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1.
Biomacromolecules ; 20(5): 2008-2014, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-30938980

RESUMO

Bioresponsive polymers can enable the development of more effective drug delivery vehicles and medical materials. Acetal-modified polysaccharides allow pH-triggered solubility switching in a versatile and effective manner, but prior work has been limited to a combination of acyclic alkoxyisopropyl and cyclic isopropylidene acetals. We describe here the preparation and characterization of spirocyclic acetal-modified dextran (SpAc-Dex), which comprises dextran decorated with cyclopentyl, cyclohexyl, or cycloheptyl acetals (SpAc5-, SpAc6-, and SpAc7-Dex, respectively). A library of materials with varying acyclic and cyclic acetal contents was synthesized, and organic-soluble materials were formed into microparticles and assessed for degradability and cytocompatibility. At high levels of modification, SpAc5-Dex degraded most quickly and SpAc7-Dex degraded most slowly. SpAc6-Dex features lower degrees of substitution but spans a wide range of degradability. These materials were found to be noncytotoxic and may find future use in biomedical applications.

2.
Integr Biol (Camb) ; 7(12): 1611-21, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26507156

RESUMO

Epithelial sheets fold into complex topographies that contribute to their function in vivo. Cells can sense and respond to substrate topography in their immediate vicinity by modulating their interfacial mechanics, but the extent to which these mechanical properties contribute to their ability to sense substrate topography across length scales larger than a single cell has not been explored in detail. To study the relationship between the interfacial mechanics of single cells and their collective behavior as tissues, we grew cell-sheets on substrates engraved with surface features spanning macroscopic length-scales. We found that many epithelial cell-types sense and respond to substrate topography, even when it is locally nearly planar. Cells clear or detach from regions of local negative curvature, but not from regions with positive or no curvature. We investigated this phenomenon using a finite element model where substrate topography is coupled to epithelial response through a balance of tissue contractility and adhesive forces. The model correctly predicts the focal sites of cell-clearing and epithelial detachment. Furthermore, the model predicts that local tissue response to substrate curvature is a function of the surrounding topography of the substrate across long distances. Analysis of cell-cell and cell-substrate contact angles suggests a relationship between these single-cell interfacial properties, epithelial interfacial properties, and collective epithelial response to substrate topography. Finally, we show that contact angles change upon activation of oncogenes or inhibition of cell-contractility, and that these changes correlate with collective epithelial response. Our results demonstrate that in mechanically integrated epithelial sheets, cell contractility can be transmitted through multiple cells and focused by substrate topography to affect a behavioral response at distant sites.


Assuntos
Adesão Celular/fisiologia , Células Epiteliais/fisiologia , Modelos Biológicos , Animais , Apoptose , Fenômenos Biomecânicos , Células CACO-2 , Engenharia Celular , Proliferação de Células , Cães , Epitélio/fisiologia , Matriz Extracelular/fisiologia , Análise de Elementos Finitos , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Madin Darby de Rim Canino , Propriedades de Superfície , Engenharia Tecidual
3.
Proc Natl Acad Sci U S A ; 112(7): 2287-92, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25633040

RESUMO

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo- and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.


Assuntos
Comunicação Celular , Glândulas Mamárias Humanas/citologia , Células Epiteliais/citologia , Matriz Extracelular , Humanos
4.
Nat Methods ; 10(12): 1203-5, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24122039

RESUMO

Precise control over interfacial chemistry between nanoparticles and other materials remains a major challenge that limits broad application of nanotechnology in biology. To address this challenge, we used 'steric exclusion' to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping each QD with a functionalized oligonucleotide. We demonstrated the utility of these QDs as modular and nonperturbing imaging probes by tracking individual Notch receptors on live cells.


Assuntos
Microscopia de Fluorescência/métodos , Nanopartículas/química , Nanotecnologia/métodos , Pontos Quânticos , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Luz , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/instrumentação , Oligonucleotídeos/química , Oligonucleotídeos Fosforotioatos/química , Distribuição de Poisson , Espalhamento de Radiação , Compostos de Sulfidrila/química
5.
J Am Chem Soc ; 134(2): 765-8, 2012 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-22176556

RESUMO

Cell adhesion organizes the structures of tissues and mediates their mechanical, chemical, and electrical integration with their surroundings. Here, we describe a strategy for chemically controlling cell adhesion using membrane-anchored single-stranded DNA oligonucleotides. The reagents are pure chemical species prepared from phosphoramidites synthesized in a single chemical step from commercially available starting materials. The approach enables rapid, efficient, and tunable cell adhesion, independent of proteins or glycans, by facilitating interactions with complementary labeled surfaces or other cells. We demonstrate the utility of this approach by imaging drug-induced changes in the membrane dynamics of non-adherent human cells that are chemically immobilized on a passivated glass surface.


