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1.  Dual-Purpose Magnetic Micelles for MRI and Gene Delivery 
Gene therapy is a promising therapeutic approach for treating disease, but the efficient delivery of genes to desired locations with minimal side effects remains a challenge. In addition to gene therapy, it is also highly desirable to provide sensitive imaging information in patients for disease diagnosis, screening and post-therapy monitoring. Here, we report on the development of dual-purpose chitosan and polyethyleneimine (PEI) coated magnetic micelles (CP-mag-micelles) that can deliver nucleic acid-based therapeutic agents and also provide magnetic resonance imaging (MRI). These ‘theranostic’ CP-mag-micelles are composed of monodisperse hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) loaded into the cores of micelles that are self-assembled from a block copolymer of poly (D, L-lactide) (PLA) and monomethoxy polyethylene glycol (mPEG). For efficient loading and protection of the nucleic acids the micelles were coated with cationic polymers, such as chitosan and PEI. The morphology and size distribution of the CP-mag-micelles were characterized and their potential for use as an MRI-probe was tested using an MRI scanner. The T2 relaxivity of micelles was similar to CP-mag-micelles confirming that coating with cationic polymers did not alter magnetism. Nanoparticles coated with chitosan:PEI at a weight ratio of 5:5 showed higher transfection efficiency in HEK293, 3T3 and PC3 cells than with weight ratios of 3:7 or 7:3. CP-mag-micelles are biocompatible, can be delivered to various organs and are safe. A single injection of CP-mag-micelles carrying reporter plasmids in vivo expressed genes for at least one week. Collectively, our results demonstrate that a structural reinforcement of SPIONs loaded in the core of an mPEG-PLA micelle coated with cationic polymers provides efficient DNA delivery and enhanced MRI potential, and affords a promising candidate for theranostics in the future.
doi:10.1016/j.jconrel.2012.04.030
PMCID: PMC3632302  PMID: 22561339
Chitosan and PEI coated magnetic micelles (CP-mag-micelles); magnetic resonance imaging (MRI); super paramagnetic iron oxide nanoparticles (SPIONs); gene delivery; theranostics
2.  A novel Poly(ε-caprolactone)-Pluronic-Poly(ε-caprolactone) grafted Polyethyleneimine(PCFC-g-PEI), Part 1, synthesis, cytotoxicity, and in vitro transfection study 
BMC Biotechnology  2009;9:65.
Background
Polyethyleneimine (PEI), a cationic polymer, is one of the successful and widely used vectors for non-viral gene transfection in vitro. However, its in vivo application was greatly limited due to its high cytotoxicity and short duration of gene expression. To improve its biocompatibility and transfection efficiency, PEI has been modified with PEG, folic acid, and chloroquine in order to improve biocompatibility and enhance targeting.
Results
Poly(ε-caprolactone)-Pluronic-Poly(ε-caprolactone) (PCFC) was synthesized by ring-opening polymerization, and PCFC-g-PEI was obtained by Michael addition reaction with GMA-PCFC-GMA and polyethyleneimine (PEI, 25 kD). The prepared PCFC-g-PEI was characterized by 1H-NMR, SEC-MALLS. Meanwhile, DNA condensation, DNase I protection, the particle size and zeta potential of PCFC-g-PEI/DNA complexes were also determined. According to the results of flow cytometry and MTT assay, the synthesized PCFC-g-PEI, with considerable transfection efficiency, had obviously lower cytotoxicity against 293 T and A549 cell lines compared with that of PEI 25 kD.
Conclusion
The cytotoxicity and in vitro transfection study indicated that PCFC-g-PEI copolymer prepared in this paper was a novel gene delivery system with lower cytotoxicity and considerable transfection efficiency compared with commercial PEI (25 kD).
doi:10.1186/1472-6750-9-65
PMCID: PMC2717081  PMID: 19607728
3.  Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells 
Background
To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs) more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG) and a series of its mannosylated derivatives.
Methods
PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs) using flow cytometry.
Results
PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation.
Conclusion
These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system.
doi:10.2147/IJN.S31760
PMCID: PMC3384368  PMID: 22745554
dendritic cells; DCs; mannose; polyethyleneimine; PEI; gene delivery
4.  Thiolated chitosan nanoparticles for enhancing oral absorption of docetaxel: preparation, in vitro and ex vivo evaluation 
The aim of this study was to prepare and evaluate mucoadhesive core-shell nanoparticles based on copolymerization of thiolated chitosan coated on poly methyl methacrylate cores as a carrier for oral delivery of docetaxel. Docetaxel-loaded nanoparticles with various concentrations were prepared via a radical emulsion polymerization method using cerium ammonium nitrate as an initiator. The physicochemical properties of the obtained nanoparticles were characterized by: dynamic light-scattering analysis for their mean size, size distribution, and zeta potential; scanning electron microscopy and transmission electron microscopy for surface morphology; and differential scanning calorimetry analysis for confirmation of molecular dispersity of docetaxel in the nanoparticles. Nanoparticles were spherical with mean diameter below 200 nm, polydispersity of below 0.15, and positive zeta potential values. The entrapment efficiency of the nanoparticles was approximately 90%. In vitro release studies showed a sustained release characteristic for 10 days after a burst release at the beginning. Ex vivo studies showed a significant increase in the transportation of docetaxel from intestinal membrane of rat when formulated as nanoparticles. Cellular uptake of nanoparticles was investigated using fluoresceinamine-loaded nanoparticles. Docetaxel nanoparticles showed a high cytotoxicity effect in the Caco-2 and MCF-7 cell lines after 72 hours. It can be concluded that by combining the advantages of both thiolated polymers and colloidal particles, these nanoparticles can be proposed as a drug carrier system for mucosal delivery of hydrophobic drugs.
