Transferrin-mediated PEGylated nanoparticles for delivery of DNA/PLL

The purpose of this work was to determine the stability of pDNA/poly(L-lysine) complex (DNA/PLL) during microencapsulation, prepare transferrin (TF) conjugated PEGylated nanoparticles (TF-PEG-NP) loading DNA/PLL, and assess its physicochemical characteristics and in vitro transfection efficiency. The DNA/PLL was prepared by mixing plasmid DNA (pDNA) in deionized water with various amounts of PLL. PEGylated nanoparticles (PEG-NP) loading DNA/PLL were prepared by a water-oil-water double emulsion solvent evaporation technique. TF-PEG-NP was prepared by coupling TF with PEG-NP. The physicochemical characteristics of TF-PEG-NP and in vitro transfection efficiency on K562 cells were measured. The results showed that free pDNA reserved its double supercoiled form (dsDNA) for only on average 25.7% after sonification, but over 70% of dsDNA was reserved after pDNA was contracted with PLL. The particle size range of TF-PEG-NP loading DNA/PLL was 150-450?nm with entrapment efficiency over 70%. TF-PEG-NP exhibited the low burst effect (<10%) within 1 day. After the first phase, DNA/PLL displayed a sustained release. The amount of cumulated DNA/PLL release from TF-PEG-NP with 2% polymer over 7 days was 85.4% for DNA/PLL (1:0.3 mass ratio), 59.8% and 43.1% for DNA/PLL (1:0.6) and DNA/PLL (1:1.0), respectively. To TF-PEG-NP loading DNA/PLL without chloroquine, the percentage of EGFP expressing cells was 28.9% for complexes consisting of DNA/PLL (1:0.3), 38.5% and 39.7% for DNA/PLL (1:0.6) and DNA/PLL (1:1.0), respectively. In TF-PEG-NP loading DNA/PLL with chloroquine, more cells were transfected, the percentage of positive cells was 37.6% (DNA/PLL, 1:0.3), 47.1% (DNA/PLL, 1:0.6) and 45.8% (DNA/PLL, 1:1.0), which meant that the transfection efficiency of pDNA was increased by over 50 times when PLL and TF-PEG-NP were jointly used as a plasmid DNA carrier, in particular, the maximal percentage of positive cells (47.1%) from TF-PEG-NP loading DNA/PLL (1:0.6) was about 70 times the transfection efficiency of free plasmid DNA. The average cell viability of TF-PEG-NP loading DNA/PLL was about 90%, which meant that TF-PEG-NP appeared to be safer than PLL alone. As a result, TF-PEG-NP loading DNA/PLL could be a more effective non-viral vector for the delivery of pDNA.     

Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studies

In the present study, the effect of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto branched poly(ethylenimine) (PEI) with different grafting degree was examined for gene delivery applications. The DMAEMA-grafted-PEI conjugates were characterized and complexed with plasmid DNA (pDNA) at various concentrations, and the physicochemical properties, cell viability, and in vitro transfection efficiency of the complexes were evaluated in HEK 293T cells. Computational techniques were used to analyze the interaction energies and possible binding modes between DNA and conjugates at different grafting degrees. The cytotoxicity analysis and in vitro transfection efficiency of the conjugate/pDNA complexes exhibited a beneficial effect of DMAEMA conjugation when compared to PEI alone. The computational results revealed that the DNA/vector interaction energy decreases with increasing grafting degree, which can be associated to an enhanced release of the pDNA from the carrier once inside cells. The results indicate the significance of DMAEMA conjugation onto PEI as a promising approach for gene delivery applications.     

