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ABSTRACT: We have previously described the development of novel sterically stabilized F3-targeted pH-sensitive liposomes, which exhibited the ability to target both cancer and endothelial cells. Herein, the therapeutic potential of those liposomes was assessed upon encapsulation of a siRNA against a well-validated molecular target, PLK1. Treatment of prostate cancer (PC3) and angiogenic endothelial (HMEC-1) cells with F3-targeted liposomes containing anti-PLK1 siRNA resulted in a significant decrease in cell viability, which was mediated by a marked PLK1 silencing, both at the mRNA and protein levels. Furthermore, pre-treatment of PC3 cells with F3-targeted liposomes containing anti-PLK1 siRNA enabled a 3-fold reduction of paclitaxel IC50 and a 2.5-fold augment of the percentage of cancer cells in G2/mitosis arrest, which ultimately culminated in cell death. Overall, the F3-targeted nanocarrier containing an anti-PLK1 siRNA might constitute a valuable system for prostate cancer treatment, either applied in a single schedule or combined with conventional chemotherapy.
European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 05/2013; · 3.15 Impact Factor
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ABSTRACT: The main goal of this work was to assess in vitro the potential of Polo-like kinase gene (PLK-1) as a molecular target within the tumor microenvironment, namely in both cancer cells of tumors of different histological origin and endothelial cells from angiogenic blood vessels, upon silencing with anti-PLK-1 siRNA. In addition, the effect of Plk-1 downregulation on the cancer cells chemosensitization to paclitaxel was further assessed. Downregulation of Plk-1 reduced cancer cells viability from 40 to 85% and up to 59% in endothelial cells. Regarding the latter, it compromised their ability to form new tube-like structures, decreasing the formation of network projections up to 46%. This suggested for the first time, PLK-1 as a valuable angiogenic molecular target. In combination with paclitaxel, anti-PLK-1 siRNA chemosensitized non-small cell lung cancer (NSCLC) and prostate carcinoma cell lines, leading up to a 2-fold increase in the drug cytotoxic effect. Moreover, the sequential incubation of anti-PLK-1 siRNA and paclitaxel led to a decrease in the IC50 of the latter up to 2.7- and 4.1-fold, in A-549 and PC-3 cells, respectively. The combination of anti-PLK-1 siRNA with paclitaxel led to cell cycle arrest, increasing the number of cells at the G2/M and S phases to 1.5 and 1.3-fold in PC-3 cells, and to 1.6 and 1.4-fold in A-549 cells, respectively. Overall, it has been demonstrated that PLK-1 silencing with siRNA can impact multiple cellular players of tumor aggressiveness, thus enabling the opportunity to interfere with different hallmarks of cancer, in tumors with diverse histological origin.
Current Gene Therapy 03/2013; 13(3). · 3.39 Impact Factor
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ABSTRACT: The main goal of this work was to assess in vitro the potential of Polo-like kinase gene (PLK-1) as a molecular
target within the tumor microenvironment, namely in both cancer cells of tumors of different histological origin and endothelial
cells from angiogenic blood vessels, upon silencing with anti-PLK-1 siRNA. In addition, the effect of Plk-1 downregulation
on the cancer cells chemosensitization to paclitaxel was further assessed.
Downregulation of Plk-1 reduced cancer cells viability from 40 to 85% and up to 59% in endothelial cells. Regarding the
latter, it compromised their ability to form new tube-like structures, decreasing the formation of network projections up to
46%. This suggested for the first time, PLK-1 as a valuable angiogenic molecular target. In combination with paclitaxel,
anti-PLK-1 siRNA chemosensitized non-small cell lung cancer (NSCLC) and prostate carcinoma cell lines, leading up to
a 2-fold increase in the drug cytotoxic effect. Moreover, the sequential incubation of anti-PLK-1 siRNA and paclitaxel led
to a decrease in the IC50 of the latter up to 2.7- and 4.1-fold, in A-549 and PC-3 cells, respectively. The combination of
anti-PLK-1 siRNA with paclitaxel led to cell cycle arrest, increasing the number of cells at the G2/M and S phases to 1.5
and 1.3-fold in PC-3 cells, and to 1.6 and 1.4-fold in A-549 cells, respectively. Overall, it has been demonstrated that
PLK-1 silencing with siRNA can impact multiple cellular players of tumor aggressiveness, thus enabling the opportunity
to interfere with different hallmarks of cancer, in tumors with diverse histological origin.
