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ABSTRACT: High-density lipoprotein (HDL) is a key mediator in reverse cholesterol transport and is involved in a mechanism known as 'selective lipid uptake', a process mediated by scavenger receptor B type I (SR‑BI), which is a HDL receptor. The aim of the present study was to investigate the therapeutic effect of the SR‑BI gene when delivered by combining cationic liposomal microbubbles (CLMs) and ultrasound (US) in hypercholesterolemic rats. Hypercholesterolemia was induced by administration of excessive doses of vitamin D3 and cholesterol in rats. The CLMs consisted of perfluoropropane gas encapsulated in a phospholipid shell using the sonication‑lyophilization method. The SR‑BI gene, mixed with the self‑made microbubbles, was transfected into hypercholesterolemic rat arteries using therapeutic US. SR‑BI protein expression was determined by western blot analysis 2 days post-transfection. Two weeks after transfection, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and HDL serum concentrations were measured. Transfection efficiency of the SR‑BI gene in the SR‑BI + US/CLM group increased 6‑7‑fold compared with the SR‑BI group. Two weeks after transfection, plasma lipid levels in treated hypercholesterolemic rats were observed to be significantly reduced compared with rats that did not receive treatment. However, no significant change was observed in the SR‑BI group compared with that in the SR‑BI + US/CLM group. Results of the present study indicate that the combination of US and CLMs loaded with the SR‑BI gene may exert a protective role in hypercholesterolemia.
Molecular Medicine Reports 04/2013; · 0.42 Impact Factor
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ABSTRACT: BACKGROUND: Non-viral lipid-based gene delivery vectors have been shown to possess better stability and longer circulation time after surface PEG modification. However, surface PEGylation may decrease the transfection efficiency dramatically. Here, we address the hypothesis that down regulating the lysosomal processing with a clinical available proton pump inhibitor omeprazole might decrease sequestration of PEGylated lipid-mu-DNA (LMD) in intracellular organelles, thereby increasing their transfection efficiencies. METHODS: LMD nanoparticles were prepared by the self-assembling of cationic liposomes, peptide mu and plasmid DNA. The characteristics of LMD lipopolyplexes were detected by scanning electron microscopy (TEM), photon correlation spectroscopy (PCS) and DNA gel electrophoresis. They were added into cultured cells with or without omeprazole pretreatment. The detailed cellular uptake and subcellular distribution were followed by flow cytometry and confocal microscopy. The gene transfection efficiencies were evaluated in four cell lines as well as in xenograft tumor models. RESULTS: Lysosome staining revealed that 0.1 mg/ml (290 μM) omeprazole raised the pH value of intracellular acidic organelles and induced alterations in the lysosomal compartments. Confocal microscopy showed that more gene materials distributed from intracellular organelles to cytoplasms with omeprazole treatment. Luciferase gene transfection assay showed that omeprazole at probably non-toxic concentration increased PEGylated LMD transfection efficiency significantly in human (NCI-H1299, HT-1080 and A375) and mouse (4 T1) cell lines, as well as in tumors of H1299 xenograft tumor models (p < 0.05). CONCLUSIONS: Omeprazole might be used as a helper to improve gene transfection efficiencies of some PEGylated gene delivery vectors in vitro and in vivo. This article is protected by copyright. All rights reserved.
The Journal of Gene Medicine 04/2013; · 2.48 Impact Factor
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ABSTRACT: PURPOSE: Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for treatments of numerous diseases. However, the progress towards broad application of siRNA requires the development of safe and effective vectors that target to specific cells. In this study, we developed a novel recombinant high density lipoprotein (rHDL) vector with high siRNA encapsulation efficiency. METHODS: They were prepared by condensing siRNA with various commercial cationic polymers and coating the polyplex with a layer of lipids and apolipoprotein AI (apo AI). The rHDL nanoparticles were used to transfect SMMC-7721 hepatoma cells with stable luciferase expression. The uptake and intracellular trafficing of siRNA were also investigated. RESULTS: Characterization studies revealed these rHDL nanoparticles had similar physical properties as natural HDLs. The various rHDL formulations had high silencing efficiency (more than 70% knockdown) in hepatocytes with minimum cytotoxicity. Moreover, the uptake of rHDL by SMMC-7721 was confirmed to be mediated through the natural HDL uptake pathway. CONCLUSIONS: The work described here demonstrated the optimized rHDL nanoparticles may offer a promising tool for siRNA delivery to the liver.