doi:10.2147/IJN.S15500
PMCID: PMC3026577  PMID: 21289989
poly methyl methacrylate; mucoadhesive; cytotoxicity
5.  Comparisons of three polyethyleneimine-derived nanoparticles as a gene therapy delivery system for renal cell carcinoma 
Background
Polyethyleneimine (PEI), which can interact with negatively charged DNA through electrostatic interaction to form nanocomplexes, has been widely attempted to use as a gene delivery system. However, PEI has some defects that are not fit for keeping on gene expression. Therefore, some modifications against PEI properties have been done to improve their application value in gene delivery. In this study, three modified PEI derivatives, including poly(ε-caprolactone)-pluronic-poly(ε-caprolactone) grafted PEI (PCFC-g-PEI), folic acid-PCFC-isophorone diidocyanate-PEI (FA-PEAs) and heparin-PEI (HPEI), were evaluated in terms of their cytotoxicity and transfection efficiency in vitro and in vivo in order to ascertain their potential application in gene therapy.
Methods
MTT assay and a marker GFP gene, encoding green fluorescent protein, were used to evaluate cell toxicity and transfection activity of the three modified PEI in vitro. Renal cell carcinoma (RCC) models were established in BALB/c nude mice inoculated with OS-RC-2 cells to detect the gene therapy effects using the three PEI-derived nanoparticles as gene delivery vehicles. The expression status of a target gene Von Hippel-Lindau (VHL) in treated tumor tissues was analyzed by semiquantitative RT-PCR and immunohistochemistry.
Results
Each of three modified PEI-derived biomaterials had an increased transfection efficiency and a lower cytotoxicity compared with its precursor PEI with 25-kD or 2-kD molecule weight in vitro. And the mean tumor volume was obviously decreased 30% by using FA-PEAs to transfer VHL plasmids to treat mice RCC models. The VHL gene expression was greatly improved in the VHL-treated group. While there was no obvious tumor inhibition treated by PCFC-g-PEI:VHL and HPEI:VHL complexes.
Conclusions
The three modified PEI-derived biomaterials, including PCFC-g-PEI, FA-PEAs and HPEI, had an increased transfection efficiency in vitro and obviously lower toxicities compared with their precursor PEI molecules. The FA-PEAs probably provide a potential gene delivery system to treat RCC even other cancers in future.
doi:10.1186/1479-5876-9-46
PMCID: PMC3108928  PMID: 21513541
Polyethyleneimine; nanoparticle; gene delivery; VHL; renal cell carcinoma
6.  Dual-degradable disulfide-containing PEI–Pluronic/DNA polyplexes: transfection efficiency and balancing protection and DNA release 
Polymeric gene-delivery vectors to achieve lack of toxicity and a balance between protection and DNA release remains a formidable challenge. Incorporating intracellular environment-responsive degradable bonds is an appreciable step toward developing safer transfection agents. In this study, novel, dual-degradable polycation copolymers (Pluronic-diacrylate [PA]–polyethyleneimine [PEI]–SS) were synthesized through the addition of low molecular weight (800 Da) PEI cross-linked with SS (PEI-SS) to PA. Three PA-PEI-SS copolymers (PA-PEI-SS1, 2, and 3) with different PEI-SS to Pluronic molar ratios were investigated and found to strongly condense plasmid DNA into positively charged nanoparticles with an average particle size of approximately 200 nm and to possess higher stability against DNase I digestion and sodium heparin. Disulfide and ester bonds of the copolymers were susceptible to intracellular redox conditions. In vitro experiments demonstrated that the PA-PEI-SS copolymers had significantly lower cytotoxicity and higher transfection efficiency in both BGC-823 and 293T cell lines than the controls of degradable PEI-SS and nondegradable 25 kDa PEI. Transfection activity was influenced by the PEI-SS content in the polymers and PA-PEI-SS1 showed the highest efficiency of the three copolymers. These studies suggest that these dual-degradable copolymers could be used as potential biocompatible gene delivery carriers.
doi:10.2147/IJN.S49595
PMCID: PMC3792845  PMID: 24109182
Pluronic; PEI; gene vector; dual-degradable; disulfide-containing linker
7.  PEI-PEG-Chitosan Copolymer Coated Iron Oxide Nanoparticles for Safe Gene Delivery: synthesis, complexation, and transfection** 
Advanced functional materials  2009;19(14):2244-2251.
