Gene Transfer with Three Amphiphilic Glycol Chitosans—the Degree of Polymerisation is the Main Controller of Transfection Efficiency

A number of studies have examined the possibility of delivering genes for the treatment of genetic diseases using various polymers and lipids. We have previously demonstrated the gene transfer ability of amphiphilic polymers (a soluble amine polymer covalently bound to lipid pendant groups). In the current communication we explore the gene transfer activity of amphiphilic glycol chitosans. Glycol chitosan was acid depolymerised to give polymers of various molecular weights. Palmitoyl or hexadecyl and in some cases additional N-methyl quaternary ammonium groups were attached to the polymers. DNA binding was studied by measuring the reduced fluorescence of ethidium bromide and the polyplex particle size and zeta potential. Biological characterisation of the polyplexes involved haemolysis, cytotoxicity and gene transfer assays. For the 22 polymers tested, DNA binding was optimum at a nitrogen to phosphate ratio of 2:1 and above. Polyplexes were 200–500 nm in diameter with a neutral or positive zeta potential. The haemolytic activity of the N-methyl polymers was studied and no haemolysis was detected up to a concentration of 10 mg ml^?1. Cytotoxicity studies showed that the biocompatibility of glycol chitosan was adversely affected by a combination of a palmitoyl group and depolymerisation and that biocompatibility was subsequently restored with the introduction of N-methyl groups. In vitro transfection efficiency superior to the cationic lipid formulation N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP) was seen with depolymerised glycol chitosan in the A431 cell line only and with the depolymerised N-methyl quaternary ammonium amphiphilic derivatives in both the A431 and A549 cell lines. Degree of polymerisation (DP) was the most important controller of transfection efficiency and transfection resided within polymers with a DP of 73–171. High DP polymers diminished DNA–cell association, the first step in the cellular gene transfer process, thus apparently diminishing cell uptake. In vivo transfection with the N-methyl quaternary ammonium amphiphile was best at a DP of 86 and this glycol chitosan amphiphile gave superior liver and heart gene expression levels when compared to both Exgen 500 (linear polyethylenimine) and Superfect (a polyamidoamine dendrimer).     

Tumour gene expression from C12 spermine amphiphile gene delivery systems

Gene therapy requires safe and efficient gene delivery systems. Towards this aim both the gene formulation and tumour transfection ability of C12 spermine amphiphiles were tested. Five amphiphiles were synthesised and characterised: 1-[N,N-bis(3-aminopropyl)-1,4-butane diamine] dodecane (12G0--a C12 spermine amphiphile), a poly(ethylene glycol) (PEG, MW = 2 kDa) derivative of 12G0, 1,12-[N,N-bis(3-aminopropyl)-1,4-butane diamine] dodecane (12G1--a C12 spermine bolaamphiphile) and N-methyl quaternary ammonium derivatives of both 12G0 (12QG0) and 12G1 (12QG1). All amphiphiles except 12G0, which precipitates, yield nanoparticles in aqueous media with and without DNA. Thus when 12G0 is substituted with either quaternary ammonium or PEG groups it forms nanoparticles both with and without DNA. The minimum nitrogen, phosphate ratio required to completely condense DNA (NP) was inversely proportional to the particles' zeta potential (zeta), NP = 1626/zeta(0.98). Biological testing showed that both PEG and quaternary ammonium groups diminished the membrane lytic ability of these C12 amphiphiles. On intratumoural injection, while PEG groups hamper gene transfer, the quaternary ammonium amphiphile (12QG0) produces tumour confined gene expression that is 80% of that produced by linear poly(ethylenimine) (LPEI, MW = 22 kDa); while the intratumoural injection of LPEI produced significant gene expression in the liver and lung, making 12QG0 suitable for the administration of cytotoxic tumouricidal genes.     