Current Gene Therapy 03/2013; 13(3). · 3.39 Impact Factor
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ABSTRACT: Aim: The design of novel F3-targeted liposomes with adequate features for systemic administration, to enable efficient intracellular delivery of siRNA toward both cancer and endothelial cells from angiogenic blood vessels. Materials & methods: Cellular association studies were performed by flow cytometry. Gene silencing was evaluated with eGFP-overexpressing cells, by flow cytometry and real-time reverse-transcription PCR. Safety and immunogenicity was assessed in CD1 mice. Results: A strong improvement on siRNA internalization by the target cells was achieved, which was correlated with effective downregulation of eGFP. In addition, the F3-targeted liposomes were nonimmunogenic, even in a multiadministration schedule. Conclusion: Overall, the developed F3-targeted nanocarrier constitutes a valuable tool for the specific and safe systemic delivery of siRNA to solid tumors. Original submitted 19 April 2012; Revised submitted 12 September 2012.
Nanomedicine 02/2013; · 5.05 Impact Factor
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ABSTRACT: The present work aimed at designing a lipid-based nanocarrier for siRNA delivery toward two cell sub-populations within breast tumors, the cancer and the endothelial cells from angiogenic tumor blood vessels. To achieve such goal, the F3 peptide, which is specifically internalized by nucleolin overexpressed on both those sub-populations, was used as a targeting moiety. The developed F3-targeted stable nucleic acid lipid particles presented adequate features for systemic administration. In addition, the attachment of the F3 peptide onto the liposomal surface enabled an internalization by both cancer and endothelial cells from angiogenic blood vessels that was significantly higher than the one observed with non-cancer cells. Sequence-specific downregulation of enhanced green fluorescent protein (eGFP) in eGFP-overexpressing human cancer cell lines, both at the protein and mRNA levels, was further observed upon delivery of anti-eGFP siRNA by F3-targeted liposomes, in contrast with the non-targeted counterpart. This effect was highly dependent on the content of poly(ethylene glycol) (PEG), as evidenced by the co-localization studies between the siRNA and the lysosomes. Overall, the present work represents an important contribution toward a nanoparticle with multi-targeting capabilities in breast cancer, both at the cellular and molecular level.
International journal of pharmaceutics 05/2012; 434(1-2):9-19. · 2.96 Impact Factor
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ABSTRACT: RNA interference (RNAi) is a specific gene-silencing mechanism that can be mediated by the delivery of chemical synthesized small-interfering RNA (siRNA). RNAi might constitute a novel therapeutic approach for cancer treatment because researchers can easily design siRNA molecules to inhibit, specifically and potently, the expression of any protein involved in tumor initiation and progression. Despite all the potential of siRNA as a novel class of drugs, the limited cellular uptake, low biological stability, and unfavorable pharmacokinetics of siRNAs have limited their application in the clinic. Indeed, blood nucleases easily degrade naked siRNAs, and the kidneys rapidly eliminate these molecules. Furthermore, at the level of target cells, the negative charge and hydrophilicity of siRNAs strongly impair their cellular internalization. Therefore, the translation of siRNA to the clinical setting is highly dependent on the development of an appropriate delivery system, able to ameliorate siRNA pharmacokinetic and biodistribution properties. In this regard, major advances have been achieved with lipid-based nanocarriers sterically stabilized by poly(ethylene glycol) (PEG), such as the stabilized nucleic acid lipid particles (SNALP). However, PEG has not solved all the major problems associated with siRNA delivery. In this Account, the major problems associated with PEGylated lipid-based nanoparticles, and the different strategies to overcome them are discussed. Although PEG has revolutionized the field of nanocarriers, cumulative experience has revealed that upon repeated administration, PEGylated liposomes lose their ability to circulate over long periods in the bloodstream, a phenomenon known as accelerated blood clearance. In addition, PEGylation impairs the internalization of the siRNA into the target cell and its subsequent escape from the endocytic pathway, which reduces biological activity. An interesting approach to overcome such limitations relies on the design of novel exchangeable PEG-derivatized lipids. After systemic administration, these lipids can be released from the nanoparticle surface. Moreover, the design and synthesis of novel cationic lipids that are more fusogenic and the use of internalizing targeting ligands have contributed to the emergence of novel lipid-based nanoparticles with remarkable transfection efficiency.