Gene therapy offers the potential of mediating disease through modification of specific cellular functions of target cells. However, effective transport of nucleic acids to target cells with minimal side effects remains a challenge despite the use of unique viral and non-viral delivery approaches. Here we present a non-viral nanoparticle gene carrier that demonstrates effective gene delivery and transfection both in vitro and in vivo. The nanoparticle system (NP-CP-PEI) is made of a superparamagnetic iron oxide nanoparticle (NP), which enables magnetic resonance imaging, coated with a novel copolymer (CP-PEI) comprised of short chain polyethylenimine (PEI) and poly(ethylene glycol) (PEG) grafted to the natural polysaccharide, chitosan (CP), which allows efficient loading and protection of the nucleic acids. The function of each component material in this nanoparticle system is illustrated by comparative studies of three nanoparticle systems of different surface chemistries, through material property characterization, DNA loading and transfection analyses, and toxicity assessment. Significantly, NP-CP-PEI demonstrates an innocuous toxic profile and a high level of expression of the delivered plasmid DNA in a C6 xenograft mouse model, making it a potential candidate for safe in vivo delivery of DNA for gene therapy.
doi:10.1002/adfm.200801844
PMCID: PMC2756666  PMID: 20160995
biomaterials; superparamagnetic nanoparticles; DNA; drug delivery; gene therapy
8.  N-Alkyl-PEI Functional Iron Oxide Nanocluster for Efficient siRNA Delivery** 
Small interfering RNA (siRNA) is an emerging class of therapeutics, working by regulating the expression of a specific gene involved in disease progression. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. In this study, a non-viral nanoparticle gene carrier has been developed and its efficiency for siRNA delivery and transfection has been validated at both in vitro and in vivo levels. Such a nanocarrier, abbreviated as Alkyl-PEI2k-IO, was constructed with a core of iron oxide (IO) and a shell of alkylated PEI2000 (Alkyl-PEI2k). It was found to be able to bind with siRNA, resulting in well-dispersed nanoparticles with a controlled clustering structure and narrow size distribution. Electrophoresis studies showed that the Alkyl-PEI2k-IOs could retard siRNA completely at N/P ratios above 10, protect siRNA from enzymatic degradation in serum and release complexed siRNA efficiently in the presence of polyanionic heparin. The knockdown efficiency of the siRNA loaded nanocarriers was assessed with 4T1 cells stably expressing luciferase (fluc-4T1) and further, with a fluc-4T1 xenograft model. Significant downregulation of luciferase was observed, and unlike the high molecular weight analogs, the Alkyl-PEI2k coated IOs showed a good biocompatibility. In conclusion, Alkyl-PEI2k-IOs demonstrate highly efficient delivery of siRNA and an innocuous toxic profile, making it a potential carrier for gene therapy.
doi:10.1002/smll.201100825
PMCID: PMC3759164  PMID: 21861295
Biomaterials; Superparamagnetic nanoparticles; Polytehyleneimine; Small interfering RNA
9.  PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells 
Nanoscale Research Letters  2011;6(1):447.
The biggest challenge in the field of gene therapy is how to effectively deliver target genes to special cells. This study aimed to develop a new type of poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles for gene delivery, which are capable of overcoming the disadvantages of polyethylenimine (PEI)- or cationic liposome-based gene carrier, such as the cytotoxicity induced by excess positive charge, as well as the aggregation on the cell surface. The PLGA-based nanoparticles presented in this study were synthesized by emulsion evaporation method and characterized by transmission electron microscopy, dynamic light scattering, and energy dispersive spectroscopy. The size of PLGA/PEI nanoparticles in phosphate-buffered saline (PBS) was about 60 nm at the optimal charge ratio. Without observable aggregation, the nanoparticles showed a better monodispersity. The PLGA-based nanoparticles were used as vector carrier for miRNA transfection in HepG2 cells. It exhibited a higher transfection efficiency and lower cytotoxicity in HepG2 cells compared to the PEI/DNA complex. The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes. Moreover, nanocomplex shows better serum compatibility than commercial liposome. PLGA nanocomplexes obviously accumulate in tumor cells after transfection, which indicate that the complexes contribute to cellular uptake of pDNA and pronouncedly enhance the treatment effect of miR-26a by inducing cell cycle arrest. Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.
