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1.
PLoS One ; 19(3): e0297769, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38547243

RESUMO

Mesenchymal stromal cells (MSCs) are promising therapeutic agents for cartilage regeneration, including the potential of cells to promote chondrogenesis in vivo. However, process development and regulatory approval of MSCs as cell therapy products benefit from facile in vitro approaches that can predict potency for a given production run. Current standard in vitro approaches include a 21 day 3D differentiation assay followed by quantification of cartilage matrix proteins. We propose a novel biophysical marker that is cell population-based and can be measured from in vitro monolayer culture of MSCs. We hypothesized that the self-assembly pattern that emerges from collective-cell behavior would predict chondrogenesis motivated by our observation that certain features in this pattern, namely, topological defects, corresponded to mesenchymal condensations. Indeed, we observed a strong predictive correlation between the degree-of-order of the pattern at day 9 of the monolayer culture and chondrogenic potential later estimated from in vitro 3D chondrogenic differentiation at day 21. These findings provide the rationale and the proof-of-concept for using self-assembly patterns to monitor chondrogenic commitment of cell populations. Such correlations across multiple MSC donors and production batches suggest that self-assembly patterns can be used as a candidate biophysical attribute to predict quality and efficacy for MSCs employed therapeutically for cartilage regeneration.


Assuntos
Condrogênese , Células-Tronco Mesenquimais , Humanos , Cartilagem/metabolismo , Diferenciação Celular , Doadores de Tecidos , Células Cultivadas
2.
Sci Adv ; 9(24): eadf3120, 2023 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-37327346

RESUMO

Human cord blood-derived γδ T cells (CBγδ) display a highly diverse TCRγδ repertoire and have a unique subtype composition different from fetal or adult peripheral blood counterparts. We expanded CBγδ in vitro using an irradiated Epstein-Barr virus-transformed feeder cell-based modified rapid expansion protocol (REP). Single-cell RNA sequencing tracked progressive differentiation of naïve CBγδ into cells expressing neoantigen-reactive tumor-infiltrating lymphocyte as well as tissue-resident memory precursor-like and antigen-presenting cell-like gene signatures. TCRγδ clonal tracing revealed a bias toward cytotoxic effector differentiation in a much larger proportion of Vδ2- clones compared to Vδ2+ clones, resulting in the former being more cytotoxic at the population level. These clonotype-specific differentiation dynamics were not restricted to REP and were recapitulated upon secondary nonviral antigen stimulations. Thus, our data showed intrinsic cellular differences between major subtypes of human γδ T cells already in operation at early postnatal stage and highlighted key areas of consideration in optimizing cell manufacturing processes.


Assuntos
Infecções por Vírus Epstein-Barr , Linfócitos T , Adulto , Humanos , Sangue Fetal , Herpesvirus Humano 4 , Receptores de Antígenos de Linfócitos T gama-delta/genética
3.
Leukemia ; 33(6): 1542, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30787431

RESUMO

In the original version of this article there was a mistake in the spelling of the author Sujoy Ghosh, originally spelt Sujoy Gosh. This has now been corrected in both the PDF and HTML versions of the article.

4.
Leukemia ; 33(6): 1487-1500, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30575819

RESUMO

The study of myelodysplastic syndromes (MDS) in murine models has now indicated the possible involvement of the bone marrow microenvironment in the generation of dysplastic hematopoietic cells. However, there is scant work on patient samples and the role of hypomethylating agents on the bone marrow stromal cells of MDS patients is unclear. We show that human MDS-MSCs exhibit phenotypic, transcriptomic and epigenetic abnormalities. Stimuli provided by MDS-MSCs impaired the growth and function of healthy HSPCs, which is further sustained autonomously in HSPCs for significant periods of time resulting in a failure for active hematopoietic engraftment across primary and secondary transplant recipients (chimerism: 0.34-91% vs 2.78%, engraftment frequencies: at 0.06 ± 0.02 vs full engraftment for MDS-MSC vs healthy groups, respectively). Hypomethylation of MDS-MSCs improved overall engraftment in most of the MDS-MSC groups tested (2/7 with p < 0.01, 3/7 with p < 0.05 and 2/7 with no significant difference). MDS-MSCs that fail to respond to hypomethylating therapy are associated with patients with rapid adverse disease transformation and this further suggests that MDS-MSCs may be an integral part of disease progression and have prognostic value as well as potential as a therapeutic target.


