High Molecular Weight Polyethylenimine Research Articles

Dihydroxyphenylalanine-conjugated high molecular weight polyethylenimine for targeted delivery of Plasmid

High molecular weight polyethylenimine (HMW PEI; branched 25 kDa PEI) has been widely investigated for gene delivery due to its high transfection efficiency. However, the toxicity and lack of targeting to specific cells have limited its clinical application. In the present investigation, L-3, 4‐dihydroxyphenylalanine (L-DOPA) was conjugated on HMW PEI in order to target L-type amino acid transporter 1 (LAT-1) and modulate positive charge density on the surface of polymer/plasmid complexes (polyplexes). The results of biophysical characterization revealed that the PEI conjugates are able to form nanoparticles ≤ 180 nm with the zeta potential ranging from + 9.5–12.4 mV. These polyplexes could condense plasmid DNA and protect it against nuclease digestion at the carrier to plasmid ratios higher than 4. L-DOPA conjugated PEI derivatives were complexed with a plasmid encoding human interleukin-12 (hIL-12). Targeted polyplexes showed up to 2.5 fold higher transfection efficiency in 4T1 murine mammary cancer cell line, which expresses LAT-1, than 25 kDa PEI polyplexes prepared in the same manner. The cytotoxicity of these polyplexes was also substantially lower than the unmodified parent HMW PEI. These results support the use of L-3, 4‐dihydroxyphenylalanine derivatives of PEI in any attempt to develop a LAT-1 targeted gene carrier.

Synthesis and Application of Low Molecular Weight PEI-Based Copolymers for siRNA Delivery with Smart Polymer Blends.

Polyethylenimine (PEI) is a commonly used cationic polymer for small-interfering RNA (siRNA) delivery due to its high transfection efficiency at low commercial cost. However, high molecular weight PEI is cytotoxic and thus, its practical application is limited. In this study, different formulations of low molecular weight PEI (LMW-PEI) based copolymers polyethylenimine-g-polycaprolactone (PEI-PCL) (800Da-40kDa) and PEI-PCL-PEI (5-5-5kDa) blended with or without polyethylene glycol-b-polycaprolactone (PEG-PCL) (5kDa-4kDa) are investigated to prepare nanoparticles via nanoprecipitation using a solvent displacement method with sizes ≈100nm. PEG-PCL can stabilize the nanoparticles, improve their biocompatibility, and extend their circulation time in vivo. The nanoparticles composed of PEI-PCL-PEI and PEG-PCL show higher siRNA encapsulation efficiency than PEI-PCL/PEG-PCL based nanoparticles at low N/P ratios, higher cellular uptake, and a gene silencing efficiency of ≈40% as a result of the higher molecular weight PEI blocks. These results suggest that the PEI-PCL-PEI/PEG-PCL nanoparticle system could be a promising vehicle for siRNA delivery at minimal synthetic effort.

Dual-functional amino-quinone coatings on cotton fabrics assembled with different molecular weight polyethyleneimine and dopamine

The bonding of textiles and surface coatings was often assisted by the use of chemical cross-linking agents. Inspired by mussel chemistry, we propose that the strategy of co-assembly of quinone compound (dopamine, DA) and amino compound (polyethyleneimine, PEI) to form amino-quinone network (AQN) coating can effectively solve the problem. In this work, the molecular weight of PEI was used to regulate the surface coating morphology and its properties on cotton fabrics.The results showed that the roughness (Ra) of AQN coatings formed on the surface of glass slides by DA and low, medium or high molecular weight PEI were 3.94, 24.1 or 71.4 nm, respectively. In addition, the antibacterial rates of AQN-coated cotton fabric against E. coli and S. aureus were above 98 %, and the ultraviolet protection factor (UPF) value was above 50. After several washes, the antibacterial and UPF values only decreased by about 1 %, which indicates that AQN coating gives cotton fabric good antibacterial and UV protection properties, as well as strong bonds with cotton fabric.This provides insight into the wide application of AQN coating technology in the field of textile modification.

