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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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A small interfering RNA (siRNA) nanovector with dual targeting specificity and dual therapeutic effect is developed for targeted cancer imaging and therapy. The nanovector is comprised of an iron oxide magnetic nanoparticle core coated with three different functional molecules: polyethyleneimine (PEI), siRNA, and chlorotoxin (CTX). The primary amine group of PEI is blocked with citraconic anhydride that is removable at acidic conditions, not only to increase its biocompatibility at physiological conditions but also to elicit a pH-sensitive cytotoxic effect in the acidic tumor microenvironment. The PEI is covalently immobilized on the nanovector via a disulfide linkage that is cleavable after cellular internalization of the nanovector. CTX as a tumor-specific targeting ligand and siRNA as a therapeutic payload are conjugated on the nanovector via a flexible and hydrophilic PEG linker for targeted gene silencing in cancer cells. With a size of ~ 60 nm, the nanovector exhibits long-term stability and good magnetic property for magnetic resonance imaging. The multifunctional nanovector exhibits both significant cytotoxic and gene silencing effects at acidic pH conditions for C6 glioma cells, but not at physiological pH conditions. Our results suggest that this nanovector system could be safely used as a potential therapeutic agent for targeted treatment of glioma as well as other cancers.

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.

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.

Polyethylenimine-cyclodextrin-tegafur (PEI-CyD-tegafur) conjugate was synthesized as a novel multifunctional prodrug of tegafur for co-delivery of chemotherapeutic agent tegafur and enhanced green fluorescent protein (EGFP) reporter plasmid DNA. Conjugation of tegafur to PEI-CyD via chemical linkage was characterized by 1H NMR spectrometry and ultraviolet (UV) spectrometry. PEI-CyD-tegafur was able to condense plasmid DNA into complexes of around 150 nm with positive charge at the N/P ratio of 25, in accordance with electron microscopy observation of compact and monodisperse nanoparticles. The results of in vitro experiments showed enhanced cytotoxicity and considerable transfection efficiency in B16F10 cell line. Therefore, PEI-CyD-tegafur may have great potential as a co-delivery system with anti-cancer activity and potential for gene delivery.

Polyethylenimine (PEI), especially PEI 25 kDa, has been widely studied for delivery of nucleic acid drugs both in vitro and in vivo. However, it lacks degradable linkages and is too toxic for therapeutic applications. Hence, low-molecular-weight PEI has been explored as an alternative to PEI 25 kDa. To reduce cytotoxicity and increase transfection efficiency, we designed and synthesized a novel small-molecular-weight PEI derivative (PEI-Et, Mn: 1220, Mw: 2895) with ethylene biscarbamate linkages. PEI-Et carried the ability to condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed complete condensation of pDNA at w/w ratios that exceeded three. The particle size of polymer/pDNA complexes was between 130 nm and 180 nm and zeta potential was 5–10 mV, which were appropriate for cell endocytosis. The morphology of PEI-Et/pDNA complexes observed by atomic force microscopy (AFM) was spherically shaped with diameters of 110–190 nm. The transfection efficiency of polymer/pDNA complexes as determined with the luciferase activity assay as well as fluorescence-activated cell-sorting analysis (FACS) was higher than commercially available PEI 25 kDa and Lipofectamine 2000 in various cell lines. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration in three cell lines. Therefore, our results indicated that the PEI-Et would be a promising candidate for safe and efficient gene delivery in gene therapy.

In the present study, we evaluated polyethylenimine (PEI) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting nondifferentiated COS-1 (green monkey fibroblasts) and well-differentiated human submucosal airway epithelial cells (Calu-3). Studying the effect of particle size, zeta potential, presence of serum proteins or chloroquine, it appeared that transfection efficiency depends on the experimental conditions and not on the MW of the PEI used. Comparing transfection efficiencies in both cell lines, we found that PEI was 3 orders of magnitude more effective in COS-1 than in Calu-3 cells, because Calu-3 cells are differentiated and secrete mucins, which impose an additional barrier to gene delivery. Transfection efficiency was strongly correlated to PEI cytotoxicity. Also, some evidence for PEI-induced apoptosis in both cell lines was found. In conclusion, our results indicate that PEI is a useful vector for nonviral transfection in undifferentiated cell lines. However, results from studies in differentiated bronchial epithelial cells suggest that PEI has yet to be optimized for successful gene therapy of cystic fibrosis (CF).

Development of micro- and nanotechnology for the study of living cells, especially in the field of drug delivery, has gained interest in recent years. Although several studies have reported successful results in the internalization of micro- and nanoparticles in phagocytic cells, when nonphagocytic cells are used, the low internalization efficiency represents a limitation that needs to be overcome. It has been reported that covalent surface modification of micro- and nanoparticles increases their internalization rate. However, this surface modification represents an obstacle for their use as drug-delivery carriers. For this reason, the aim of the present study was to increase the capability for microparticle internalization of HeLa cells through the use of noncovalently bound transfection reagents: polyethyleneimine (PEI) Lipofectamine™ 2000 and FuGENE 6®. Both confocal microscopy and flow cytometry techniques allowed us to precisely quantify the efficiency of microparticle internalization by HeLa cells, yielding similar results. In addition, intracellular location of microparticles was analyzed through transmission electron microscopy and confocal microscopy procedures. Our results showed that free PEI at a concentration of 0.05 mM significantly increased microparticle uptake by cells, with a low cytotoxic effect. As determined by transmission electron and confocal microscopy analyses, microparticles were engulfed by plasma-membrane projections during internalization, and 24 hours later they were trapped in a lysosomal compartment. These results show the potential use of noncovalently conjugated PEI in microparticle internalization assays.