Formulation and characterization of naked DNA and complexed DNA loaded polymer films

Sustained release of DNA from polymeric films is of considerable interest for enhanced and prolonged gene therapy. This report describes the detailed studies of the formulation of naked plasmid DNA (pDNA) and complexed DNA-incorporated polymer films and in vitro release of the DNA. The effect of hydrophilic polymers (PEG, HA) also studied to modulate the release of pDNA and complexed DNA (lipoplex) from slow biodegradable polymer (PCL) films. The polymer system consists of a biodegradable semi-crystalline polymer (PCL) blended with a hydrophilic polymer such as poly (ethylene glycol) (PEG) or hyaluronic acid (HA). For the release of pDNA (naked DNA), a burst effect was always seen, and the addition of HA and PEG did not suppress the burst release of pDNA from PCL films. For complexed pDNA (lipoplex), the release was slow, but it could be accelerated using additives such as PEG or HA. The transfection efficiency of the complexed DNA and the naked pDNA was determined in vitro using COS 7 cells to evaluate the bioactivity of the released DNA. Transfection was observed from released lipoplexes samples from PCL/HA film. Overall, this work suggested that these polymeric DNA delivery systems are promising for the local sustained release of DNA from implanted films.     

Charge shielding effects on gene delivery of polyethylenimine/DNA complexes: PEGylation and phospholipid coating

Polyethylenimine (PEI) is an efficient cationic polymer for gene delivery, but defective in biocompatibility. In this study, we developed two different strategies to shield the positively charged PEI/DNA complexes: PEGylation and lipid coating. The physicochemical properties, cytotoxicity and transfection efficiency of the two gene delivery systems were investigated. Both PEGylation and lipid coating succeeded in reducing the zeta-potential of the complexes. Lipid-coated PEI/DNA complexes (LPD complexes) and PEI/DNA complexes exhibited similar cytotoxicity, whereas PEG-PEI/DNA complexes showed lower cytotoxicity, especially at high N/P ratios. LPD complexes were less efficient in transfection compared to PEG-PEI/DNA complexes. The transfection efficiency was influenced remarkably by cytotoxicity and surface charge of the complexes. Intracellular processes studies revealed that endosomal release might be one of the rate-limiting steps in cell transfection with PEI as a gene delivery carrier.     

Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery

The covalent functionalization of graphene oxide (GO) with chitosan (CS) is successfully accomplished via a facile amidation process. The CS-grafted GO (GO-CS) sheets consist of about 64 wt.% CS, which imparts them with a good aqueous solubility and biocompatibility. Additionally, the physicochemical properties of GO-CS are studied. As a novel nanocarrier, GO-CS is applied to load a water-insoluble anticancer drug, camptothecin (CPT), via π-π stacking and hydrophobic interactions. It is demonstrated that GO-CS possesses a superior loading capacity for CPT, and the GO-CS-CPT complexes show remarkably high cytotoxicity in HepG2 and HeLa cell lines compared to the pure drug. At the same time, GO-CS is also able to condense plasmid DNA into stable, nanosized complexes, and the resulting GO-CS/pDNA nanoparticles exhibit reasonable transfection efficiency in HeLa cells at certain nitrogen/phosphate ratios. Therefore, the GO-CS nanocarrier is able to load and deliver both anticancer drugs and genes.     

Cationic liposomes-polyallylamine-plasmid nanocomplexes for gene delivery

The aim of the present study was to prepare, characterise and evaluate the transfection efficiency of ternary complexes (lipopolyplexes) composed of cationic liposome, polyallylamine (PAA), plasmid DNA (pDNA). PAA was reacted with a varying amount of a linker, 6-bromohexanoic acid (6-bromo-HA), to prepare a series of modified polymers. Lipopolyplexes consisting of cationic liposome, PAA (or modified PAA), pDNA were prepared. The nanoparticles, so formed, were characterised by their size and zeta potential and were subsequently evaluated for their cytotoxicity and transfection ability on Neuro2A cells. Mean size of prepared complexes ranged from 170 to 280 nm. All lipopolylexes showed a positive zeta potential. Highest transfection efficiency was for lipopolyplex containing PAA 15 kDa-modified polymer and liposome at C/P ratio of 0.5. High molecular weight PAA was more toxic than PAA 15 kDa for Neuro2A cells especially in higher C/P ratio. The results indicate that using the hydrophobic modified PAA in the structure of lipopolyplexes is an effective strategy for improving transfection efficiency.     

