Systemic tumor-targeted sodium iodide symporter (NIS) gene therapy of hepatocellular carcinoma mediated by B6 peptide polyplexes.

BACKGROUND: Nonviral polymer-based gene transfer represents an adaptable system for tumor-targeted gene therapy because various design strategies of shuttle systems, together with the mechanistic concept of active tumor targeting, lead to improved gene delivery vectors resulting in higher tumor specificity, efficacy and safety. METHODS: Using the sodium iodide symporter (NIS) as a theranostic gene, nonviral gene delivery vehicles based on linear polyethylenimine (LPEI), polyethylene glycol (PEG) and coupled to the synthetic peptide B6 (LPEI-PEG-B6), which specifically binds to tumor cells, were investigated in a hepatocellular carcinoma xenograft model for tumor selectivity and transduction efficiency. RESULTS: In vitro incubation of three different tumor cell lines with LPEI-PEG-B6/NIS resulted in significant increase in iodide uptake activity compared to untargeted and empty vectors. After establishment of subcutaneous HuH7 tumors, NIS-conjugated nanoparticles were injected intravenously followed by analysis of radioiodide biodistribution using 123 I-scintigraphy showing significant perchlorate-sensitive iodide accumulation in tumors of LPEI-PEG-B6/NIS-treated mice (8.0 ± 1.5% ID/g 123 I; biological half-life of 4 h). After four cycles of repetitive polyplex/131 I applications, a significant delay of tumor growth was observed, which was associated with markedly improved survival in the therapy group. CONCLUSIONS: These results clearly demonstrate that systemic in vivo NIS gene transfer using nanoparticle vectors coupled to B6 tumor targeting ligand is capable of inducing tumor-specific radioiodide uptake. This promising gene therapy approach opens the exciting prospect of NIS-mediated radionuclide therapy in metastatic cancer, together with the possibility of combining several targeting ligands to enhance selective therapeutic efficacy in a broad field of cancer types with various receptor expression profiles.

Toward Artificial Immunotoxins: Traceless Reversible Conjugation of RNase A with Receptor Targeting and Endosomal Escape Domains.

The specific transport of bioactive proteins into designated target cells is an interesting and challenging perspective for the generation of innovative biopharmaceuticals. Natural protein cytotoxins perform this task with outstanding efficacy. They enter cells with receptor-targeted specificity, respond to changing intracellular microenvironments, and by various mechanisms translocate their cytotoxic protein subunit into the cytosol. Here we imitate this toxin-based delivery strategy in an artificial setting, by bioreversible conjugation of a cytotoxic cargo protein (RNase A) with receptor-targeting PEG-folate and the pH-specific endosomolytic peptide INF7 as synthetic delivery domains. Covalent modification of the cargo protein was achieved using the pH-labile AzMMMan linker and copper-free click chemistry with DBCO-modified delivery modules. This linkage is supposed to enable traceless intracellular release of the RNase A after exposure to the endosomal weakly acidic environment. Delivery of RNase A via this polycation-free delivery strategy resulted in high cytotoxicity against receptor-positive KB tumor cells only when both PEG-folate and INF7 were attached.

pH-Reversible Cationic RNase A Conjugates for Enhanced Cellular Delivery and Tumor Cell Killing.

Intracellularly-acting therapeutic proteins are considered promising alternatives for the treatment of various diseases. Major limitations of their application are low efficiency of intracellular delivery and possible reduction of protein activity during derivatization. Herein, we report pH-sensitive covalent modification of proteins with a histidine-rich cationic oligomer (689) for efficient intracellular transduction and traceless release of functional proteins. Enhanced Green fluorescent protein (EGFP), as model for the visualization of protein transduction, and RNase A, as therapeutic protein with antitumoral effect, were modified with the pH-sensitive bifunctional AzMMMan linker and varying amounts of cationic oligomer. The modification degree showed impact on the internalization and cellular distribution of EGFP as well as the biological effect of RNase A conjugates, which mediated considerable toxicity against cancer cells at optimal ratio. The presented conjugates demonstrate their qualification to achieve efficient intracellular delivery and controlled release without protein inactivation and potential prospective applications in protein-based therapies.

Combination of sequence-defined oligoaminoamides with transferrin-polycation conjugates for receptor-targeted gene delivery.

