Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Photosynthesis induction in Eucalyptus urograndis seedlings and cuttings measured by Open Photoacoustics

In this work we study the photosynthetic induction in Eucalyptus urograndis leaves using the Open Photoacoustic Cell Technique. In vivo and in situ measurements were performed in leaves of four months-old E. urograndis seedlings and C041 cuttings previously dark-adapted for at least 10 h. Experimental results for the gas exchange component of the photoacoustic (PA) signal are interpreted considering that a gas uptake component would have a phase angle nearly opposite to that of the oxygen evolution component. Analysis of the photosynthetic induction data shows that seedlings present a net oxygen evolution before cuttings, but cuttings reach a higher steady-state photosynthetic activity.

Photosynthetic induction in Eucalyptus urograndis seedlings and cuttings measured by an open photoacoustic cell

Photosynthetic induction in leaves of four-month-old Eucalyptus urograndis seedlings and of cuttings obtained from adult trees that were previously dark-adapted was studied by the in vivo and in situ Open Photoacoustic Cell Technique, Results for the gas exchange component of the photoacoustic (PA) signal were interpreted considering that the gas uptake component would have a phase angle nearly opposite to that of the oxygen evolution component. By subtracting the thermal component from the total PA signal, we studied the competition between gas uptake and oxygen evolution during the photosynthetic induction. Seedlings presented a net oxygen evolution prior to cuttings, but cuttings reached a higher steady-state photosynthetic activity. The chlorophyll (Chl) a/b ratio and the Chl fluorescence induction characteristic F-v/F-m were significantly higher for cuttings, while there was no difference between samples in stomata density and leaf thickness. Thus the differences in PA signals of seedlings and cuttings are associated to differences between the photosystem 2 antenna systems of these samples.

Influência do armazenamento de ramas para plantio em algumas características agronômicas da mandioca

Nas condições do Estado de São Paulo é freqüente o armazenamento de ramas de mandioca (Manihot esculenta Crantz) para aguardar a melhor oportunidade de efetuar o plantio. Utilizam-se os sistemas de empilhamento horizontal e vertical, sendo mais comum o primeiro. Raramente, empregam-se ramas recém-colhidas para plantio. 0 presente trabalho teve por objetivo estudar o efeito do material de plantio em alguns parâmetros fitotécnicos da cultura, em função dos diferentes estádios fisiológicos causados pelo armazenamento ou não das ramas. Num esquema fatorial de 2 x 3, em blocos ao acaso com quatro repetições, utilizaram-se dois cultivares - IAC 12-829 e SRT 1287-Fibra, de alta e baixa capacidade de ramificação respectivamente - e três tipos de manivas, originárias de ramas recém-colhidas e de ramas conservadas por 105 dias nos sistemas de empilhamento horizontal e vertical. O experimento foi desenvolvido no ano agrícola 1991/92, na Estação Experimental do Vale do Paranapanema (IAC), Assis (SP), em latossolo vermelho-escuro, álico, textura média. Os resultados mais relevantes mostraram que: (a) O armazenamento de ramas em pilhas horizontais provocou aumento no número de hastes primárias dos dois cultivares e aumentou o peso da parte aérea do SRT 1287-Fibra; (b) O SRT 1287-Fibra foi mais influenciado pelo armazenamento da rama na posição horizontal que o IAC 12-829; (c) Este, em média, foi superior na produção de raízes, não diferindo, entretanto, do primeiro quando as manivas provinham de ramas armazenadas na posição horizontal; (d) As ramas procedentes do armazenamento na posição vertical comportaram-se similarmente às não armazenadas.

Sclerocorneal limbal stem cell autograft transplantation in dogs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cardiac function in dogs with chronic Chagas cardiomyopathy undergoing autologous stem cell transplantation into the coronary arteries

This study assessed the effects of a single intracoronary injection of autologous stem cells on the cardiac function of dogs with Chagas cardiomyopathy. Bone-marrow-derived stem cells were delivered into the right and left coronary arteries of 5 mature dogs with mildly compromised cardiac function due to chronic Chagas cardiomyopathy. Blood pressure and electrocardiographic and echocardiographic parameters were recorded at monthly intervals for 6 mo in the 3 dogs that survived. Although no changes were observed in the electrocardiogram and blood pressure, there was a significant increase in peak velocity of aortic flow 3 mo after stem cell transplantation. Pre-ejection period, isovolumic relaxation time, and the Tei index of myocardial performance were reduced significantly 4 mo after the procedure. All significant changes persisted to the end of the study. The results suggest that the transplantation of autologous bone-marrow-derived stem cells into the coronary arteries of dogs with Chagas cardiomyopathy may have a beneficial effect but the small number of dogs studied was a limitation.