Assuntos
Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Membrana Celular/química , Oligonucleotídeos/química , Animais , Linhagem Celular , Membrana Celular/metabolismo , DNA/química , Vidro , Humanos , Propriedades de Superfície
6.
Bioconjug Chem ; 22(5): 949-57, 2011 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-21476603

RESUMO

Biotherapeutic delivery is a rapidly growing field in need of new materials that are easy to modify, are biocompatible, and provide for triggered release of their encapsulated cargo. Herein, we report on a particulate system made of a polysaccharide-based pH-sensitive material that can be efficiently modified to display mannose-based ligands of cell-surface receptors. These ligands are beneficial for antigen delivery, as they enhance internalization and activation of APCs, and are thus capable of modulating immune responses. When compared to unmodified particles or particles modified with a nonspecific sugar residue used in the delivery of antigens to dendritic cells (DCs), the mannosylated particles exhibited enhanced antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. This represents the first demonstration of a mannosylated particulate system that enables enhanced MHC I antigen presentation by DCs in vitro. Our readily functionalized pH-sensitive material may also open new avenues in the development of optimally modulated vaccine delivery systems.


Assuntos
Apresentação do Antígeno/imunologia , Dextranos/química , Sistemas de Liberação de Medicamentos/métodos , Imunomodulação/imunologia , Manose/química , Manose/metabolismo , Nanopartículas/química , Antígenos/imunologia , Antígenos/metabolismo , Configuração de Carboidratos , Sobrevivência Celular , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Relação Estrutura-Atividade
7.
Chem Commun (Camb) ; 47(2): 665-7, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21113530

RESUMO

Acid-degradable microcapsules were prepared via an interfacial polymerization. Degradation of the thin wall of the capsules leads to all-or-nothing cargo release. The only byproducts of degradation are acetone, and a non-toxic triamide. Proof-of-concept experiments showed that cargo can be delivered to and released in cells.


Assuntos
Ácidos/química , Cápsulas/química , Portadores de Fármacos/química , Materiais Biocompatíveis/química , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Cinética , Paclitaxel/administração & dosagem
8.
J Am Chem Soc ; 133(4): 756-8, 2011 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-21171594

RESUMO

Dextran, a water-soluble, biocompatible polymer of glucose, was modified at its hydroxyls with arylboronic esters to make it soluble in common organic solvents, allowing for the facile preparation of oxidation-sensitive dextran (Oxi-DEX) carrier microparticles. These particles were found to release their payload with a half-life of 36 min at 1 mM H2O2, which can be compared with a half-life of greater than 1 week in the absence of H2O2. When used in a model vaccine application, Oxi-DEX particles loaded with ovalbumin (OVA) increased the presentation to CD8+ T-cells 27-fold relative to OVA encapsulated in a classical vehicle not sensitive to oxidation. No presentation was observed from cells incubated with unencapsulated OVA. Additionally, Oxi-DEX was found to be nontoxic in preliminary in vitro cytotoxicity assays. Because it is easy to prepare, sensitive to biological oxidation, and biocompatible, this material may represent an attractive new platform for selective delivery applications.


Assuntos
Materiais Revestidos Biocompatíveis/química , Dextranos/química , Portadores de Fármacos/química , Animais , Apresentação do Antígeno/imunologia , Materiais Revestidos Biocompatíveis/toxicidade , Células Dendríticas/imunologia , Dextranos/toxicidade , Portadores de Fármacos/toxicidade , Células HeLa , Humanos , Hidróxidos/química , Camundongos , Ovalbumina/química , Ovalbumina/imunologia , Oxirredução , Solubilidade , Solventes/química
10.
Mol Pharm ; 7(3): 826-35, 2010 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-20230025

RESUMO

Toll-like receptor (TLR) agonists induce potent innate immune responses and can be used in the development of novel vaccine adjuvants. However, access to TLRs can be challenging as exemplified by TLR 7, which is located intracellularly in endosomal compartments. To increase recognition and subsequent stimulatory effects of TLR 7, imiquimod was encapsulated in acetalated dextran (Ac-DEX) microparticles. Ac-DEX, a water-insoluble and biocompatible polymer, is relatively stable at pH 7.4, but degrades rapidly under acidic conditions, such as those found in lysosomal vesicles. To determine the immunostimulatory capacity of encapsulated imiquimod, we compared the efficacy of free versus encapsulated imiquimod in activating RAW 264.7 macrophages, MH-S macrophages, and bone marrow derived dendritic cells. Encapsulated imiquimod significantly increased IL-1 beta, IL-6, and TNF-alpha cytokine expression in macrophages relative to the free drug. Furthermore, significant increases were observed in classic macrophage activation markers (iNOS, PD1-L1, and NO) after treatment with encapsulated imiquimod over the free drug. Also, bone marrow derived dendritic cells produced significantly higher levels of IL-1 beta, IL-6, IL-12p70, and MIP-1 alpha as compared to their counterparts receiving free imiquimod. These results suggest that encapsulation of TLR ligands within Ac-DEX microparticles results in increased immunostimulation and potentially better protection from disease when used in conjunction with vaccine formulations.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Dextranos/química , Nanopartículas/química , Adjuvantes Imunológicos/química , Aminoquinolinas/administração & dosagem , Aminoquinolinas/química , Animais , Linhagem Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Imiquimode , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Microscopia Eletrônica de Varredura , Nanopartículas/ultraestrutura , Reação em Cadeia da Polimerase
11.
J Am Chem Soc ; 131(30): 10360-1, 2009 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-19591467