PEI-based vesicle-polymer hybrid gene delivery system with improved biocompatibility

Wider use of the transfection agent polymer polyethylenimine (PEI) in vivo has been hampered by its toxicity. In order to examine whether material combining properties of polymers and lipid type of carriers would have improved characteristics, four PEI derivatives were synthesised: The methylation of the branched PEI (25 kDa) created a permanently charged quaternary ammonium derivative. Acylation of these backbones using pendant palmitic acid chains created amphiphilic PEI variants which formed nanoparticles or vesicles. Finally hydrophilic groups were added to the polymer backbone by PEGylation. The materials were characterised and their in vitro and in vivo properties were tested. The modifications improved the materials biocompatibility markedly when compared to the starting material but also reduced transfection efficiency. The material bearing ammonium and palmitoyl groups was 10x less toxic while retaining about 30% of the transfection efficiency in vitro. After intravenous administration in a mouse model the materials also gave rise to GFP transgene expression in the liver. The synthetic strategy altered complex physicochemistry and improved biocompatibility while maintaining in vitro gene expression for most formulations. The strategy of combination of complementary properties of cationic lipids and polymers into a hybrid material may also be applicable to other materials.     

Polymeric Chitosan-based Vesicles for Drug Delivery

A simple carbohydrate polymer glycol chitosan (degree of polymerization 800 approx.) has been investigated for its ability to form polymeric vesicle drug carriers. The attachment of hydrophobic groups to glycol chitosan should yield an amphiphilic polymer capable of self-assembly into vesicles. Chitosan is used because the membrane-penetration enhancement of chitosan polymers offers the possibility of fabricating a drug delivery system suitable for the oral and intranasal administration of gut-labile molecules. Glycol chitosan modified by attachment of a strategic number of fatty acid pendant groups (11–16mol%) assembles into unilamellar polymeric vesicles in the presence of cholesterol. These polymeric vesicles are found to be biocompatible and haemocompatible and capable of entrapping water-soluble drugs. By use of an ammonium sulphate gradient bleomycin (MW 1400), for example, can be efficiently loaded on to these polymeric vesicles to yield a bleomycin-to-polymer ratio of 0.5 units mg^?1. Previously polymers were thought to assemble into vesicles only if the polymer backbone was separated from the membrane-forming amphiphile by a hydrophilic side-arm spacer. The hydrophilic spacer was thought to be necessary to decouple the random motion of the polymer backbone from the ordered amphiphiles that make up the vesicle membrane. However, stable polymeric vesicles for use in drug delivery have been prepared from a modified carbohydrate polymer, palmitoyl glycol chitosan, without this specific architecture. These polymeric vesicles efficiently entrap water-soluble drugs.     

Synthesis and evaluation of functional hyperbranched polyether polyols as prospected gene carriers

Hyperbranched polyether polyols have been partially functionalized with quaternary or tertiary ammonium groups. Five derivatives have been prepared bearing 4, 8 and 12 quaternary or 4 and 21 tertiary ammonium groups. The resulting dendritic polymers interact with plasmid DNA affording the corresponding polyplexes. The complexes were physicochemically characterized while their transfection ability was assessed by gel retardation assay, ethidium bromide exclusion assay and cell culture transfection. All the investigated polymers were shown to have marginal to low cytotoxicity in mammalian cells. Transfection efficiency comparable to that of polyethylenimine was exhibited by selected quaternized polymers. However, the introduction of tertiary amino groups on polyglycerol did not improve the transfection of the ineffective parent polymer, despite the fact that the derivatives obtained exhibited additional buffering capacity (sponge effect). The observed transfection efficiency for the quaternized polymers has been attributed to the destabilization of the lysosomal membrane originating from the interaction between the cationic polymers and the anionic moieties located at the membrane. These results are encouraging for the prospective application of these polyols as gene delivery vectors.     

Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate

The ammonium glycyrrhizinate-loaded chitosan nanoparticles were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The particle size and zeta potential of nanoparticles were determined, respectively, by dynamic light scattering (DLS) and a zeta potential analyzer. The effects, including chitosan molecular weight, chitosan concentration, ammonium glycyrrhizinate concentration and polyethylene glycol (PEG) on the physicochemical properties of the nanoparticles were studied. These nanoparticles have ammonium glycyrrhizinate loading efficiency. The encapsulation efficiency decreased with the increase of ammonium glycyrrhizinate concentration and chitosan concentration. The introduction of PEG can decrease significantly the positive charge of particle surface. These studies showed that chitosan can complex TPP to form stable cationic nanoparticles for subsequent ammonium glycyrrhizinate loading.     

