Comparison of the efficiency and toxicity of sonoporation with branched polyethylenimine-mediated gene transfection in various cultured cell lines

Objective. The objective of this study is to evaluate transfection efficiency and safety for gene delivery by sonoporation in comparison with cationic polymer gene carrier branched polyethylenimine (BPEI).

Methods. The cDNA expressing VEGF¹⁶⁵ was cloned under chicken β-actin promoter. The plasmid DNA was transfected into the CHO, HEK293, and NIH3T3 cells using microbubble-based sonoporation and BPEI (25 kDa) under various conditions. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expressed protein level. Cytotoxicities of transfection methods were compared by Cell Counting Kit-8.

Results. At 1 MHz intensity, transfection efficiency of sonoporation was enhanced by microbubble concentration with no detrimental effects. By contrast, BPEI exacerbated cell viability, despite its high transgene expression efficiency.

Conclusion. Sonoporation gene therapy might be the safest technique to be used in actual clinical practice.     

Myristic acid-conjugated polyethylenimine for brain-targeting delivery: in vivo and ex vivo imaging evaluation

To investigate the potential of myristic acid (MC) to mediate brain delivery of polyethylenimine (PEI) as a gene delivery system, a covalent conjugate (MC-PEI) of MC, and PEI was synthesized. A near-infrared fluorescence probe, IR820 was conjugated to MC-PEI to explore its in vivo distribution after intravenous (i.v.) administration in mice. The brain targeting ability of MC-PEI was evaluated by near-infrared fluorescence imaging and analyzed semiquantitatively by fluorescence intensity, respectively. Significant NIR fluorescent signal was detected in the brain 12?h after i.v. administration and further confirmed by imaging the whole brain and brain slices. Semiquantitative results from fluorescence intensity further supported the successful brain delivery of MC-PEI which led to a very significant increase (～200%) in the brain uptake after i.v. injection in comparison with unmodified PEI. The capability of MC-PEI to condense DNA was further confirmed using agarose gel retardation assay, indicating its potential for gene delivery. The significant in vivo and ex vivo results suggest that MC-PEI is a promising brain-targeting drug delivery system, especially for gene delivery.     

DNA delivery to the mitochondria sites using mitochondrial leader peptide conjugated polyethylenimine

Some genetic diseases are associated with the defects of the mitochondrial genome. Direct DNA delivery to the mitochondrial matrix has been suggested as an approach for mitochondrial gene therapy for these diseases. We hypothesized that a mitochondrial leader peptide (LP) conjugated polyethylenimine (PEI) could deliver DNA to the mitochondrial sites. PEI-LP was synthesized by the conjugation of LP to PEI using disulfide bond. The complex formation of PEI-LP with DNA was confirmed by a gel retardation assay. In this study, DNA was completely retarded at a 0.4/1 PEI-LP/DNA weight ratio. In vitro delivery tests into isolated mitochondria or living cells were performed with rhodamin-labeled DNA and PEI-LP. In vitro cell-free delivery assay with isolated mitochondria showed that PEI-LP/DNA complexes were localized at mitochondria sites. Furthermore, the PEL-LP/DNA complexes were localized at the mitochondrial sites in living cells. However, a control carrier, PEI, did not show this effect. In addition, MTT assay showed that PEI-LP showed lower cytotoxicity than PEI. These results suggest that PEI-LP can deliver DNA to the mitochondrial sites and may be useful for the development of mitochondrial gene therapy.     

The efficiency of a novel delivery system (PEI600-Tat) in development of potent DNA vaccine using HPV16 E7 as a model antigen

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. The mode of plasmid DNA delivery is critical to make progress in DNA vaccination. Using human papillomavirus type 16 E7 as a model antigen, this study evaluated the effect of peptide-polymer hybrid including PEI600-Tat conjugate as a novel gene delivery system on the potency of antigen-specific immunity in mice model. At ratio of 10:50 PEI-Tat/E7DNA (w/w), both humoral and cellular immune responses were significantly enhanced as compared with E7DNA construct and induced Th1 response. Therefore, this new delivery system could have promising applications in gene therapy.     

Investigating siRNA delivery to chronic myeloid leukemia K562 cells with lipophilic polymers for therapeutic BCR-ABL down-regulation

RNAi represents a new alternative for treatment of chronic myeloid leukemia (CML) to overcome the difficulties of current drug treatments such as the acquired resistance. However, potent carriers that can overcome delivery barriers to RNAi agents and have therapeutic efficacy especially in difficult-to-transfect CML cells are needed. Here, we explored the use of lipid-modified polyethylenimines (PEI) of low molecular weights (0.6, 1.2 and 2.0 kDa) in K562 cells and showed that the delivery efficiency was dependent on the type of lipid used for polymer modification, degree of lipid substitution and polymer molecular weight. Among the lipid-substituted polymers investigated, palmitic acid (PA)-substituted 1.2 kDa PEI (~ 2 lipids/PEI) has proven to be highly efficient in delivering siRNA and silencing of the reporter gene green fluorescent protein (GFP). The silencing efficacy achieved with this polymer was found to be higher than the 25 kDa PEI and is similar to commercial reagent Lipofectamine™ 2000. Moreover, when BCR-ABL protein was targeted in K562 cells, a reduction in the corresponding mRNA levels was observed, as well as an induction of early and late stage apoptosis. The results of this study demonstrated that PA-substitutions on low MW polymers could be useful for siRNA delivery in CML cells for therapeutic purposes.     