BACKGROUND: Transferrin receptor (TfR), over-expressed on a majority of malignant cells, has been widely studied as a target for drug, protein and gene delivery. Both stable nucleic acid compact ability and efficient cytosol gene release capability are essential for the success of gene delivery. METHODS: In the present study, a novel nonviral TfR-targeted gene delivery system was developed based on sequence-defined cationic oligoaminoamide oligomers with endolysosomal buffer capacity and DNA binding transferrin (Tf) polycation conjugates. RESULTS: Gene transfer activities were significantly increased in a series of TfR over-expressing human tumour cell lines (K562, DU145 and KB) with mixed ternary polyplexes containing Tf-conjugates and 3-arm (386 and 689) or 4-arm (577, 579 and 607) sequence-defined oligomers. Especially polyplexes containing a histidine-rich 4-arm oligomer (607) and Tf-PEG-PEI achieved a 100-fold increase in gene expression compared to previously established formulations. Tf competition experiments indicate enhanced polyplex internalization via TfR as prerequisite for the high transfection activity. The additional histidines in the oligoaminoamide oligomer structure are required for more effective endolysosomal escape of the gene delivery vehicle. Polyplexes formed by first mixing pDNA with the oligomer as a cationic core, followed by the addition of the Tf-polycation conjugate for presentation at the exterior nanoparticles, exhibited the highest transfection activity. CONCLUSIONS: Utilizing the synergistic effects of Tf for receptor targeting and oligomer for packaging and endolysosomal escape, an efficient gene delivery carrier was developed. The mixed polyplex containing Tf-polycation conjugates and histidinylated 4-arm oligomer with succinoyl tetraethylene pentamine or glutaroyltriethylene tetramine building blocks exhibited the highest gene transfection efficiency in TfR over-expressing human tumour cell lines.

Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes.

The cationizable nature of 'proton-sponge' transfection agents facilitates pDNA delivery in several steps. Protonated amines account for electrostatic DNA binding and cellular uptake, buffering amines mediate polyplex escape from acidifying intracellular vesicles. As demonstrated with a sequence-defined library of oligo(ethanamino)amides containing selected oligoethanamino acids and histidines, the total protonation capacity as well as the cationization pH profile within the endolysosomal range have critical impact on gene transfer. Building blocks with even numbered amine groups (Gtt, Sph) exhibited higher total endolysosomal buffer capacity than odd number (Stp) analogs. Within the endolysosomal range, Gtt has the highest buffer capacity around pH5, whereas Stp has its maximum around pH7. Histidines increased the total buffer capacity, resulted in a more continuous cationization pH profile and greatly improved transgene expression in vitro and in vivo. Using receptor targeted and polyethylene glycol shielded polyplexes, better endosomal escape and >100-fold enhanced transfection was detected. FROM THE CLINICAL EDITOR: Proton-sponge transfection agents for pDNA delivery are characterized in this study, demonstrating over 100-fold enhanced transection and better endosomal escape by using receptor targeted and polyethylene glycol shielded polyplexes.

Adenoviral vectors coated with PAMAM dendrimer conjugates allow CAR independent virus uptake and targeting to the EGF receptor.

Adenovirus type 5 (Ad) is an efficient gene vector with high gene transduction potential, but its efficiency depends on its native cell receptors coxsackie- and adenovirus receptor (CAR) for cell attachment and α(v)ß(3/5) integrins for internalization. To enable transduction of CAR negative cancer cell lines, we have coated the negatively charged Ad by noncovalent charge interaction with cationic PAMAM (polyamidoamine) dendrimers. The specificity for tumor cell infection was increased by targeting the coated Ad to the epidermal growth factor receptor using the peptide ligand GE11, which was coupled to the PAMAM dendrimer via a 2 kDa PEG spacer. Particles were examined by measuring surface charge and size, the degree of coating was determined by transmission electron microscopy. The net positive charge of PAMAM coated Ad enhanced cellular binding and uptake leading to increased transduction efficiency, especially in low to medium CAR expressing cancer cell lines using enhanced green fluorescent protein or luciferase as transgene. While PAMAM coated Ad allowed for efficient internalization, coating with linear polyethylenimine induced excessive particle aggregation, elevated cellular toxicity and lowered transduction efficiency. PAMAM coating of Ad enabled successful transduction of cells in vitro even in the presence of neutralizing antibodies. Taken together, this study clearly proves noncovalent, charge-based coating of Ad vectors with ligand-equipped dendrimers as a viable strategy for efficient transduction of cells otherwise refractory to Ad infection.