负载pVAX1-wapA的壳聚糖及季铵化壳聚糖纳米粒的制备研究

目的 制备负载抗龋DNA疫苗pVAX1-wapA质粒的壳聚糖和季铵化壳聚糖纳米粒,优化其制备工艺,测定其细胞转染效率。 方法 以包封率和粒径为主要指标,单因素法考察载体浓度、pH值、N/P、TPP浓度等因素的影响,Realtime-PCR检测细胞对质粒编码蛋白的转录表达水平以评价载质粒纳米粒的促转染作用。 结果 制得的载DNA疫苗纳米粒粒径均一,形态圆整。壳聚糖(CS)纳米粒粒径为(219.2±18.2) nm,Zeta电位为(24.7±3.5) mV,包封率为91.24%。季铵化壳聚糖(CSTM)纳米粒粒径为(222.5±15.6) nm,Zeta电位为(19.6±1.2) mV,包封率为87.66%。纳米粒可以促进pVAX1-wapA进入细胞,并成功被转录。 结论 制备的包载pVAX1-wapA的季铵化壳聚糖纳米粒可用于重组基因疫苗的运送。     

Preparation and evaluation of chitosan-DNA-FAP-B nanoparticles as a novel non-viral vector for gene delivery to the lung epithelial cells

Gene delivery using cationic polymers such as chitosan shows good biocompatibility, but reveals low transfection efficiency. Fibronectin Attachment Protein of Mycobacterium bovis (FAP-B) which is responsible for the attachment of many Mycobacteria on the Fibronectin molecule of epithelial cell membrane can be considered as a new targeting ligand and can improve transfection rates in epithelial cells. In this study, chitosan-DNA nanoparticles were prepared using coacervation process. The effect of stirring speed and charge ratio (N/P) on the size and zeta potential of nanoparticles were evaluated. FAP-B ligand was added to nanoparticles at the specific condition to form chitosan-DNA-FAP-B nanoparticles via electrostatic attraction. Transfection efficiency of the final nanoparticles was investigated in A549 (alveolar epithelial cells). Cell viability was investigated using MTT assay. The optimum speed of stirring which was yielded the smallest chitosan-DNA nanoparticles with a narrow distribution (227±43 nm), was 500 rpm with the corresponding N/P ratio of 20. Chitosan-DNA-FAP-B nanoparticles presented the size of 279±27 nm with transfection efficiency about 10-fold higher than chitosan-DNA nanoparticles and resulted in 97.3% cell viability compared to 71.7% using Turbofect controls. Chitosan-DNA-FAP-B nanoparticles showed good transfection efficiency without cell toxicity. They have small particle size around 279 nm which make them a promising candidate as a novel non-viral gene vector for gene delivery to lung epithelial cells.     

In vitro evaluation of chitosan-EDTA conjugate polyplexes as a nanoparticulate gene delivery system

It was the purpose of this study to evaluate the potential of different molecular-weight chitosan-EDTA conjugates as a carrier matrix for nanoparticulate gene delivery systems. Covalent binding of EDTA to more than one chitosan chain provides a cross-linked polymer that is anticipated to produce stabilized particles. pDNA/chitosan-EDTA particles, generated via coazervation, were characterized in size and zeta potential by electrophoretic light scattering and electron microscopy. Stability was investigated at different pH values by enzymatic degradation and subsequent gel retardation assay. Lactate dehydrogenase assay was performed to determine toxicity. Furthermore, transfection efficiency into Caco-2 cells was assessed using a beta-galactosidase reporter gene. Chitosan-EDTA produced from low-viscous chitosan with 68% amino groups being modified by the covalent attachment of EDTA showed the highest complexing efficacy resulting in nanoparticles of 43 nm mean size and exhibiting a zeta potential of +6.3 mV. These particles were more stable at pH 8 than chitosan control particles. The cytotoxicity of chitosan-EDTA particles was below 1% over a time period of 4 hours. These new nanoplexes showed 35% improved in vitro transfection efficiency compared with unmodified chitosan nanoparticles. According to these results, the chitosan-EDTA conjugate may be a promising polymer for gene transfer.     