Mechanically tuneable physical nanocomposite hydrogels from polyelectrolyte complex templated silica nanoparticles for anionic therapeutic delivery

Hydrogels have shown great promise for drug delivery and tissue engineering but can be limited in practical applications by poor mechanical performance. The incorporation of polymer grafted silica nanoparticles as chemical or physical crosslinkers in in situ polymerised nanocomposite hydrogels has been widely researched to enhance their mechanical properties. Despite the enhanced mechanical stiffness, tensile strength, and self-healing properties, there remains a need for the development of simpler and modular approaches to obtain nanocomposite hydrogels. Herein, we report a facile protocol for the polyelectrolyte complex (PEC) templated synthesis of organic-inorganic hybrid poly(ethylenimine) functionalised silica nanoparticles (PEI-SiNPs) and their use as multifunctional electrostatic crosslinkers with hyaluronic acid (HA) to form nanocomposite hydrogels. Upon mixing, electrostatic interactions between cationic PEI-SiNPs and anionic HA resulted in the formation of a coacervate nanocomposite hydrogel with enhanced mechanical stiffness that can be tuned by varying the ratios of PEI-SiNPs and HA present. The reversible electrostatic interactions within the hydrogel networks also enabled self-healing and thixotropic properties. The excess positive charge present within the PEI-SiNPs facilitated high loading and retarded the release of the anionic anti-cancer drug methotrexate from the nanocomposite hydrogel. Furthermore, the electrostatic complexation of PEI-SiNP and HA was found to mitigate haemotoxicity concerns associated with the use of high molecular weight PEI. The method presented herein offers a simpler and more versatile strategy for the fabrication of coacervate nanocomposite hydrogels with tuneable mechanical stiffness and self-healing properties for drug delivery applications.

High molecular weight polyethylenimine encapsulated into a porous polymer monolithic by one-step polymerization for CO2 capture

A low-cost porous polymer monolithic with a well-interconnected 3D structure and high amino efficiency for CO2 capture.

Supramolecular self-assemblies of engineered polyethylenimines as multifunctional nanostructures for DNA transportation with excellent antimicrobial activity

In this study, indole-3-butanoic acid (IBA), a biologically and environmentally safe entity, has been grafted onto low and high molecular weight (1.8 and 25 kDa) polyethylenimines (PEI) mainly through primary amines to obtain amphiphilic indole-3-butanoyl-polyethylenimines (IBPs). Two series of IBPs (IBP1.8 and IBP25) were prepared which, on self-assembly in aqueous medium, yielded multifunctional nanomicellar structures (IBP1.8 and IBP25) capable of transporting genetic material in vitro and exhibiting other biological activities. Physicochemical characterization showed the size of IBP1.8 and IBP25 nanostructures in the range of ~332–234 nm and ~283–166 nm, respectively, with zeta potential varying from ~+29–17 mV and ~+37–25 mV. DNA release assay demonstrated higher release of plasmid DNA from IBP nanostructures as compared to native PEIs. Cytotoxicity showed a decreasing pattern with increasing degree of grafting of IBA onto PEIs making these nanostructures non-toxic. pDNA complexes of these nanostructures (both IBPs1.8 and IBPs25) displayed considerably higher transfection efficiency, however, IBP1.8/pDNA complexes performed much better (~7–9 folds) as compared to native PEI/pDNA and Lipofectamine/pDNA complexes on mammalian cells. CLSM analysis revealed that these complexes entered nucleus in sufficient amounts suggesting higher uptake and efficient internalization of the complexes. Besides, these supramolecular nanostructures not only exhibited excellent antimicrobial potential (MIC ~49–100 µg/ml) against clinical as well as resistant pathogenic strains but also found to possess antioxidant property. Overall, the projected low molecular weight PEI-based vectors could serve as more effective multifunctional nanomaterials having promising potential for future gene therapy applications with capability to provide protection against other bacterial infections.

Targeted double domain nanoplex based on galactosylated polyethylenimine enhanced the delivery of IL-12 plasmid.