Assuntos
Azacitidina/farmacologia , Biomarcadores Tumorais/metabolismo , Metilação de DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Síndromes Mielodisplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Proliferação de Células , Epigênese Genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Stem Cell Res Ther ; 9(1): 268, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30352620

RESUMO

BACKGROUND: Efficient and sustained hematopoietic recovery after hematopoietic stem cell or bone marrow transplantation is supported by paracrine signaling from specific subpopulations of mesenchymal stromal cells (MSCs). Here, we considered whether in vitro mechanopriming of human MSCs could be administered to predictively and significantly improve in vivo hematopoietic recovery after irradiation injury. METHODS: First, we implemented regression modeling to identify eight MSC-secreted proteins that correlated strongly with improved rescue from radiation damage, including hematopoietic recovery, in a murine model of hematopoietic failure. Using these partial least squares regression (PLSR) model parameters, we then predicted recovery potential of MSC populations that were culture expanded on substrata of varying mechanical stiffness. Lastly, we experimentally validated these predictions using an in vitro co-culture model of hematopoiesis and using new in vivo experiments for the same irradiation injury model used to generate survival predictions. RESULTS: MSCs grown on the least stiff (elastic moduli ~ 1 kPa) of these polydimethylsiloxane (PDMS) substrata secreted high concentrations of key proteins identified in regression modeling, at concentrations comparable to those secreted by minor subpopulations of MSCs shown previously to be effective in supporting such radiation rescue. We confirmed that these MSCs expanded on PDMS could promote hematopoiesis in an in vitro co-culture model with hematopoietic stem and progenitor cells (HSPCs). Further, MSCs cultured on PDMS of highest stiffness (elastic moduli ~ 100 kPa) promoted expression of CD123+ HSPCs, indicative of myeloid differentiation. Systemic administration of mechanoprimed MSCs resulted in improved mouse survival and weight recovery after bone marrow ablation, as compared with both standard MSC expansion on stiffer materials and with biophysically sorted MSC subpopulations. Additionally, we observed recovery of white blood cells, platelets, and red blood cells, indicative of complete recovery of all hematopoietic lineages. CONCLUSIONS: These results demonstrate that computational techniques to identify MSC biomarkers can be leveraged to predict and engineer therapeutically effective MSC phenotypes defined by mechanoprimed secreted factors, for translational applications including hematopoietic recovery.


Assuntos
Dimetilpolisiloxanos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos da radiação , Mecanotransdução Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos da radiação , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos , Plaquetas/citologia , Plaquetas/fisiologia , Diferenciação Celular , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Dimetilpolisiloxanos/química , Módulo de Elasticidade , Eritrócitos/citologia , Eritrócitos/fisiologia , Raios gama , Expressão Gênica , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Hematopoese/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucócitos/citologia , Leucócitos/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Análise de Regressão , Análise de Sobrevida , Alicerces Teciduais , Irradiação Corporal Total
6.
Immunity ; 48(2): 364-379.e8, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29466759

RESUMO

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.


Assuntos
Células da Medula Óssea/fisiologia , Neutrófilos/fisiologia , Animais , Células da Medula Óssea/imunologia , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Linhagem da Célula , Movimento Celular , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Camundongos , Neoplasias Experimentais/imunologia , Neutrófilos/imunologia
7.
Stem Cells Transl Med ; 7(5): 376-393, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29392885

RESUMO

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure-activity-relationship studies of SB203580, a known p38-MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393.


Assuntos
Antígenos CD34/metabolismo , Azóis/farmacologia , Sangue Fetal/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Integrina alfa6/metabolismo , Células-Tronco/efeitos dos fármacos , Antígenos Thy-1/metabolismo , Animais , Células Cultivadas , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Imidazóis/farmacologia , Camundongos , Camundongos SCID , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Células-Tronco/metabolismo , Relação Estrutura-Atividade
8.
ACS Biomater Sci Eng ; 3(12): 3292-3306, 2017 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33445371