Effect of wounds applied to the bases of herbaceous cuttings on the rooting of four Japanese apricot clones (Prunus mume Sieb. et Zucc.) in an intermittent mist system

The Japanese apricot (Prunus mume Sieb. et Zucc.) is a fruit tree of the Rosaceae family which produces very acid and bitter fruits, highly appreciated by Orientals. In Brazil, this species has been studied as a rootstock for peach and nectarine trees, its main advantage being the reduction in plant vigour, which can favour the production of compact trees and orchard cultural treatments. This study was conducted in the Vegetable Production Department of FCAV/UNESP, Jaboticabal Campus, São Paulo State, Brazil, and the objective was to examine the effect of wounding the herbaceous cutting bases on the rooting of four Japanese apricot clones. The clones were obtained from plants under cultivation in the Instituto Agronomico de Campinas, Brazil, and were identified as Clones 02, 05, 10 and 15. The stock plants, obtained through herbaceous cuttings, were maintained under lath house conditions (50% of natural light). Cuttings 12 cm long with 3 to 5 leaves were collected from these clones and prepared. The experiment was carried out in a completely randomised design with 4 repetitions of 20 cuttings per replication, in a factorial 4 x 2 design, the clone factor having 4 levels (Clones 02, 05, 10 and 15) and the wounding factor at 2 levels of incisions into the cutting base (with and without). All the cuttings were treated with 2000 mg.L-1 of IBA for five seconds. Differences between the clones were observed concerning the rooting percentage, dead cuttings, number and length of roots. The incision (wound) at the base of the herbaceous cuttings of the Japanese apricot increased the number of roots and improved the distribution of these in the damaged tissue but the results were not considered sufficiently beneficial to make the treatment worthwhile.

Evaluation and development of passion fruit (Passiflora alata Dryander) propagated by cutting and seet in conditions of commercial orchard

The present work was conducted in a fruit tree propagation area of the Plant Production Department of the Faculdade de Ciencias Agrarias e Veterinarias, Universidade Estadual Paulista (FCAV/UNESP) in Jaboticabal, SP, and also in a commercial orchard in Araguari, MG, with the objective to verify the potential of vegetative growth (stem diameter, height of plants and leaf number) of plants of passion fruit (Passiflora alata Dryander), gotten by cutting and seed, comparing the initial development of plants in the field. This experiment was carried out from January 2002 to February 2003. The experiment using seeds was conducted at a shadow house, and the one that used cuttings in an intermitent mist. The cuttings and seeds were collected from adult plants which came from Passifloraceae Active Germoplasm Bank (BAG) of the Plant Production Department of FCAC/UNESP. For the cuttings, it was used the intermediate part of the branches in stadium of vegetative growth. The seeds, in order to obtain the seedlings, had been sown in plastic trays. Cuttings and seedlings were transplanted to plastic bags with substrate in shadow house and with daily irrigation. They were acclimatized and planted on field, after 60 days. on field, the stem diameter, plant height and number of leaves were better for cuttings than for seedlings in Jaboticabal, SP. In Araguari, MG, stem diameter was larger in the seedlings, which plant heights and number of leaves were larger on cuttings.

Tolerance to iron chlorosis in non-grafted quince seedlings and in pear grafted onto quince plants

Grafting is a technique that may affect plant tolerance to iron chlorosis in plants cultivated for their fruit. Therefore, the objective of this study was to evaluate the tolerance of non-grafted quince seedlings and pear grafted onto quince plants cultivated in pots with alkaline soil. The experiment was conducted in a greenhouse at the University of Cordoba, Spain, in pots (3 L) filled with alkaline soil, with one plant per pot. The treatments consisted of two genotypes, quince (Cydonia oblonga Mill) semi-woody rooted cuttings, cultivar BA29, and pear (Pyrus Communis L.), cultivar Ercolini, grafted onto quince cultivar BA29 (rootstock), and two nutrient solutions with and without iron (80 mu M Fe-EDDHA) arranged in a completely random design with eight repetitions. Each pot received 250 mL of the nutrient solution on June 3rd, 2010. Chlorophyll indirect measurements and the main stem length were evaluated for six weeks after the commencement of the treatments. During the last week, the main stem dry matter weight and the leaf total iron content were determined. It was found that grafting pear seedlings onto quince rootstock resulted in a higher tolerance to iron deficiency than when quince was not grafted. Non-grafted quince plants without iron in the nutrient solution, compared to the results with its application, showed low SPAD (Soil-Plant Analyses Development) values and resulted in plants with a lower leaf iron content and lower dry matter production; however, decreased seedling stem growth was observed only in the last week of cultivation.

