Hydrolysis Of Ester Bond Research Articles

Carboxymethyl chitosan-based nanogels via acid-labile ortho ester linkages mediated enhanced drug delivery.

This work described the preparation of two type nanogels based on the crosslinking between carboxymethyl chitosan (CMCS) and two different crosslink agents, an acid-labile cyclic ortho ester compound with dual epoxy end groups (OEDe) or corresponding non-sensitive ethyleneglycol diglycidyl ether (EGDE). The particle size, zeta potential, and micromorphology were characterized by dynamic light scattering and electron microscopy, respectively. Nanogels' stability was also investigated at physiological environments. Doxorubicin hydrochloride as a therapeutic drug model was efficiently embedded into nanogels. The pH-triggered size changing, degradation and drug release were then investigated at three different pH values. Cellular uptake and cytotoxicity evaluation demonstrated that NG1/DOX could be successfully degraded and efficiently release DOX in acid cell organelles, leading to higher cytotoxicity than NG2/DOX. The accumulation and penetration of these DOX-loaded nanogel were then investigated by tumor-like multicellular spheroids (MCTS). The results indicated that the acid-degradable nanogels can deliver more DOX into the inner of MCTS by the hydrolysis of ortho ester bonds, thus efficiently inhibit the growth of MCTS. All results suggested that the acid-degradable nanogels could be degraded in mildly acidic conditions and remain stable at physiological environment, which indicated that the acid-degradable nanogels would be potentially useful as drug carriers.

Preparation and anticancer activity evaluation of self-assembled paclitaxel conjugated MPEG-PCL micelles on 4T1 cells

In this study, we prepared self-assembled micelles with methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) (MPEG-PCL) for paclitaxel (PTX) delivery. MPEG-PCL was synthesized by ring-opening polymerization in which the hydroxyl group was substituted for a carboxyl group by succinic anhydride. PTX was then coupled to carboxylated MPEG-PCL through esterification (MPEG-PCL-PTX). MPEG-PCL-PTX micelles were prepared by a solvent evaporation method. Conjugation of PTX to MPEG-PCL-PTX was confirmed by 1H NMR. MPEG-PCL-PTX micelles self-assembled into micellar nanoparticles at concentrations above 2.5×10−2mg/mL, as evaluated by fluorescence spectrometry. DLS and TEM analyses revealed that the micelles were globular in shape with an average size of 111nm. The drug release studies under three pH conditions demonstrated that PTX release from MPEG-PCL-PTX micelles was entirely dependent upon the hydrolysis of ester bond between PTX and MPEG-PCL copolymer, therefore, no severe initial burst release but pH sensitive profile was observed. In vitro tests with the 4T1 cell line showed that conjugation of PTX to MPEG-PCL led to improved cellular uptake and apoptosis compared to free PTX, supporting the feasibility of using the micelles as drug carriers for cancer therapy.

Synthesis, Characterization and Biodegradation of Poly(?-Methyl ?-Propiolactone)

Poly(α-methyl β-propiolactone) (PmPL) was synthesized through anionic ring-opening polymerization of the corresponding lactone. Structural characterization of PmPL was performed by using FTIR, 1H-NMR and MALDI-MS spectrometry. Thermal behavior of the polymer was investigated by using DSC and TGA. Glass transition, melting and thermal degradation temperatures of PmPL were determined as -36.7, 95.5 and 242.4 oC, respectively. Biodegradation studies were conducted at 37 oC in three different media, that is, pH 7.4 phosphate buffered saline (PBS), PBS containing pancreatic trypsin and PBS containing pancreatic lipase. The biodegradation kinetics and mechanism of the polymer were followed by gravimetric measurements and LC-MS analysis. Up to 65% mass loss in 5-week period was recorded for the buffer media in the presence of lipase and trypsin. LC-MS analysis of the residues in the media revealed that biodegradation undergoes mainly through ester bond hydrolysis.

Tumor-Microenvironment-Responsive Nanoconjugate for Synergistic Antivascular Activity and Phototherapy.