Pharmaceutical Research 12/2012; · 4.09 Impact Factor
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ABSTRACT: The heat-inducible lysyl-tRNA synthetase from Escherichia coli (LysU: EC6.1.1.6) converts ATP to diadenosine tri- and tetraphosphates (Ap(3) A/Ap(4) A) in the presence of L-lysine/Mg(2+) /Zn(2+) . To understand LysU in more detail, 26 mutants were prepared: six of E264, four of R269 and sixteen mutants by alanine-scanning of the inner shell/motif 2 loop. In the presence of glycerol and absence of exogenously added Zn(2+) /L-lysine we unexpectedly found that E264K catalysed the production of glycerol-3-phosphate, powered by ATP turnover to ADP. E264Q and E264N are also capable of this activity but all three show little formation of Ap(4) A/Ap(3) A under normal conditions (additional Zn(2+) /L-lysine/Mg(2+) ). By contrast, wild type LysU has a weaker glycerol kinase-like capability in the absence of Zn(2+) and is dominated by Ap(4) A/Ap(3) A synthesis in its presence. Kinetic and isothermal titration calorimetry results suggest that E264 is a crucial residue for Zn(2+) promotion of Ap(4) A/Ap(3) A synthesis. This is consistent with the hypothesis that E264 provides an anchor point for a Zn(2+) ion complexed to the active site, with simultaneous coordination to the enzyme bound lysyl-adenylate intermediate and secondary substrate ATP/ADP. The glycerol kinase-like activity is uncovered on disruption of this specific coordination. © 2012 The Authors Journal compilation © 2012 FEBS.
FEBS Journal 11/2012; · 3.79 Impact Factor
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ABSTRACT: For gene-based therapeutic approaches that require transfection of specific cell targets in vivo, it is important to design the delivery system to have optimized tissue distribution. Surface PEGylation of liposomes and polymer nanoparticles has been shown to lead to prolonged blood circulation and also preferential accumulation in tumor sites resulting from the enhanced permeability and retention (EPR) effect. The aim of this study was to investigate the effect of different surface PEGylation densities on resulted biodistribution profiles of Lipid-Mu peptide-DNA (LMD) nanoparticles. LMD particles containing the near infrared fluorescent lipid dye, Cy5.5-DSPE, were injected intravenously, and whole-body fluorescence images of the live animal were recorded. Analysis of these time series of images indicated that LMDs with different surface PEG(2000) densities had distinctively different biodistribution and tumor accumulation profiles. LMDs containing approximately 15-25% of surface PEG(2000) were shown to have the highest accumulation and longest residence time in tumor. LMD distribution and pharmacokinetic profiles in other organs were also observed to be different and were analyzed.
Journal of Liposome Research 10/2012; · 1.71 Impact Factor
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ABSTRACT: BACKGROUND: A major obstacle in applying gene therapy methods in experimental and clinical practice is the lack of safe and efficient gene delivery systems. Electroporation has been shown to an effective physical delivery method. A variety of factors have been shown to affect the electroporation mediated gene delivery efficiency. Here we assess the usefulness of noncoding short-fragment DNA (sf-DNA) for facilitating electroporation mediated gene transfer. METHODS: The plasmid pGL3-control encoding firefly luciferase was injected into tissues together with or without sf-DNA. Immediately after injection, the tissues were electroporated and the level of luciferase activity was assessed 24 hours later. Different types of DNA fragments with different molecular weight, structure and dose were compared. The transfection efficiencies of sf-DNA mediated electroporation in different tissues or with different electric field strengths were examined. RESULTS: Plasmid DNA formulated with 300 bp sf-DNA resulted in significant improvement in electroporation mediated gene transfer efficiency. The effect is dose dependent and also affected by the DNA fragment length and structures. It was useful for intramuscular electroporation application as well as intratumor applications with various pulse voltage parameters. CONCLUSIONS: Our data indicated that sf-DNA can be used as a helper molecule to improve the electroporation mediated gene transfection efficiency. Copyright © 2012 John Wiley & Sons, Ltd.