PolyIC GE11 polyplex inhibits EGFR-overexpressing tumors.

Phage display has identified the dodecapeptide YHWYGYTPQNVI (GE11) as a ligand that binds to the epidermal growth factor receptor (EGFR) but does not activate the receptor. Here, we compare the EGFR binding affinities of GE11, EGF, and their polyethyleneimine-polyethyleneglycol (PEI-PEG) conjugates. We found that although GE11 by itself does not exhibit measurable affinity to the EGFR, tethering it to PEI-PEG increases its affinity markedly, and complex formation with polyinosine/cytosine (polyIC) further enhances the affinity to the submicromolar range. PolyIC/PPGE11 has a similar strong antitumor effect against EGFR overexpressing tumors in vitro and in vivo, as polyIC/polyethyleneimine-polyetheleneglycol-EGF (polyIC/PP-EGF). Absence of EGFR activation, as previously shown by us and easier production of GE11 and GE11 conjugates, confer polyIC/PPGE11 a significant advantage over similar EGF-based polyplexes as a potential therapy of EGFR overexpressing tumors.

Poly(I:C)-mediated tumor growth suppression in EGF-receptor overexpressing tumors using EGF-polyethylene glycol-linear polyethylenimine as carrier.

PURPOSE: To develop a novel polyethylenimine (PEI)-based polymeric carrier for tumor-targeted delivery of cytotoxic double-stranded RNA polyinosinic:polycytidylic acid, poly(I:C). The novel carrier should be chemically less complex but at least as effective as a previously developed tetra-conjugate containing epidermal growth factor (EGF) as targeting ligand, polyethylene glycol (PEG) as shielding spacer, 25 kDa branched PEI as RNA binding and endosomal buffering agent, and melittin as endosomal escape agent. METHODS: Novel conjugates were designed employing a simplified synthetic strategy based on 22 kDa linear polyethylenimine (LPEI), PEG spacers, and recombinant EGF. The efficacy of various conjugates (different PEG spacers, with and without targeting EGF) in poly(I:C)-mediated cell killing was evaluated in vitro using two human U87MG glioma cell lines. The most effective polyplex was tested for in vivo activity in A431 tumor xenografts. RESULTS: Targeting conjugate LPEI-PEG2 kDa-EGF was found as most effective in poly(I:C)-triggered killing of tumor cells in vitro. The efficacy correlated with glioma cell EGFR levels. Repeated intravenous administration of poly(I:C) polypexes strongly retarded growth of A431 human tumor xenograft in mice. CONCLUSIONS: The optimized LPEI-PEG2 kDa-EGF conjugate displays reduced chemical complexity and efficient poly(I:C)-mediated killing of EGFR overexpressing tumors in vitro and in vivo.

Synthesis of Polyethylenimine-Based Nanocarriers for Systemic Tumor Targeting of Nucleic Acids.

Nucleic acid-based therapies offer the option to treat tumors in a highly selective way, while toxicity towards healthy tissue can be avoided when proper delivery vehicles are used. We have recently developed carrier systems based on linear polyethylenimine, which after chemical coupling of protein- or peptide-based ligands can form nanosized polyplexes with plasmid DNA (pDNA) or RNA and deliver their payload into target cells by receptor-mediated endocytosis. This chapter describes the synthesis of LPEI from a precursor polymer and the current coupling techniques and purification procedure for peptide conjugates with linear polyethylenimine. A protocol is also given for the formation and characterization of polyplexes formed with LPEI conjugate and pDNA.

Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors.

The transferrin (TfR) and epidermal growth factor receptors (EGFR) are known to be overexpressed on the surface of a wide variety of tumor cells. Therefore, the peptides B6 (TfR specific) and GE11 (targeted to the EGFR) were linked to the PAMAM (polyamidoamine) structure via a polyethylenglycol (PEG) 2 kDa chain with the aim of improving the silencing capacity of the PAMAM-based dendriplexes. The complexes showed an excellent binding capacity to the siRNA with a maximal condensation at nitrogen/phosphate (N/P) 2. The nanoparticles formed exhibited hydrodynamic diameters below 200 nm. The zeta potential was always positive, despite the complexes containing the PEG chain in the structure showing a drop of the values due to the shielding effect. The gene silencing capacity was assayed in HeLa and LS174T cells stably transfected with the eGFPLuc cassette. The dendriplexes containing a specific anti luciferase siRNA, assayed at different N/P ratios, were able to mediate a mean decrease of the luciferase expression values of 14% for HeLa and 20% in LS174T cells, compared to an unspecific siRNA-control. (p < 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%.

