RESUMO
Tumor heterogeneity, within and between tumors, may have severe implications for tumor therapy, especially for targeted gene therapy, where single-targeted approaches often result in limited efficacy and therapy resistance. Polymer-formulated nonviral vectors provide a potent delivery platform for cancer therapy. To improve applicability for future clinical use in a broad range of patients and cancer types, a dual-targeting approach was performed. Synthetic LPEI-PEG2kDa-based polymer backbones were coupled to two tumor-specific peptide ligands GE11 (EGFR-targeting) and cMBP (cMET-targeting). The dual-targeting approach was used to deliver the theranostic sodium iodide symporter (NIS) gene to hepatocellular cancer. NIS as auspicious theranostic gene allows noninvasive imaging of functional NIS gene expression and effective anticancer radioiodide therapy. Enhanced tumor-specific transduction efficiency of dual-targeted polyplexes compared to single-targeted polyplexes was demonstrated in vitro using tumor cell lines with different EGFR and cMET expression and in vivo by 124I-PET-imaging. Therapeutic efficacy of the bispecific concept was mirrored by significantly reduced tumor growth and perfusion, which was associated with prolonged animal survival. In conclusion, the dual-targeting approach highlights the benefits of a bifunctional strategy for a future clinical translation of the bioimaging-based NIS-mediated radiotherapy allowing efficient targeting of heterogeneic tumors with variable receptor expression levels.
Assuntos
Carcinoma Hepatocelular/genética , Terapia Genética/métodos , Nanomedicina Teranóstica/métodos , Animais , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/análise , Receptores ErbB/genética , Feminino , Expressão Gênica/genética , Técnicas de Transferência de Genes , Heterogeneidade Genética , Xenoenxertos , Humanos , Ligantes , Neoplasias Hepáticas/genética , Camundongos , Camundongos Nus , Peptídeos/síntese química , Peptídeos/genética , Polímeros , Proteínas Proto-Oncogênicas c-met/análise , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
BACKGROUND: Developing new drug delivery carriers addressing chemoresistance is still full of challenges and opportunities. As the rapid development of small interfering RNA (siRNA) provides promising therapeutic perspectives, nanocarriers for drug and siRNA co-delivery present new alternatives for cancer therapy. METHODS: A co-delivery nanosystem for methotrexate (MTX) or gamma-glutamylated derivatives (gE2 -MTX and gE5 -MTX) and antitumoral EG5 siRNA has been developed utilizing the sequence defined cationic lipo-oligomers 454, 1021 and 1027. Based on a lipo-oligomer-MTX-siRNA core, an epidermal growth factor receptor (EGFR) targeted delivery system was established via post modification with the GE11 targeting peptide. RESULTS: Almost 100% MTX derivative incorporation was achieved in gE2 -MTX or gE5 -MTX siRNA/454 polyplexes, whereas the particle sizes (100-150 nm) and siRNA binding abilities were well maintained. Our co-delivery system greatly increased the MTX sensitivity of MTX resistant KB cells. Enhanced cellular internalization of GE11 siRNA/454 polyplexes incorporating either gE2 -MTX or gE5 -MTX was observed and attributed to GE11-mediated targeting of EGFR overexpressing KB cells. GE11 modified gE2 -MTX or gE5 -MTX EG5 siRNA polyplexes illustrated the highest anti-tumoral activities compared to free MTX or nontargeted polyplexes. The His-containing gE2 -MTX or gE5 -MTX siRNA/1027 polyplexes showed increased tumor cell killing compared to the His-free analogous 1021 polyplexes. CONCLUSIONS: A new strategy for co-delivering negatively charged MTX and cytotoxic siRNA has been developed by utilizing sequence defined cationic lipo-oligomers. Mediated by the combined effect of antifolate MTX, antimitotic EG5 siRNA and EGFR targeting by GE11, superior tumor cell killing was obtained with GE11 gE2 -MTX or gE5 -MTX EG5 siRNA/454 polyplexes.
Assuntos
Metotrexato/farmacologia , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Receptores ErbB/genética , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Metotrexato/administração & dosagem , Nanopartículas , Peptídeos/química , RNA Interferente Pequeno/administração & dosagemRESUMO
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.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Oligopeptídeos/genética , Simportadores/química , Simportadores/genética , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Radioisótopos do Iodo/química , Camundongos , Camundongos Nus , Polietilenoglicóis/química , Polietilenoimina/química , Polímeros/química , Distribuição TecidualRESUMO
The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g (123)I, biological half-life 3 hours) by (123)I-scintigraphy. Therapy studies with three cycles of polyplexes and (131)I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.