doi:10.1186/1556-276X-6-447
PMCID: PMC3211866  PMID: 21749688
10.  Linear polyethylenimine produced by partial acid hydrolysis of poly(2-ethyl-2-oxazoline) for DNA and siRNA delivery in vitro 
Polyethylenimines (PEIs) are the most efficient synthetic vectors for gene delivery available to date. With its high charge density and strong proton-buffering effect, PEI has an ability to condense DNA and small interfering RNA at physiologic pH. However, the polymer suffers from the disadvantage of high cellular toxicity. To reduce its cellular toxicity, we synthesized linear PEIs by partial hydrolysis of poly(2-ethyl-2-oxazoline). Three linear PEIs with different hydrolysis percentages (30%, 70%, and 96%, respectively) were produced as PEI30, PEI70, and PEI96. PEI30 and PEI96 cannot be considered as suitable transfection agents because of low transfection efficiency (PEI30) or high cellular toxicity (PEI96). PEI70 displayed very weak cell toxicity. The charge density of this polymer (PEI70) was strong enough to condense DNA and small interfering RNA at a physiologic pH of 7.4. Our results also show that PEI70 was highly efficient in DNA delivery and small interfering RNA-mediated knockdown of target genes. Thus, polymers such as PEI70 appear to be very promising vectors for gene delivery.
doi:10.2147/IJN.S47413
PMCID: PMC3817027  PMID: 24204139
nonviral vector; polyethylenimine; gene delivery; DNA; small interfering RNA
11.  Intracellular Delivery of siRNA by Polycationic Superparamagnetic Nanoparticles 
Journal of Drug Delivery  2012;2012:218940.
The siRNA transfection efficiency of nanoparticles (NPs), composed of a superparamagnetic iron oxide core modified with polycationic polymers (poly(hexamethylene biguanide) or branched polyethyleneimine), were studied in CHO-K1 and HeLa cell lines. Both NPs demonstrated to be good siRNA transfection vehicles, but unmodified branched polyethyleneimine (25 kD) was superior on both cell lines. However, application of an external magnetic field during transfection (magnetofection) increased the efficiency of the superparamagnetic NPs. Furthermore, our results reveal that these NPs are less toxic towards CHO-K1 cell lines than the unmodified polycationic-branched polyethyleneimine (PEI). In general, the external magnetic field did not alter the cell's viability nor it disrupted the cell membranes, except for the poly(hexamethylene biguanide)-modified NP, where it was observed that in CHO-K1 cells application of the external magnetic field promoted membrane damage. This paper presents new polycationic superparamagnetic NPs as promising transfection vehicles for siRNA and demonstrates the advantages of magnetofection.
doi:10.1155/2012/218940
PMCID: PMC3437298  PMID: 22970377
12.  The investigation of polymer-siRNA nanoparticle for gene therapy of gastric cancer in vitro 
Small interfering RNA (siRNA) molecules have significant therapeutic promise for the genetic treatment of cancer. To overcome instability and low transfection efficiency, polyethylene glycol-polyethyleneimine (PEG-PEI) was synthesized and investigated as a non-viral carrier of siRNA targeting CD44v6 in gastric carcinoma cells. The size, surface charge using zeta potential, and morphology via scanning electron microscopy (SEM) of PEG-PEI/siRNA nanoparticles was characterized, and their cytotoxicity, transfection efficiency, and interaction with SGC7901 human gastric carcinoma cells was evaluated. The transfection efficiency of PEG-PEI/siRNA nanocomplexes was dependant on the charge ratio between amino groups of PEG-PEI and phosphate groups of siRNA (N/P) values, which reflected the molar ratio of PEG-PEI to siRNA during complex formation. The transfection efficiency of PEG-PEI/siRNA at N/P 15 was 72.53% ± 2.38%, which was higher than that observed using Lipofectamine 2000 and PEI as delivery carriers. Cytotoxicity of PEG-PEI was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and was obviously lower than that of PEI. Moreover, when N/P was below 15, PEG-PEI/siRNA was less toxic than Lipofectamine 2000/siRNA. RT-PCR (real time polymerase chain reaction) and Western blot analyses of CD44v6 expression demonstrated the gene silencing effect of PEG-PEI/siRNA at N/P 15. These data indicate that PEG-PEI may be a promising non-viral carrier for altering gene expression in the treatment of gastric cancer with many advantages, such as relatively high gene transfection efficiency and low cytotoxicity.
PMCID: PMC2841491  PMID: 20309399
siRNA; PEG-PEI; nanoparticles; CD44v6 gene; gastric carcinoma cells
13.  Synthesis and properties of a novel biodegradable poly(ester amine) copolymer based on poly(L-lactide) and low molecular weight polyethylenimine for gene delivery 
Background
Gene therapy is a promising approach to the treatment of a wide range of diseases. The development of efficient and adequate gene delivery systems could be one of the most important factors. Polyethyleneimine, a cationic polymer, is one of the most successful and widely used vectors for nonviral transfection in vitro and in vivo.
Methods
A novel biodegradable poly(ester amine) copolymer (PEA) was successfully prepared from low molecular weight polyethylenimine (PEI, 2000 Da) and poly(L-lactide) copolymers.
Results
According to the results of agarose gel electrophoresis, particle size and zeta potential measurement, and transfection efficiency, the PEA copolymers showed a good ability to condense plasmid DNA effectively into nanocomplexes with a small particle size (≤150 nm) and moderate zeta potential (≥10 mV) at an appropriate polymeric carrier/DNA weight ratio. Compared with high molecular weight PEI (25kDa), the PEA obtained showed relatively high gene transfection efficiency as well as low cytotoxicity in vitro.