RESUMO

Human mesenchymal stem cells (MSCs) exhibit morphological and phenotypic changes that correlate with mechanical cues presented by the substratum material to which those cells adhere. Such mechanosensitivity has been explored in vitro to promote differentiation of MSCs along tissue cell lineages for direct tissue repair. However, MSCs are increasingly understood to facilitate indirect tissue repair in vivo through paracrine signaling via secreted biomolecules. Here we leveraged cell-material interactions in vitro to induce human bone marrow-derived MSCs to preferentially secrete factors that are beneficial to hematopoietic cell proliferation. Specifically, we varied the viscoelastic properties of cell-culture-compatible polydimethylsiloxane (PDMS) substrata to demonstrate modulated MSC expression of biomolecules, including osteopontin, a secreted phosphoprotein implicated in tissue repair and regeneration. We observed an approximately 3-fold increase in expression of osteopontin for MSCs on PDMS substrata of lowest stiffness (elastic moduli <1 kPa) and highest ratio of loss modulus to storage modulus (tan(δ) > 1). A specific subpopulation of these cells, shown previously to express increased osteopontin in vitro and to promote bone marrow recovery in vivo, also exhibited up to a 5-fold increase in osteopontin expression when grown on compliant PDMS relative to heterogeneous MSCs on polystyrene. Importantly, this mechanically modulated increase in protein expression preceded detectable changes in the terminal differentiation capacity of MSCs. In coculture with human CD34+ hematopoietic stem and progenitor cells (HSPCs) that repopulate the blood cell lineages, these mechanically modulated MSCs promoted in vitro proliferation of HSPCs without altering the multipotency for either myeloid or lymphoid lineages. Cytokine and protein expression by human MSCs can thus be manipulated directly by mechanical cues conferred by the material substrata prior to and instead of tissue lineage differentiation. This approach enables enhanced in vitro production of both mesenchymal and hematopoietic stem and progenitor cells that aid regenerative clinical applications.

9.
Stem Cells ; 34(10): 2471-2484, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27299710

RESUMO

In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016;34:2471-2484.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Telômero/metabolismo , Animais , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Etoposídeo/farmacologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Engenharia Genética , Genoma Humano , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/transplante , Humanos , Camundongos SCID , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transplante de Células-Tronco , Telomerase/metabolismo , Encurtamento do Telômero/efeitos dos fármacos , Teratoma/genética , Teratoma/patologia
10.
Biol Blood Marrow Transplant ; 21(6): 1008-19, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25555449

RESUMO

The successful expansion of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB) for transplantation could revolutionize clinical practice by improving transplantation-related outcomes and making available UCB units that have suboptimal cell doses for transplantation. New cytokine combinations appear able to promote HSPC growth with minimal differentiation into mature precursors and new agents, such as insulin-like growth factor-binding protein 2, are being used in clinical trials. Molecules that simulate the HSPC niche, such as Notch ligand, have also shown promise. Further improvements have been made with the use of mesenchymal stromal cells, which have made possible UCB expansion without a potentially deleterious prior CD34/CD133 cell selection step. Chemical molecules, such as copper chelators, nicotinamide, and aryl hydrocarbon antagonists, have shown excellent outcomes in clinical studies. The use of bioreactors could further add to HSPC studies in future. Drugs that could improve HSPC homing also appear to have potential in improving engraftment times in UCB transplantation. Technologies to expand HSPC from UCB and to enhance the homing of these cells appear to have attained the goal of accelerating hematopoietic recovery. Further discoveries and clinical studies are likely to make the goal of true HSPC expansion a reality for many applications in future.


Assuntos
Células-Tronco Hematopoéticas/imunologia , Células-Tronco Mesenquimais/imunologia , Nicho de Células-Tronco/imunologia , Reatores Biológicos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaios Clínicos como Assunto , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Citocinas/farmacologia , Etilenodiaminas/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Proteínas de Membrana/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Niacinamida/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/imunologia
11.
Stem Cells Transl Med ; 4(1): 56-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25411477

RESUMO

Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as "cell factories" that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies.


Assuntos
Células da Medula Óssea/citologia , Medula Óssea/fisiologia , Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Técnicas Analíticas Microfluídicas/métodos , Animais , Ensaios de Triagem em Larga Escala , Humanos , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Proc Natl Acad Sci U S A ; 111(42): E4409-18, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25298531

RESUMO

The capacity to produce therapeutically relevant quantities of multipotent mesenchymal stromal cells (MSCs) via in vitro culture is a common prerequisite for stem cell-based therapies. Although culture expanded MSCs are widely studied and considered for therapeutic applications, it has remained challenging to identify a unique set of characteristics that enables robust identification and isolation of the multipotent stem cells. New means to describe and separate this rare cell type and its downstream progenitor cells within heterogeneous cell populations will contribute significantly to basic biological understanding and can potentially improve efficacy of stem and progenitor cell-based therapies. Here, we use multivariate biophysical analysis of culture-expanded, bone marrow-derived MSCs, correlating these quantitative measures with biomolecular markers and in vitro and in vivo functionality. We find that, although no single biophysical property robustly predicts stem cell multipotency, there exists a unique and minimal set of three biophysical markers that together are predictive of multipotent subpopulations, in vitro and in vivo. Subpopulations of culture-expanded stromal cells from both adult and fetal bone marrow that exhibit sufficiently small cell diameter, low cell stiffness, and high nuclear membrane fluctuations are highly clonogenic and also exhibit gene, protein, and functional signatures of multipotency. Further, we show that high-throughput inertial microfluidics enables efficient sorting of committed osteoprogenitor cells, as distinct from these mesenchymal stem cells, in adult bone marrow. Together, these results demonstrate novel methods and markers of stemness that facilitate physical isolation, study, and therapeutic use of culture-expanded, stromal cell subpopulations.