Silicalite nanoparticles that promote transgene expression

Here, we report on a new zeolite-based silicalite nanoparticle that can enhance the transfection efficiencies generated by poly ethylene imine-plasmid DNA (PEI-pDNA) complexes via a sedimentation mechanism and can enhance the transfection efficiencies of pDNA alone when surface functionalized with amine groups. The silicalite nanoparticles have a mean size of 55?nm. Functionalizing the silicalite nanoparticles with amine groups results in a clear transition in zeta potential from -25.9 ± 2.3?mV (pH 7.4) for unfunctionalized silicalite nanoparticles to 4.9 ± 0.7?mV (pH 7.4) for amine functionalized silicalite nanoparticles. We identify that silicalite nanoparticles used to promote non-viral vector acceleration to the cell surface are found in acidic vesicles or the cytoplasm but not the nucleus. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay showed that the silicalite nanoparticles were non-toxic at the concentrations tested for transfection. We show that surface functionalization of silicalite nanoparticles with amine groups results in a significant (230%) increase in transfection efficiency of pDNA when compared to unfunctionalized silicalite nanoparticles. Silicalite nanoparticles enhanced pDNA-PEI induced transfection of human embryonic kidney (HEK-293) cells by over 150%.     

Quaternized chitosan/rectorite intercalative materials for a gene delivery system

Non-viral vectors have gained increasing attention in gene therapy because of their safety, but with the shortcoming of low transfection efficiency. We have developed a hybrid material as a novel non-viral vector, which combines the advantages of both biopolymer and clay in a gene delivery system. Quaternized chitosan was intercalated into the interlayers of rectorite to obtain a new polymer/layered silicate nanocomposite. In vitro and in vivo toxicity studies revealed that the nanocomposites were biocompatible and non-toxic. At the nanocomposite:pDNA mass ratio of 8:1, they achieved 100% pDNA adsorption capacity. In vitro cell transfection revealed a transfection efficiency of 32.1% at 96?h as shown by a flow-cytometric study, and the intensive green fluorescence protein (GFP) expression could last for up to 120?h. Furthermore, an in vivo transfection study showed that the most prominent GFP expression was observed in the gastric and duodenum mucosa, and good transfection efficiency was also obtained when injected into the muscle. All the results suggest that quaternized chitosan/rectorite nanocomposite is a novel and potential non-viral gene carrier.     

Oxidation‐Responsive Nanoassemblies for Light‐Enhanced Gene Therapy

Microenvironment‐responsive supramolecular assemblies have attracted great interest in the biomedical field due to their potential applications in controlled drug release. In this study, oxidation‐responsive supramolecular polycationic assemblies named CPAs are prepared for nucleic acid delivery via the host–guest interaction of β‐cyclodextrin based polycations and a ferrocene‐functionalized zinc tetraaminophthalocyanine core. The reactive oxygen species (ROS) can accelerate the disassembly of CPA/pDNA complexes, which would facilitate the release of pDNA in the complexes and further benefit the subsequent transfection. Such improvement in transfection efficiency is proved in A549 cells with high H₂O₂ concentration. Interestingly, the transfection efficiencies mediated by CPAs are also different in the presence or absence of light in various cell lines such as HEK 293 and 4T1. The single oxygen (¹O₂), produced by photosensitizers in the core of CPAs under light, increases the ROS amount and accelerates the disassembly of CPAs/pDNA complexes. In vitro and in vivo studies further illustrate that suppressor tumor gene p53 delivered by CPAs exhibits great antitumor effects under illumination. This work provides a promising strategy for the design and fabrication of oxidation‐responsive nanoassemblies with light‐enhanced gene transfection performance.     

