Non‐viral gene delivery for cancer immunotherapy

Recent decades have witnessed the revolutionary development of cancer immunotherapies, which boost cancer‐specific immune responses for long‐term tumor regression. However, immunotherapy still has limitations, including off‐target side effects, long processing times and limited patient responses. These disadvantages of current immunotherapy are being addressed by improving our understanding of the immune system, as well as by establishing combinational approaches. Advanced biomaterials and gene delivery systems overcome some of these delivery issues, harnessing adverse effects and amplifying immunomodulatory effects, and are superior to standard formulations with respect to eliciting antitumor immunity. Nucleic acid‐based nanostructures have diverse functions, ranging from gene expression and gene regulation to pro‐inflammatory effects, as well as the ability to specifically bind different molecules. A brief overview is provided of the recent advances in the non‐viral gene delivery methods that are being used to activate cancer‐specific immune responses. Furthermore, the tumor microenvironment‐responsive synergistic strategies that modulate the immune response by targeting various signaling pathways are discussed. Nanoparticle‐based non‐viral gene delivery strategies have great potential to be implemented in the clinic for cancer immunotherapy.     

Efficacy of immobilized polyplexes and lipoplexes for substrate‐mediated gene delivery

Non‐viral gene delivery by immobilization of complexes to cell‐adhesive biomaterials, a process termed substrate‐mediated delivery, has many in vitro research applications such as transfected cell arrays or models of tissue growth. In this report, we quantitatively investigate the efficiency of gene delivery by surface immobilization, and compare this efficiency to the more typical bolus delivery. The ability to immobilize vectors while allowing cellular internalization is impacted by the biomaterial and vector properties. Thus, to compare this efficiency between vector types and delivery methods, transfection conditions were initially identified that maximized transgene expression. For surface delivery from tissue culture polystyrene, DNA complexes were immobilized to pre‐adsorbed serum proteins prior to cell seeding, while for bolus delivery, complexes were added to the media above adherent cells. Mathematical modeling of vector binding, release, and cell association using a two‐site model indicated that the kinetics of polyplex binding to cells was faster than for lipoplexes, yet both vectors have a half‐life on the surface of approximately 17 min. For bolus and surface delivery, the majority of the DNA in the system remained in solution or on the surface, respectively. For polyplexes, the efficiency of trafficking of cell‐associated polyplexes to the nucleus for surface delivery is similar or less than bolus delivery, suggesting that surface immobilization may decrease the activity of the complex. The efficiency of nuclear association for cell‐associated lipoplexes is similar or greater for surface delivery relative to bolus. These studies suggest that strategies to enhance surface delivery for polyplexes should target the vector design to enhance its potency, whereas enhancing lipoplex delivery should target the material design to increase internalization. Biotechnol. Bioeng. 2009;102: 1679–1691. © 2008 Wiley Periodicals, Inc.     

Synthesis of aminated polysorbate 80 for polyplex‐mediated gene transfection

To develop novel gene delivery carriers, aminated polysorbate 80 (P80‐NH₂) was synthesized with strong positively charged properties through the introduction of three amine groups. The resulting P80‐NH₂ and DNA polyplex exhibited superb condensation abilities due to the high densities of positively charged amines groups. Size and surface charge of polyplex were shown to be well suited for cellular internalization. In addition, the P80‐NH₂/DNA polyplex demonstrated acceptable transfection efficiency in HeLa cells and was nontoxic relative to the conventional 25‐kDa polyethyleneimine system. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Virus‐inspired and mimetic designs in non‐viral gene delivery

Virus‐inspired mimics for nucleic acid transportation have attracted much attention in the past decade, especially the derivative microenvironment stimuli‐responsive designs. In the present mini‐review, the smart designs of gene carriers that overcome biological barriers and realize an efficient delivery are categorized with respect to the different “triggers” provided by tumor cells, including pH, redox potentials, ATP, enzymes and reactive oxygen species. Some dual/multi‐responsive gene vectors have also been introduced that show a more precise and efficient delivery in the complicated environment of human body. In addition, inspired by the special recognition mechanisms and components of viruses, improvements in the design of carriers relating to targeting/penetration properties, as well as chemical component evolution, are also addressed.     

Acid‐activated nonviral peptide vector for gene delivery

Nonviral vector–based gene therapy is a promising strategy for treating a myriad of diseases. Cell‐penetrating peptides are gaining increasing attention as vectors for nucleic acid delivery. However, most studies have focused more on the transfection efficiency of these vectors than on their specificity and toxicity. To obtain ideal vectors with high efficiency and safety, we constructed the vector stearyl‐TH by attaching a stearyl moiety to the N‐terminus of the acid‐activated cell penetrating peptide TH in this study. Under acidic conditions, stearyl‐TH could bind to and condense plasmids into nanoparticle complexes, which displayed significantly enhanced cellular uptake and transfection efficiencies. In contrast, stearyl‐TH lost the capacities of DNA binding and transfection at physiological pH. More importantly, stearyl‐TH and the complexes formed by stearyl‐TH and plasmids displayed no obvious toxicity at physiological pH. Consequently, the high transfection efficiency under acidic conditions and low toxicity make stearyl‐TH a potential nucleic acid delivery vector for gene therapy.     