Conclusion
These results indicate that such PEA might have potential application as a gene delivery system.
doi:10.2147/IJN.S21141
PMCID: PMC3160950  PMID: 21904454
polyethylenimine; poly(L-lactide); gene delivery; cytotoxicity; transfection efficiency
14.  A Reducible Polycationic Gene Vector Derived from Thiolated Low Molecular Weight Branched Polyethyleneimine Linked by 2-Iminothiolane 
Biomaterials  2010;32(4):1193-1203.
To improve transfection efficiency and reduce the cytotoxicity of polymeric gene vectors, reducible polycations (RPC) were synthesized from low molecular weight (MW) branched polyethyleneimine (bPEI) via thiolation and oxidation. RPC (RPC-bPEI0.8kDa) possessed a MW of 5 kDa~80 kDa, and 50%~70% of the original proton buffering capacity of bPEI0.8kDa was preserved in the final product. The cytotoxicity of RPC-bPEI0.8kDa was 8~19 times less than that of the gold standard of polymeric transfection reagents, bPEI25kDa. Although bPEI0.8kDa exhibited poor gene condensing capacities (~2 µm at a weight ratio (WR) of 40), RPC-bPEI0.8kDa effectively condensed plasmid DNA (pDNA) at a WR of 2. Moreover, RPC-bPEI0.8kDa/pDNA (WR ≥ 2) formed 100~200 nm-sized particles with positively charged surfaces (20~35 mV). In addition, the results of the present study indicated that thiol/polyanions triggered the release of pDNA from RPC-bPEI0.8kDa/pDNA via the fragmentation of RPC-bPEI0.8kDa and ion-exchange. With negligible polyplex-mediated cytotoxicity, the transfection efficiencies of RPC-bPEI0.8kDa/pDNA were approximately 1200~1500-fold greater than that of bPEI0.8kDa/pDNA and were equivalent or superior (~7-fold) to that of bPEI25kDa/pDNA. Interestingly, the distribution of high MW RPC-bPEI0.8kDa/pDNA in the nucleus of the cell was higher than that of low MW RPC-bPEI0.8kDa/pDNA. Thus, the results of the present study suggest that RPC-bPEI0.8kDa has the potential to effectively deliver genetic materials with lower levels of toxicity.
doi:10.1016/j.biomaterials.2010.08.079
PMCID: PMC2992579  PMID: 21071079
15.  Magnetofection: A Reproducible Method for Gene Delivery to Melanoma Cells 
BioMed Research International  2013;2013:209452.
Magnetofection is a nanoparticle-mediated approach for transfection of cells, tissues, and tumors. Specific interest is in using superparamagnetic iron oxide nanoparticles (SPIONs) as delivery system of therapeutic genes. Magnetofection has already been described in some proof-of-principle studies; however, fine tuning of the synthesis of SPIONs is necessary for its broader application. Physicochemical properties of SPIONs, synthesized by the co-precipitation in an alkaline aqueous medium, were tested after varying different parameters of the synthesis procedure. The storage time of iron(II) sulfate salt, the type of purified water, and the synthesis temperature did not affect physicochemical properties of SPIONs. Also, varying the parameters of the synthesis procedure did not influence magnetofection efficacy. However, for the pronounced gene expression encoded by plasmid DNA it was crucial to functionalize poly(acrylic) acid-stabilized SPIONs (SPIONs-PAA) with polyethyleneimine (PEI) without the adjustment of its elementary alkaline pH water solution to the physiological pH. In conclusion, the co-precipitation of iron(II) and iron(III) sulfate salts with subsequent PAA stabilization, PEI functionalization, and plasmid DNA binding is a robust method resulting in a reproducible and efficient magnetofection. To achieve high gene expression is important, however, the pH of PEI water solution for SPIONs-PAA functionalization, which should be in the alkaline range.
doi:10.1155/2013/209452
PMCID: PMC3686069  PMID: 23862136
16.  Patterned Immobilization of Antibodies within Roll-to-Roll Hot Embossed Polymeric Microfluidic Channels 
PLoS ONE  2013;8(7):e68918.
This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R2 = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays.