The objective of the present investigation was to design a targeted polyethylenimine (PEI)-based polyplex by conjugating lactose bearing galactose groups on low molecular weight PEI (LMW PEI) grafted to a high molecular weight PEI (HMW PEI) via a succinic acid linker in order to restore the amine content of the whole conjugate used for ligand conjugation. The PEI conjugate was synthesized and characterized in terms of buffering capacity, particle size, zeta potential, plasmid condensation ability, and protection of DNA against degrading enzymes. Also, the transfection efficiency and cytotoxicity were evaluated in the cell line over-expressing asialoglycoprotein receptors (ASGPRs) and compared with the cells lacking the receptors. The results demonstrated the ability of PEI conjugate in condensation of plasmid DNA and protection against enzyme degradation. The PEI conjugate formed nanoparticles of around 75 nm with higher buffering capacity compared with unmodified PEI. The polyplexes prepared by the modified PEI could increase the level of transgene up to four folds in the cells over-expressing the receptor. The results demonstrated the separation of targeting and delivery domains could be considered as a strategy to restore the amine content of the PEI molecule utilized for targeting ligand conjugation.

Polyethylenimine-Modified Zeolite 13X for CO2 Capture: Adsorption and Kinetic Studies.

A class of high molecular weight polyethylenimine (PEI)-modified zeolite 13X adsorbents were synthesized by varying the concentration of imines and screened for preliminary investigation of CO2 capture studies. The impregnated molecular amine zeolite composite was characterized and CO2 adsorption performance was investigated through TGA in the presence of atmospheric pure CO2 gas at 25, 50, 75, and 100 °C, respectively, using 20–80 wt % of PEI-loaded zeolite 13X adsorbents. This paper reports on the effects of temperature and amine (PEI) loading on CO2 adsorption capacity and estimated kinetic parameters through modeling of selected models which represent the reaction rate and diffusion rate models. The studied adsorbents showed the highest adsorption capacity at 75 °C with 60 wt % PEI loading. Thus, the optimum temperature of 75 °C and optimal loading of 60 wt % was observed from the current studies for CO2 capture. From modeling study, it was found that Avrami’s fractional order and dual kinetic models (DKM) described well the adsorption behavior of CO2 on PEI-impregnated zeolite 13X at all temperatures accurately and up to 75 °C, respectively. Besides, intraparticle diffusion was found to be the rate-limiting step when compared with the film diffusion model.

MR molecular imaging of HCC employing a regulated ferritin gene carried by a modified polycation vector.

Purpose: Early diagnosis is essential for reducing liver cancer mortality, and molecular diagnosis by magnetic resonance imaging (MRI) is an emerging and promising technology. The chief aim of the present work is to use the ferritin gene, modified by the alpha-fetoprotein (AFP) promoter, carried by a highly safe vector, to produce signal contrast on T2-weighted MR imaging as an endogenous contrast agent, and to provide a highly specific target for subsequent therapy.Methods: Polyethyleneimine-β-cyclodextrin (PEI-β-CD, PC) was synthesized as a novel vector. The optimal nitrogen/phosphorus ratio (N/P) of the PC/plasmid DNA complex was determined by gel retardation, biophysical properties and transmission electron microscopy morphological analysis. The transfection efficiency was observed under a fluorescence microscope and analyzed by flow cytometry. Cellular iron accumulation caused by ferritin overexpression was verified by Prussian blue staining, and the resulting contrast imaging effect was examined by MRI.Results: The modified cationic polymer PC was much safer than high molecular weight PEI, and could condense plasmid DNA at an N/P ratio of 50 with suitable biophysical properties and a high transfection efficiency. Overexpression of ferritin enriched intracellular iron. The short-term iron imbalance initiated by AFP promoter regulation only occurred in hepatoma cells, resulting in signal contrast on MRI. The specific target TfR was also upregulated during this process.Conclusion: These results illustrate that the regulated ferritin gene carried by PC can be used as an endogenous contrast agent for MRI detection of hepatocellular carcinoma (HCC). This molecular imaging technique may promote safer early diagnosis of HCC, and provide a more highly specific target for future chemotherapy drugs.