Assuntos
Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Adulto , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Fenômenos Biofísicos , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Linhagem da Célula , Membrana Celular/metabolismo , Proliferação de Células , Tamanho Celular , Células Cultivadas , Citoplasma/metabolismo , Feto/metabolismo , Humanos , Técnicas Analíticas Microfluídicas , Análise Multivariada , Membrana Nuclear/metabolismo , Estresse Mecânico
13.
ACS Nano ; 8(10): 9767-80, 2014 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-25198246

RESUMO

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.


Assuntos
Antineoplásicos/administração & dosagem , DNA Antissenso/química , Portadores de Fármacos , Microscopia Eletrônica de Transmissão
14.
Adv Healthc Mater ; 3(6): 867-75, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24124132

RESUMO

Current treatment options for debilitating bone diseases such as osteosarcoma, osteoporosis, and bone metastatic cancer are suboptimal and have low efficacy. New treatment options for these pathologies require targeted therapy that maximizes exposure to the diseased tissue and minimizes off-target side effects. This work investigates an approach for generating functional and targeted drug carriers specifically for treating primary osteosarcoma, a disease in which recurrence is common and the cure rate has remained around 20%. This approach utilizes the modularity of Layer-by-Layer (LbL) assembly to generate tissue-specific drug carriers for systemic administration. This is accomplished via surface modification of drug-loaded nanoparticles with an aqueous polyelectrolyte, poly(acrylic acid) (PAA), side-chain functionalized with alendronate, a potent clinically used bisphosphonate. Nanoparticles coated with PAA-alendronate are observed to bind and internalize rapidly in human osteosarcoma 143B cells. Encapsulation of doxorubicin, a front-line chemotherapeutic, in an LbL-targeted liposome demonstrates potent toxicity in vitro. Active targeting of 143B xenografts in NCR nude mice with the LbL-targeted doxorubicin liposomes promotes enhanced, prolonged tumor accumulation and significantly improved efficacy. This report represents a tunable approach towards the synthesis of drug carriers, in which LbL enables surface modification of nanoparticles for tissue-specific targeting and treatment.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Resinas Acrílicas/química , Alendronato/administração & dosagem , Alendronato/química , Animais , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/química , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Meia-Vida , Humanos , Lipossomos/química , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/metabolismo , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Radiografia , Transplante Heterólogo
15.
Biomacromolecules ; 13(5): 1315-22, 2012 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-22376183

RESUMO

One of the major obstacles that delay the clinical translation of polymeric micelle drug delivery systems is whether these self-assembled micelles can retain their integrity in blood following intravenous (IV) injection. The objective of this study was to evaluate the impact of core functionalization on the thermodynamic and kinetic stability of polymeric micelles. The combination of ring-opening polymerization of N-carboxyanhydride (NCA) with highly efficient "click" coupling has enabled easy and quick access to a family of poly(ethylene glycol)-block-poly(γ-R-glutamate)s with exactly the same block lengths, for which the substituent "R" is tuned. The structures of these copolymers were carefully characterized by (1)H NMR, FT-IR, and GPC. When pyrene is used as the fluorescence probe, the critical micelle concentrations (CMCs) of these polymers were found to be in the range of 10(-7)-10(-6) M, which indicates good thermodynamic stability for the self-assembled micelles. The incorporation of polar side groups in the micelle core leads to high CMC values; however, micelles prepared from these copolymers are kinetically more stable in the presence of serum and upon SDS disturbance. It was also observed that these polymers could effectively encapsulate paclitaxel (PTX) as a model anticancer drug, and the micelles possessing better kinetic stability showed better suppression of the initial "burst" release and exhibited more sustained release of PTX. These PTX-loaded micelles exerted comparable cytotoxicity against HeLa cells as the clinically approved Cremophor PTX formulation, while the block copolymers showed much lower toxicity compared to the cremophor-ethanol mixture. The present work demonstrated that the PEG-b-PPLG can be a uniform block copolymer platform toward development of polymeric micelle delivery systems for different drugs through the facile modification of the PPLG block.