Enhancing polysaccharide-mediated delivery of nucleic acids through functionalization with secondary and tertiary amines

Chitosan is a polysaccharide that has generated significant interest as a non-viral gene delivery vehicle due to its cationic and biocompatible characteristics. However, transfection efficiency of chitosan is significantly lower compared to other cationic gene delivery agents, e.g. polyethyleneimine (PEI), dendrimers or cationic lipids. This is primarily attributed to its minimal solubility and low buffering capacity at physiological pH leading to poor endosomal escape of the gene carrier and inefficient cytoplasmic decoupling of the complexed nucleic acid. Here we have developed an imidazole acetic acid (IAA)-modified chitosan to introduce secondary and tertiary amines to the polymer in order to improve its endosomal buffering and solubility. The modified polymer was characterized by ninhydrin and (1)H NMR assays for degree of modification, while buffering and solubility were analyzed by acid titration. Nanocomplex formation, studied at various polymer-nucleic acid ratios, showed an increase in particle zeta potential for chitosan-IAA, as well as an increase in the effective diameter. Up to 100-fold increase in transfection efficiency of pDNA was seen for chitosan-IAA as compared to native chitosan, nearly matching that of PEI. In addition, transfection of siRNA by the modified polymers showed efficient gene knockdown equivalent to commercially available siPORT Amines. Collectively, these results demonstrate the potential of the imidazole-grafted chitosan as a biocompatible and effective delivery vehicle for both pDNA and siRNA.     

Thiolated Chitosan/DNA Nanocomplexes Exhibit Enhanced and Sustained Gene Delivery

Purpose Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative.Methods Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo.Results Thiolated chitosan–DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio ≥ 2.5:1. CSH360/DNA nanocomplexes induced significantly (P < 0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days.Conclusions Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.     

Design of Amine-Modified Graft Polyesters for Effective Gene Delivery Using DNA-Loaded Nanoparticles

PURPOSE: The purpose of this study was the design of a polymeric platform for effective gene delivery using DNA-loaded nanoparticles. METHODS: The polymers were synthesized by carbonyldiimidazole (CDI)-mediated coupling of diamines diethylaminopropylamine (DEAPA), dimethylaminopropylamine (DMAPA) or diethylaminoethylamine (DEAEA) to poly(vinyl alcohol) (PVA) with subsequent grafting of D,L-lactide and glycolide (1:1) in the stoichiometric ratios of 1:10 and 1:20 (free hydroxyl groups/monomer units). The polymers were characterized by 1H-NMR, gel permeation chromatography-multiple-angle laser-light-scattering, and differential scanning calorimetry. DNA-loaded nanoparticles prepared by a modified solvent displacement method were characterized with regard to their zeta (zeta)-potential and size. The transfection efficiency was assessed with the plasmid DNA pCMV-luc in L929 mouse fibroblasts. RESULTS: The polymers were composed of highly branched, biodegradable cationic polyesters exhibiting amphiphilic properties. The amine modification enhanced the rapid polymer degradation and resulted in the interaction with DNA during particle preparation. The nanoparticles exhibited positive zeta-potentials up to +42 mV and high transfection efficiencies, comparable to polyethylenimine (PEI) 25 kDa/DNA complexes at a nitrogen to phosphate ratio of 5. CONCLUSIONS: The polymers combined amine-functions and short poly(D,L-lactic-co-glycolic acid) (PLGA) chains resulting in water-insoluble polymers capable of forming biodegradable DNA nanoparticles through coulombic interactions and polyester precipitation in aqueous medium. The high transfection efficiency was based on fast polymer degradation and the conservation of DNA bioactivity.     