Enhanced uptake of plasmid at boronic acid decorated linear polyethylenimines results in higher transfection efficiency.

High molecular weight polyethylenimines (PEIs) are considered as gold standard for transfection studies; however, cytotoxicity associated with branched ones and lower charge density on linear PEIs as well as lower uptake of the resulting deoxyribonucleic acid (DNA) complexes have limited their applications in clinical studies. In order to address these concerns and improve the uptake efficiency of the DNA complexes of linear polyethylenimine (25 kDa), the polymer was grafted with variable amounts of butylboronic acid to obtain a small series of linear polyethylenimine-butylboronic acid polymers. These modified polymers were allowed to interact with plasmid DNA and the resulting complexes were characterized by physicochemical techniques. Dynamic light scattering data showed the formation of nanosized complexes with positive zeta potential values. Furthermore, when these complexes were evaluated in vitro, they not only showed enhanced cell viability but also exhibited higher transfection efficiency as compared to native linear and branched PEIs and a commercially available standard transfection reagent, Lipofectamine 2000.

Facile Gene Delivery Derived from Branched Low Molecular Weight Polyethylenimine by High Efficient Chemistry.

Convenient methods for the preparation of gene delivery platforms based on branched low molecular weight polyethylenimine (PEI) were described. Firstly, PEI lipids, with a low molecular weight PEI headgroup and hexadecyl chain tail group, were prepared through a highly efficient ring-opening reaction of glycidyl hexadecyl ether (EpoxyC16) by amine from PEI. Then, the PEI lipids were used as a component of cationic liposomes and as a surfactant for the preparation of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticle (NP) via solvent extraction/evaporation method. As potential effective gene delivery platforms, their preparation, size, size distribution, toxicities, plasmid DNA loading, in vitro transfection and intracellular trafficking were studied. Both facile platforms showed less toxicity and higher transfection efficacy when compared to high molecular weight PEI in vitro, and may have further versatile applications in the gene delivery field.

Tailoring flame-retardancy and strength of papers via layer-by-layer treatment of cellulose fibers

The layer-by-layer (LbL) technology was used to adsorb polyelectrolyte multilayers consisting of cationic polyethylenimine (PEI) and anionic sodium hexametaphosphate (SHMP) onto cellulose fibers in order to enhance the flame-retardancy and tensile strength of paper sheets made from these fibers. The fundamental effect of PEI molecular mass on the build-up of the multilayer film was investigated using model cellulose surfaces and a quartz crystal microbalance technique. The adsorption of a low (LMw) and a high molecular weight (HMw) PEI onto cellulose fibers and carboxymethylated (CM) cellulose fibers was investigated using polyelectrolyte titration. The fibers were consecutively treated with PEI and SHMP to deposit 3.5 bilayers (BL) on the fiber surfaces, and the treated fibers were then used to prepare sheets. In addition, a wet-strength paper sheet was prepared and treated with the same LbL coatings. Thermal gravimetric analysis of LbL-treated fibers showed that the onset temperature for cellulose degradation was lowered and that the amount of residue at 800 °C increased. A horizontal flame test and a vertical flame test were used to evaluate the combustion behavior of the paper sheets. Papers prepared from both cellulose fibers and CM-cellulose fibers treated with HMw-PEI/SHMP LbL-combination self-extinguished in a horizontal configuration despite the rather low amounts of adsorbed polymer which form very thin films (wet thickness of ca. 17 nm). The tensile properties of handsheets showed that 3.5 BL of HMw-PEI and SHMP increased the stress at break by 100% compared to sheets prepared from untreated cellulose fibers.

Cholic acid-modified polyethylenimine: in vitro and in vivo studies.