EGFR-targeted nonviral NIS gene transfer for bioimaging and therapy of disseminated colon cancer metastases.

Liver metastases present a serious problem in the therapy of advanced colorectal cancer (CRC), as more than 20% of patients have distant metastases at the time of diagnosis with less than 5% being cured. Consequently, new therapeutic approaches are of major need together with high-resolution imaging methods that allow highly specific detection of small metastases. The unique combination of reporter and therapy gene function of the sodium iodide symporter (NIS) may represent a promising theranostic strategy for CRC liver metastases allowing non-invasive imaging of functional NIS expression and therapeutic application of 131I. For targeted NIS gene transfer polymers containing linear polyethylenimine (LPEI), polyethylene glycol (PEG) and the epidermal growth factor receptor (EGFR)-specific ligand GE11 were complexed with human NIS DNA (LPEI-PEG-GE11/NIS). Tumor specificity and transduction efficiency were examined in high EGFR-expressing LS174T metastases by non-invasive imaging using 18F-tetrafluoroborate (18F-TFB) as novel NIS PET tracer. Mice that were injected with LPEI-PEG-GE11/NIS 48 h before 18F-TFB application showed high tumoral levels (4.8±0.6% of injected dose) of NIS-mediated radionuclide uptake in comparison to low levels detected in mice that received untargeted control polyplexes. Three cycles of intravenous injection of EGFR-targeted NIS polyplexes followed by therapeutic application of 55.5 MBq 131I resulted in marked delay in metastases spread, which was associated with improved animal survival. In conclusion, these preclinical data confirm the enormous potential of EGFR-targeted synthetic polymers for systemic NIS gene delivery in an advanced multifocal CRC liver metastases model and open the exciting prospect of NIS-mediated radionuclide therapy in metastatic disease.

Targeted siRNA Delivery Using a Lipo-Oligoaminoamide Nanocore with an Influenza Peptide and Transferrin Shell.

Developing RNA-interference-based therapeutic approaches with efficient and targeted cytosolic delivery of small interfering RNA (siRNA) is remaining a critical challenge since two decades. Herein, a multifunctional transferrin receptor (TfR)-targeted siRNA delivery system (Tf&INF7) is designed based on siRNA complexes formed with the cationic lipo-oligoamino amide 454, sequentially surface-modified with polyethylene glycol-linked transferrin (Tf) for receptor targeting and the endosomolytic peptide INF7 for efficient cytosolic release of the siRNA. Effective Tf&INF7 polyplex internalization and target gene silencing are demonstrated for the TfR overexpressing tumor cell lines (K562, D145, and N2a). Treatment with antitumoral EG5 siRNA results in a block of tumor cell growth and triggered apoptosis. Tf-modified polyplexes are far more effective than the corresponding albumin- (Alb) or nonmodified 454 polyplexes. Competition experiments with excess of Tf demonstrate TfR target specificity. As alternative to the ligand Tf, an anti-murine TfR antibody is incorporated into the polyplexes for specific targeting and gene silencing in the murine N2a cell line. In vivo distribution studies not only demonstrate an enhanced tumor residence of siRNA in N2a tumor-bearing mice with the Tf&INF7 as compared to the 454 polyplex group but also a reduced siRNA nanoparticle stability limiting the in vivo performance.

Evaluation of improved PAMAM-G5 conjugates for gene delivery targeted to the transferrin receptor.

The transfection activity of non-viral vectors is highly dependent on the delivery capacity of the carriers. Therefore, the aim of this work was to evaluate the activity of a new PAMAM dendrimer-Transferrin conjugate (P-Tf) with improved gene delivery activity to cancer cells. The formulations containing the novel P-Tf were able to bind pDNA and protect it from the activity of DNAse I enzyme. Moreover, it formed nanoparticles with positive surface charge, although the presence of Tf led to a decrease of the zeta potential to almost electroneutral values. This new vector, formulated at N/P 6, exhibited excellent transfection efficacy in HeLa, HepG2 and CT26 cell lines, whereas in Neuro2A no improvement was achieved. Compared to control complexes with branched polyethylenimine (bPEI), targeted dendriplexes (complexes formed by cationic polymeric dendrimers and DNA) were more efficient in HepG2 and HeLa cells. Cellular viability was always kept over 80% in these cell lines with higher values than bPEI control polyplexes. The uptake via receptor-mediated endocytosis was ensured by a competition assay, by adding an excess of free Tf, which led to a decrease in the transfection activity of targeted dendriplexes.