An E3‐14.7K Peptide that Promotes Microtubules‐Mediated Transport of Plasmid DNA Increases Polyplexes Transfection Efficiency

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non‐viral vectors. Here, the interaction between E3‐14.7K and FIP‐1 to favor migration of pDNA along microtubules is exploited. E3‐14.7K is an early protein of human adenoviruses that interacts via FIP‐1 (Fourteen.7K Interacting Protein 1) protein with the light‐chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79‐98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non‐directional movements in the cytoplasm. Remarkably, P79‐98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.     

Enhanced gene transfer with multilayered polyplexes assembled with layer-by-layer technique

Successful gene therapy asks for multifunctional vectors which can not only protect DNA from degradation but also transfer it into nuclear and subsequently express the loaded gene. Here we reported a novel multilayered delivery system constructed with DNA, protamine (Pro) and polyethylenimine (PEI) via lay-by-layer (LbL) technique, which posed multifunctions. DNA was previously condensed into a compact core with Pro which also contained nuclear localisation signals (NLS) domains for nuclear transfer. Then additional DNA was deposited as the first layer onto the cationic core via the electrostatic attraction which would increase the loading dose of DNA. At last, PEI was absorbed as the outmost layer to achieve the endosomal escape. Therefore a quaternary polyplexes which offered high loading of DNA, nuclear transfer ability and endosomal escape capability was constructed with the LbL technique. The obtained quaternary polyplexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA protection capability. Compared with commercially available PEI/DNA complexes, the novel multifuctional vector exhibited not only lower cytotoxicity (P<0.05) but also higher transfection efficiency in HepG2 and HeLa cells (P<0.05) in vitro test.     

Galactosylation of chitosan-graft-spermine as a gene carrier for hepatocyte targeting in vitro and in vivo

Polyethyleneimine (PEI) has been described as a highly efficient gene carrier due to its efficient proton sponge effect within endosomes. However, many studies have demonstrated that PEI is toxic and associated with a lack of cell specificity despite high transfection efficiency. In order to minimize the toxicity of PEI, we prepared chitosan-graft-spermine (CHI-g-SPE) in a previous study. CHI-g-SPE showed low toxicity and high transfection efficiency. However, this compound also had limited target cell specificity. In the present study, we synthesized galactosylated CHI-g-SPE (GCS) because this modified GCS could be delivered specifically into the liver due to hepatocyte-specific galactose receptors. The DNA-binding properties of GCS at various copolymer/DNA weight ratios were evaluated by a gel retardation assay. The GCS copolymer exhibited significant DNA-binding ability and efficiently protected DNA from nuclease attack. Using energy-filtered transmission electron microscopy (EF-TEM), we observed dense spherical, nano-sized GCS/DNA complexes with a homogenous distribution. Most importantly, GCS was associated with remarkably low cytotoxicity compared to PEI in HepG2, HeLa, and A549 cells. Moreover, GCS carriers specifically delivered the gene-of-interest into hepatocytes in vitro as well as in vivo. Our results suggest that the novel GCS described here is a safe and highly efficient carrier for hepatocyte-targeted gene delivery.     

Design of novel polysaccharidic nanostructures for gene delivery

The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10?kDa (HAO) and chitosan 125 (CS) or 10-12?(CSO)?kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230?nm, a positive zeta potential of +10 to +32?mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK?293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In?conclusion, HA:CS nanoparticles are promising carriers for gene delivery.     

Formulation of chitosan-TPP-pDNA nanocapsules for gene therapy applications

The encapsulation of DNA inside nanoparticles meant for gene delivery applications is a challenging process where several parameters need to be modulated in order to design nanocapsules with specific tailored characteristics. The purpose of this study was to investigate and improve the formulation parameters of plasmid DNA (pDNA) loaded in chitosan nanocapsules using tripolyphosphate (TPP) as polyanionic crosslinker. Nanocapsule morphology and encapsulation efficiency were analyzed as a function of chitosan degree of deacetylation and chitosan-TPP ratio. The manipulation of these parameters influenced not only the particle size but also the encapsulation and release of pDNA. Consequently the transfection efficiency of the nanoparticulated systems was also enhanced with the optimization of the particle characteristics. Overall, the differently formulated nanoparticulated systems possess singular properties that can be employed according to the desired gene delivery application.     