Low-molecular-weight polyethylenimine has lower cytotoxicity than high molecular weight polyethylenimine, but it is not an efficient transfection agent because of limitations of DNA delivery into the cytoplasm. Therefore, in the present study, the hydrophobic modification of low-molecular-weight polyethylenimine (PEI 2 kDa [PEI2]) by cholic acid (ChA) was performed to form PEI2-ChA, and in vitro and in vivo studies were performed. Results indicate that the nanoplexes of PEI2-ChA with gWIZ-GFP have greater transfection efficiency (27%) in NT8e cell lines as evaluated by flow cytometry and also observed by fluorescence imaging. The present study concluded that the transferrin-containing nanoplexes of PEI2-ChA conjugates with plasmid p53 warrant clinical trials in humans after exhaustive animal studies for use as a novel gene delivery system.

Charge reduction: an efficient strategy to reduce toxicity and increase the transfection efficiency of high molecular weight polyethylenimine

With high potency in treatment of various diseases, gene therapy is mainly hindered by lack of safe and efficient gene delivery vectors. The current study was aimed to develop an efficient non-viral vector with adequate cytotoxicity. To this end, alkylcarboxylate chains (6C, 10C, 16C) were exploited to ameliorate the characteristics of PEI 750 kDa. Briefly, alkylcarboxylate chains with three different lengths (6C, 10C, 16C) were chemically grafted to the primary amine groups of PEI 750 kDa in three percentages (10, 50, and 100%). After evaluating the physicochemical properties of prepared vectors including surface charge, size, buffering capacity, and DNA condensing, their transfection efficiency and cytotoxicity were investigated in Neuro2A cells. The polyplexes size were 158.9–264.5 nm and their zeta potentials were 14–30 mV, while their buffering capacity and DNA condensing were not significantly decreased. The highest transfection efficiency in term of C/P ratio was observed in PEI750-10C-68%, PEI750-10C-7%, and PEI750-6C-7% at C/P ratios of 2, 4, and 6, respectively. Altogether, the decanoylcarboxylate-modified PEI with medium grafting percentages showed promising results as gene delivery vector. To sum up, the modification of high molecular weight PEIs by alkylcarboxylate chains is an efficient approach for development of more efficient non-viral vectors.

Aromatic Modification of Low Molecular Weight PEI for Enhanced Gene Delivery.

Low molecular weight polyethylenimine (1800 Da, also referred to as oligoethylenimines, OEI) was modified with amino acids, including two aromatic amino acids (tryptophan, phenylalanine) and an aliphatic amino acid (leucine). The substitution degree of amino acids could be controlled by adjusting the feeding mole ratio of the reactants. Fluorescence spectroscopy and circular dichroism experiments demonstrated that the indole ring of tryptophan may intercalate into the DNA base pairs and contribute to efficient DNA condensation. In vitro gene expression results revealed that the modified OEIs (OEI-AAs) may provide higher transfection efficiency even than high molecular weight polyethylenimine (25 kDa, PEI), especially the aromatic tryptophan substituted OEI. Moreover, OEI-AAs exhibited excellent serum tolerance, and up to 137 times higher transfection efficiency than PEI 25 kDa that was obtained in the presence of serum. The cytotoxicity of OEI-AAs is much lower than PEI 25 kDa. This study may afford a new method for the development of low molecular weight oligomeric non-viral gene vectors with both high efficiency and biocompatibility.

MoS2-LA-PEI nanocomposite carrier for real-time imaging of ATP metabolism in glioma stem cells co-cultured with endothelial cells on a microfluidic system

Stimuli-responsive carriers have extensively attracted attention in recent years. However, long-term and real-time tracking ability with stimuli-responsive carrier is still in its infant stage due to the limitations such as, low efficacy, instability and cytotoxicity in a bio-environment. In this work, we developed a reduction-sensitive carrier composed of lipoic acid-modified low molecular weight polyethyleneimine (LA-PEI) and large surface ratio MoS2 nanosheet integrated via disulfide bond to mimic a high molecular weight PEI. The positively charged carriers loading negatively charged aptamer enter the cells for a real time long-term tracking of adenosine triphosphate (ATP) metabolism in glioma stem cells (GSCs) when stimulated by TGFβ factor secreted from HUVECs. We envision that MoS2-LA-PEI carrier has a promising potential for delivery and monitoring the changes in live cells with low cytotoxicity and high efficiency.