Insufficient oxygen supply (hypoxia), short half-life (<40 ns) of singlet oxygen, and up-regulation of the heat shock protein expression in solid tumors impede the photodynamic and photothermal therapeutic efficacy. Herein, a near-infrared carrier-free nanoconjugate direct-acting antiviral (DAA) with synergistic antivascular activity and pH-responsive photodynamic/photothermal behavior was designed and synthesized to improve cancer treatment efficacy. Obtained by the self-assembly approach, the biocompatible DAA nanoparticles (NPs) displayed amplifying pH-responsive photodynamic/photothermal performance in an acidic tumor microenvironment due to the protonation of diethylaminophenyl units. Most important, the antivascular agent 5,6-dimethylxanthenone-4-acetic acid, targeting the vascular endothelial growth factor, can be smartly released from the pro-drug DAA via ester bond hydrolysis at the subacid endocytosis organelles in the endothelial cells, which can effectively destroy the vascular region to prevent tumor proliferation and metastasis. Hence, DAA NPs can specifically target vascular endothelial cells and tumorous lysosomes with desired cellular damage properties in vitro. Therefore, the tumors can be ablated completely with no recurrence and side effects in vivo, which implies that DAA NPs provide a promising approach for cancer treatment via synergistic antivascular activity and photodynamic/photothermal therapy.

RETRACTED ARTICLE: Substrates specificity of tannase from Streptomyces sviceus and Lactobacillus plantarum

Tannases can catalyze the hydrolysis of galloyl ester and depside bonds of hydrolysable tannins to release gallic acid and glucose, but tannases from different species have different substrate specificities. Our prior studies found that tannase from Lactobacillus plantarum (LP-tan) performed a higher esterase activity, while the tannase from Streptomyces sviceus (SS-tan) performed a higher depsidase activity; but the molecular mechanism is not elucidated. Based on the crystal structure of LP-tan and the amino acid sequences alignment between LP-tan and SS-tan, we found that the sandwich structure formed by Ile206-substrate-Pro356 in LP-tan was replaced with Ile253-substrate-Gly384 in SS-tan, and the flap domain (amino acids: 225–247) formed in LP-tan was missed in SS-tan, while a flap-like domain (amino acids: 93–143) was found in SS-tan. In this study, we investigated the functional role of sandwich structure and the flap (flap-like) domain in the substrate specificity of tannase. Site-directed mutagenesis was used to disrupt the sandwich structure in LP-tan (P356G) and rebuilt it in SS-tan (G384P). The flap in LP-tan and the flap-like domain in SS-tan were deleted to construct the new variants. The activity assay results showed that the sandwich and the flap domain can help to catalytic the ester bonds, while the flap-like domain in SS-tan mainly worked on the depside bonds. Enzymatic characterization and kinetics data showed that the sandwich and the flap domain can help to catalytic the ester bonds, while the flap-like domain in SS-tan may worked on the depside bonds.

Development of simultaneous quantification method of loteprednol etabonate (LE) and its acidic metabolites, and analysis of LE metabolism in rat

Loteprednol etabonate (LE) is a soft corticosteroid with two labile ester bonds at 17α- and 17β-positions. Its corticosteroidal activity disappears upon hydrolysis of either ester bond. Hydrolysis of both ester bonds produces the inactive metabolite, Δ1-cortienic acid (Δ1-CA).The simple high-performance liquid chromatography method using acetic acid gradient was developed for the simultaneous determination of LE and its acidic metabolites.LE was hydrolyzed in rat plasma with a half-life of 9 min. However, LE hydrolysis was undetectable in rat liver and intestine. LE hydrolysis in rat plasma was completely inhibited by paraoxon and bis(p-nitrophenyl) phosphate, thus identifying carboxylesterase as the LE hydrolase. Rat plasma carboxylesterase had a Km of 6.7 μM for LE.In contrast to the disappearance rate of LE in rat plasma, the formation rate of 17α-monoester and Δ1-CA was markedly low, and a main hydrolysate of LE was not detected in rat plasma.The metabolism of LE proceeded via different pathways in human and rat plasma. LE was slowly hydrolyzed by paraoxonase in human plasma to 17α-monoester with a half-life of 12 h, but by carboxylesterase in rat plasma to yield undetectable products, presumed to include the unstable 17β-monoester.