The Journal of Gene Medicine 08/2012; · 2.48 Impact Factor
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ABSTRACT: PURPOSE: To develop a liposome formulation incorporating antigen-presenting cells (APCs) membrane microdomains with enriched epitope/MHC complexes to evaluate the activities of these liposomes (RAFTsomes) to activate T cells and prime immune responses. METHODS: We isolated membrane microdomain structures that contained the epitope/MHC complexes from ovalbumin (OVA) primed dendritic cells (DCs), and reconstituted them on liposomes surface by detergent dialysis. The resulted RAFTsomes were purified by density gradient centrifugation. Their T cell activation functions were evaluated by IL-2 secreting and proliferation assays in vitro. In vivo immune responses and the protective effect against OVA expressing EG.7 tumor challenge were also examined. RESULTS: Membrane microdomains containing enriched epitope/MHC complexes can be reconstituted into liposomes with defined size and composition. The integrity and activities of these complexes after reconstitution were confirmed by in vitro T cell assays. OVA epitope loaded RAFTsomes injected in vivo resulted in high anti-OVA IgG production (predominantly IgG1). The immunized mice were protected from EG.7 tumor cell inoculation challenge. CONCLUSIONS: Based on these findings, we propose that RAFTsomes can be prepared with unique properties that may be used as an antigen delivery system for immunotherapeutic applications.
Pharmaceutical Research 08/2012; · 4.09 Impact Factor
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ABSTRACT: Natural high-density lipoproteins (HDL) possess important physiological functions to the transport of cholesterol from the peripheral tissues to the liver for metabolic degradation and excretion in the bile.
In this work, we took advantage of this pathway and prepared two different gadolinium (Gd)-DTPA-labeled cholesterol-containing recombinant HDL nanoparticles (Gd-chol-HDL) and Gd-(chol)(2)-HDL as liver-specific magnetic resonance imaging (MRI) contrast agents. The reconstituted HDL nanoparticles had structural similarity to native HDL, and could be taken up by HepG2 cells via interaction with HDL receptors in vitro. In vivo MRI studies in rats after intravenous injections of 10 μmol gadolinium per kg of recombinant HDL nanoparticles indicated that both nanoparticles could provide signal enhancement in the liver and related organs. However, different T(1)-weighted image details suggested that they participated in different cholesterol metabolism and excretion pathways in the liver.
Such information could be highly useful to differentiate functional changes as well as anatomic differences in the liver. These cholesterol-derived contrast agents and their recombinant HDL preparations may warrant further development as a new class of contrast agents for MRI of the liver and related organs.
International Journal of Nanomedicine 01/2012; 7:3751-68. · 3.13 Impact Factor
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ABSTRACT: Huperzine A is a bioactive compound derived from traditional Chinese medicine plant Qian Ceng Ta (Huperzia serrata), and was found to have multiple neuroprotective effects. In addition to being a potent acetylcholinesterase inhibitor, it was thought to act through other mechanisms such as antioxidation, antiapoptosis, etc. However, the molecular targets involved with these mechanisms were not identified. In this study, we attempted to exam the interactome of Huperzine A using a cDNA phage display library and also mammalian brain tissue extracts. The drugs were chemically linked on the surface of magnetic particles and the interactive phages or proteins were collected and analyzed. Among the various cDNA expressing phages selected, one was identified to encode the mitochondria NADH dehydrogenase subunit 1. Specific bindings between the drug and the target phages and target proteins were confirmed. Another enriched phage clone was identified as mitochondria ATP synthase, which was also panned out from the proteome of mouse brain tissue lysate. These data indicated the possible involvement of mitochondrial respiratory chain matrix enzymes in Huperzine A's pharmacological effects. Such involvement had been suggested by previous studies based on enzyme activity changes. Our data supported the new mechanism. Overall we demonstrated the feasibility of using magnetic biopanning as a simple and viable method for investigating the complex molecular mechanisms of bioactive molecules.