Enhancement on oral absorption of paclitaxel by multifunctional pluronic micelles

The aim of the present study is to synthesize Pluronic F127-polyethylenimine-folate (PF127-PEI-FA) copolymer, construct a mixed micelle system with PF127-PEI-FA copolymer and Pluronic P123 (PP123) and to evaluate the potential of these mixed micelles as an oral drug delivery system for paclitaxel (PTX). The results of intestinal absorption revealed that the PTX-loaded micelles displayed superior permeability across intestinal barrier than free drug and PF127-PEI-FA/PP123 mixed micelles exhibited the strongest permeability across intestinal barrier. These results were also proved by the studies on cytotoxicity and cell uptake tests. The mechanism was demonstrated in connection with inhibition of the efflux mediated by intestinal P-glycoprotein (P-gp) and enhancement of the electrostatic interaction of positive micelles with the negative intestinal epithelial cells, thereby promoting the permeation across the intestinal wall. The presence of verapamil and Pluronic both improved the intestinal absorption of PTX, which further certified the effect of Pluronic on P-gp inhibition. Pharmacokinetic study demonstrated that the area under the plasma concentration-time curve (AUC_{0→36 h}) of PTX-loaded micelles was three times greater than the PTX solution (dissolved in a 50/50 (vol/vol) mixture of Cremophore EL/dehydrated ethanol) (p < 0.05). In general PF127-PEI-FA/PP123 mixed micelles were proved to be potential oral drug delivery system for PTX.     

Photochemically Enhanced Gene Delivery of EGF Receptor-targeted DNA Polyplexes

Epidermal growth factor receptor (EGFR) targeted DNA polyplexes, containing polyethylenimine (PEI) conjugated with EGF protein as cell-binding ligand for endocytosis and polyethylene glycol (PEG) for masking the polyplex surface charge, mediated a 3- to 30-fold higher luciferase gene expression in HUH7, HepG2 and A431 cell transfections than analogous untargeted PEG–PEI polyplexes. Transfection levels can be further enhanced by treatment of cells with amphiphilic photosensitizers followed by illumination. In this process photosensitizers localized in membranes of endocytic vesicles are activated by light, resulting in the destruction of endocytic membrane structures and releasing co-endocytosed polyplexes into the cell cytosol. Photochemical enhanced gene expression was observed in all cell lines, with the magnitude of enhancement depending on the particular PEI polyplex formulation and cell line, ranging between 2- and 600-fold. Importantly, improved gene transfer retained EGF receptor specificity, as demonstrated by comparison with ligand-free polyplexes and by receptor antibody or ligand competition experiments. These results suggest that this combined procedure enables a dual mode of targeting polyplexes: biological targeting via EGFR interaction, combined with physical targeting with light to direct a photochemical delivery of therapeutic genes to a desired location.     

Mucoadhesive polyethylenimine–dextran sulfate nanoparticles containing Punica granatum peel extract as a novel sustained-release antimicrobial

Abstract

Mucoadhesive polyethylenimine–dextran sulfate nanoparticles (PDNPs) were developed for local oral mucosa delivery. Punica granatum peel extract (PGE) was loaded into PDNPs for oral malodor reduction and caries prevention. PDNPs were constructed using the polyelectrolyte complexation technique employing oppositely charged polymers polyethylenimine (PEI) and dextran sulfate (DS), with PEG 400 as a stabilizer. Under optimal conditions, spherical particles of ～500?nm with a zeta potential of ～+28?mV were produced. Up to 98%, drug entrapment efficiency was observed. The mass ratio of PEI:DS played a significant role in controlling particle size and entrapment efficacy. PDNPs shown to be a good mucoadhesive drug delivery system as confirmed by ex vivo wash off test. In vitro dissolution studies revealed that PGE-loaded PDNPs manifested a prolong release characteristic with a burst release within 5?min. In addition, they remained effectively against oral bacteria.     

Emerging inorganic nanomaterials for pancreatic cancer diagnosis and treatment

Pancreatic cancer is a devastating disease with incidence increasing at an alarming rate and survival not improved substantially during the past three decades. Although enormous efforts have been made in early detection and comprehensive treatment for this disease, little or no survival improvement was obtained, which necessitates the development of novel strategies. Emerging inorganic nanomaterials, such as carbon nanotubes, quantum dots, mesoporous silica/gold/supermagnetic nanoparticles, have been widely used in biomedical research with great optimism for cancer diagnosis and therapy. Such nanoparticles possess unique optical, electrical, magnetic and/or electrochemical properties. With such properties along with their impressive nano-size, these particles can be targeted to cancer cells, tissues, and ligands efficiently and monitored with extreme precision in real-time. In additional to liposome, dendrimer, and polymeric nanoparticles, they are considered the most promising nanomaterials with the capability of both cancer detection and multimodality treatment. Emerging approaches to harness nanotechnology to optimize the existing diagnostic and therapeutic tools for pancreatic cancer have been extensively explored during the recent years. Future options for early detection, individual therapy and monitoring responses of pancreatic cancer are focused on multifunctional nanomedicine. In this review, we present the recent development of clinically applicable inorganic nanoparticles, with focus on the diagnosis and treatment of pancreatic cancer. Furthermore, their advantages in theranostic nanomedicine, and challenges of translation to clinical practice, are discussed.