Synthesis and cellular properties of Near-IR BODIPY–PEG and carbohydrate conjugates

A series of red and near-IR fluorescent BODIPY conjugates, containing either one or two indolylstyryl groups at the 3- and/or 5-positions and a low molecular weight PEG or carbohydrate group, were synthesized using a Cu(I)-catalyzed azide–alkyne Huisgen cycloaddition (‘click’ reaction). All BODIPY conjugates show emission and fluorescence quantum yields in the ranges of 642–732 nm and 0.24–0.56, respectively, and Stokes' shifts of ca. 30 nm. In vitro cellular investigations using human carcinoma HEp2 cells showed that all BODIPYs are non-toxic, both in the absence and presence of light (1 J/cm2), up to 100 μM concentrations. The PEG and galactose conjugates were more efficient at cell internalization than the unconjugated BODIPYs. The mono-indolylstyryl–BODIPYs showed higher cellular uptake (about five-fold) compared with the di-indolylstyryl–BODIPYs, and higher fluorescence quantum yields.

Characterization of glycated lysozyme with galactose, galactooligosaccharides and galactan: Effect of glycation on structural and functional properties of conjugates

Glycated lysozyme (LZM) with galactose, galactooligosaccharides (GOSs) and galactan, through the Maillard reaction, were produced and characterized structurally and functionally. The extent of glycation of LZM was evaluated by the measurement of the blocked lysine and the formed furosine. The results indicated a high initial reactivity of galactose as compared to GOSs and galactan. In the presence of GOSs, the oxidative and cross-linking side reactions competed with the initial formation of Amadori product. The longer chain carbohydrate galactan exhibited the lowest initial rate of glycation with no significant cross-linking side reaction. Electrospray ionization mass spectrometry revealed the formation of glycated LZM containing up to eight galactose moieties; while only mono- to tetra-glycated LZM with GOSs were detected. 2–3 mol of galactan were conjugated to 1 mol of LZM. Galactan:LZM conjugates exhibited higher solubility, thermal stability and emulsion stability as compared to the unmodified LZM and LZM:galactose conjugates. LZM:GOS conjugates demonstrated the most improvement in the emulsion stability than the other conjugates. Furthermore, the glycation of LZM with galactose/GOSs/galactan decreased its immunoreactivity.

Preparation and characterization of polymeric nanoparticles composed of poly(dl-lactide-co-glycolide) and poly(dl-lactide-co-glycolide)-co-poly(ethylene glycol)-10%-Triblock end-capped with a galactose moiety

This work aimed at the synthesis of novel type of targetable polymeric conjugates with a sugar moiety (galactose) with potential application for liver-specific drug delivery. Simple and effective synthesis of poly(dl-lactide-co-glycolide)-galactose conjugate and poly(dl-lactide-co-glycolide)-co-poly(ethylene glycol)-10%-Triblock-galactose conjugate by esterification of the end carboxyl groups has been reported. Spherical hydrophilic nanoparticles with elevated galactose content on the surface were prepared from these polymeric conjugates, and their physical properties were determined by DLS, SEM, XPS and DSC.

Multifunctional multivalency: a focused library of polymeric cholera toxin antagonists

Structural pre-organization of the multivalent ligands is important for successful interaction with multimeric proteins. Polymer-based heterobifunctional ligands that contain pendant groups prearranged into heterodimers can be used to probe the active site and surrounding area of the receptor. Here we describe the synthesis and activities of a series of galactose conjugates on polyacrylamide and dextran. Conjugation of a second fragment resulted in nanomolar inhibitors of cholera toxin, while the galactose-only progenitors showed no detectable activity.