Novel hyaluronic acid-chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis

Gene therapy is a promising new treatment strategy for common joint-disorders such as osteoarthritis. The development of safe, effective, targeted non-viral gene carriers is important for the clinical success of gene therapy. The present work describes the use of hybrid hyaluronic acid (HA)/chitosan (CS) nanoparticles as novel non-viral gene delivery vectors capable of transferring exogenous genes into primary chondrocytes for the treatment of joint diseases. HA/CS plasmid-DNA nanoparticles were synthesized through the complex coacervation of the cationic polymers with pEGFP. Particle size and zeta potential were related to the weight ratio of CS to HA, where increases in nanoparticle size and decreases in surface charge were observed as HA content increased. The particle size and the zeta potential varied according to pH. Transfection of primary chondrocytes was performed under different conditions to examine variations in the pH of the transfection medium, different N/P ratios, different plasmid concentrations, and different molecular weights of chitosan. Transfection efficiency was maximized for a medium pH of approximately 6.8, an N/P ratio of 5, plasmid concentration of 4 μg/ml, and a chitosan molecular weight of 50 kDa. The transfection efficiency of HA/CS-plasmid nanoparticles was significantly higher than that of CS-plasmid nanoparticles under the same conditions. The average viability of cells transfected with HA/CS-plasmid nanoparticles was over 90%. These results suggest that HA/CS-plasmid nanoparticles could be an effective non-viral vector suitable for gene delivery to chondrocytes.     

Cationic lipids containing cyclen and ammonium moieties as gene delivery vectors

In this study, two novel cationic lipids containing protonated cyclen and quaternary ammonium moieties were designed and synthesized as non-viral gene delivery vectors. The structures of the two lipids differ in their hydrophobic region (cholesterol or diosgenin). Cationic liposomes were easily prepared from the lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidylethanolamine. Several studies including DLS, gel retardation assay, and ethidium bromide intercalation assay suggest that these amphiphilic molecules are able to bind and compact DNA into nanometer particles which can be used as non-viral gene delivery agents. Our results from in vitro transfection show that in association with dioleoylphosphatidylethanolamine, two cationic lipids can induce effective gene transfection in human embryonic kidney 293 cells, although the gene transfection efficiencies of two cationic lipids were found to be lower than that of lipofectamine 2000(TM) . Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vectors.     

Arginine-chitosan/DNA self-assemble nanoparticles for gene delivery: In vitro characteristics and transfection efficiency

Chitosan (Cs) is a natural cationic polysaccharide that has shown potential as non-viral vector for gene delivery because of its biocompatibility and low toxicity. However, chitosan used for gene delivery is limited due to its poor water solubility and low transfection efficiency. The purpose of this work was to prepare Arginine-chitosan (Arg-Cs)/DNA self-assemble nanoparticles (ACSNs), and determine their in vitro characteristics and transfection efficiency against HEK 293 and COS-7 cells. Our experimental results showed that the particle size and zeta potential of ACSNs prepared with different N/P ratios were 200-400nm and 0.23-12.25mV, respectively. The in vitro transfection efficiency of ACSNs showed dependence on pH of transfection medium, and the highest expression efficiency was obtained at pH 7.2. The transfection efficiency increased with the ratio of chitosan-amine/DNA phosphate (N/P ratio) from 1 to 5, and reached the highest level with the N/P ratio 5. Effect of plasmid dosage on the transfection efficiency showed the highest transfection efficiency was obtained at 4microg/well for HEK 293 cells and 6microg/well for COS-7 cells. The transfection efficiency of ACSNs was much higher than that of Cs/DNA self-assemble nanoparticles (CSNs). The average cell viability of ACSNs was over 90%. These results suggested that ACSNs could be a safe and effective non-viral vector for gene delivery.     

Self-assembled human serum albumin-coated complexes for gene delivery with improved transfection

The efficiency and safety of gene delivery vectors were important factors for gene therapy. To enhance gene transfection efficiency and to incorporate biocompatible components to the polyamidoamine (PAMAM) dendrimer mediated gene delivery systems, human serum albumin (HSA) was introduced to dendriplexes of PAMAM dendrimer and DNA via electrostatic interactions to form self-assembled PAMAM/DNA/HSA complexes (HSA-dendriplexes). The self-assembled complexes were characterized by gel retardation assay and particle size and zeta potential analysis. Meanwhile, the toxicity of HSA-dendriplexes was evaluated by the MTT assay and haemolysis test, which indicated that the complexes exhibited decreased cytotoxicity with the incorporation of HSA. As compared to dendriplexes, the ternary HSA-dendriplexes increased the enhanced green fluorescent protein gene (EGFP) expression in vitro by 1.7-fold. In addition, HSA-dendriplexes showed a significantly higher luciferase gene expression than dendriplexes or naked DNA in the liver, kidney, lungs and spleen of mice. Our results demonstrated that HSA-dendriplexes increases PAMAM mediated gene transfection efficiency and decreases the cytotoxicity and haemolysis, which made the ternary complexes a promising targeting gene delivery system.     