Non‐viral gene delivery transfection profiles influence neuronal architecture in an in vitro co‐culture model

Gene delivery from tissue engineering scaffolds can induce expression of tissue inductive factors to stimulate the cellular processes required for regeneration. Transfected cells secrete diffusible proteins that can create local concentration gradients, depending on the number, distribution, and expression level of transfected cells. These gradients are linked to cellular organization and tissue architecture during embryogenesis. In this report, we investigate neuronal architecture and neurite guidance in response to the concentration gradients achieved by localized secretion of a neurotrophic factor from transfected cells. A co‐culture model was employed to examine neuronal responses to multiple transfection profiles, which affects the local concentration of secreted nerve growth factor (NGF). Neuronal architecture, as defined by number of neurites per neuron and length of neurites, was influenced by the transfection profile. Low levels of NGF production by few transfected cells produced longer primary neurites with less branching relative to the higher expression levels or increased numbers of transfected cells. Furthermore, for low NGF production by few transfected cells, the growth cone of the axons was marked by longer extensions and larger surface area, suggesting the presence of a guidance cue. Control studies with varying NGF concentrations did not substantially alter the neuronal architecture, further supporting an effect of localized concentration gradients, and not simply the concentration. Mathematical modeling of NGF diffusion was employed to predict the concentration gradients produced by the transfection profiles, and the resultant gradients were correlated to the cellular response. This report connects the transfection profile, concentration gradients, and the resulting cellular architecture, suggesting a critical design consideration for the application of gene delivery to regenerative medicine. Biotechnol. Bioeng. 2009;103: 1023–1033. © 2009 Wiley Periodicals, Inc.     

Electro‐mediated gene transfer and expression are controlled by the life‐time of DNA/membrane complex formation

Background

Electroporation is a physical method used to transfer molecules into cells and tissues. Clinical applications have been developed for antitumor drug delivery. Clinical trials of gene electrotransfer are under investigation. However, knowledge about how DNA enters cells is not complete. By contrast to small molecules that have direct access to the cytoplasm, DNA forms a long lived complex with the plasma membrane and is transferred into the cytoplasm with a considerable delay.

Methods

To increase our understanding of the key step of DNA/membrane complex formation, we investigated the dependence of DNA/membrane interaction and gene expression on electric pulse polarity and repetition frequency.

Results

We observed that both are affected by reversing the polarity and by increasing the repetition frequency of pulses. The results obtained in the present study reveal the existence of two classes of DNA/membrane interaction: (i) a metastable DNA/membrane complex from which DNA can leave and return to external medium and (ii) a stable DNA/membrane complex, where DNA cannot be removed, even by applying electric pulses of reversed polarity. Only DNA belonging to the second class leads to effective gene expression.

Conclusions

The life‐time of DNA/membrane complex formation is of the order of 1 s and has to be taken into account to improve protocols of electro‐mediated gene delivery. Copyright © 2009 John Wiley & Sons, Ltd.     

Delivery of small interfering RNA with a synthetic collagen poly(Pro‐Hyp‐Gly) for gene silencing in vitro and in vivo

Silencing gene expression by small interfering RNAs (siRNAs) has become a powerful tool for the genetic analysis of many animals. However, the rapid degradation of siRNA and the limited duration of its action in vivo have called for an efficient delivery technology. Here, we describe that siRNA complexed with a synthetic collagen poly(Pro‐Hyp‐Gly) (SYCOL) is resistant to nucleases and is efficiently transferred into cells in vitro and in vivo, thereby allowing long‐term gene silencing in vivo. We found that the SYCOL‐mediated local application of siRNA targeting myostatin, coding a negative regulator of skeletal muscle growth, in mouse skeletal muscles, caused a marked increase in the muscle mass within a few weeks after application. Furthermore, in vivo administration of an anti‐luciferase siRNA/SYCOL complex partially reduced luciferase expression in xenografted tumors in vivo. These results indicate a SYCOL‐based non‐viral delivery method could be a reliable simple approach to knockdown gene expression by RNAi in vivo as well as in vitro.     

Synthesis and characterization of dexamethasone‐conjugated linear polyethylenimine as a gene carrier

Linear polyethylenimine (25 kDa, LPEI25k) has been shown to be an effective non‐viral gene carrier with higher transfection and lower toxicity than branched polyethylenimine (BPEI) of comparable molecular weight. In this study, dexamethasone was conjugated to LPEI25k to improve the efficiency of gene delivery. Dexamethasone is a synthetic glucocorticoid receptor ligand. Dexamethasone‐conjugated LPEI25k (LPEI–Dexa) was evaluated as a gene carrier in various cells. Gel retardation assays showed that LPEI–Dexa completely retarded plasmid DNA (pDNA) at a 0.75:1 weight ratio (LPEI/pDNA). LPEI–Dexa had the highest transfection efficiency at a 2:1 weight ratio (LPEI–Dexa/DNA). At this ratio, the size of the LPEI–Dexa/pDNA complex was approximately 125 nm and the zeta potential was 35 mV. LPEI–Dexa had higher transfection efficiency than LPEI and Lipofectamine 2000. In addition, the cytotoxicity of LPEI–Dexa was much lower than that of BPEI (25 kDa, BPEI25k). In conclusion, LPEI–Dexa has a high transfection efficiency and low toxicity and can therefore be used for non‐viral gene delivery. J. Cell. Biochem. 110: 743–751, 2010. © 2010 Wiley‐Liss, Inc.