β-Cyclodextrin hydroxypropyl methylcellulose hydrogels for bisphenol A adsorption

Crosslinking of βcyclodextrin (βCD) and hydroxypropyl methylcellulose (HPMC) was mediated by esterification with citric acid. The resulting networks presented high chemical stability in aqueous medium in the pH range from one to nine; pH > 10 led to the hydrolysis of ester bonds. The freeze-dried networks presented pores with size ranging from 10 μm to 200 μm. The amount of βCD bound to the HPMC network was estimated indirectly as 5.6 wt% with basis on the inclusion of phenolphthalein assay. Fourier transform infrared vibrational spectroscopy in the attenuated total reflectance mode evidenced the esterification between βCD, HPMC and citric acid molecules. HPMC and HPMC-βCD hydrogels presented similar swelling degrees for water of ~18 g per g of cryogel. The inclusion constant for the βCD-BPA complex was determined at 25 °C by 1H NMR spectroscopy and the Benesi-Hildebrand method as (100 ± 20) L mol−1. The adsorption kinetics of bisphenol A (BPA) on HPMC-βCD hydrogels fitted well to the pseudo-second order model, yielding rate constant of 0.00879 g mg−1 min−1, and to the intraparticle diffusion model. The latter revealed a two stage process: a fast one with diffusion rate of 1.185 mg g−1 min−0.5 related to the diffusion of BPA from bulk to hydrogel external area and a slower one related to the mass transport into the hydrogel internal area. The adsorption isotherms fitted well the Freundlich and Dubinin-Radushkevitch (D-R) models, the latter yielded maximum adsorption of 14.6 mg g−1 and mean adsorption energy E of 7.9 kJ mol−1. The hydrogels could be recycled five times without losing adsorption efficiency.

An industrial‐scale synthesis of biodegradable soluble in organic solvents butyric–acetic chitin copolyesters

AbstractThe article presents the results of study related to upscaling chitin esterification reaction to the industrial scale. The obtained results allowed for creation of a product which can later be used in manufacturing biologically active dressing materials. The implementation of the laboratory scale showed that there is a possibility of upscaling the procedure to make it suitable for the needs of industry. Problems and risks associated with the transition to an industrial scale, such as process temperature control, time of the reaction and the method of isolation of product (emptying the industrial reactor from the reaction mixture), have been presented and discussed. The results of the tests showed the interdependence between the stages of production process, both in laboratory and in industrial scale. The realization of the presented project enabled solving the problems arising both from the preparation to the synthesis, carrying out the process itself, and obtaining the final product. An important factor in this case was to obtain the same physicochemical properties of the final product on both laboratory and industrial scale. The tests of biodegradation of butyric–acetic chitin copolyester in fresh human plasma and blood serum confirmed that two enzymatic degradation processes occur due to the presence of the enzymes in these body fluids. At the same time, enzymatic hydrolysis of ester bonds and enzymatic hydrolysis of the glycoside bonds of the main polysaccharide chain take place.

Ultrasensitive detection of trypsin activity and inhibitor screening based on the electron transfer between phosphorescence copper nanocluster and cytochrome c

Trypsin, as one of important proteases, is specific for catalyzing the hydrolysis of peptide and ester bonds containing lysine and arginine residues at the C-terminus. The level of trypsin in biological fluids can serve as a reliable and specific diagnostic biomarker for pancreatic function and its pathological changes. Herein, we demonstrate the application of phosphorescent Cu NCs for trypsin detection for the first time depending on the electron transfer between Cu NCs and cyt c. Cyt c and Cu NCs were selected as the quencher and the fluorophore, respectively. Cu NCs could bind to the positively charged cyt c through electrostatic and hydrophobic interactions, and the phosphorescence of Cu NCs was efficiently quenched by the metal-containing heme of cyt c. In the presence of trypsin, cyt c was digested, thus phosphorescence of Cu NCs remained. Therefore, a new and continuous phosphorescence assay for the detection of trypsin activity and its inhibitor screening was established. The plot of relative fluorescence versus trypsin concentration obtains a good linear detection range from 0 to 20 ng/mL (R2 = 0.9657), and a detection limit of 2 ng/mL, which is much lower than 20 ng/mL of the sensor in buffer solution because of urine amplifying the phosphorescence signal of Cu NCs based on the FRET strategy. This assay still has been successfully applied to trypsin inhibitor screening, demonstrating its potential application in drug discovery.

Synthetic and Natural Lipase Inhibitors.