An antibody-directed nonviral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles and a gastric cancer-associated CD44v6 single-chain variable fragment (scFvCD44v6,-PEG-g-PEI-SPION), was constructed as a gastric cancer-targeting and magnetic resonance imaging (MRI)-visible nanocarrier for small interfering RNA (siRNA) delivery. Biophysical characterization of PEG-g-PEI-SPION and scFvCD44v6-PEG-g-PEI-SPION was carried out, including siRNA condensation capacity, cell viability, and transfection efficiency. Both the targeting and nontargeting nanocarriers were effective for transferring siRNA in vitro. The cellular uptake and distribution of nanoparticles complexed with siRNA was analyzed by fluorescence imaging and immunofluorescent staining. Moreover, the gastric cancer-targeting effect was verified in vivo by MRI and histology analysis. These results indicate that scFvCD44v6-PEG-g-PEI-SPION is a promising nonviral vector for gastric cancer gene therapy and diagnosis.

Background

A successful gene delivery system needs to breakthrough several barriers to allow efficient transgenic expression. In the present study, successive targeting liposomes (STL) were constructed by integrating various targeting groups into a nanoparticle to address this issue.

Methods

Polyethylenimine (PEI) 1800-triamcinolone acetonide (TA) with nuclear targeting capability was synthesized by a two-step reaction. Lactobionic acid was connected with cholesterol to obtain a compound of [(2-lactoylamido) ethylamino]formic acid cholesterol ester (CHEDLA) with hepatocyte-targeting capability. The liposome was modified with PEI 1800-TA and CHEDLA to prepare successive targeting liposome (STL). Its physicochemical properties and transfection efficiency were investigated both in vitro and in vivo.

Results

The diameter of STL was approximately 100 nm with 20 mV of potential. The confocal microscopy observation and potential assay verified that lipid bilayer of STL was decorated with PEI 1800-TA. Cytotoxicity of STL was significantly lower than that of PEI 1800-TA and PEI 25K. The transfection efficiency of 10% CHEDLA STL in HepG2 cells was the higher than of the latter two with serum. Its transfection efficiency was greatly reduced with excessive free galactose, indicating that STL was absorbed via galactose receptor-mediated endocytosis. The in vivo study in mice showed that 10% CHEDLA STL had better transgenic expression in liver than the other carriers.

Conclusions

STL with multi-ligand was able to overcome the various barriers to target nucleus and special cells and present distinctive transgenic expression. Therefore, it has a great potential for gene therapy as a nonviral carrier.

This study aimed to examine the applicability of polyethyleneimine (PEI)-modified magnetic nanoparticles (GPEI) as a potential vascular drug/gene carrier to brain tumors. In vitro, GPEI exhibited high cell association and low cell toxicity – properties which are highly desirable for intracellular drug/gene delivery. In addition, a high saturation magnetization of 93 emu/g Fe was expected to facilitate magnetic targeting of GPEI to brain tumor lesions. However, following intravenous administration, GPEI could not be magnetically accumulated in tumors of rats harboring orthotopic 9L-gliosarcomas due to its poor pharmacokinetic properties, reflected by a negligibly low plasma AUC of 12 ± 3 μg Fe/ml*min. To improve “passive” GPEI presentation to brain tumor vasculature for subsequent “active” magnetic capture, we examined the intra-carotid route as an alternative for nanoparticle administration. Intra-carotid administration in conjunction with magnetic targeting resulted in 30-fold (p = 0.002) increase in tumor entrapment of GPEI compared to that seen with intravenous administration. In addition, magnetic accumulation of cationic GPEI (ζ-potential = + 37.2 mV) in tumor lesions was 5.2-fold higher (p = 0.004) than that achieved with slightly anionic G100 (ζ-potential = −12 mV) following intra-carotid administration, while no significant accumulation difference was detected between the two types of nanoparticles in the contra-lateral brain (p = 0.187). These promising results warrant further investigation of GPEI as a potential cell-permeable, magnetically-responsive platform for brain tumor delivery of drugs and genes.

Background

Systemic delivery of small interfering RNA (siRNA) is limited by its poor stability and limited cell-penetrating properties. To overcome these limitations, we designed an efficient siRNA delivery system using polyethyleneimine-coated virus-like particles derived from adeno-associated virus type 2 (PEI-AAV2-VLPs).

Methods

AAV2-VLPs were produced in insect cells by infection with a baculovirus vector containing three AAV2 capsid genes. Using this method, we generated well dispersed AAV2-VLPs with an average diameter of 20 nm, similar to that of the wild-type AAV2 capsid. The nanoparticles were subsequently purified by chromatography and three viral capsid proteins were confirmed by Western blot. The negatively charged AAV2-VLPs were surface-coated with PEI to develop cationic nanoparticles, and the formulation was used for efficient siRNA delivery under optimized transfection conditions.

Results

PEI-AAV2-VLPs were able to condense siRNA and to protect it from degradation by nucleases, as confirmed by gel electrophoresis. siRNA delivery mediated by PEI-AAV2-VLPs resulted in a high transfection rate in MCF-7 breast cancer cells with no significant cytotoxicity. A cell death assay also confirmed the efficacy and functionality of this novel siRNA formulation towards MCF-7 cancer cells, in which more than 60% of cell death was induced within 72 hours of transfection.

Conclusion

The present study explores the potential of virus-like particles as a new approach for gene delivery and confirms its potential for breast cancer therapy.