Accounts of Chemical Research 05/2012; 45(7):1163-71. · 21.64 Impact Factor
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ABSTRACT: Chemically diverse oxysterols were prepared and evaluated for cytotoxicity, aiming to push forward potency and selectivity. They were tested against seven cancer (HT-29, HepG2, A549, PC3, LAMA-84, MCF-7, and SH-SY5Y) and two noncancerous cell lines (ARPE-19 and BJ). The influence of the oxidation pattern on rings A and B was studied. Oxygen functionalities on ring B, such as oxo, oxime, acetamide, acetate, and alkoxy, were evaluated. Most oxysterols were cytotoxic in the low micromolar range, with emphasis to the tetrols 14 and 34, the 6β methoxy and acetoxy derivatives 21 and 45, and the oxime 28. In general, the oxysterols were more toxic to cancer cells and a set of compounds (9, 14, 21, 28, 45) with very high selectivity was identified. The cytotoxicity of 3β-acetates was lower than that of the parent alcohols, although incubation for a longer period rendered them equally cytotoxic, pointing them as potential prodrugs of oxysterols.
Journal of Medicinal Chemistry 08/2011; 54(18):6375-93. · 4.80 Impact Factor
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ABSTRACT: Limiting tumor invasion to the surrounding healthy tissues has proven to be clinically relevant for anticancer treatment options. We have demonstrated that, within a solid tumor, it is possible to achieve such a goal with the same nanoparticle by intracellular and triggered targeted drug delivery to more than one cell population. We have identified the nucleolin receptor in endothelial and cancer cells in tissue samples from breast cancer patients, which enabled the design of a F3-peptide-targeted sterically stabilized pH-sensitive liposome. The clinical potential of such strategy was demonstrated by the successful specific cellular association by breast cancer cells harvested from tumors of patients submitted to mastectomy. In vitro, the nanoparticle targeted the nucleolin receptor on a cell and ligand-specific manner and improved cytotoxicity of doxorubicin (used as a model drug) towards breast cancer and endothelial cells by 177- and 162-fold, respectively, relative to the commercially available non-targeted non-pH-sensitive liposomes. Moreover, active accumulation of F3-targeted pH-sensitive liposomes into human orthotopic tumors, implanted in the mammary fat pad of nude mice, was registered for a time point as short as 4 h, reaching 48% of the injected dose/g of tissue. Twenty-four hours post-injection the accumulation of the dual-targeted pH-sensitive nanoparticle in the tumor tissue was 33-fold higher than the non-targeted non-pH-sensitive counterpart. In mice treated with the developed targeted nanoparticle significant decrease of the tumor viable rim area and microvascular density, as well as limited invasion to surrounding healthy tissues were observed (as opposed to other tested controls), which may increase the probability of tumors falling in the category of "negative margins" with reduced risk of relapse.
Breast Cancer Research and Treatment 07/2011; 133(1):61-73. · 4.43 Impact Factor
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ABSTRACT: Chronic myeloid leukemia (CML) is triggered by the BCR-ABL oncogene. Imatinib is the first-line treatment of CML; however imatinib resistance and intolerance have been detected in many patients. Therefore, new therapeutic approaches are required. The present work aimed at the development and application of transferrin receptor (TrfR) targeted liposomes co-encapsulating anti-BCR-ABL siRNA and imatinib at different molar ratios. The encapsulation yields and drug loading of each molecule was evaluated. Anti-leukemia activity of the developed formulations co-encapsulating siRNA and imatinib and of the combination of Trf-liposomes carrying siRNA and free imatinib under two different treatment schedules of pre-sensitization was assessed. The results obtained demonstrate that the presence of imatinib significantly decreases the encapsulation yields of siRNA, whereas imatinib encapsulation yields are increased by the presence of siRNA. Cytotoxicity assays demonstrate that the formulations co-encapsulating siRNA and imatinib promote a 3.84-fold reduction on the imatinib IC(50) (from 3.49 to 0.91 µM), whereas a 8.71-fold reduction was observed for the pre-sensitization protocols (from 42.7 to 4.9 nM). It was also observed that the formulations with higher siRNA to imatinib molar ratios promote higher cell toxicity. Thus, the present work describes a novel triple targeting strategy with one single system: cellular targeting (through the targeting ligand, transferrin) and molecular targeting at the BCR-ABL mRNA and Bcr-Abl protein level.