Lipases are enzymes that catalyse the hydrolysis of ester bonds of triglycerides ranging among biocatalysts of considerable physiological significance and industrial potential. Better understanding of the catalytic functions and achieving the possibility to control the biocatalysis process, in particular exploring some activators and inhibitors of lipases, seems to be crucial in the context of novel applications. The lipase activity is a function of interfacial composition: the enzyme can be there activated as well as denaturated or deactivated and the interface is an appropriate site for modulating lipolysis. Lipase inhibitor, interacts directly with the enzyme and inhibits lipase action. Alternatively, some compounds can postpone the lipolytic reaction via adsorption to the interphase or to the substrate molecules. The aim of this review is to summarise the current knowledge concerning human, animal and microbial lipase inhibitors, which were grouped into two categories: synthetic lipase inhibitors (including phosphonates, boronic acids and fats analogues) and natural compounds (including β-lactones and some botanical foodstuffs - plant extracts and plant metabolites, mainly polyphenols and saponins as well as peptides and some dietary fibers). The topics discussed include also inhibition issues from the viewpoint of obesity treatment. Among natural compounds able to inhibit lipase activity are β- lactones including orlistat. Orlistat is the only registered drug for obesity treatment in many countries and lipases are essential enzymes for lipid absorption - thus fat absorption or obesity can be controlled by lipase inhibition, especially pancreatic lipase which is responsible for the hydrolysis of over 80% of total dietary fats. Its effectiveness in obesity treatment was also described.

Growth associated degradation of aliphatic-aromatic copolyesters by Cryptococcus sp. MTCC 5455

The degradation of aliphatic-aromatic copolyesters, poly[(butylene succinate/terephthalate/isophthalate)-co-(lactate)] (PBSTIL) and poly(butylene adipate co-terephthalate) (PBAT) under mesophilic conditions was investigated using the yeast, Cryptococcus sp. MTCC 5455. Complete degradation of PBSTIL and PBAT films was observed at 25 °C within 96 and 216 h, respectively. Lipase was produced during the course of degradation and the encoding gene was expressed in Escherichia coli BL21 (DE3). The production of lipase was enhanced by cumulative induction with 0.1 mM IPTG and 1.5 mM lactose for 24 h at 25 °C, which resulted in a maximum lipase activity of 27.75 ± 1.0 U/mL. Spectral studies of the degraded polymeric films confirmed the hydrolysis of ester bonds by the lipase. Scanning electron microscopy revealed the formation of cracks and holes on the surface of the polymeric films during degradation. The results indicated the feasibility of the process towards degradation of polymeric waste at ambient temperatures within a short period of time.

Novel approach for a PTX/VEGF dual drug delivery system in cardiovascular applications-an innovative bulk and surface drug immobilization.

The successive incorporation of several drugs into the polymeric bulk of implants mostly results in loss of considerable quantity of one drug, and/or the loss in quality of the coating and also in changes of drug release time points. A dual drug delivery system (DDDS) based on poly-L-lactide (PLLA) copolymers combining the effective inhibition of smooth muscle cell proliferation while simultaneously promoting re-endothelialization was successfully developed. To overcome possible antagonistic drug interactions and the limitation of the polymeric bulk material as release system for dual drugs, a novel concept which combines the bulk and surface drug immobilization for a DDDS was investigated. The advantage of this DDDS is that the bulk incorporation of fluorescein diacetate (FDAc) (model drug for paclitaxel (PTX)) via spray coating enhanced the subsequent cleavable surface coupling of vascular endothelial growth factor (VEGF) via the crosslinker bissulfosuccinimidyl suberate (BS3). In the presence of the embedded FDAc, the VEGF loading and release are about twice times higher than in absence. Furthermore, the DDDS combines the diffusion drug delivery (FDAc or PTX) and the chemical controlled drug release, VEGF via hydrolysable ester bonds, without loss in quantity and quality of the drug release curves. Additionally, the performed in vitro biocompatibility study showed the bimodal influences of PTX and VEGF on human endothelial EA.hy926 cells. In conclusion, it was possible to show the feasibility to develop a novel DDDS which has a high potential for the medical application due to the possible easy and short modification of a polymer-based PTX delivery system.

Hydrolytic Stability of Boronate Ester‐Linked Covalent Organic Frameworks

AbstractThe stability of covalent organic frameworks (COFs) is essential to their applications. However, the common boronate ester‐linked COFs are susceptible to attack by nucleophiles (such as water molecules) at the electron‐deficient boron sites. To provide an understanding of the hydrolytic stability of the representative boronate ester‐linked COF‐5 and of the associated hydrolysis mechanisms, density functional theory (DFT) calculations were performed to characterize the hydrolysis reactions of the molecule formed by the condensation of 1,4‐phenylenebis(boronic acid) (PBBA) and 2,3,6,7,10,11‐hexahydroxytriphenylene (HHTP) monomers; two cases were considered, one dealing with the freestanding molecule and the other with the molecule interacting with COF layers. It was found that the boronate ester (B–O) bond dissociation, which requires one H2O molecule, has a relatively high energy barrier of 22.3 kcal mol−1. However, the presence of an additional H2O molecule significantly accelerates hydrolysis by reducing the energy barrier by a factor of 3. Importantly, the hydrolysis of boronate ester bonds situated in a COF environment follows reaction pathways that are different and have increased energy barriers. These results point to an enhanced hydrolytic stability of COF‐5 crystals.

