Define baseline levels of segments per beam for intensity-modulated radiation therapy delivery for brain, head and neck, thoracic, abdominal, and prostate applications.

The purpose of this study was to evaluate the number of segments per beam for intensity-modulated radiation therapy (IMRT) treatments and its effects on the plan quality, treatment delivery time, machine quality assurance, and machine maintenance. We have retrospectively analyzed 24 patients treated with IMRT. Five were selected within each of the following regions: head and neck, thoracic, abdomen, and prostate. Four patients were optimized within the brain region. The clinically treated plans were re-optimized using Philips Pinnacle3 v. 8 with the direct machine parameter optimization algorithm. The number of segments per beam from the treated plan was systematically reduced by 80%, 60%, 40%, and 30%, and the following statistics have been analyzed for plan quality: target min, mean, and max doses; critical structure doses; and integral dose. We have attempted to define the smallest number of segments per beam for IMRT treatment plans. Results indicate that IMRT plans can be delivered with acceptable quality with approximately 3-6 segments per beam for the anatomical regions analyzed. A reduction in the number of segments decreases treatment delivery time, reduces machine wear and tear, and minimizes the amount of time the patient is on the treatment table, which in turn reduces the chances of intrafractional uncertainties.

Effects of cell-penetrating peptides and pegylation on transfection efficiency of polyethylenimine in mouse lungs.

BACKGROUND: Cell-penetrating peptides (CPPs) could potentially be used as vectors for intracellular delivery of proteins, peptides and nucleic acids. The present study examined different CPPs, such as TAT-derived and arginine rich sequences, as well as model amphiphilic peptide, with respect to transfection efficiency of pegylated polyethylenimine (PEI) in A549, Calu-3 cells and in mice after intra-tracheal administration. METHODS: The conjugates were prepared by the coupling of CPPs to PEI via a heterobifunctional polyethylene glycol (PEG) linker, resulting in the bioconjugates CPP-PEG-PEI. Structures were successfully confirmed by (1)H-nuclear magnetic resonance and diffusion-ordered spectroscopy. Unmodified PEI 25 kDa was compared with pegylated PEI, and aggregation tendency in cell culture medium, interaction with mucin and stability against heparin was assayed. After evaluating transfection efficiency of the polymers in two different lung cell lines, luciferase reporter gene expression was determined in mouse lungs. RESULTS: All conjugates showed superior transfection efficiency compared to unmodified PEI 25 kDa. The conjugates sizes were generally < 300 nm, thus enabling them to penetrate through the mucus lining of the lung and reach the target cells. Coupling of CPPs to PEG-PEI, however, did not significantly improve transfection efficiency in A549 cells, calu-3 cells and in mouse lungs. CONCLUSIONS: We show that small and stable polyplex size achieved by pegylation is favourable for successful pulmonary gene delivery. Compared to PEI 25 kDa, pegylated PEI and CPP-PEG-PEI displayed enhanced transfection efficiency both in vitro and in vivo.

HER2 targeted polyplexes: the effect of polyplex composition and conjugation chemistry on in vitro and in vivo characteristics.

Knowledge of the influence of targeting ligands on pharmacokinetics and biodistribution of polymeric nonviral vectors is presently limited. We investigated the properties of three structurally different conjugates of polyethylenglycol-modified polyethylenimine coupled to the HER2 specific antibody Trastuzumab. Unlike polyethylenimine, conjugates formed small (100-230 nm) DNA polyplexes with zeta-potentials of +/- 2 mV at a broad range of N/P ratios. Stability as assessed by heparin displacement was slightly improved compared to unmodified copolymers. Erythrocyte aggregation and hemolysis were strongly reduced with conjugates. Conjugate polyplexes showed significant differences in specificity and transfection efficiency in vitro. These could be attributed to differences in cell binding and uptake assessed by flow cytometry. Pharmacokinetics of conjugates in mice revealed significant improvements over free plasmid DNA and polyethylenimine. Area under the plasma level-time curve of conjugates was increased up to 48% or 114% compared to that of polyethylenimine or free plasmid DNA, respectively. Deposition of conjugate polyplexes in lung and spleen was significantly reduced compared to that of polyethylenimine. Differences could be attributed to antibody conjugation since no significant differences in pharmacokinetics and biodistribution were found between conjugates. These findings demonstrate that conjugated antibodies not only confer active targeting but also significantly improve in vivo properties of polyplexes.

Bioreversibly crosslinked polyplexes of PEI and high molecular weight PEG show extended circulation times in vivo.