壳聚糖纳米粒用作基因递送载体的初步研究

目的初步研究基因壳聚糖纳米粒的性质和转染活性。方法用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;用荧光分光光度法测定基因包封率;用凝胶阻滞分析和荧光扫描测定基因在纳米粒中的位置;用体外基因转染实验定性评价纳米粒的转染活性。结果纳米粒形态多呈球形,平均粒径为218.9 nm,多分散度为0.276,zeta电位为+21.2 mV;基因包封率为99.6%;凝胶阻滞分析和荧光扫描表明基因几乎全部被包裹在纳米粒内部,表面吸附很少;体外基因转染实验表明基因壳聚糖纳米粒能够转染人胚胎肾细胞(HEK293)和肝癌细胞(HepG2),基因能够在这两种细胞中表达。结论壳聚糖纳米粒能将基因递送到细胞内并且基因能够表达,因此可以用作基因药物载体。     

Imidazolyl-PEI modified nanoparticles for enhanced gene delivery

The derivatives of polyethylenimine (PEI 25 and 750kDa) were synthesized by partially substituting their amino groups with imidazolyl moieties. The series of imidazolyl-PEIs thus obtained were cross-linked with polyethylene glycol (PEG) to get imidazolyl-PEI-PEG nanoparticles (IPP). The component of hydrophobicity was introduced by grafting the lauryl groups in the maximal substituted IPP nanoparticles (IPPL). The nanoparticles were characterized with respect to DNA interaction, hydrodynamic diameter, zeta potential, in vitro cytotoxicity and transfection efficiency on model cell lines. The IPP and IPPL nanoparticles formed a loose complex with DNA compared to the corresponding native PEI, leading to more efficient unpackaging of DNA. The DNA loading capacity of IPP and IPPL nanoparticles was also lower compared to PEI. The imidazolyl substitution improved the gene delivery efficiency of PEI (750kDa) by nine- to ten-fold and PEI (25kDa) by three- to four-fold. At maximum transfection efficiency, the zeta potential of nanoparticles was positive after forming a complex with DNA. The maximum level of reporter gene expression was mediated by IPPL nanoparticles in both the series. The cytotoxicity, another pertinent problem with cationic polymers, was also negligible in case of IPP and IPPL nanoparticles.     

Efficacy Interactions of PEG–DOX–N-acetyl Glucosamine Prodrug Conjugate for Anticancer Therapy

Present investigation is exploring structure–biocompatibility interaction of tumour targeted polyethylene glycol (PEG) based drug conjugate of doxorubicin using N-acetyl glucosamine as targeting ligand. The synthesized polymer drug conjugate was evaluated for particle size, zeta potential, molecular weight, haemolysis activity, cytotoxicity, protein binding and in vitro receptor (lectin) binding study. The particle size of synthesized conjugate was observed to be around 30 nm with polydispersability index of 0.213 indicating mono-disperse particles. Fluorescence quenching assay addressed relatively lower binding interactions of polymer drug conjugate to bovine serum albumin in comparison with free doxorubicin which may be governed to the hydrophilicity of polyethylene glycol and N-acetyl glucosamine. The cell compatibility and haemolysis study showed that PEG drug conjugate was nontoxic and biocompatible, which recommends the suitability of polymer drug conjugates for delivering biological active agents systemically. In vitro ligand–lectin receptor binding assays of synthesized targeted polymer conjugate suggest the possibility of promising interaction of N-acetyl glucosamine in vivo. Thus, the study indicated the suitability of N-acetyl glucosamine anchored targeted polymer drug conjugate in delivering bio-therapeutics for specifically targeting to tumour tissues.