doi:10.1371/journal.pone.0068918
PMCID: PMC3715544  PMID: 23874811
17.  Inhalable Large Porous Microspheres of Low Molecular Weight Heparin: In Vitro and In Vivo Evaluation 
This study tests the feasibility of large porous particles as long-acting carriers for pulmonary delivery of low molecular weight heparin (LMWH). Microspheres were prepared with a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), by a double-emulsion–solvent-evaporation technique. The drug entrapment efficiencies of the microspheres were increased by modifying them with three different additives—polyethyleneimine (PEI), Span 60 and stearylamine. The resulting microspheres were evaluated for morphology, size, zeta potential, density, in vitro drug-release properties, cytotoxicity, and for pulmonary absorption in vivo. Scanning electron microscopic examination suggests that the porosity of the particles increased with the increase in aqueous volume fraction. The amount of aqueous volume fraction and the type of core-modifying agent added to the aqueous interior had varying degrees of effect on the size, density and aerodynamic diameter of the particles. When PEI was incorporated in the internal aqueous phase, the entrapment efficiency was increased from 16.22±1.32% to 54.82±2.79%. The amount of drug released in the initial burst phase and the release-rate constant for the core-modified microspheres were greater than those for the plain microspheres. After pulmonary administration, the half-life of the drug from the PEI- and stearylamine-modified microspheres was increased by 5- to 6-fold compared to the drug entrapped in plain microspheres. The viability of Calu-3 cells was not adversely affected when incubated with the microspheres. Overall, the data presented here suggest that the newly developed porous microspheres of LMWH have the potential to be used in a form deliverable by dry-powder inhaler as an alternative to multiple parenteral administrations of LMWH.
doi:10.1016/j.jconrel.2008.03.013
PMCID: PMC2556066  PMID: 18471921
Large porous particles; microspheres; Low molecular weight heparin; pulmonary delivery
18.  Polymeric Nanoparticles for siRNA Delivery and Gene Silencing 
International journal of pharmaceutics  2008;367(1-2):195-203.
Gene silencing using small interfering RNA (siRNA) has several potential therapeutic applications. In the present study, we investigated nanoparticles formulated using the biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA) for siRNA delivery. A cationic polymer, polyethylenimine (PEI), was incorporated in the PLGA matrix to improve siRNA encapsulation in PLGA nanoparticles. PLGA-PEI nanoparticles were formulated using double emulsion-solvent evaporation technique and characterized for siRNA encapsulation and in vitro release. The effectiveness of siRNA-loaded PLGA-PEI nanoparticles in silencing a model gene, fire fly luciferase, was investigated in cell culture. Presence of PEI in PLGA nanoparticle matrix increased siRNA encapsulation by about 2-fold and also improved the siRNA release profile. PLGA-PEI nanoparticles carrying luciferase-targeted siRNA enabled effective silencing of the gene in cells stably expressing luciferase as well as in cells that could be induced to overexpress the gene. Quantitative studies indicated that presence of PEI in PLGA nanoparticles resulted in 2-fold higher cellular uptake of nanoparticles while fluorescence microscopy studies showed that PLGA-PEI nanoparticles delivered the encapsulated siRNA in the cellular cytoplasm; both higher uptake and greater cytosolic delivery could have contributed to the gene silencing effectiveness of PLGA-PEI nanoparticles. Serum stability and lack of cytotoxicity further add to the potential of PLGA-PEI nanoparticles in gene silencing-based therapeutic applications.
doi:10.1016/j.ijpharm.2008.09.039
PMCID: PMC2660441  PMID: 18940242
RNA interference; siRNA; poly(d,l-lactide-co-glycolide); luciferase; sustained release
19.  Low-weight polyethylenimine cross-linked 2-hydroxypopyl-β-cyclodextrin and folic acid as an efficient and nontoxic siRNA carrier for gene silencing and tumor inhibition by VEGF siRNA 
Background
Targeted delivery of small interfering RNA (siRNA) has been regarded as one of the most important technologies for the development of siRNA therapeutics. However, the need for safe and efficient delivery systems is a barrier to further development of RNA interference therapeutics. In this work, a nontoxic and efficient siRNA carrier delivery system of low molecular weight polyethyleneimine (PEI-600 Da) cross-linked with 2-hydroxypopyl-β-cyclodextrin (HP-β-CD) and folic acid (FA) was synthesized for biomedical application.
Methods
The siRNA carrier was prepared using a simple method and characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The siRNA carrier nanoparticles were characterized in terms of morphology, size and zeta potential, stability, efficiency of delivery, and gene silencing efficiency in vitro and in vivo.
Results
The siRNA carrier was synthesized successfully. It showed good siRNA binding capacity and ability to protect siRNA. Further, the toxicity of the carrier measured in vitro and in vivo appeared to be negligible, probably because of degradation of the low molecular weight PEI and HP-β-CD in the cytosol. Flow cytometry and confocal microscopy confirmed that the FA receptor-mediated endocytosis of the FA-HP-β-CD-PEI/siRNA complexes was greater than that of the HP-β-CD-PEI/siRNA complexes in FA receptor-enriched HeLa cells. The FA-HP-β-CD-PEI/siRNA complexes also demonstrated excellent gene silencing efficiency in vitro (in the range of 90%), and reduced vascular endothelial growth factor (VEGF) protein expression in the presence of 20% serum. FA-HP-β-CD-PEI/siRNA complexes administered via tail vein injection resulted in marked inhibition of tumor growth and reduced VEGF protein expression in the tumors.