Propagação vegetativa da alfavaca-cravo utilizando diferentes tipos de estacas e substratos

A propagação vegetativa é considerada importante ferramenta para o melhoramento de espécies lenhosas e herbáceas e vem sendo amplamente utilizada, visando melhorar e manter variedades de importância econômica e medicinal. Neste contexto utilizou-se estacas de alfavaca-cravo de 15 cm para apicais e 25 cm para as medianas, sendo estas com e sem folhas. As estacas foram plantadas em bandejas de isopor de 72 células, utilizando-se 10 substratos. Após 35 dias, as estacas foram retiradas com cuidado, sendo as partes aéreas e radiculares separadas e acondicionadas em saco de papel e mantidas em estufa a 70ºC por 48 h para posterior avaliação da matéria seca. Analisou-se a percentagem de enraizamento, número de folhas na estaca, peso de matéria seca de folha e de raiz. Não houve interação significativa entre os diferentes tipos de substrato e estaca para percentagem de enraizamento, sendo que os substratos de modo geral diferiram pouco entre si. Entre os tipos de estaca destacaram-se para esta variável a mediana sem folha e a apical com folha. Para as demais variáveis houve interação significativa entre o tipo de estaca e o substrato. A melhor estaca foi a mediana sem folhas plantadas em substrato arisco + esterco + húmus e vermiculita nas seguintes proporções: 20%:40%:40%; 40%:20%:40% e 40%:40%.

Stem diameter and height of chrysanthemum cv Yokoono as affected by gibberellic acid

The effect of gibberellic acid has been shown mainly to promote cell division and elongation. This study was aimed to evaluate the development of height and diameter of the stems of chrysanthemum cultivar Yoko ono by the applications of gibberellic acid (GA(3)) in the field. The treatments were composed of four doses (0, 40, 80 and 120 mg L(-1)) at 15 and 30 days after transplanting. From the findings, It can be concluded that GA(3) significantly affected the diameter of stem at higher doses, and was unable to affect the height of stem.

Coffee leaf and stem anatomy under boron deficiency

A deficiência de B é muito comum nos cafezais brasileiros, mas as respostas do cafeeiro ao B têm sido erráticas, dependendo do ano, do modo e época de aplicação e, ainda, da fonte de B empregada. Um melhor entendimento dos efeitos do B na fisiologia e anatomia do cafeeiro é importante para o desenvolvimento de um programa racional de adubação boratada, uma vez que a anatomia da planta pode influenciar a translocação do nutriente. Neste experimento, plantas de dois cultivares foram cultivadas em soluções nutritivas com 0,0 (deficiente), 5,0 (adequado) e 25,0 µM (alto) de B. Quando os primeiros sintomas de deficiência apareceram, as folhas foram cortadas e tiveram suas paredes celulares isoladas e analisadas quanto aos teores de B e Ca. Cortes foram feitos em folhas novas e no ápice de ponteiros e fotografados em microscópio eletrônico de varredura. A resposta dos dois cultivares ao B foi semelhante, não tendo sido observados sintomas de toxidez. O teor de B nas paredes celulares foi aumentado com o incremento da concentração desse elemento na solução, enquanto o teor de Ca não foi afetado. A relação Ca/B decresceu com o aumento da concentração de B na solução. Com deficiência de B, os tecidos vasculares foram desorganizados e as paredes do xilema ficaram mais finas. Folhas de café com deficiência deste nutriente apresentaram menos estômatos, os quais se encontravam.

STEM ANATOMY and DEVELOPMENT of SUCCESSIVE CAMBIA IN THE NEOTROPICAL LIANA SECURIDACA RIVINIFOLIA (POLYGALACEAE)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aerial stem and leaf morphoanatomy of some species of Smilax

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enxertia de mesa de Passiflora edulis Sims f. flavicarpa Deg. sobre Passiflora alata Curtis, em ambiente de nebulização intermitente

O maracujazeiro é uma cultura típica de países tropicais, sendo estes responsáveis por cerca de 90% da produção mundial. A propagação vegetativa do maracujazeiro permite a obtenção de pomares uniformes, bem como de porta-enxertos altamente produtivos ou resistentes a doenças. O experimento teve como objetivo verificar a viabilidade da enxertia de mesa do maracujazeiro-amarelo (Passiflora edulis Sims f. flavicarpa Deg.) sobre o maracujazeiro-doce (Passiflora alata Curtis), conduzido em telado com 50% de sombreamento, dotado de sistema de nebulização intermitente. Foi conduzido de agosto de 2001 a maio de 2002, na Fazenda de Ensino e Pesquisa da UNESP - Câmpus de Ilha Solteira, localizada no município de Selvíria - MS. O delineamento experimental utilizado foi blocos ao acaso, esquema de parcelas subdivididas no tempo, totalizando quatro tratamentos (T1 - enxertia tipo fenda cheia, com o garfo do ponteiro dos ramos; T2 - enxertia tipo inglês simples, com o garfo do ponteiro dos ramos; T3 - enxertia tipo fenda cheia, com o garfo da parte mediana dos ramos; T4 - enxertia tipo inglês simples, com o garfo da parte mediana dos ramos), cinco repetições e 25 estacas enxertadas por parcela. Foram avaliados: a) porcentagem de sobrevivência das estacas enxertadas; b) porcentagem de estacas enxertadas enraizadas; c) número de brotos emitidos por estaca enxertada; d) número de folhas emitidas por estaca enxertada; e) massa da matéria seca da raiz, caule, folha e planta. Com base nos resultados, pode-se concluir que: a técnica de enxertia de mesa, quando aplicada ao maracujazeiro, mostrou-se viável, com excelente porcentagem de sobrevivência e enraizamento.