Structural Mechanism for the Temperature-Dependent Activation of the Hyperthermophilic Pf2001 Esterase

Lipases and esterases constitute a group of enzymesthat catalyze the hydrolysis or synthesis of ester bonds. A major biotechnological interest corresponds to thermophilic esterases, due to their intrinsic stability at high temperatures. The Pf2001 esterase from Pyrococcus furiosus reaches its optimal activity between 70°C and 80°C. The crystal structure of the Pf2001 esterase shows two different conformations: monomer and dimer. The structures reveal important rearrangements in the "cap" subdomain between monomer and dimer, by the formation of an extensive intertwined helical interface. Moreover, the dimer interface is essential for the formation of the hydrophobic channel for substrate selectivity, as confirmed by mutagenesis and kinetic analysis. We also provide evidence for dimer formation at high temperatures, a process that correlates with its enzymatic activation. Thus, we propose a temperature-dependent activation mechanism of the Pf2001 esterase via dimerization that is necessary for the substrate channel formation in the active-site cleft.

A supramolecular hydrogel for spatial-temporal release of auxin to promote plant root growth.

Short peptide-based hydrogels have attracted extensive research interests in drug delivery because of their responsive properties. So far, most drug molecules have been conjugated with short peptides via an amide bond, restricting the release of the native drug molecules. In this study, we demonstrated the effectiveness of an auxin-based hydrogelator linked by a hydrolysable ester bond. Hydrogel I, formed by the gelator (NAA-G'FFY) linked with an ester bond, was able to release 1-naphthaleneacetic acid (NAA), whereas hydrogel II, formed by the gelator without an ester bond (NAA-GFFY), was not. By mixing NAA-G'FFY with Fmoc-GFFY to form a two-component hydrogel, the spatial and temporal release of NAA was achieved, promoting on-site auxin responses including primary root elongation and lateral root formation in the model plant Arabidopsis thaliana. The strategy of using a hydrolysable ester bond to connect drug molecules and self-assembling peptides could lead to the development of supramolecular hydrogels with more controllable drug release profiles.

Integration of phospholipid-hyaluronic acid-methotrexate nanocarrier assembly and amphiphilic drug-drug conjugate for synergistic targeted delivery and combinational tumor therapy.

Combinational cancer therapy has been considered as a promising strategy to achieve synergetic therapeutic effects and suppression of multidrug resistance. Herein, we adopted a combination of methotrexate (MTX), an antimetabolite acting on cytoplasm, and 10-hydroxycamptothecin (HCPT), an alkaloid acting on nuclei, to treat cancer. Given the different solubilities, membrane permeabilities, and anticancer mechanisms of both drugs, we developed a dual-targeting delivery system based on 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-hyaluronic acid (a principal ligand of CD44 receptors)-MTX (a selective ligand of folate receptors) nanoparticles, which was exploited to carry HCPT-MTX conjugate for synergistically boosting dual-drug co-delivery. The HCPT-MTX conjugate was synthesized by a blood-stable yet intracellularly hydrolysable ester bond. The core-shell-corona DSPE-HA-MTX nanoparticles encapsulating HCPT-MTX (HCPT-MTX@DHM) exhibited high drug entrapment efficiency (∼91.8%) and pH/esterase-controlled release behavior. Cellular uptake studies confirmed significant increase in the efficiency of selective internalization of HCPT-MTX@DHM via CD44/folate receptors compared with those of DSPE-HA nanoparticles encapsulating HCPT-MTX (HCPT-MTX@DH), both drugs, or each individual drug. Furthermore, in vivo near-infrared fluorescence and photoacoustic dual-modal imaging indicated that DiR-doped HCPT-MTX@DHM nanoparticles efficiently accumulated at the tumor sites through passive-plus-active targeting. Finally, the synergistic active targeting and synchronous dual-drug release at a synergistic drug-to-drug ratio resulted in highly synergetic tumor cell-killing and tumor growth inhibition in MCF-7 tumor-bearing mice. Therefore, HCPT-MTX@DHM nanoparticles can be an efficient and smart platform for tumor-targeting therapy.