Combined Delivery of a Lipopolysaccharide-Binding Peptide and the Heme Oxygenase-1 Gene Using Deoxycholic Acid-Conjugated Polyethylenimine for the Treatment of Acute Lung Injury.

A ternary complex comprising plasmid DNA, lipopolysaccharide-binding peptide (LBP), and deoxycholic acid-conjugated polyethylenimine (PEI-DA) is prepared for combinational therapy of acute lung injury (ALI). The LBP is designed as an anti-inflammatory peptide based on the lipopolysaccharide (LPS)-binding domain of HMGB-1. In vitro cytokine assays show that LBP reduces levels of proinflammatory cytokines by inhibiting LPS. PEI-DA is synthesized as the gene carrier by conjugation of deoxycholic acid to low-molecular weight polyethylenimine (2 kDa, PEI2k). PEI-DA has higher transfection efficiency than high-molecular weight polyethylenimine (25 kDa, PEI25k). The ternary complex of an HO-1 plasmid (pHO-1), PEI-DA, and LBP is prepared as a combinational system to deliver the therapeutic gene and peptide. The transfection efficiency of the ternary complex is higher than that of the pHO-1/PEI-DA binary complex. The ternary complex also reduces TNF-α secretion in LPS-activated Raw264.7 macrophage cells. Administration of the ternary complex into the lungs of an animal ALI model by intratracheal injection induces HO-1 expression and reduces levels of proinflammatory cytokines more efficiently than the pHO-1/PEI-DA binary complex or LBP alone. In addition, the ternary complex reduces inflammation in the lungs. Therefore, the pHO-1/PEI-DA/LBP ternary complex may be an effective treatment for ALI.

High performance and stable naphthalene diimide based n-channel organic field-effect transistors by polyethylenimine doping

In this paper, we study the effect of the structure and molecular weight of polyethylenimine (PEI) on doping efficiency of n-type organic semiconductors based on poly[[N, N 9-bis(2-octyldodecyl)-naphthalene-1,4,5,8-bis(dicarboximide)-2,6-diyl]-alt-5,59-(2,29-bithiophene)] (P(NDI2OD-T2)). Linear and branched PEI with low (Mw = 2 and 3 kDa) and high molecular weights (Mw = 25 kDa) are used. It is found that the low molecular weight PEI shows higher doping efficiency than high molecular weight PEI due to better mix-ability between the dopant and the host. Doped P(NDI2OD-T2) OFETs show highly improved mobility up to 0.85 cm2/V·s (linear) at 0.1 wt% doping concentration from 0.21 cm2/V·s for pristine devices with enhanced air and operational stability.

High molecular weight polyethyleneimine functionalized three dimensional mesoporous silica for regenerable CO2 separation

Polyethylenimine functionalized mesoporous silica is a potential alternative of amine based absorption process for CO2 capture from flue gas. By impregnation of high molecular weight polyethylenimine (PEI) into three dimensional mesoporous KIT-6, a novel sorbent was prepared for highly CO2/N2 selective separation at moderate temperature and low partial pressure with high stability. TEM micrograph shows that, pore is covered with PEI and also a layer of PEI is formed on the surface of adsorbent without disturbing its original structure. TGA analysis confirmed that with increasing molecular weight of PEI, thermal stability of adsorbent was increased. The sorption capacity was increased with increasing the temperature form 30 to 120°C and the adsorption mechanism is elucidated with the help of the nature of CO2 equilibrium isotherms. The very high CO2/N2 selective adsorbent follows the order K/250PEI<K/12PEI<K/8PEI with maximum operating sorption capacity 3.0 mmol CO2/g of K/8PEI at 105°C and 100kPa. Adsorbent showed stable sorption performance at moderate temperature (90–105)A/100D°C during 20 adsorption/desorption cycles. However, K/250PEI gives the maximum benefits in terms of stable sorption performance (2.4mmolCO2/g adsorbent at 105°C and 100kPa) and the necessary thermal stability for practical application.