Conclusion
Our results suggest that the FA-HP-β-CD-PEI complex is a nontoxic and highly efficient gene carrier with the potential to deliver siRNA for cancer gene therapy effectively in vitro and in vivo.
doi:10.2147/IJN.S42440
PMCID: PMC3678862  PMID: 23766646
polyethyleneimine; 2-hydroxypropyl-β-cyclodextrin; folic acid; siRNA carrier; vascular endothelial growth factor; gene silencing
20.  Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides 
Antisense oligonucleotides (AOs) have been shown to induce dystrophin expression in muscles cells of patients with Duchenne Muscular Dystrophy (DMD) and in the mdx mouse, the murine model of DMD. However, ineffective delivery of AOs limits their therapeutic potential. Copolymers of cationic poly(ethylene imine) (PEI) and non-ionic poly(ethylene glycol) (PEG) form stable nanoparticles when complexed with AOs, but the positive surface charge on the resultant PEG-PEI-AO nanoparticles limits their biodistribution. We adapted a modified double emulsion procedure for encapsulating PEG-PEI-AO polyplexes into degradable polylactide-co-glycolic acid (PLGA) nanospheres. Formulation parameters were varied including PLGA molecular weight, ester end-capping, and sonication energy/volume. Our results showed successful encapsulation of PEG-PEI-AO within PLGA nanospheres with average diameters ranging from 215 to 240 nm. Encapsulation efficiency ranged from 60 to 100%, and zeta potential measurements confirmed shielding of the PEG-PEI-AO cationic charge. Kinetic measurements of 17 kDa PLGA showed a rapid burst release of about 20% of the PEG-PEI-AO, followed by sustained release of up to 65% over three weeks. To evaluate functionality, PEG-PEI-AO polyplexes were loaded into PLGA nanospheres using an AO that is known to induce dystrophin expression in dystrophic mdx mice. Intramuscular injections of this compound into mdx mice resulted in over 300 dystrophin-positive muscle fibers distributed throughout the muscle cross-sections, approximately 3.4 times greater than for injections of AO alone. We conclude that PLGA nanospheres are effective compounds for the sustained release of PEG-PEI-AO polyplexes in skeletal muscle and concomitant expression of dystrophin, and may have translational potential in treating DMD.
doi:10.1186/1477-3155-7-1
PMCID: PMC2671478  PMID: 19351396
21.  Using poly(lactic-co-glycolic acid) microspheres to encapsulate plasmid of bone morphogenetic protein 2/polyethylenimine nanoparticles to promote bone formation in vitro and in vivo 
Repair of large bone defects is a major challenge, requiring sustained stimulation to continually promote bone formation locally. Bone morphogenetic protein 2 (BMP-2) plays an important role in bone development. In an attempt to overcome this difficulty of bone repair, we created a delivery system to slowly release human BMP-2 cDNA plasmid locally, efficiently transfecting local target cells and secreting functional human BMP-2 protein. For transfection, we used polyethylenimine (PEI) to create pBMP-2/PEI nanoparticles, and to ensure slow release we used poly(lactic-co-glycolic acid) (PLGA) to create microsphere encapsulated pBMP-2/PEI nanoparticles, PLGA@pBMP-2/PEI. We demonstrated that pBMP-2/PEI nanoparticles could slowly release from the PLGA@pBMP-2/PEI microspheres for a long period of time. The 3–15 μm diameter of the PLGA@pBMP-2/PEI further supported this slow release ability of the PLGA@pBMP-2/PEI. In vitro transfection assays demonstrated that pBMP-2/PEI released from PLGA@pBMP-2/PEI could efficiently transfect MC3T3-E1 cells, causing MC3T3-E1 cells to secrete human BMP-2 protein, increase calcium deposition and gene expressions of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), SP7 and I type collagen (COLL I), and finally induce MC3T3-E1 cell differentiation. Importantly, in vivo data from micro-computed tomography (micro-CT) and histological staining demonstrated that the human BMP-2 released from PLGA@pBMP-2/PEI had a long-term effect locally and efficiently promoted bone formation in the bone defect area compared to control animals. All our data suggest that our PLGA-nanoparticle delivery system efficiently and functionally delivers the human BMP-2 cDNA and has potential clinical application in the future after further modification.
doi:10.2147/IJN.S45184
PMCID: PMC3748902  PMID: 23990717
gene therapy; bone regeneration; biodegradable polymer; human BMP-2
22.  Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells 
Background
Polyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors.
Methods
PEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression.
Results
PEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependant, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.
Conclusion
PEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors.
doi:10.1186/1479-0556-5-11
PMCID: PMC2211466  PMID: 18036259
23.  Photothermal ablation of pancreatic cancer cells with hybrid iron-oxide core gold-shell nanoparticles 
Purpose
Photothermal ablation is a minimally invasive approach, which typically involves delivery of photothermal sensitizers to targeted tissues. The purpose of our study was to demonstrate that gold nanoparticles are phagocytosed by pancreatic cancer cells, thus permitting magnetic resonance imaging (MRI) of sensitizer delivery and photothermal ablation.
