Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes

Substrate-mediated gene delivery describes the immobilization of gene therapy vectors to a biomaterial, which enhances gene transfer by exposing adhered cells to elevated DNA concentrations within the local microenvironment. Surface chemistry has been shown to affect transfection by nonspecifically immobilized complexes using self-assembled monolayers (SAMs) of alkanethiols on gold. In this report, SAMs were again used to provide a controlled surface to investigate whether the presence of oligo(ethylene glycol) (EG) groups in a SAM could affect complex morphology and enhance transfection. EG groups were included at percentages that did not affect cell adhesion. Nonspecific complex immobilization to SAMs containing combinations of EG- and carboxylic acid-terminated alkanethiols resulted in substantially greater transfection than surfaces containing no EG groups or SAMs composed of EG groups combined with other functional groups. Enhancement in transfection levels could not be attributed to complex binding densities or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance and could indicate the ability of these surfaces to preserve complex morphology upon binding. The ability to control the morphology of the immobilized complexes and influence transfection levels through surface chemistry could be translated to scaffolds for gene delivery in tissue engineering and diagnostic applications.     

Surface adsorption of DNA to tissue engineering scaffolds for efficient gene delivery

Gene delivery from tissue engineering scaffolds has potential to promote localized transgene expression that can induce the formation of functional tissues. Substrate-mediated delivery, an alternative delivery strategy to sustained release, is based on immobilization of DNA complexes to the polymer surface for subsequent delivery to cells cultured on the substrate. We investigate polyethylenimine (PEI)/DNA complex immobilization and subsequent cellular transfection on tissue engineering scaffolds fabricated from poly(lactide-co-glycolide) (PLG). The properties of the substrate and the complex affect both immobilization and cellular transfection. PLG promotes binding of PEI/DNA complexes, with percent bound independent of the N/P ratio or the DNA dosage. The levels of transgene expression are similar to or greater than control studies based on bolus DNA delivery, with orders of magnitude less DNA. Immobilization also homogeneously distributes the DNA throughout the scaffold, resulting in large numbers of transfected cells (>60%) at low surface quantities (<50 ng). Importantly, this approach can be employed to transfect cells throughout a three-dimensional scaffold. Tissue engineering scaffolds that are prefabricated into various shapes from a range of materials could potentially employ this strategy for numerous applications.     

Self-assembling peptide-lipoplexes for substrate-mediated gene delivery

The efficiency of biomaterial-based gene delivery is determined by the interaction of the material and the vector. For lipoplexes, surface immobilization has been used to transfect cells for applications such as cell arrays and model tissue formation through patterned transfection. Further increases in the delivery efficiency are limited by cellular internalization, which may be overcome by altering the material/vector interactions. In this report, we investigated the modification of the lipoplex physical properties through self-assembly with cationic peptides, and subsequently quantified cellular association, internalization and nuclear accumulation of DNA and transfection. Relative to lipid alone, peptide-lipoplexes enhanced transfection by up to 4.6-fold. The presence of the peptide in the lipoplex increased internalization efficiency by up to 4.5-fold, decreased the percentage of lysosomal DNA by 2.1-fold and increased the efficiency of nuclear accumulation by 3.0-fold. In addition, an analysis of internalization pathways for peptide-lipoplexes indicated a greater role of clathrin and caveolae-mediated endocytosis relative to macropinocytosis, which was not observed for peptide-free lipoplexes. These results demonstrate peptide-induced enhancement of gene transfer by surface immobilization due to increased cellular internalization and nuclear accumulation, which has numerous applications ranging from cell-based assays to regenerative medicine.     

Patterned transgene expression in multiple-channel bridges after spinal cord injury

Patterning of gene delivery on sub-millimeter length scales within tissue engineering scaffolds is fundamental to recreating the complex architectures of tissues. Surface-mediated delivery of lipoplexes mixed with fibronectin was investigated to pattern vectors within 250 μm channels in poly(lactide-co-glycolide) (PLG) bridges. Initial studies performed in vitro on PLG surfaces indicated that a DNA density of 0.07 μg mm^–2 inside each channel with a weight ratio of DNA to fibronectin of 1:20 maximized the number of transfected cells and the levels of transgene expression. Patterned vectors encoding for nerve growth factor (NGF) resulted in localized neurite extension within the channel. Translation to three-dimensional multiple-channel bridges enabled patterned transfection of different vectors throughout the channels for DNA:fibronectin ratios of 1:4 and multiple DNA depositions, with a large increase of neural cell bodies and neurite extension for delivery of DNA encoding for NGF. In vivo, the immobilization of non-viral vectors within the channels resulted in localized transfection within the pore structure of the bridge immediately around the channels of the bridge containing DNA. This surface immobilization strategy enables patterned gene delivery in vitro and in vivo on length scales of hundreds of microns and may find utility in strategies aimed at regenerating tissues with complex architectures.     