A delicate balance when substituting a small hydrophobe onto low molecular weight polyethylenimine to improve its nucleic acid delivery efficiency

Event Abstract Back to Event A delicate balance when substituting a small hydrophobe onto low molecular weight polyethylenimine to improve its nucleic acid delivery efficiency Deniz Meneksedag-Erol1, 2*, Remant Bahadur Kc2, Tian Tang1, 3* and Hasan Uludag1, 2, 4* 1 University of Alberta, Department of Biomedical Engineering, Canada 2 University of Alberta, Department of Chemical & Materials Engineering, Canada 3 University of Alberta, Department of Mechanical Engineering, Canada 4 University of Alberta, Faculty of Pharmacy and Pharmaceutical Sciences, Canada Introduction: High molecular weight polyethylenimine (PEI) is an effective non-viral gene carrier extensively studied in gene therapies. However, its considerable amount of toxicity encouraged a search for non-toxic and non-immunogenic alternatives. Due to the low toxicity profile of low molecular weight (LMW) PEI, research has focused on improving its performance via hydrophobic modifications. In this study, we systematically investigated, via experiments and molecular dynamics simulations, how the hydrophobic modification of LMW (1.2 kDa) PEI with a short propionic acid (PrA) influences the complexation mechanism of short interfering RNA (siRNA), as well as transfection efficacy and silencing capability of polyplexes. Materials and Methods: 1.2 kDa PEI was modified with PrA via N-acylation, a similar procedure described in[1]. Composition of modified PEIs was analyzed by 1H-NMR. The PrA substitution amounts were α = 0.26, 0.69, 1.14 and 1.59 PrA/PEI. A chronic myeloid leukemia K562 cells was used as a cell model. siRNA delivery efficacy was assessed by flow cytometry with FAM-labeled siRNA. Green fluorescence protein (GFP) silencing in GFP-expressing K562 cells was investigated for silencing activity. For computational studies, a branched PEI (MW: 1205 Da) and an siRNA sequence against myeloid cell leukemia 1 (Mcl-1) were used. Four systems were designed with varying PrA substitutions; α = 0.25, 0.75, 1 and 2. Simulations were performed with NAMD[2] for 200 ns, and trajectories were analyzed with Visual Molecular Dynamics (VMD). Results and Discussion: Substitution of PrA at low amounts, α = 0.26 and 0.69, resulted in substantially higher (>10 fold) delivery than native PEI. Increasing the substitution amount α further to 1.14 and 1.59 abolished the uptake efficiency of siRNA (Figure 1a). Native PEI (α = 0) showed ~5% silencing, whereas α = 0.26 and α = 0.69 yielded ~10% and ~33% silencing, respectively. Further increase in the substitution extent decreased siRNA silencing down to 4% (Figure 1b). MD simulations on radial distribution function of the PrA Cs from the center of mass of polyplexes revealed a new assembly mechanism: preferential mobilization of PrA moieties to the core at high substitution. This caused a non-monotonic change in the surface PrA density of the polyplexes (Figure 2a), with α = 1 having the most hydrophobic surface. Migration of PrAs into the core dragged the associated PEIs, and caused higher cationic surface charge at intermediate substitutions (α = 0.75), while further increasing the substitution significantly reduced the surface cationic charge density (Figure 2b). Conclusions: Small amount of hydrophobic modification is beneficial to enhance the surface hydrophobicity of the polyplexes; while excess hydrophobic groups resulted in preferential mobilization of these groups to the polyplex core, lowering the surface hydrophobicity as well as cationic charge. This phenomenon results in less effective polyplexes for siRNA delivery. Compute Canada; National Institute for Nanotechnology, Edmonton, Canada; Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research; NSERC CREATE Program for Regenerative Medicine (NCPRM); Alberta Innovates-Technology Futures; Canada Foundation for Innovation