PLoS ONE 01/2012; 7(5):e37098. · 4.09 Impact Factor
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ABSTRACT: We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.
Bioorganic & medicinal chemistry letters 12/2011; 21(23):7175-9. · 2.65 Impact Factor
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ABSTRACT: Computed tomography (CT) imaging is a valuable tool for the diagnosis of colorectal diseases. However, the colonic wall depiction in 2D CT images is usually poor because of the low contrast between the colonic wall and the luminal content. In order to improve the visualization of the colonic wall and any abnormality on its surface, we report in this paper the development of an oil-free foam fluid negative contrast medium for improved CT imaging of the colon. The foam fluid negative contrast medium was prepared by dispersing and stabilizing microbubbles in a polymeric solution. The stabilities of both the individual bubbles and the foam fluid system were optimized by incorporating bovine serum albumin and gluconolactone as the stabilization agent. The medium had a mean X-ray density of -120 Hounsfield units (close to that of the extraluminal tissues), and enabled clear 2D visualization of the colonic wall in both ex vivo and in vivo imaging studies. The measured colonic wall thicknesses at different segments in a beagle dog based on the 2D CT images obtained with the negative contrast medium accurately reflected the anatomical values, as compared with the values based on air-contrasted images. In vivo study of a simulated polyps pig model demonstrated sensitive detection of 11 out of 12 polyps with the smallest one 2 mm in diameter. We believe this new and safe foam fluid negative medium would enable the implementation of CT imaging as a convenient and useful tool for diagnosis of colon cancer, especially in the elderly population.
Contrast Media & Molecular Imaging 11/2011; 6(6):465-73. · 3.33 Impact Factor
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ABSTRACT: The development of safe and efficient avian influenza vaccines for human and animal uses is essential for preventing virulent outbreaks and pandemics worldwide. In this study, we constructed a recombinant (pgsA-HA1 gene fusion) Lactococcus lactis strain that expresses and displays the avian influenza virus HA1 antigens on its surface. The vectors were administered by oral delivery with or without the addition of cholera toxin subunit B (CTB). The resulting immune responses were analyzed, and the mice were eventually challenged with lethal doses of H5N1 viruses. Significant titers of hemagglutinin (HA)-specific serum IgG and fecal IgA were detected in the group that also received CTB. Cellular immunities were also shown in both cell proliferation and gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays. Most importantly, the mice that received the L. lactis pgsA-HA1 strain combined with CTB were completely protected from lethal challenge of the H5N1 virus. These findings support the further development of L. lactis-based avian influenza virus vaccines for human and animal uses.
Clinical and vaccine immunology: CVI 06/2011; 18(7):1046-51. · 2.37 Impact Factor
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ABSTRACT: Traditional phosphodiester lipid vesicles (liposomes) are not stable and could be easily degraded in the gastrointestinal (GI) tract. We prepared a novel lipid based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and tested their immunogenic potentials as oral vaccine delivery vehicles. Our study showed that the archaeosomes had significant superior stability in simulated gastric and intestinal fluids, and would help fluorescent labeled antigens to reside longer time in the GI tract after oral administration. The resulted immune responses against model antigen ovalbumin (OVA) were greatly improved, eliciting substantial IgG response systemically as well as IgA response mucosally. In addition, the archaeosomes also facilitated antigen specific CD8(+) T cell proliferation. These data indicate that archaeosomes may be a potential vaccine carrier and adjuvant for effective oral immunization.