Decaarginine-PEG-artificial lipid/DNA complex for gene delivery: nanostructure and transfection efficiency

Oligoarginine conjugates are highly efficient vectors for the delivery of plasmid DNA into cells. Decaarginine-conjugated lipid (Arg10-PEG-lipid) was synthesized and the effects of Arg10-PEG-lipid concentration at a fixed DNA concentration on transfection efficiency and the structure of the complexes were studied below and above critical micelle concentration (CMC), and at the lipid nitrogen/DNA phosphate (N/P) ratio corresponding to transfection, respectively. Arg10-PEG-lipid at the concentration below CMC showed stronger interaction with DNA by fluorescence intensity distribution analysis, and significantly higher luciferase and green fluorescent protein expression than that above CMC. A phase-contrast cryo-transmission electron microscope (cryo-TEM) experiment showed that the morphology of the complexes depended on the N/P ratio. At a low N/P ratio corresponding to that in transfection at a lipid concentration below CMC, a net-like structure developed in which plasmid DNA was involved. A further increase in the N/P ratio, a large fibrous nanostructure of complexes, was also observed. Without DNA, these structures were not obtained. The cellular uptake mechanism of complexes using flow cytometry with inhibitors suggested that complexes with two different morphologies showed similar cellular uptake and uptake mechanism, macropinocytosis. Differences in transfection efficiency of the complexes may be explained by a large fibrous nanostructure inhibiting the cellular internalization of complexes or the release of DNA from macropinosomes into cytoplasm. Arg10-PEG-lipid/DNA complexes formed a favorable nanostructure for gene delivery, depending on the N/P ratio in water.     

The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes: a comparative study

Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class of vector. To investigate the general validity of these criteria, we studied the efficacy of a variety of DNA delivery vehicles including liposomes (DOTAP, SAINT2) with and without helper lipid (DOPE), the polymer polyethyleneimine (PEI), and cationic nanoparticles (Si26H, PLGA/chitosan) in a comparative manner. Sizes of the DNA complexes varied between 100 and 500 nm for PEI polyplexes and DOTAP/DOPE lipoplexes, respectively. The zeta potential was positive for PEI, Si26H, and DOTAP based complexes, while it was neutral for SAINT2-DNA complexes and negative for PLGA/chitosan-DNA complexes. The latter finding was elucidated by AFM, showing a layer of DNA adsorbed onto the nanoparticles. Transfection activity was negligible for PLGA/chitosan nanospheres, moderate for Si26H nanospheres and high for all other complexes, PEI being the most active carrier. The liposomal preparations were of low (DOTAP) or moderate (SAINT2) stability in serum, resulting in a pronounced reduction of gene expression, which was partially restored by the addition of chloroquine. In conclusion, transfection efficiency (i) seems to require a positive or neutral zeta potential, (ii) is depending on size, e.g., is higher for smaller particles, and (iii) requires a vector that is stable in serum.     

Anionic Lipid,pH‐Sensitive Liposome‐Gold Nanoparticle Hybrids for Gene Delivery – Quantitative Research of the Mechanism

Gene therapy is a potential method for treating a large range of diseases. Gene vectors are widely used in gene therapy for promoting the gene delivery efficiency to the target cells. Here, gold nanoparticles (AuNPs) coated with dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) are synthesized using a facile method for a new gene vector (DODAB/DOPE‐AuNPs), which possess 3‐ and 1.5‐fold higher transfection efficiency than those of DODAB‐AuNPs and a commercial transfection agent, respectively. Meanwhile, it is nontoxic with concentrations required for effective gene delivery. Imaging and quantification studies of cellular uptake reveal that DOPE increases gene copies in cells, which may be attributed to the smaller size of AuNPs/DNA complexes. The dissociation efficiency of DNA from the endocytic pathway is quantified by incubating with different buffers and investigated directly in the cells. The results suggest that DOPE increases the internalization of AuNPs/DNA complexes and promotes DNA release from early endosomes for the vector is sensitive to the anionic lipid membrane and the decreasing pH along the endocytic pathway. The new vector contains the potential to be the new alternative as gene delivery vector for biomedical applications.     