Patients and methods
Iron-oxide core/gold-shell nanoparticles (GoldMag®, 30 nm diameter; Xi’an GoldMag Biotechnology Co, Xi’an, People’s Republic of China) were used. In a 96-well plate, 3 × 104 PANC-1 (human pancreatic cancer cell line) cells were placed. GoldMag (0, 25, or 50 μg/mL) was added to each well and 24 hours allowed for cellular uptake. Samples were then divided into two groups: one treated with photothermal ablation (7.9 W/cm2) for 5 minutes, the other not treated. Photothermal ablation was performed using laser system (BWF5; B&W Tek, Inc, Newark, DE, USA). Intraprocedural temperature changes were measured using a fiber optic temperature probe (FTP-LN2; Photon Control Inc, Burnaby, BC, Canada). After 24 hours, the remaining number of viable cells was counted using trypan blue staining; cell proliferation percentage was calculated based on the total number of viable cells after treatment compared with control. MRI of GoldMag uptake was performed using a 7.0T ClinScan system (Bruker BioSpin, Ettlingen, Germany).
Results
Temperature curves demonstrated that with increased GoldMag uptake, laser irradiation produced higher temperature elevations in the corresponding samples; temperature elevations of 12.89°C, 35.16°C, and 79.51°C were achieved for 0, 25, and 50 μg/mL GoldMag. Without photothermal ablation, the cell proliferation percentage changed from 100% to 71.3% and 47.0% for cells treated with 25 and 50 μg/mL GoldMag. Photothermal ablation of PANC-1 cells demonstrated an effective treatment response, specifically a reduction to only 61%, 21.9%, and 2.3% cell proliferation for cells treated with 0, 25, and 50 μg/mL GoldMag. MRI was able to visualize GoldMag uptake within PANC-1 cells.
Conclusion
Our findings suggest that photothermal ablation may be effective in the treatment of pancreatic cancer. GoldMag nanoparticles could serve as photothermal sensitizers, and MRI is feasible to quantify delivery.
doi:10.2147/IJN.S47585
PMCID: PMC3771851  PMID: 24039426
photothermal ablation therapy; hybrid nanoparticles; magnetic resonance imaging
24.  A peptide-mediated targeting gene delivery system for malignant glioma cells 
Glioblastoma multiforme (GBM) is the most common and malignant glioma. Although there has been considerable progress in treatment strategies, the prognosis of many patients with GBM remains poor. In this work, polyethylenimine (PEI) and the VTWTPQAWFQWV (VTW) peptide were modified and synthesized into GBM-targeting nanoparticles. The transfection efficiency of U-87 (human glioblastoma) cells was evaluated using fluorescence microscopy and flow cytometry. Cell internalization was investigated to verify the nanoparticle delivery into the cytoplasm. Results showed that the methods of polymer conjugation and the amount of VTW peptide were important factors to polymer synthesis and transfection. The PEI-VTW20 nanoparticles increased the transfection efficiency significantly. This report describes the use of VTW peptide-based PEI nanoparticles for intracellular gene delivery in a GBM cell-specific manner.
doi:10.2147/IJN.S44990
PMCID: PMC3790891  PMID: 24101872
glioblastoma; polyethylenimine; nanoparticles; drug-delivery systems; gene transfer techniques
25.  Alternating Magnetic Field Controlled, Multifunctional Nano-Reservoirs: Intracellular Uptake and Improved Biocompatibility 
Nanoscale Research Letters  2009;5(1):195-204.
Biocompatible magnetic nanoparticles hold great therapeutic potential, but conventional particles can be toxic. Here, we report the synthesis and alternating magnetic field dependent actuation of a remotely controllable, multifunctional nano-scale system and its marked biocompatibility with mammalian cells. Monodisperse, magnetic nanospheres based on thermo-sensitive polymer network poly(ethylene glycol) ethyl ether methacrylate-co-poly(ethylene glycol) methyl ether methacrylate were synthesized using free radical polymerization. Synthesized nanospheres have oscillating magnetic field induced thermo-reversible behavior; exhibiting desirable characteristics comparable to the widely used poly-N-isopropylacrylamide-based systems in shrinkage plus a broader volumetric transition range. Remote heating and model drug release were characterized for different field strengths. Nanospheres containing nanoparticles up to an iron concentration of 6 mM were readily taken up by neuron-like PC12 pheochromocytoma cells and had reduced toxicity compared to other surface modified magnetic nanocarriers. Furthermore, nanosphere exposure did not inhibit the extension of cellular processes (neurite outgrowth) even at high iron concentrations (6 mM), indicating minimal negative effects in cellular systems. Excellent intracellular uptake and enhanced biocompatibility coupled with the lack of deleterious effects on neurite outgrowth and prior Food and Drug Administration (FDA) approval of PEG-based carriers suggest increased therapeutic potential of this system for manipulating axon regeneration following nervous system injury.
doi:10.1007/s11671-009-9465-9
PMCID: PMC2894335  PMID: 20652104
Magnetic actuation; Biomaterials; Thermo-sensitive polymers; Nano-biotechnology; Biocompatibility; Neuron

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