Portraying the role of halo ligands and the auxiliary part of ligands of mononuclear manganese(iii)-Schiff base complexes in catalyzing phospho–ester bond hydrolysis

The influences of the auxiliary part of ligands and halo ligands have been explored towards phosphatase like activity using a series of manganese(iii)-Schiff base complexes as catalysts.

Bacteria-mediated phthalic acid esters degradation and related molecular mechanisms.

Phthalic acid esters (PAEs) have long been known as the most widely used plasticizer with a broad range of industrial application. PAEs are ubiquitous in different environments and our daily life due to their large and widespread application. Recent PAEs research mainly focused on their environmental fate (including leaching, migration, transformation) and toxicology and risk assessment. With the comprehensive recognition of their potential hazard, the elimination of PAEs has attracted worldwide concerns. Although many factors may contribute to the degradation of PAEs, the dominant role of biodegradation was widely reported. Many PAEs-degrading bacteria were isolated, metabolites and metabolic pathways were proposed, and enzymes involved in the degradation were identified. The current paper presents an overview of available reports about PAEs-degrading bacteria and related molecular mechanisms. The metabolic pathways deduced from the identified intermediates were presented. The upstream and downstream pathways of PAEs metabolism were summarized, including the aerobic and anaerobic pathways of phthalic acid (PA) degradation. Known enzymes involved in the hydrolysis of ester bonds were characterized according to their properties. Based on phylogenetic analysis, all these enzymes were distributed in four families of esterases and one unknown family. For these five families, conserved sequence motifs were identified and the biological properties of these motifs were characterized. Challenges and emerging opportunities are also discussed.

Fungal feruloyl esterases: Functional validation of genome mining based enzyme discovery including uncharacterized subfamilies

Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharification for biofuel and biochemical production. A limited number of FAEs have been functionally characterized compared to over 1000 putative fungal FAEs that were recently predicted by similarity-based genome mining, which divided phylogenetically into different subfamilies (SFs). In this study, 27 putative and six characterized FAEs from both ascomycete and basidiomycete fungi were selected and heterologously expressed in Pichia pastoris and the recombinant proteins biochemically characterized to validate the previous genome mining and phylogenetical grouping and to expand the information on activity of fungal FAEs. As a result, 20 enzymes were shown to possess FAE activity, being active towards pNP-ferulate and/or methyl hydroxycinnamate substrates, and covering 11 subfamilies. Most of the new FAEs showed activities comparable to those of previously characterized fungal FAEs.

Fullerenol-Based Intracellular Delivery of Methotrexate: A Water-Soluble Nanoconjugate for Enhanced Cytotoxicity and Improved Pharmacokinetics.

Derivatization of fullerenes to polyhydroxylated fullerenes, i.e., fullerenols (FLU), dramatically decreases their toxicity and has been reported to enhance the solubility as well as cellular permeability. In this paper, we report synthesis of FLU as nanocarrier and subsequent chemical conjugation of Methotrexate (MTX) to FLU with a serum-stable and intracellularly hydrolysable ester bond between FLU and MTX. The conjugate was characterized for physiochemical attributes, micromeritics, drug-loading, and drug-release and evaluated for cancer cell-toxicity, cellular-uptake, hemocompatibility, protein binding, and pharmacokinetics. The developed hemocompatible FL-MTX offered lower protein binding vis-à-vis naïve drug and substantially higher drug loading. The conjugate offered pH-dependent release of 38.20 ± 1.19% at systemic pH and 85.67 ± 3.39% at the cancer cell pH. FLU-MTX-treated cells showed significant reduction in IC50 value vis-à-vis the cells treated with pure MTX. Analogously, the results from confocal scanning laser microscopy also confirmed the easy access of the dye-tagged FLU-MTX conjugate to the cell interiors. In pharmacokinetics, the AUC of MTX was enhanced by approx. 6.15 times and plasma half-life was enhanced by 2.45 times, after parenteral administration of single equivalent dose in rodents. FLU-MTX offered enhanced availability of drug to the biological system, meanwhile improved the cancer-cell cytotoxicity, sustained the effective plasma drug concentrations, and offered substantial compatibility to erythrocytes.