Assuntos
Antineoplásicos/química , Paclitaxel/química , Peptídeos/química , Polietilenoglicóis/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Células HeLa , Humanos , Micelas , Estrutura Molecular , Paclitaxel/farmacologia , Tamanho da Partícula , Peptídeos/síntese química , Peptídeos/farmacologia , Polietilenoglicóis/síntese química , Polietilenoglicóis/farmacologia , Relação Estrutura-Atividade , Propriedades de Superfície , Células Tumorais Cultivadas
16.
ACS Nano ; 5(6): 4284-92, 2011 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-21513353

RESUMO

Inspired by the simplicity and versatility of layer-by-layer (LbL) assembly, we applied multilayered polyelectrolyte assemblies on nanoparticles to create viable systemic delivery systems. Focusing on tumor-specific delivery, LbL nanoparticles that exhibit a pH-sensitive outer stealth layer are demonstrated to target and be retained in hypoxic tumor regions. The neutral layers shed in response to acidity to reveal a charged nanoparticle surface that is readily taken up by tumor cells. The first in vivo demonstration of this mechanism of targeting is presented, as well as an initial examination of the mechanism of uptake of the nanoparticles. We further demonstrate that this concept for tumor targeting is potentially valid for a broad range of cancers, with applicability for therapies that target hypoxic tumor tissue.


Assuntos
Nanopartículas/química , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Eletrólitos , Feminino , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Hipóxia , Camundongos , Camundongos Nus , Nanotecnologia/métodos , Transplante de Neoplasias , Neoplasias/patologia , Tamanho da Partícula , Polietilenoglicóis/química , Propriedades de Superfície , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia
17.
Nano Lett ; 11(5): 2096-103, 2011 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-21524115

RESUMO

This paper demonstrates the generation of systemically deliverable layer-by-layer (LbL) nanoparticles for cancer applications. LbL-based nanoparticles designed to navigate the body and deliver therapeutics in a programmable fashion are promising new and alternative systems for drug delivery, but there have been very few demonstrations of their systemic delivery in vivo due to a lack of knowledge in building LbL nanofilms that mimic traditional nanoparticle design to optimize delivery. The key to the successful application of these nanocarriers in vivo requires a systematic analysis of the influence of film architecture and adsorbed polyelectrolyte outer layer on their pharmacokinetics, which has thus far not been examined for this new approach to nanoparticle delivery. Herein, we have taken the first steps in stabilizing and controlling the systemic distribution of multilayer nanoparticles. Our findings highlight the unique character of LbL systems; the electrostatically assembled nanoparticles gain increased stability in vivo with larger numbers of deposited layers, and the final layer adsorbed generates a critical surface cascade, which dictates the surface chemistry and biological properties of the nanoparticle. This outer polyelectrolyte layer dramatically affects not only the degree of nonspecific particle uptake, but also the nanoparticle biodistribution. For hyaluronic acid (HA) outer layers, a long blood elimination half-life (∼9 h) and low accumulation (∼10-15% recovered fluorescence/g) in the liver were observed, illustrating that these systems can be designed to be highly appropriate for clinical translation.


Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas/química , Nanotecnologia/métodos , Eletricidade Estática , Adsorção , Animais , Humanos , Ácido Hialurônico/química , Fígado/efeitos dos fármacos , Teste de Materiais , Camundongos , Camundongos Endogâmicos BALB C , Preparações Farmacêuticas/administração & dosagem , Propriedades de Superfície , Distribuição Tecidual
18.
Nanomedicine ; 7(2): 201-9, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20816874

RESUMO

A novel linear-dendritic block copolymer has been synthesized and evaluated for targeted delivery. The use of the dendron as the micellar exterior block in this architecture allows the presentation of a relatively small quantity of ligands in clusters for enhanced targeting, thus maintaining a long circulation time of these "patchy" micelles. The polypeptide linear hydrophobic block drives formation of micelles that carry core-loaded drugs, and their unique design gives them extremely high stability in vivo. We have found that these systems lead to extended time periods of increased accumulation in the tumor (up to 5 days) compared with nontargeted vehicles. We also demonstrate a fourfold increase in efficacy of paclitaxel when delivered in the targeted nanoparticle systems, while significantly decreasing in vivo toxicity of the chemotherapy treatment. FROM THE CLINICAL EDITOR: A micellar vehicle using dendrons as the exterior block in combination with a polypeptide hydrophobic block was shown to incorporate and deliver paclitaxel to xenograft tumors with a four-fold increase in efficacy and reduced toxicity.


Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Portadores de Fármacos/uso terapêutico , Micelas , Nanopartículas/uso terapêutico , Paclitaxel/administração & dosagem , Animais , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Portadores de Fármacos/metabolismo , Estabilidade de Medicamentos , Humanos , Injeções Intravenosas , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanomedicina , Paclitaxel/metabolismo , Paclitaxel/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
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