Assuntos
Técnicas de Transferência de Genes , Polímeros , Proteínas Proto-Oncogênicas c-met/genética , Simportadores/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Polietilenoglicóis/química , Polímeros/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Nanomedicina Teranóstica , TransfecçãoRESUMO
Sequence-defined lipo-oligomers generated via solid-phase assisted synthesis have been developed as siRNA delivery systems for RNA-interference (RNAi) based gene silencing. Here, novel siRNA lipo-polyplexes were established, which were postmodified with monovalent or bivalent DBCO-PEG24 agents terminated with peptide GE11 (YHWYGYTPQNVI) for epidermal growth factor receptor (EGFR)-targeted siRNA delivery into EGFR-positive tumor cells. Lipo-oligomers containing eight cationizable succinoyltetraethylene-pentamine (Stp) units mediated higher siRNA nanoparticle core stability than those containing four Stp units, and the incorporation of histidines for enhanced endosomal buffer capacity resulted in an improved gene silencing efficiency. Lipo-polyplexes modified with monovalent or bivalent PEG-GE11 via the copper-free click reaction possessed significantly enhanced cellular internalization and transfection efficiency in EGF receptor-positive human cervical KB and hepatoma Huh7 cells in comparison with the corresponding lipo-polyplexes shielded with PEG24 without targeting. Furthermore, modification with the bivalent DBCO-PEG24-GE11 ligand resulted in higher gene silencing efficiency than modification with the same equivalents of the monovalent DBCO-PEG24-GE11 ligand.
Assuntos
Receptores ErbB , Inativação Gênica , Linhagem Celular Tumoral , Receptores ErbB/genética , Humanos , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
For successful nonviral gene delivery, cationic polymers are promising DNA carrier, which need to comprise several functionalities. The current work focuses on the postincorporation of epidermal growth factor receptor (EGFR) targeted PEGylation agents onto lipopolyplexes for pDNA delivery. T-shaped lipo-oligomers are previously found to be effective sequence-defined carriers for pDNA and siRNA. Here, the bis-oleoyl-oligoaminoethanamide 454 containing tyrosine trimer-cysteine ends is applied for complex formation with pDNA coding for luciferase or sodium iodide symporter (NIS). In a second step, the lipopolyplexes are modified via disulfide formation with sequence-defined monovalent or bivalent PEGylation agents containing one or two 3-nitro-2-pyridinesulfenyl (NPys)-activated cysteines, respectively. For targeting, the polyethylene glycol (PEG) agents comprise the EGFR targeting peptide GE11. In comparison of all transfection complexes, 454 lipopolyplexes modified with the bidentate PEG-GE11 agent show the best, EGFR-dependent uptake as well as luciferase and NIS gene expression into receptor-positive tumor cells.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Radioisótopos do Iodo/química , Plasmídeos/genética , Polietilenoglicóis/química , Polímeros/química , Polímeros/uso terapêutico , RNA Interferente Pequeno/química , RNA Interferente Pequeno/uso terapêutico , Simportadores/química , Simportadores/uso terapêutico , TransfecçãoRESUMO
Nucleic acid molecules are important therapeutic agents in the field of antisense oligonucleotide, RNA interference, and gene therapies. Since nucleic acids are not able to cross cell membranes and enter efficiently into cells on their own, the development of efficient, safe, and precise delivery systems is the crucial challenge for development of nucleic acid therapeutics. For the delivery of nucleic acids to their intracellular site of action, either the cytosol or the nucleus, several extracellular and intracellular barriers have to be overcome. Multifunctional carriers may handle the different special requirements of each barrier. The complexity of such macromolecules however poses a new hurdle in medical translation, which is the chemical production in reproducible and well-defined form. Solid-phase assisted synthesis (SPS) presents a solution for this challenge. The current review provides an overview on the design and SPS of precise sequence-defined synthetic carriers for nucleic acid cargos.
Assuntos
Portadores de Fármacos/química , Ácidos Nucleicos/administração & dosagem , Técnicas de Síntese em Fase Sólida/métodos , Animais , Endossomos , Terapia Genética/métodos , Humanos , Ligantes , Camundongos , Ácidos Nucleicos/química , Peptídeos/químicaRESUMO
Due to its minimal size and lack of bacterial backbone sequences, minicircle (MC) DNA presents a promising alternative to plasmid DNA (pDNA) for non-viral gene delivery in terms of biosafety and improved gene transfer. Here, luciferase pDNA (pCMV-luc) and analogous MC DNA (MC07.CMV-luc) were formulated into polyplexes with c-Met targeted, PEG-shielded sequence-defined oligoaminoamides, or linear PEI (linPEI) as standard transfection agent. Distinct physicochemical and biological characteristics were observed for polyplexes formed with either pDNA or MC DNA as vectors. The carriers were found to dominate the shape of polyplexes, whereas the DNA type was decisive for the nanoparticle size. c-Met-targeted, tyrosine trimer-containing polyplexes were optimized into compacted rod structures with a size of 65-100 nm for pDNA and 35-40 nm for MC. Notably, these MC polyplexes display a lack of cell cycle dependence of transfection and a â¼200-fold enhanced gene transfer efficiency in c-Met-positive DU145 prostate carcinoma cultures over their tyrosine-free pDNA analogues.