Copolymers consisting of branched PEI 25 kDa grafted with high molecular weight PEG at a low degree of substitution were successfully synthesized using a simple two-step procedure. The resulting AB-type and ABA-type copolymers were tested for cytotoxicity and DNA condensation and complexation properties. Their polyplexes with plasmid DNA were characterized in terms of DNA size and surface charge, transfection efficiency and blood compatibility. Pharmacokinetic profiles of the complexes containing (32)P-labeled plasmid were assessed before and after surface crosslinking. A set of four copolymers containing one or two PEG 20 kDa or PEG 30 kDa chains was obtained. The cytotoxicity of PEI was strongly reduced after copolymerization. The copolymer polyplexes showed hydrodynamic diameters of less than 200 nm, comparable to PEI 25. Similarly, no reduction in DNA condensation and complexation properties was found. In fact, PEI-PEG(30 k) copolymers exhibited better condensation and complexation properties than PEI 25. The transfection efficiency of copolymer polyplexes was increased 10-fold compared to PEI 25 control and the hemolytic activity was markedly reduced. After intravenous injection into mice, plasmids complexed to PEI-PEG(30 k) copolymers resulted in significantly increased circulation times. After stabilizing the polyplexes with a redox sensitive, biodegradable crosslinker, blood levels of plasmid could be further increased up to 125% compared to PEI. These results demonstrate that polyplexes prepared using a combined strategy of surface crosslinking and PEGylation seem to provide promising properties as stable, long circulating vectors.

Crosslinked nanocarriers based upon poly(ethylene imine) for systemic plasmid delivery: in vitro characterization and in vivo studies in mice.

Crosslinked poly(ethylene imine) (PEI) polyplexes for intracellular DNA release were generated using a low molecular weight crosslinking reagent, Dithiobis(succinimidyl propionate) (DSP). Disulfide bonds of the crosslinked polyplexes were susceptible to intracellular redox conditions and DNA release was observed using an ethidium bromide exclusion assay and dynamic light scattering. Transfection experiments were performed to elucidate the effect of extra- and intracellular redox conditions. Pharmacokinetics and organ accumulation of uncrosslinked and crosslinked polyplexes were compared and gene expression patterns were measured in mice 24 h after intravenous injection. Crosslinked PEI and plasmid DNA formed stable polyplexes in a size range of 100-300 nm, with zeta potentials between +16.4 and +26.1 mV. DNA release occurred after cleavage of the disulfide bonds. Cell culture experiments under reducing conditions as well as with glutathione loaded cells confirmed the proposed intracellular activation. A significant influence of the intracellular glutathione status on the transfection efficiency was observed. Pharmacokinetic profiles of crosslinked PEI/DNA polyplexes in mice after intravenous administration showed higher blood levels for crosslinked polyplexes. These polyplexes accumulated mainly in the liver and the lungs. In vivo transfection data revealed significantly reduced (unwanted) lung transfection while liver transfection predominated. These studies suggest that crosslinked polyplexes are more stable in circulation and retain their transfection efficiency after intravenous administration.

Stabilized nanocarriers for plasmids based upon cross-linked poly(ethylene imine).

Stabilized PEI/DNA polyplexes were generated by cross-linking PEI with biodegradable disulfide bonds. The reaction conversion of different PEIs with the amine reactive cross-linker dithiobis(succinimidyl propionate) (DSP) was investigated, and the molecular weight of the reaction products was identified. Light scattering and microelectrophoresis were employed to assess size and zeta potential of the resulting polyplexes. Polyplex morphology and mechanic stability were investigated using atomic force microscopy. Finally, albumin and erythrocyte interactions and stability against polyanions and high ionic strength were checked. Polyplexes of PEI and DNA were prepared by two different formulation methods, either using pre-cross-linked polymers or by cross-linking polyplexes after complexation. Only the latter method yielded small (100-300 nm) polyplexes with a positive zeta potential when HMW PEI was used, whereas cross-linked LMW PEI resulted in polyplexes with increased size (>1000 nm) and zeta potentials down to -20 mV. In addition, only cross-linking after polyplex formation was able to enhance resistance against polyanion exchange and high ionic strength. AFM images revealed no changes in the morphology of cross-linked HWM PEI polyplexes, and indentation force measurements using AFM significantly increased mechanical stability of cross-linked HMW PEI polyplexes. These polyplexes also displayed significantly reduced interactions with major blood components like albumin and erythrocytes. The resulting biocompatible particles offer a means of combining enhanced polyplex stability with redox-triggered activation for in vivo application.

Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes.

Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.

Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives.

The continually increasing wealth of knowledge about the role of genes involved in acquired or hereditary diseases renders the delivery of regulatory genes or nucleic acids into affected cells a potentially promising strategy. Apart from viral vectors, non-viral gene delivery systems have recently received increasing interest, due to safety concerns associated with insertional mutagenesis of retro-viral vectors. Especially cationic polymers may be particularly attractive for the delivery of nucleic acids, since they allow a vast synthetic modification of their structure enabling the investigation of structure-function relationships. Successful clinical application of synthetic polycations for gene delivery will depend primarily on three factors, namely (1) an enhancement of the transfection efficiency, (2) a reduction in toxicity and (3) an ability of the vectors to overcome numerous biological barriers after systemic or local administration. Among the polycations presently used for gene delivery, poly(ethylene imine), PEI, takes a prominent position, due to its potential for endosomal escape. PEI as well as derivatives of PEI currently under investigation for DNA and RNA delivery will be discussed. This review focuses on structure-function relationships and the physicochemical aspects of polyplexes which influence basic characteristics, such as complex formation, stability or in vitro cytotoxicity, to provide a basis for their application under in vivo conditions. Rational design of optimized polycations is an objective for further research and may provide the basis for a successful cationic polymer-based gene delivery system in the future.