Vaccine 05/2011; 29(32):5260-6. · 3.77 Impact Factor
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ABSTRACT: In the development of therapeutic vaccines against cancer, it is important to design strategies for antigen cross-presentation to stimulate cell-mediated immune responses against tumor antigens.
We developed a polyethyleneimine (PEI)-based protein antigen delivery system to promote cross-presentation through the major histocompatibility complex (MHC) I pathway using ovalbumin (OVA) as a model antigen. PEIs formed nanoparticles with OVA by electrostatic interactions, as demonstrated by electrophoresis analysis, scanning electron microscopy, and photon correlation spectroscopy analysis.
The nanoparticles were used to stimulate mouse bone marrow-derived dendritic cells in vitro and resulted in significantly more OVA(257-264)/MHC I complex presentation on dendritic cell surfaces. The activated dendritic cells interacted specifically with RF33.70 to stimulate interleukin-2 secretion. The cross-presentation promoting effect was more prominent in dendritic cells that had been cultured for longer periods of time (13 days). Further studies comparing the antigen presentation efficacies by other polyanionic agents, such as PLL or lysosomotropic agents, suggested that the unique "proton sponge effect" of PEI facilitated antigen escape from the endosome toward the MHC I pathway.
Such a PEI-based nanoparticle system may have the potential to be developed into an effective therapeutic vaccine delivery system.
International Journal of Nanomedicine 01/2011; 6:77-84. · 3.13 Impact Factor
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ABSTRACT: To develop a lipid-stabilized contrast formulation containing gadobenate dimeglumine for clear visualization of the mucosal surfaces of the larynx and trachea for early diagnosis of disease by magnetic resonance imaging.
The contrast formulation was prepared by loading gadobenate dimeglumine into egg phosphotidylcholine, cholesterol, and sterylamine nanoliposomes using the dehydration-rehydration method. The liposomal contrast formulation was ultrasonically nebulized, and the deposition and coating pattern on explanted pig laryngeal and tracheal segments was examined by inductively coupled plasma atomic emission spectroscopy. The sizes of the nebulized droplets were characterized by photon correlation spectroscopy. The contrast-enhanced mucosal surface images of the larynx and trachea were obtained in a 3.0T magnetic resonance scanner using a T1-weighted spectral presaturation inversion recovery sequence.
Various cationic liposome formulations were compared for their stabilization effects on the droplets containing gadobenate dimeglumine. The liposomes composed of egg phosphotidylcholine, cholesterol, and sterylamine in a molar ratio of 1:1:1 were found to enable the most efficient nebulization and the resulting droplet sizes were narrowly distributed. They also resulted in the most even coating on the laryngeal and tracheal lumen surfaces and produced significant contrast enhancement along the mucosal surface. Such contrast enhancement could help clearer visualization of several disease states, such as intraluminal protrusions, submucosal nodules, and craters.
This lipid-stabilized magnetic resonance imaging contrast formulation may be useful for improving mucosal surface visualization and early diagnosis of disease originating in the mucosal surfaces of the larynx and trachea.
International Journal of Nanomedicine 01/2011; 6:3383-91. · 3.13 Impact Factor
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ABSTRACT: Edible vaccines that can be made widely available and easily administered could bring great benefit to the worldwide battle against pandemic viral infections. They can be used not only for the vaccination of humans and domesticated animals, but also for wild herds and live stock which are otherwise difficult to vaccinate. In this study, we report the development of an edible mini-capsule form of live, non-persisting, recombinant Lactococcus lactis (L. lactis) vaccine against the highly virulent influenza H5N1 strain. Recombinant L. lactis-based H5N1 HA antigen expression constructs were made and shown to be able to induce higher levels of HA-specific serum IgG and fecal IgA antibody production after oral administration. The vectors were then formulated into a mini-capsule dosage form and fed to mouse. Four doses of oral administration rendered complete protection of the mouse against lethal challenges of H5N1 virus.