Biomaterial-mediated retroviral gene transfer using self-assembled monolayers

Biomaterial-mediated gene delivery has recently emerged as a promising alternative to conventional gene transfer technologies that focus on direct delivery of viral vectors or DNA-polymer/matrix complexes. However, biomaterial-based strategies have primarily targeted transient gene expression vehicles, including plasmid DNA and adenovirus particles. This study expands on this work by characterizing biomaterial properties conducive to the surface immobilization of retroviral particles and subsequent transduction of mammalian cells at the cell-material interface. Self-assembled monolayers (SAMs) of functionally-terminated alkanethiols on gold were used to establish biomaterial surfaces of defined chemical composition. Gene transfer was observed to be greater than 90% on NH(2)-terminated surfaces, approximately 50% on COOH-functionalized surfaces, and undetectable on CH(3)-terminated SAMs, similar to controls of tissue culture-treated polystyrene. Gene delivery via the NH(2)-SAM was further characterized as a function of retrovirus coating time, virus concentration, and cell seeding density. Finally, SAM-mediated gene delivery was comparable to fibronectin- and poly-l-lysine-based methods for gene transfer. This work is significant to establishing safe and effective gene therapy strategies, developing efficient methods for gene delivery, and supporting recent progress in the field of biomaterial-mediated gene transfer.     

Silk fibroin layer-by-layer microcapsules for localized gene delivery

Herein, we describe the delivery of plasmid DNA (pDNA) using silk fibroin (SF) layer-by-layer assembled microcapsules. Deposition of fluorescently labeled SF onto polystyrene (PS) template particles resulted in increasing fluorescence intensity and decreasing surface charge in correlation to SF layer number. After removal of the PS core, hollow, monodisperse, and structurally stable SF microcapsules of variable size and shell thickness were obtained. Plasmid DNA encoding for enhanced green fluorescent protein (eGFP) was loaded onto 1 or 4 μm capsules, either by incorporation of pDNA within the innermost layer of the shell or by adsorption to the microcapsules surface, and in vitro pDNA release, cytotoxicty and eGFP expression were studied. Sustained pDNA release over 3 days was observed using both loading techniques, being accelerated in the presence of protease. DNA loaded SF microcapsules resulted in efficient cell transfection along with low cytotoxicity after 3 days incubation compared to treatment with pDNA/branched polyethylenimine complexes. Among the tested conditions highest transfection efficiencies were achieved using 1 μm capsules where pDNA was adsorbed to the capsule surface. Our results suggest that SF microcapsules are suitable for the localized delivery of pDNA, combining low cytotoxicity and high transfection efficiency.     

Cell patterning using molecular vapor deposition of self-assembled monolayers and lift-off technique

This paper reports a precise, live cell-patterning method by means of patterning a silicon or glass substrate with alternating cytophilic and cytophobic self-assembled monolayers (SAMs) deposited via molecular vapor deposition. Specifically, a stack of hydrophobic heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane SAMs and a silicon oxide adhesion layer were patterned on the substrate surface, and a hydrophilic SAM derived from 3-trimethoxysilyl propyldiethylenetriamine was coated on the remaining non-treated areas on the substrate surface to promote cell growth. The primary characteristics of the reported method include: (i) single-cell resolution; (ii) easy alignment of the patterns with the pre-existing patterns on the substrate; (iii) easy formation of nanoscale patterns (depending on the exposure equipment); (iv) long shelf life of the substrate pattern prior to cell culturing; (v) compatibility with conventional, inverted, optical microscopes for simple visualization of patterns formed on a glass wafer; and (vi) the ability to support patterned cell (osteoblast) networks for at least 2 weeks. Here, we describe the deposition technique and the characterization of the deposited layers, as well as the application of this method in the fabrication of multielectrode arrays supporting patterned neuronal networks.     