Assuntos
DNA Circular , Técnicas de Transferência de Genes , Vetores Genéticos , Plasmídeos , Polímeros , Animais , Ciclo Celular , Linhagem Celular Tumoral , DNA Circular/química , DNA Circular/genética , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/genética , Humanos , Nanopartículas , Tamanho da Partícula , Peptídeos/química , Plasmídeos/química , Plasmídeos/genética , Polietilenoimina/química , Polímeros/química , Transfecção , TransgenesRESUMO
Cationic polymers are promising components of the versatile platform of non-viral nucleic acid (NA) delivery agents. For a successful gene delivery system, these NA vehicles need to comprise several functionalities. This work focuses on the modification of oligoaminoamide carriers with hydrophilic oligomer blocks mediating nanoparticle shielding potential, which is necessary to prevent aggregation or dissociation of NA polyplexes in vitro, and hinder opsonization with blood components in vivo. Herein, the shielding agent polyethylene glycol (PEG) in three defined lengths (12, 24, or 48 oxyethylene repeats) is compared with two peptidic shielding blocks composed of four or eight repeats of sequential proline-alanine-serine (PAS). With both types of shielding agents, we found opposing effects of the length of hydrophilic segments on shielding and compaction of formed plasmid DNA (pDNA) nanoparticles. Two-arm oligoaminoamides with 37 cationizable nitrogens linked to 12 oxyethylene units or four PAS repeats resulted in very compact 40â»50 nm pDNA nanoparticles, whereas longer shielding molecules destabilize the investigated polyplexes. Thus, the balance between sufficiently shielded but still compact and stable particles can be considered a critical optimization parameter for non-viral nucleic acid vehicles based on hydrophilic-cationic block oligomers.
RESUMO
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.
RESUMO
Artificial oligoamino acids with appropriate protecting groups can be used for the sequential assembly of oligoaminoamides on solid-phase. With the help of these oligoamino acids multifunctional nucleic acid (NA) carriers can be designed and produced in highly defined topologies. Here we describe the synthesis of the artificial oligoamino acid Fmoc-Stp(Boc3)-OH, the subsequent assembly into sequence-defined oligomers and the formulation of tumor-targeted plasmid DNA (pDNA) polyplexes.
Assuntos
Aminoácidos/química , DNA/genética , Poliaminas/síntese química , Amidas , Técnicas de Transferência de Genes , Terapia Genética , Neoplasias/genética , Neoplasias/terapia , Plasmídeos/genética , Poliaminas/químicaRESUMO
Sophisticated drug delivery systems are coated with targeting ligands to improve the specific adhesion to surface receptors on diseased cells. In our study, we developed a method with which we assessed the potential of peptide ligands to specifically bind to receptor overexpressing target cells. Therefore, a microfluidic setup was used where the cellular adhesion of nanoparticles with ligand and of control nanoparticles was observed in parallel under the same experimental conditions. The effect of the ligand on cellular binding was quantified by counting the number of adhered nanoparticles with ligand and differently labeled control nanoparticles on single cells after incubation under flow conditions. To provide easy-to-synthesize, stable and reproducible nanoparticles which mimic the surface characteristics of drug delivery systems and meet the requirements for quantitative analysis, latex beads based on amine-modified polystyrene were used as model nanoparticles. Two short peptides were tested to serve as targeting ligand on the beads by increasing the specific binding to HuH7 cells. The c-Met binding peptide cMBP2 was used for hepatocyte growth factor receptor (c-Met) targeting and the peptide B6 for transferrin receptor (TfR) targeting. The impact of the targeting peptide on binding was investigated by comparing the beads with ligand to different internal control beads: 1) without ligand and tailored surface charge (electrostatic control) and 2) with scrambled peptide and similar surface charge, but a different amino acid sequence (specificity control). Our results demonstrate that the method is very useful to select suitable targeting ligands for specific nanoparticle binding to receptor overexpressing tumor cells. We show that the cMBP2 ligand specifically enhances nanoparticle adhesion to target cells, whereas the B6 peptide mediates binding to tumor cells mainly by nonspecific interactions. All together, we suggest that cMBP2 is a suitable choice for specific receptor targeting whereas the peptide B6 should not be considered as specific targeting moiety.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas/metabolismo , Neoplasias/metabolismo , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores da Transferrina/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Humanos , Dispositivos Lab-On-A-Chip , Ligantes , Nanopartículas/química , Peptídeos/químicaRESUMO
Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.