Polyplex

Takae S, Miyata K, Oba M, Ishii T, Nishiyama N, Itaka K, Yamasaki Y, Koyama H, Kataoka K (2008) PEG-detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors. J Am Chem Soc 130 6001-6009... 

Functions Required Within Polyplexes for Overcoming Delivery Barriers... 

Several of these problems can be solved by polyplex modification with polyethylene glycol (PEG). PEGylation has been broadly explored for surface shielding ( stealthing ) of many liposomal and nanoparticulate carriers. In the case of cationic polymers, Plank et al. [62] demonstrated that complement activation can be reduced when the polymers are PEGylated. Such a modification can be... 

Shielded polyplexes with improved blood circulating properties are interesting tools for systemic cancer therapy (see Sect. 4.2). Nanoparticles can take advantage of the enhanced permeability and retention (EPR effect) [89] for passive tumor targeting. The EPR effect is based on the leakiness of tumor vasculature, due to neovascularization in growing tumors, combined with an inadequate lymphatic drainage. Nanoparticles with an elongated plasma circulation time can extravasate and passively accumulate at the tumor site. 

Polyplex surface shielding solves several crucial problems, but may also create new problems. Shielding can strongly reduce the efficiency of subsequent cellular steps of the delivery process [68, 69], and also can negatively alter other polyplex characteristics. For pDNA/PEI polyplexes with optimum medium size of PEI, PEG was found to reduce the polyplex stability in vivo [64, 65, 81]. For a discussion of these aspects see Sect. 3.1. 

The neoangiogenic tumor vasculature overexpresses certain integrins and other surface markers, which can also be used for targeting of polyplexes. The RGD peptide motif has been successfully applied for integrin-targeted pDNA [125-128] and siRNA [129, 130] delivery. In many cases, the PEG motif-containing peptide was attached to the polycation via a PEG spacer. For RGD-PEG-PEI/pDNA polyplexes, an optimum grafting with RGD-PEG was required because transfection... 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

A highly stable and shielded polyplex should circulate in the blood stream without undesired interactions until it reaches the target cell. At that location, specific interactions with the cell surface should trigger intracellular uptake. While lipid membrane interaction is undesired at the cell surface, it should happen subsequently within the endosomal vesicle and mediate polyplex delivery into the cytosol. During or after intracellular transport to the site of action, the polyplex stability should be weakened to an extent that the nucleic acid is accessible to exert its function. 

The following sections discuss how polymers and polyplexes can be chemically designed to be bioresponsive in three key delivery functions (1) polyplex surface shielding, (2) interaction with lipid bilayers, and (3) polyplex stability. 

Fig. 1 Bioresponsive polyplexes. (a) Systemic circulation of shielded polyplexes in blood stream and attachment to cell surface receptor (b) endocytosis into endosomes, deshielding by cleavage of PEG linkers and activation of membrane-destabilizing component by acidic pH or other means (c) endosomal escape into cytosol (d) siRNA transfer to form a cytosolic RNA-induced silencing complex complex (e) cytosolic migration and intranuclear import of pDNA (/) presentation of pDNA in accessible form to the transcription machinery...

Fig. 2 Deshielding of polyplexes. After endocytosis of polyplexes into endosomes, deshielding by cleavage of PEG hydrazone or acetal linkers...

A different pH-triggered deshielding concept with hydrophilic polymers is based on reversing noncovalent electrostatic bonds [78, 195, 197]. For example, a pH-responsive sulfonamide/PEl system was developed for tumor-specific pDNA delivery [195]. At pH 7.4, the pH-sensitive diblock copolymer, poly(methacryloyl sulfadimethoxine) (PSD)-hZocA -PEG (PSD-b-PEG), binds to DNA/PEI polyplexes and shields against cell interaction. At pH 6.6 (such as in a hypoxic extracellular tumor environment or in endosomes), PSD-b-PEG becomes uncharged due to sulfonamide protonation and detaches from the nanoparticles, permitting PEI to interact with cells. In this fashion PSD-b-PEG is able to discern the small difference in pH between normal and tumor tissues. 

As outlined in previous sections, escape of polyplexes from endosomes to the cytosol can be a major bottleneck in delivery. Membrane-active polymer domains or other conjugated molecules can help to overcome this barrier (see Sect. 2.3), but they may trigger cytotoxicity when acting extracellularly or at the cell surface. Therefore membrane-crossing agents either have to be inherently specific for endo-somal compartments (for example by pH-specificity), or they have to be modified to be activated in endosomes. For example, the reducing stimulus of intracellular vesicles has been used to activate formulations containing less active disulfide precursors of LLO [163] or Mel [170]. 

On the other hand, pDNA/PEI polyplexes were found to be not stable enough in the extracellular in vivo environment. Unpackaging of PEI and PEG-PEI polyplexes was observed [64, 65, 81], for example by serum proteins, soluble glycosaminoglycans, or extracellular matrix components. The situation is even worse in the case of siRNA polyplexes, where PEI polyplexes are dissociated in full human serum, as monitored by fluorescence fluctuation spectroscopy [66, 67]. 

Since the design of the first targeted polyplexes more than 20 years ago [97, 134], numerous efforts have been made to develop polyplexes for use in medical products, both in pharmacological animal studies and in human studies. Therapeutic modalities include ex vivo treatment of isolated human patient cells, localized in vivo treatments, and - currently the most challenging delivery scenario - in vivo targeted intravenous delivery. 

First clinical human gene therapy trials with polyplexes were performed using cancer vaccines based on autologous patient tumor cells. These were modified ex vivo with interleukin-2 pDNA. To obtain high level transfection rates of patient s primary tumor cells, Tf-PLL/pDNA polyplexes linked with inactivated endosomolytic adenovirus particles were applied [221]. Polymer-based in vivo human gene transfer studies were performed with PEGylated PLL polyplexes, delivering CFTR pDNA to the airway epithelium of cystic fibrosis patients [222],... 

Intravesical infusion of linear PEI/pDNA polyplexes was evaluated in patients with superficial bladder cancer where intravesical therapy with bacillus Calmette-Guerin had failed [6, 224]. Patients had low grade superficial bladder cancer, which expressed H19. The therapeutic pDNA contains H19 gene regulatory sequences that drive the expression of an intracellular toxin. Escalating doses of 2-20 mg plasmid per intravesical treatment were applied, with responders continuing to receive polyplexes once a month every month for 1 year. The treatment resulted in complete ablation of the marker tumor, without any new tumors in four of the 18 patients (22% overall complete response rate). Eight of the 18 patients (44%) had complete marker tumor ablation or a 50% reduction of the marker lesion. 

Systemic targeting of pDNA and siRNA polyplexes has been demonstrated in several animal models. In continuation of the work with localized antiproliferative and immunostimulatory poly(I C) RNA, intravenous systemic delivery of EGER-targeted PEG-modified polyplexes were successfully used for human carcinoma treatment in mice [225]. The therapeutic effect was most pronounced when intravenous delivery of poly(I C) polyplexes was followed by intraperitoneal injection of peripheral blood mononuclear cells [226]. This induced the complete cure of SCID mice with pre-established disseminated EGFR-overexpressing tumors, without adverse toxic effects. Due to the chemokines produced by the internalized poly (I C) in the tumor cells, the immune cells home to the tumors of the treated animal and contribute to the tumor destruction. 

Various researchers have applied the receptor-targeted strategy in pharmacological models for tumor-targeted delivery of pDNA expressing tumor necrosis factor alpha (TNFa). For example, Tf- or Tf-PEG-shielded PEI polyplexes have been used... 

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