Biotechnology and Bioengineering 12/2010; 107(5):884-93. · 3.95 Impact Factor
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ABSTRACT: The cytotoxicity of oxysterols was systematically studied in tumor and normal cells. Synthetic strategies to prepare this library included oxidations at ring B and a new method to yield 6β-hemiphthalates directly from Δ(5)-steroids. Most oxysterols were cytotoxic and showed selectivity toward cancer cells, LAMA-84 cells (leukemia) being particularly sensitive to 4, 8, 22, and 27 (IC(50) < 5.6 μM). The structural requirements to induce selective toxicity are discussed to shed light on the development of new anticancer drugs.
Journal of Medicinal Chemistry 10/2010; 53(21):7632-8. · 4.80 Impact Factor
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ABSTRACT: Chemical transformation studies were conducted on betulin and betulinic acid, common plant-derived lupane-type triterpenes. The concise synthesis, via a stepwise approach, of betulin and betulinic acid carbamate and N-acylheterocyclic containing derivatives is described. All new compounds, as well as betulinic acid were tested in vitro for their cytotoxic activity. Most of the compounds have shown a better cytotoxic profile than betulinic acid, including the synthesized betulin derivatives. Compounds 25 and 32 were the most promising derivatives, being up to 12-fold more potent than betulinic acid against human PC-3 cell lines (IC(50) values of 1.1 and 1.8 microM, respectively).
Bioorganic & medicinal chemistry 06/2010; 18(12):4385-96. · 2.82 Impact Factor
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ABSTRACT: Anticancer systemic gene silencing therapy has been so far limited by the inexistence of adequate carrier systems that ultimately provide an efficient intracellular delivery into target tumor cells. In this respect, one promising strategy involves the covalent attachment of internalizing-targeting ligands at the extremity of PEG chains grafted onto liposomes. Therefore, the present work aims at designing targeted liposomes containing nucleic acids, with small size, high encapsulation efficiency and able to be actively internalized by SCLC cells, using a hexapeptide (antagonist G) as a targeting ligand. For this purpose, the effect of the liposomal preparation method, loading material (ODN versus siRNA) and peptide-coupling procedure (direct coupling versus post-insertion) on each of the above-mentioned parameters was assessed. Post-insertion of DSPE-PEG-antagonist G conjugates into preformed liposomes herein named as stabilized lipid particles, resulted in targeted vesicles with a mean size of about 130 nm, encapsulation efficiency close to 100%, and a loading capacity of approximately 5 nmol siRNA/mumol of total lipid. In addition, the developed targeted vesicles showed increased internalization in SCLC cells, as well as in other tumor cells and HMEC-1 microvascular endothelial cells. The improved cellular association, however, did not correlate with enhanced downregulation of the target protein (Bcl-2) in SCLC cells. These results indicate that additional improvements need to be performed in the future, namely by ameliorating the access of the nucleic acids to the cytoplasm of the tumor cells following receptor-mediated endocytosis.
Biochimica et Biophysica Acta 12/2009; 1798(3):433-41. · 4.66 Impact Factor
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ABSTRACT: The present work aimed at the development and application of transferrin receptor (TrfR)-targeted sterically stabilized liposomes encapsulating anti-BCR-ABL siRNA or asODN. Transferrin was coupled to the surface of liposomes encapsulating siRNA or asODN through the postinsertion method. Cell association and internalization were assessed by flow cytometry and confocal microscopy, respectively. BCR-ABL mRNA and Bcr-Abl protein levels were evaluated by qRT-PCR and Western blot, respectively. Cell viability was assessed using the resazurin reduction method. The amount of coupled transferrin and the size and stability over time of the liposomes were very satisfactory and reproducible. The siRNA encapsulation yield was dependent on the concentration of the encapsulation buffer used (20 or 300 mM), as opposed to asODN encapsulation yield which was high for both concentrations tested. Cell association and internalization studies were performed in leukemia cell lines treated with liposomes coupled to Trf (Trf-liposomes) or albumin (BSA-liposomes) or with nontargeted liposomes (NT-liposomes) encapsulating fluorescently labeled siRNA (Cy3-siRNA). These experiments clearly indicated that BSA- and NT-liposomes have no ability to promote the delivery of the encapsulated nucleic acids and that the Trf-liposomes deliver the nucleic acids by a Trf receptor-dependent mechanism. The Trf-liposomes encapsulating siRNA or asODN promote sequence-specific down-regulation of the BCR-ABL mRNA, although a certain extent of nonspecific sequence effects at the protein and cell viability level were observed. Overall, our results indicate that Trf-liposomes encapsulating gene silencing tools allow combining molecular and cellular targeting, which is a valuable approach for cancer treatment.
