In Vivo Applications of Polyplexes

For targeted delivery into a distant organ, the following factors have to be taken into account (Fig. 1): ideally 1) DNA polyplexes are stable and inert in blood; 2) they must be able to reach their target tissue and therefore to cross different biologicals barriers, including vascular endothelium, extracellular matrix, and others; 3) once reaching the target cell they should internalize; 4) should disassemble at the right moment, but still protect the DNA against intracellular degradation; 5) release the DNA into the nuclear compartment; 6) they must elicit as low an inflammatory or immune response as possible. Although no polyplex or other nonviral gene transfer system exists which would fulfill all these requirements, targeted delivery to the lung, the liver, or tumors has already been achieved in experimental animals (Table 1).

Several strategies have been evaluated for gene transfer to the lung. Systemic application of PEI polyplexes resulted in very high gene transfer to the lung.[9] In this application, the linear polymer form of PEI mediates a much higher transfection activity than branched PEI of similar molecular weight.[10] However, a positive charge ratio (cation of polymer to DNA phosphate anion ratio) is required, with a narrow window between efficiency and severe toxicity, as PEI/DNA activates the lung endothe-lium and forms small aggregates.

As an alternative approach, Ferkol and colleagues generated polylysine polyplexes for targeting the polymeric immunoglobulin receptor by using an antibody Fab fragment as ligand conjugated with polylysine. Systemic delivery of these polyplexes in rats resulted in reporter gene expression in cells of the airway epithelium and submucosal glands.[11] Also, different lung-targeted ligand-polylysine polyplexes were evaluated, using a q-DNA#

Ligand

Polycation Shielding agent Endosomal release agent

Endothelial cells

Blood vessel

Ligand

Polycation Shielding agent Endosomal release agent

Endothelial cells

Blood vessel

, Ligand/Receptor targeting

, Ligand/Receptor targeting

Fig. 1 Targeting opportunities for DNA polyplexes. Polyplex formation with polymer compacting DNA into particles, shielding the polyplex against degradation and undesired interactions, enabling migration to the target tissue, enhancing cell binding and intracellular delivery into cytoplasm and the nucleus. (View this art in color at www.dekker.com.)

Fig. 1 Targeting opportunities for DNA polyplexes. Polyplex formation with polymer compacting DNA into particles, shielding the polyplex against degradation and undesired interactions, enabling migration to the target tissue, enhancing cell binding and intracellular delivery into cytoplasm and the nucleus. (View this art in color at www.dekker.com.)

synthetic peptide ligand for the serpin-enzyme complex receptor. These polyplexes upon nasal administration were able to transiently correct the chloride transport defect in the nasal epithelium of CF mice.[12]

Targeted gene transfer to the liver was reported by Wu and Wu.[13] DNA/asialoorosomucoid-polylysine complexes were administered for targeting to the hepato-cyte-specific asialoglycoprotein receptor, and intravenous (i.v.) injection in rats resulted in marker gene expression in rat livers. This work was the very first successful in vivo application of a targeted polyplex system for gene transfer. Using the same type of formulation, Nagase analbuminemic rats were injected with a human albumin expression plasmid, followed by partial hepatectomy.[14] Circulating human albumin increased in concentration to a maximum by 2 weeks postinjection and remained stable for further 2 weeks.

A synthetic hepatocyte-directed, multifunctional polyplex system was applied by the group of Nishikawa et al.,[15] consisting of DNA complexed with polyorni-thine which was modified with galactose (to serve as asialoglycoprotein receptor ligand) and a fusogenic peptide derived from the influenza virus hemagglutinin subunit HA2 (to serve as endosomal release domain). Upon intravenous injection in mice, a large amount of a marker gene product was detected in the liver, with the hepatocytes contributing more than 95% of the total activity in all tissues. In another approach, a conjugate of low-molecular weight PEI with Pluronic 123 (a block co-polymer of polyethylene oxide and polypropylene oxide) was synthesized by the group of Nguyen et al.[16] In combination with unmodified Pluronic 123 and DNA, the conjugate forms small and stable complexes which after i.v. injection into mice exhibit highest gene expression in liver.

Targeting tumors might present a unique opportunity to reach and attack multiple-spread metastases. Direct intra-tumoral delivery of PEI polyplexes has been investigated. Expression levels were low, and a special form of administration, the local infusion of PEI polyplexes into the tumor mass by a micropump,[17] had to be applied to obtain satisfactory results. As an alternative for lung tumors, Gautam and colleagues successfully delivered PEI polyplexes to lung metastases of melanoma as aerosol through the airways.[18]

A series of targeted DNA polyplex formulations with the potential of systemically targeting tumors have been established. A charge-neutral surface of the DNA particles is essential to minimize nonspecific interactions with blood components, allowing greater intravenous circulation time for the vector to reach its target, and also reducing vector toxicity.[4] The hydrophilic shielding agents investigated include the serum protein transferrin and hydrophilic polymers such as hydroxy-propyl methacrylate or polyethylene glycol (PEG). For example, transferrin-shielded polyplexes[19] or PEG-shielded polyplexes[20,21] demonstrated potential for systemic in vivo targeting of tumors. Intravenous injection resulted in gene transfer into distant subcutaneous neuroblastoma tumors of syngeneic mice[19-21] with luciferase marker gene expression levels in tumor tissues approximately 100-fold higher than in other organ tissues. Specificity was confirmed by luciferase imaging in living mice.[22] In analogous manner, EGF-PEG-coated polyplexes were successfully applied for systemic targeting of human hepatocellular carcinoma xenografts in SCID mice.[23] Similar observations of in vivo hepatoma targeting were made with polylysine polyplexes linked with EGF-derived peptides and an endosomally active peptide.[24]

Table 1 In vivo delivery of polyplexes

Polyplex system Delivery mode Target organ Results Reference

Polyplex system Delivery mode Target organ Results Reference

Table 1 In vivo delivery of polyplexes

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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