Synthesis of polyethylenimine-based nanocarriers for systemic tumor targeting of nucleic acids.

Nucleic acid-based therapies offer the option to treat tumors in a highly selective way, while toxicity towards healthy tissue can be avoided when proper delivery vehicles are used. We have recently developed carrier systems based on linear polyethylenimine, which after chemical coupling of proteinous or peptidic ligands can form nanosized polyplexes with plasmid DNA or RNA and deliver their payload into target cells by receptor-mediated endocytosis. This chapter describes the synthesis of linear PEI (LPEI) from a precursor polymer and the current coupling techniques and purification procedure for peptide conjugates with linear polyethylenimine. A protocol is also given for the formation and characterization of polyplexes formed with LPEI conjugate and plasmid DNA.

Controlled shielding and deshielding of gene delivery polyplexes using hydroxyethyl starch (HES) and alpha-amylase.

The non-viral delivery of nucleic acids faces many extracellular and intracellular hurdles on the way from injection site to the site of action. Among these, aggregation in the blood stream and rapid elimination by the mononuclear phagocytic system (MPS) represent strong obstacles towards successful development of these promising therapeutic modalities. Even the state-of-the-art solutions using PEGylation show low transfection efficiency due to limited uptake and hindered endosomal escape. Engineering the carriers with sheddable coats reduces aggregation and phagocytosis due to the effective shielding, while the controlled deshielding at the desired site of action enhances the uptake and intracellular release. This work reports for the first time the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. HES, with different molar masses, was grafted to polyethylenimine (PEI) and characterized using (1)H NMR, colorimetric copper-assay, and SEC. HES-PEI conjugates were used to generate polyplexes with the luciferase-expressing plasmid DNA pCMVluc, and were characterized by DLS and zeta potential measurements. Deshielding was tested in vitro by zeta potential measurements and, erythrocyte aggregation assay upon addition of α-amylase (AA) to the HES-decorated particles. The addition of AA led to gradual increase in the zeta potential of the nanoparticles over 0.5 to 1h and to a higher aggregation tendency for erythrocytes due to the degradation of the HES-coat and exposure of the polyplexes' positive charge. In vitro transfection experiments were conducted in 2 cell-lines±AA in the culture medium. The amylase-treated HES-decorated complexes showed up to 2 orders of magnitude higher transfection levels compared to the untreated HES-shielded particles, while AA had no effect on the transfection of PEG-coated or uncoated polyplexes. Finally, flow cytometry showed that the addition of AA increased the amount of delivered DNA per cell for the HES-shielded polyplexes. This study shows that decorating nanoparticles with HES can be a promising tool for the controlled shielding/deshielding of polyplexes.

Dual-targeted polyplexes: one step towards a synthetic virus for cancer gene therapy.

Incorporating ligands into nano-scale carriers for specific delivery of therapeutic nucleic acids to tumor sites is a promising approach in anti-cancer strategies. Current artificial vector systems however still suffer from efficient and specific delivery, compared to their natural counterparts and addressed receptor types rarely are exclusively expressed on target cells. In this study synthetic dual receptor targeted polyplexes were developed, mimicking biphasic cell entry characteristics of natural viruses to increase efficiency and specificity by a dual-receptor internalization mechanism. For engineering the synthetic dual targeted vector system, the transferrin targeting peptide B6 was evaluated for the first time in the context of PEGylated PEI based polyplexes. As a second ligand, arginine-glycine-aspartic acid (RGD) containing peptide was incorporated for simultaneous integrin targeting. Cellular association, cellular uptake, transfection efficiency and accordant competition experiments displayed specificity of both ligands for each targeted receptor in two prostate cancer cell lines. A clear synergy of dual targeting over the combination of single-targeted polyplexes was found, suggesting that the dual targeting strategy is one step towards safe vectors for therapeutic approaches.