Patterned PLG substrates for localized DNA delivery and directed neurite extension

Tissue engineering strategies that enable nerve regeneration will require methods that can promote and direct neurite extension across the lesion. In this report, we investigate an in vitro combinatorial approach to directed neurite outgrowth using gene delivery from topographically patterned substrates, which can induce expression of neurotrophic factors to promote neurite extension and direct the extending neurites. Poly(lactide-co-glycolide) (PLG), which has been used to fabricate conduits or bridges for regeneration, was compression molded to create channels with 100, 150, and 250 microm widths. DNA complexes were immobilized to the PLG, and cells cultured on the substrate were transfected with efficiencies dependent on channel width and DNA amount. A co-culture model consisting of primary neurons and accessory cells was employed to investigate neurite outgrowth within the channels. Localized secretion of nerve growth factor (NGF) by the accessory cells promoted neuron survival and neurite extension. Neurons cultured in channels with NGF expression exhibited longer primary neurites than in the absence of channels. Neurons cultured in smaller width PLG microchannels exhibited a greater degree of directionality and less secondary sprouting than larger channels. Finally, surface immobilization allowed for the delivery of distinct plasmids from each channel, which may enable channels to be tailored for specific nerve tracts. This approach demonstrates the ability to combine gene delivery with physical guidance, and can be tailored to target specific axonal populations with varying neurotrophic factor requirements.     

Size-dependent transfection efficiency of PEI-coated gold nanoparticles

Gold nanoparticles (Au NPs) are promising vectors for gene delivery applications. In order to gain insight on the influence of particle size on cell transfection, Au NPs were combined with poly(ethylenimine) (PEI) to prepare two sets of PEI-coated Au NPs having particle-size distributions centered at about 6 nm (<10 nm Au-PEI NPs) or 70 nm (<100 nm Au-PEI NPs), respectively. Au-PEI NPs were coupled to a variety of plasmids carrying reporter or suicide genes to prepare Au-PEI NPs/DNA complexes, and human osteosarcoma Saos-2 cells were used to investigate the performance of the Au-PEI NPs as transfection vectors in serum-containing media. The conjugates of DNA with both types of Au-PEI NPs were found to be negatively charged. In spite of the electrostatic repulsion that occurs between the surface of the cell and the surface of the plasmid-conjugated NPs, cell internalization was observed for both kinds of Au-PEI NPs. Cells were efficiently transfected with complexes derived from <10 nm Au-PEI NPs, but not with the <100 nm Au-PEI NPs. Large aggregates of NPs associated with DNA were found in endocytic vesicles of cells incubated with <100 nm Au-PEI NPs, while the success of the smaller Au-PEI NPs as transfection vectors was related to their lower agglomeration state inside cells and to endosomal escape of DNA.     

纳米材料在基因转染中的应用--Starburst PAMAM dendrimers研究现状

提高重组DNA对培养细胞及活体水平的转染效率是利用重组DNA进行预防和治疗疾病所必须解决的难题之一。一种新型阳离子多聚物00Starburst PAMAM dendrimers的出现为解决现存在难题带来了一线曙光。Starburst PAMAM dendrimers是1985年之后出现的一类新型星射状树形高分子，它们结构规整，具有呈辐射关对称的刚性球体结构，在生理条件下，Starburst PAMAM dendrimers分子具高的表面正电荷密度，能与天然状态下存在的带负电荷的生物活性物质（如核酸）发生静电相互作用形成复合物。该类树形高分子可极大增强DNA转染真核细胞的转染效率，具有许多优于其它现有转染试剂的优良特性，并具有在活体内应用的潜力。本文介绍了九十年代以来对该类树形高分子在体外培养细胞及活体水平增强DNA转染效率的研究现状并对今后的研究和应用进行了分析。     

超声可控释药体系研究进展

超声可控释药体系是一种新兴的靶向给药及基因转运方法。以超声敏感材料作为药物或基因转送的载体,当超声辐照于靶组织或靶器官时, 靶体内载体可定向释放出包裹或附着的基因或药物, 实现对负载药物的定时定量定点释放和提高药物输送效率或基因转染率的目的。文中对超声可控释药体系的作用机制、超声敏感载体材料及生物医学应用等方面进行综述,最后对该领域目前存在的问题和今后的发展方向提出了一些看法。     