Bioconjugate Chemistry 12/2009; 21(1):157-68. · 4.93 Impact Factor
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ABSTRACT: A library of diastereomerically pure epoxysterols, prepared by combining chemical and enzymatic methodologies, was evaluated for cytotoxicity toward human cancer and noncancer cell lines. Unsaturated steroids were oxidized by magnesium bis(monoperoxyphthalate) hexahydrate in acetonitrile, and the resulting epimeric epoxides were enzymatically separated using Novozym 435 or lipase AY. Some of the synthesized epoxysterols have potent cytotoxicity and higher activity on cancer cell lines HT29 and LAMA-84.
Journal of Medicinal Chemistry 06/2009; 52(13):4007-19. · 4.80 Impact Factor
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ABSTRACT: The main aim of this work was to develop novel targeted sterically stabilised pH-sensitive liposomes tailored to promote efficient intracellular delivery of therapeutic molecules into human T-leukaemia cells. Our results indicate that the targeting moiety (thiolated transferrin) was successfully coupled to the distal reactive maleimide terminus of poly(ethylene glycol)-phospholipid conjugates incorporated in the liposomal bilayer. Results from atomic force microscopy studies, performed to characterise vesicle surface topology, indicated that, to a certain extent, thiolated transferrin has the ability to associate in a non-specific manner with the lipid membrane of pegylated liposomes. This is an issue not commonly reported in the literature but which is crucial to demonstrate the targeting proof of principle. Nevertheless, fluorimetric studies together with confocal microscopy clearly demonstrate that liposomes bearing covalently coupled transferrin associate more extensively to human T-leukaemia cells in vitro than non-targeted liposomes. Cell mechanistic studies indicate that targeted liposomes bind specifically to transferrin receptors and are internalised via receptor-dependent endocytotic pathway. In addition, the biophysical features exhibited by the developed liposomes, namely their ability to promote pH-triggered cytoplasmic delivery of loaded material, make them promising delivery systems for in vivo targeting of therapeutic molecules to tumours.
European Journal of Pharmaceutics and Biopharmaceutics 03/2005; 59(2):359-66. · 4.27 Impact Factor
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ABSTRACT: Simple methods for the large-scale manufacture of ligand-targeted liposomes will be needed if clinical trials are to proceed. We tested a recently developed technology for inserting peptide ligands into preformed Stealth liposomes. Antagonist G-targeted liposomes (PLG) were prepared and loaded with doxorubicin and their cellular association and cytotoxicity were evaluated using the human small cell lung cancer H69 cell line.
The hexapeptide antagonist G was covalently coupled via a thioether bond to the terminus of polyethylene glycol (PEG) in micelles formed from maleimide-derivatized poly(ethylene glycol) (Mr 2000) distearoylphosphatidylethanolamine followed by transfer into preformed liposomes during a one-step incubation. For cellular association, we used radiolabeled liposomes. Cytotoxicity was evaluated using the MTT in vitro proliferation assay.
The postinsertion approach to the formation of peptide-targeted liposomes led to the production of PLG bearing a maximum of approximately 0.3 microg antagonist G/micromol phospholipid. These liposomes had increased cellular association to H69 cells relative to nontargeted liposomes and, when loaded with doxorubicin, they resulted in similar levels of cytotoxicity to those obtained by conventional coupling techniques.
The postinsertion technique is a simple, effective means for the production of biologically active peptide-targeted liposomes.
Pharmaceutical Research 04/2002; 19(3):265-9. · 4.09 Impact Factor