Nano-scratching and nano-machining in different environments on Cr2N/Cu multilayer thin films

Five nanostructured Cr₂N/Cu multilayer coatings were deposited by a bipolar asymmetric reactive pulsed DC magnetron sputtering system, and various bilayer periods (Λ) were achieved by controlling the holding time of Si substrates in the plasma of Cr or Cu. The hardness and elastic modulus of multilayer coatings were investigated by means of a nanoindenter. Nano-scratch and nano machining experiments on multilayered coatings were conducted using atomic force microscopy (AFM) in air and DI-water, respectively. According to the groove depth, width, and coefficient of friction (COF) obtained from nano-scratch tests, influences of scratch cycle numbers and bilayer periods on the scratchability of Cr₂N/Cu multilayered thin films were examined. It was observed that after nano-scratch experiments in air and water, the COF values and the amount of removed material increased with increasing bilayer period. After nano-machining tests in air and water, different types of the cutting chip pile-ups were observed. In this work, the surface tribological properties and machinability of Cr₂N/Cu multilayered thin films when using an AFM are discussed.     

Structural characterization of novel gemini non-viral DNA delivery systems for cutaneous gene therapy

The structural and physicochemical properties of novel cationic lipid-based DNA complexes have been investigated for the purpose of designing micro/nano-scale self-assembling delivery systems for cutaneous gene therapy. DNA/gemini surfactant (spacer n?=?3–16; chain m?=?12 or 16) complexes (1?:?10 charge ratio), with or without dioleoylphosphatidyl-ethanolamine (DOPE), designed for cellular transfection, were generally in the range of 100–200?nm as demonstrated by atomic force microscopy and particle size analysis. Small-angle X-ray scattering measurements indicated that the DNA/gemini complexes lacked long-range order, whereas DNA/gemini/DOPE complexes exhibited lamellar and polymorphic phases other than hexagonal. Correlation studies using transfection efficiency data in PAM 212 keratinocytes and in vitro skin absorption indicated that formulations containing gemini surfactants having the ability to induce structures other than lamellar in the resulting complexes, generally exhibited greater transfection activity and cutaneous absorption.     

Self-assembled micellar aggregates based monomethoxyl poly(ethylene glycol)-<Emphasis Type="Italic">b</Emphasis>-poly(ε-caprolactone)-<Emphasis Type="Italic">b</Emphasis>-poly(aminoethyl methacrylate) triblock copolymers as efficient gene delivery vectors

Amphiphilic triblock copolymers monomethoxyl poly(ethylene glycol) (mPEG)-b-poly(ε-caprolactone) (PCL)-b-poly(aminoethyl methacrylate)s (PAMAs) (mPECAs) were synthesized as gene delivery vectors. They exhibited lower cytotoxicity and higher transfection efficiency in COS-7 cells in presence of serum compared to 25 kDa bPEI. The influence of mPEG and PCL segments in mPECAs was evaluated by comparing with corresponding diblock copolymers. The studies showed the incorporation of the hydrophobic PCL segment in triblock copolymers affected the binding capability to pDNA and surface charges of complexes due to the formation of micelles increasing the local charges. The presence of mPEG segment in gene vector decreased the surface charges of the complexes and increased the stability of the complexes in serum because of the steric hindrance effect. It was also found that the combination of PEG and PCL segments into one macromolecule might lead to synergistic effect for better transfection efficiency in serum.