Virology 11/2010; 407(2):319-24. · 3.35 Impact Factor
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ABSTRACT: Salvianolic acid B is one of the effective components from the Chinese traditional drug Salvia miltiorrhiza (Danshen), which is widely used as a usual clinic drug for atherosclerosis-related disorder patients in China. But the targeting protein of salvianolic acid B is still not known. The possible targeting proteins of salvianolic acid B were explored by high throughput screening in this paper. Attached to the magnetic nanoparticles, salvianolic acid B was used for screening the high-affinity protein from the displaying cDNA peptide library phage. After biopanning, the selected protein or peptide sequences were used to explore the whole proteins containing the selected sequences in the National Center for Biotechnology Information website using blast. One of the selected phages was carried out by affinity analysis with salvianolic acid B using capillary electrophoresis (CE). The CE results indicated that the protein or peptide on the surface of the selected phages could bind the drug salvianolic acid B. The results are helpful to preliminarily explain the pharmacology of salvianolic acid B.
Applied biochemistry and biotechnology 10/2010; 162(4):1206-13. · 1.94 Impact Factor
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ABSTRACT: The aim of this study was to explore a novel gene vector for targeting gene therapy.
We conjugated a peptide ligand (named GA3) for endothelial TEK tyrosine kinase (Tie2) with polyethylenimine (PEI) to construct a GA3-PEI complex and used the vector to transfer reporter and therapeutic gene in vitro and in vivo respectively.
The results demonstrated the vehicle was able to transfer reporter genes specifically into lung cancer SPC-A1 cells and SPC-A1 xenografts highly expressing Tie2 and epithelial cells of bronchus, but not in heart, liver, spleen, kidney, lung alveolar and vascular tissues. In the gene therapy study, tumor growth was significantly inhibited in SPC-A1 xenograft-bearing mice treated with GA3-PEI/p53 complexes compared with control groups (p<0.05).
Our results indicated that GA3-PEI is an efficient gene delivery system targeting Tie2.
Anticancer research 06/2010; 30(6):1999-2004. · 1.73 Impact Factor
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ABSTRACT: Oral administration of peptide and protein drugs faces a big challenge partly due to the hostile gastrointestinal (GI) environment. Lipid-based delivery systems are attractive because they offer some protection for peptides and proteins. In this context, we prepared a special lipid-based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and explored its potential as an oral drug delivery vehicle. Our study demonstrates that archaeosomes have superior stability in simulated GI fluids, and enable fluorescent labeled peptides to reside for longer periods in the GI tract after oral administration. Although archaeosomes have little effect on the transport of insulin across the Caco-2 cell monolayers, the in vivo experiments indicated that archaeosomes containing insulin induced lower levels of blood glucose than a conventional liposome formulation. These data indicate that archaeosomes could be a potential carrier for effective oral delivery of peptide drugs.
Biochemical and Biophysical Research Communications 03/2010; 394(2):412-7. · 2.48 Impact Factor
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ABSTRACT: Hyperbranched poly(amido amine) (HPAMAM), which is structurally analogous to PAMAM dendrimers, has been proposed to be an effective agent for gene delivery. The facile synthesis of HPAMAM with scalable productivity by one-pot polymerization of monomers of methyl acrylate (MA) and diethylenetriamine (DETA) has been set up previously. In this study, the HPAMAM was further modified on the terminal amino groups with phenylalanine to various degrees (HPAMAM-PHE30, PHE45, PHE60). We showed that HPAMAM and HPAMAM-PHEs were all able to form complexes with plasmid DNA (pDNA) at various mass ratios. The cytotoxicity and transfection efficiencies of these polymers were evaluated in SMMC-7721 and COS-7 cell lines. The PHE modifications affected the cell transfection efficiency significantly. The HPAMAM-PHE60 was the most efficient, with transfection activities consistently higher than the commercial transfection reagent PEI. Our study demonstrated that HPAMAM-PHEs may be good new materials for gene delivery and other applications because of its large-scale availability, economical cost, and low toxicity.
Biomacromolecules 11/2009; 11(1):245-51. · 5.48 Impact Factor