Electrostatic immobilization of DNA polyplexes on small intestinal submucosa for tissue substrate-mediated transfection

Naturally occurring extracellular matrices (ECMs) such as small intestinal submucosa (SIS) have received significant attention for their therapeutic applications in tissue repair and regeneration. However, there have been no reports exploring the electrostatic properties of naturally occurring ECMs as a means to control transgene delivery. In the present study, we electrostatically adsorbed DNA polyplexes onto SIS for transfection upon cellular adhesion. To associate polyplexes with SIS, we first used a streaming potential method to characterize the surface charge of SIS and obtained a negative zeta potential at neutral pH, which can be attributed to the abundant glycosaminoglycan (GAG) content in SIS. We next prepared cationic polyethylenimine (PEI)/DNA polyplexes to associate with the negatively charged SIS for conjugation. Using the Cy(TM)3 dye-labeled control DNA as the reporter, we visualized the adsorption of PEI/DNA polyplexes at the SIS surface. Using luciferase, green fluorescent protein and beta-galactosidase as reporter proteins, we showed that the adsorbed PEI/DNA polyplexes were active and capable of carrying out transfection upon cellular adhesion, indicating that the electrostatic binding of polyplexes with SIS was reversible. In addition, the SIS-mediated transfection was contact-dependent: separation of SIS from the target cells via a 0.5 mm porous polyester membrane significantly reduced the efficiency of transfection in comparison to a direct seeding of cells onto SIS. We conclude that electrostatic immobilization of PEI/DNA polyplexes on SIS is capable of initiating efficient transgene delivery, which can be a useful tool in developing localized gene transfer.     

Micropatterning Polydiacetylene Supramolecular Vesicles on Glass Substrates using a Pre‐Patterned Hydrophobic Thin Film

A micropatterning method for depositing polydiacetylene (PDA) supramolecular vesicles on glass substrates, employing a pre‐patterned hydrophobic thin film, is presented. The pre‐patterned hydrophobic thin film is used to define surface‐exposed regions on hydrophilic glass substrates where selective immobilization of PDA vesicles takes place. The observation that fluorescent patterns are generated by heat treatment of the deposited PDAs demonstrates that the vesicles are successfully immobilized in the form of micropatterns. Finally, the possibility that this strategy may be used to design a vesicle‐based sensor chip system is demonstrated by the observation that fluorescence patterns are generated when the immobilized PDA vesicles interact with cyclodextrin.     

Two-Dimensional Protein Micropatterning for Sensor Applications Through Chemical Selectivity Technique

Two-dimensional protein micropatterning with immobil-ization of IgG and poly (ethylene glycol) (PEG) on patterned Au and Si surfaces was performed through a new technique. The technique for micropatterning is based on a chemical selectivity method by creating chemical bonding between protein, self-assembled monolayers (SAMs) and substrates rather than physical means. The substrates used in this study are pre-fabricated with silicon wafer patterned with arrays of gold squares. The silicon regions of the substrate are modified with polyethylene glycol (PEG) to resist protein adsorption and cell adhesion. The gold regions on the substrate are first immobilized with bifunctional SAM layers that can covalently bound adhesion proteins for individual cell attachment against a PEG background. The surface coatings are characterized by contact angle measurement, ellips-ometry, and atomic force microscopy (AFM). The patterns of fluorescence-labeled proteins are examined using fluorescence microscopy. Our study demonstrated that the PEG modified silicon region showed an effective protein reduction while the gold regions were successfully covalently bonded with proteins. This technique also demonstrated a combined feature of ensuring the activity, selectivity, and stability of the immobilized proteins. A simple lift-off microfabrication process was introduced in this study to pattern metal on silicon substrates without using expensive metal etching.     

DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach

Efficient and controlled gene delivery from biodegradable materials can be employed to stimulate cellular processes that lead to tissue regeneration. In this report, a substrate-mediated approach was developed to deliver DNA from hyaluronic acid-collagen hydrogels. The hydrogels were formed by crosslinking HA with poly(ethylene glycol) diglycidyl ether. Poly(ethylene imine)(PEI)/DNA complexes were immobilized to the substrate using either biotin/neutravidin or non-specific adsorption. Complexes were formed in the presence or absence of salt to regulate complex size, and resulted in complexes with z-average diameters of 1221.7 +/- 152.3 and 139.4 +/- 1.3 nm, respectively. During 48-h incubation in PBS or hyaluronidase, DNA was released slowly from the hydrogel substrate (<30% of immobilized DNA), which was enhanced by incubation with conditioned media (approximately 50% of immobilized DNA). Transgene expression mediated by immobilized, large diameter complexes was 3 to 7-fold greater than for small diameter complexes. However, the percentage of cells expressing the transgene was greater for small diameter complexes (48.7%) than for large diameter complexes (22.3%). Spatially controlled gene transfer was achieved by topographically patterning the hydrogel to pattern cell adhesion. Biomaterial-based gene delivery can be applicable to numerous tissue engineering applications, or as a tool to examine tissue formation.     

Contribution of hydrophobic/hydrophilic modification on cationic chains of poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) amphiphilic co-polymer in gene delivery

Nanoparticles (NPs) assembled from amphiphilic polycations have been certified as potential carriers for gene delivery. Structural modification of polycation moieties may be an efficient route to further enhance gene delivery efficiency. In this study two electroneutral monomers with different hydrophobicities, 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA), were incorporated into the cationic poly(dimethylamino ethyl methacrylate) (PDMAEMA) side-chains of amphiphilic poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) (PCD) by random co-polymerization, to obtain poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl methacrylate) (PCD-HEMA) and poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl acrylate) (PCD-HEA). Minimal HEA or HEMA moieties in PDMAEMA do not lead to statistically significant changes in particle size, zeta potential, DNA condensation properties and buffering capacity of the naked NPs. However, the incorporation of HEMA and HEA lead to reductions and increases, respectively, in the surface hydrophilicity of the naked NPs and NPs/DNA complexes, which was confirmed by water contact angle assay. These simple modifications of PDMAEMA with HEA and HEMA moieties significantly affect the gene transfection efficiency on HeLa cells in vitro: PCD-HEMA NP/DNA complexes show a much higher transfection efficiency than PCD NPs/DNA complexes, while PCD-HEA NPs/DNA complexes show a lower transfection efficiency than PCD NP/DNA complexes. Fluorescence activated cell sorter and confocal laser scanning microscope results indicate that the incorporation of hydrophobic HEMA moieties facilitates an enhancement in both cellular uptake and endosomal/lysosomal escape, leading to a higher transfection efficiency. Moreover, the process of endosomal/lysosomal escape confirmed in our research that PCD and its derivatives do not just rely on the proton sponge mechanism, but also on membrane damage due to the polycation chains, especially hydrophobic modified ones. Hence, it is proved that hydrophobic modification of cationic side-chains is a crucial route to improve gene transfection mediated by polycation NPs.     

An acid-labile temperature-responsive sol–gel reversible polymer for enhanced gene delivery to the myocardium and skeletal muscle cells

The work demonstrates the development of acid-labile temperature-responsive sol–gel reversible polymer for enhanced in vivo myocardium and skeletal muscle gene delivery. In this report, multi-block copolymers (MBCPs) synthesized from pluronic^® and di-(ethylene glycol) divinyl ether (DEGDVE) were used as a delivery vehicle for controlled and sustained release of plasmid DNA (pDNA) in in vitro as well as in vivo experiments. The non-ionic MBCP/pDNA complex showed remarkable transfection efficiencies against the myocardium cells as well as muscle cells in vivo, which is otherwise very difficult to achieve by using cationic polymers. In in vitro experimental settings, this intelligent stimuli-responsive polymer is shown to improve the transfection efficiency of branched polyethylenimine (BPEI)/pDNA complex when used together. The effect of MBCP on the surface charge and particle size of its various complexes with pDNA and BPEI was also studied. The release profile of pDNA from the MBCP gel was investigated and pH of the degraded polymer was also monitored to ascertain its non-cytotoxicity arising due to the increased acidity as observed with other PLGA-based polymers. The rapid sol–gel transition of MBCP under thermal stimuli with concomitant release of pDNA under acidic stimulation has potential for site specific, efficient and controlled transfection of therapeutic gene. In short, MBCP provides the silver lining in combat against the hurdles encountered in transfection to myocardial or other muscle cells.     

Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting

The application of non-viral gene delivery vectors is often accompanied with the poor correlation between transfection efficiency and the safety profiles of vectors. Vectors with high transfection efficiencies often suffer from high toxicities, making it unlikely to improve their efficiencies by increasing the DNA dosage. In the current study, we developed a ternary complex system which consisted of a highly membrane-active cationic helical polypeptide (PVBLG-8), a low-toxic, membrane-inactive cationic helical polypeptide (PVBLG-7) capable of mediating mannose receptor targeting, and DNA. The PVBLG-7 moiety notably enhanced the cellular uptake and transfection efficiency of PVBLG-8 in a variety of mannose receptor-expressing cell types (HeLa, COS-7, and Raw 264.7), while it did not compromise the membrane permeability of PVBLG-8 or bring additional cytotoxicities. Because of the simplicity and adjustability of the self-assembly approach, optimal formulations of the ternary complexes with a proper balance between membrane activity and targeting capability were easily identified in each specific cell type. The optimal ternary complexes displayed desired cell tolerability and markedly outperformed the PVBLG-8/DNA binary complexes as well as commercial reagent Lipofectamine™ 2000 in terms of transfection efficiency. This study therefore provides an effective and facile strategy to overcome the efficiency-toxicity poor correlation of non-viral vectors, which contributes insights into the design strategy of effective and safe non-viral gene delivery vectors.     

Multilayer mediated forward and patterned siRNA transfection using linear-PEI at extended N/P ratios

Gene delivery from a substrate depends, in part, on the vector–nucleic acid complex that is bound to the surface and the cell adhesive properties of the surface. Here, we present a method to deliver patterns of small interfering RNA (siRNA) that capitalize on a forward transfection method (transfection by introducing siRNA transfection reagent complexes onto plated cells); herein denoted as multilayer mediated forward transfection (MFT). This method separates the substrate-mediated delivery from the cell adhesive properties of the surface. pH responsive layer-by-layer (LbL) assembled multilayers were used as the delivery platform and microcontact printing technique (μCP) was used to pattern nanoparticles of transfection reagent–siRNA complexes onto degradable multilayers. Efficient MFT depend on optimal formulation of the nanoparticles. 25 kDa linear polyethylenimine (LPEI) was optimized as the siRNA transfection reagent for normal forward transfection (NFT) of the nanoparticles. A broad range of LPEI–siRNA nitrogen/phosphate (N/P) ratios (ranging from 5 to 90) was evaluated for the relative amounts of siRNA incorporated into the nanoparticles, nanoparticle size and NFT efficiencies. All the siRNA was incorporated into the nanoparticles at N/P ratio near 90. Increasing the amount of siRNA incorporated into the nanoparticles, with increasing N/P ratio correlated with a linear blue shift in the ultraviolet/visible (UV/vis) absorbance spectrum of the LPEI–siRNA nanoparticles. NFT efficiency greater than 80% was achieved with minimal cytotoxicity at N/P ratio of 30 and siRNA concentration of 200 nM. Similarly, MFT efficiency ?60% was achieved for LPEI–siRNA nanoparticles at N/P ratios greater than 30.     

The role of adenosine receptor and caveolae-mediated endocytosis in oligonucleotide-mediated gene transfer

We previously reported the preparation and characterization of ternary nanoparticles with the negative surface charge, which comprises histidine-conjugated polyallylamine (PAA-HIS)/DNA core complex and a single-stranded oligonucleotide outer layer, to transfect various cell lines. As a continued effort, here the investigations on the endocytotic mechanisms involved in the uptake of the oligonucleotide-coated PAA-HIS/DNA complexes are reported. Interestingly, these complexes showed enhanced transfection efficiency only when deoxyadenosine-containing oligonucleotides were deposited on the PAA-HIS/DNA complex surface. The addition of uncomplexed oligonucleotide, free adenosine and adenosine receptor antagonist significantly inhibited the transfection efficiency of oligonucleotide-coated PAA-HIS/DNA complexes. These results indicated that the oligonucleotide-coated PAA-HIS/DNA complexes could specifically recognize adenosine receptors on the cell surface and were taken up by adenosine receptor-mediated process. Uptake and transfection experiments with various endocytic inhibitors suggested that, after receptor/ligand binding, oligonucleotide-coated PAA-HIS/DNA/complexes were mainly internalized via caveolae-mediated pathway to result in effective intracellular processing for gene expression. In conclusion, both adenosine receptor and caveolae-mediated endocytosis play important roles in oligonucleotide-mediated gene transfer.