Monica Montanari

pDNA condensation capacity and in vitro gene delivery properties of cationic solid lipid nanoparticles

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical-chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers.

Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells

Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34+ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.

Physiological levels of 1α, 25 dihydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors

Although supraphysiological levels of 1α, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo‐ and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid‐culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38− hematopoietic stem cells, the down‐regulation of CD34 antigen, and the increase of monocyte‐committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.

The Kinetic Status of Hematopoietic Stem Cell Subpopulations Underlies a Differential Expression of Genes Involved in Self‐Renewal, Commitment, and Engraftment

The gene expression profile of CD34− hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage‐negative (Lin−) CD34−, Lin−CD34+, and Lin+CD34+ cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34− cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin‐dependent kinases (CDKs), CDK inhibitors, and growth‐arrest genes. According to the expression of their receptors and transducers, interleukin (IL)‐10 and IL‐17 showed an inhibitory effect on the clonogenic activity of CD34− cells. Conversely, CD34+ cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34− cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self‐renewal and lineage commitment. The preferential expression in CD34+ cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34+ cells. Thus, the different kinetic status of CD34− and CD34+ cells, detailed by molecular and functional analysis, significantly influences their biological behavior.

Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.

The Vitamin D3/Hox-A10 Pathway Supports MafB Function during the Monocyte Differentiation of Human CD34+ Hemopoietic Progenitors

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D3 receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D3/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

Nuclear localization of cationic solid lipid nanoparticles containing Protamine as transfection promoter.

Protamine has attracted much attention as DNA condenser and nuclear transfer enhancer although the excess of hydrophilicity and the strong DNA pack restrain its potentialities. In order to overcome this limitation, we added Protamine in the composition of solid lipid nanoparticles (SLN-Protamine) and we compared this carrier with the same kind of SLN containing Esterquat 1 instead of Protamine (SLN-EQ1). Carriers cytotoxicity was assessed on COS-I cells evaluating the cell cycle by propidium iodide test, while the transfection efficiency was studied using pEGFP as plasmid model. The cell penetrating activity of Protamine inside the lipid vectors was evaluated studying cell internalization by confocal microscopy using Red Nile-labeled carriers. SLN-Protamine:pDNA showed a mean diameter five-times smaller than the size of SLN-EQ1:pDNA and a remarkably lesser cytotoxicity. Transfection by SLN-Protamine:pDNA was seven-times more effective compared with the Protamine:pDNA polyplexes while no transfection capacity was observed for SLN-EQ1:pDNA complexes due to their inability to be internalized owing to their larger dimension. Red Nile-SLN-Protamine were localized in endocytic-like vesicles into the nuclear membrane suggesting the inclusion of Protamine in nano-lipophilic systems may enhance the reduction in the complex dimensions, the nuclear pDNA translocation and the pDNA release in the cells.

Correlation between differentiation plasticity and mRNA expression profiling of CD34+-derived CD14- and CD14+ human normal myeloid precursors

In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to trans-differentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14− and CD14+ myeloid precursors generated by ‘in vitro’ culture of human CD34+ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14− cells were bipotent granulo-monocyte precursors, while CD14+ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are co-expressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.

The role of protamine amount in the transfection performance of cationic SLN designed as a gene nanocarrier

Cationic solid lipid nanoparticles (SLN) have been recently proposed as non-viral vectors in systemic gene therapy. The aim of this study was to evaluate the effect of the protamine amount used as the transfection promoter in SLN-mediated gene delivery. Three protamine-SLN samples (Pro25, Pro100, and Pro200) prepared by adding increasing amounts of protamine were characterized for their size, zeta potential, and protamine loading level. The samples were evaluated for pDNA complexation ability by gel-electrophoresis analysis and for cytotoxicity and transfection efficiency by using different cell lines (COS-I, HepG2, and Na1300). The size of SLN was ~230 nm and only Pro200 showed few particle aggregates. Unlike the Pro25 sample with the lowest protamine loading level, the others SLN samples (Pro100 and Pro200) exhibited a good ability in complexing pDNA. A cell-line dependent cytotoxicity lower than that of the positive control PEI (polyethilenimmine) was observed for all the SLN. Among these, only Pro100, having an intermediate amount of protamine, appeared able to promote pDNA cell transfer, especially in a neuronal cell line (Na1300). In conclusion, the amount of protamine as the transfection promoter in SLN affects not only the gene delivery ability of SLN but also their capacity to transfer genes efficiently to specific cell types.

Design flexibility influencing the in vitro behavior of cationic SLN as a nonviral gene vector.

Several advanced in vitro and in vivo studies have proved the broad potential of cationic solid lipid nanoparticles (SLN) as nonviral vectors. However, a few data are available about the correlation between lipid component of the SLN structure and in vitro performance in terms of cell tolerance and transfection efficiency on different cell lines. In this paper SLN were prepared using stearic acid as main lipid component, stearylamine as cationic agent and protamine as transfection promoter and adding phosphatidylcholine (PC), cholesterol (Chol) or both to obtain three different multicomponent SLN (SLN-PC, SLN-Chol and SLN-PC-Chol, respectively). Cytotoxicity and transfection efficiency of the obtained SLN:pDNA complexes were evaluated on three different immortalized cell lines: COS-I (African green monkey kidney cell line), HepG2 (human hepatocellular liver carcinoma cell line) and Na1300 (murine neuroblastoma cell line). Samples were characterized for the exact quantitative composition, particle size, morphology, zeta potential and pDNA binding ability. All the three SLN samples were about 250-300 nm in size with a positive zeta potential, whereas SLN:pDNA complexes were about 300-400 nm in size with a less positive zeta potential, depending on the SLN composition. Concerning the cell tolerance, the three samples showed a level of cytotoxicity lower than that of the positive control polyethylenimine (PEI), regardless of the cell lines. The best transfection performance was observed for SLN-PC-Chol on COS-I cells while a transfection level lower than PEI was observed on HepG2 cells, regardless the SLN composition. On Na1300 cells, SLN-Chol showed a double efficiency with respect to PEI. Comparing these results to those obtained with the same kind of SLN without PC and/or Chol, it is possible to conclude that the addition of Chol and/or PC to the composition of cationic SLN modify the cell tolerance and the transfection efficiency of the gene vector in a manner strictly dependent on the cell type and the internalization pathways.