RESUMO

Despite the promise of precisely targeted or otherwise functionalized polymeric particulate drug delivery vehicles, typical biocompatible particles are generally not amenable to facile and selective surface modification. Herein, we report the development of a simple, mild, and chemoselective strategy for the conjugation of biologically active molecules to the surface of dextran-based microparticles. Alkoxyamine-bearing reagents were used to form stable oxime conjugates with latent aldehyde functionality present in reducing carbohydrate chain ends. We demonstrate the functionalization of dextran-based microparticles with a fluorophore as well as a cell-penetrating peptide sequence, which facilitated the delivery of cargo to nonphagocytic cells leading to a 60-fold increase in the expression of a reporter gene when plasmid DNA-loaded particles were used.


Assuntos
Portadores de Fármacos/química , Polissacarídeos/química , Acetais/química , Animais , Bovinos , Dextranos/química , Dextranos/metabolismo , Portadores de Fármacos/metabolismo , Células HeLa , Humanos , Nanopartículas/química , Polissacarídeos/metabolismo , Especificidade por Substrato , Propriedades de Superfície
12.
Mol Pharm ; 6(4): 1160-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19415922

RESUMO

Protein-based vaccines have been explored as a safer alternative to traditional weakened or killed whole organism based vaccination strategies and have been investigated for their ability to activate the immune system against certain cancers. For optimal stimulation of T lymphocytes, protein-based vaccines should deliver protein antigens to antigen presenting cells in the context of appropriate immunostimulatory signals, thus mimicking actual pathogens. In this report, we describe the synthesis, characterization, and biological evaluation of immunostimulatory acid-degradable microparticles, which are suitable delivery vehicles for use in protein-based vaccines and cancer immunotherapy. Using a 3' conjugation strategy, we optimized the attachment of immunostimulatory CpG DNA to our vaccine carriers and demonstrated that under acidic conditions similar to those found in endosomal compartments, these new particles were capable of simultaneously releasing a model protein antigen and a CpG DNA adjuvant. We found in an in vivo cytotoxicity assay that the co-encapsulation of ovalbumin, a model antigen, and immunostimulatory agent in the same particle led to superior cytotoxic T lymphocyte activity compared to particles coadministered with adjuvant in an unbound form. In addition, we investigated the ability of these acid-degradable particles to induce protective immunity in the MO5 murine melanoma model and found that they were effective until tumor escape, which appeared to result from a loss of antigen expression by the cancer cells due to in vivo selection pressure.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antineoplásicos/administração & dosagem , Vacinas Anticâncer/uso terapêutico , Melanoma Experimental/terapia , Oligodesoxirribonucleotídeos/administração & dosagem , Ovalbumina/administração & dosagem , Adjuvantes Imunológicos/farmacocinética , Adjuvantes Imunológicos/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Citocinas/metabolismo , Sistemas de Liberação de Medicamentos , Feminino , Ativação Linfocitária , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Oligodesoxirribonucleotídeos/farmacocinética , Oligodesoxirribonucleotídeos/farmacologia , Ovalbumina/farmacocinética , Ovalbumina/farmacologia , Taxa de Sobrevida , Distribuição Tecidual
13.
Proc Natl Acad Sci U S A ; 106(14): 5497-502, 2009 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-19321415

RESUMO

Materials that combine facile synthesis, simple tuning of degradation rate, processability, and biocompatibility are in high demand for use in biomedical applications. We report on acetalated dextran, a biocompatible material that can be formed into microparticles with degradation rates that are tunable over 2 orders of magnitude depending on the degree and type of acetal modification. Varying the degradation rate produces particles that perform better than poly(lactic-co-glycolic acid) and iron oxide, two commonly studied materials used for particulate immunotherapy, in major histocompatibility complex class I (MHC I) and MHC II presentation assays. Modulating the material properties leads to antigen presentation on MHC I via pathways that are dependent or independent of the transporter associated with antigen processing. To the best of our knowledge, this is the only example of a material that can be tuned to operate on different immunological pathways while maximizing immunological presentation.


Assuntos
Materiais Biocompatíveis/química , Dextranos/uso terapêutico , Imunoterapia/métodos , Animais , Apresentação do Antígeno , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II , Humanos
14.
Mol Pharm ; 5(5): 876-84, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18710254

RESUMO

Acid-degradable particles containing a model protein antigen, ovalbumin, were prepared from a polyurethane with acetal moieties embedded throughout the polymer, and characterized by dynamic light scattering and transmission electron microscopy. The small molecule degradation byproduct of the particles was synthesized and tested in vitro for toxicity indicating an LC 50 of 12,500 microg/mL. A new liquid chromatography-mass spectrometry technique was developed to monitor the in vitro degradation of these particles. The degradation byproduct inside RAW macrophages was at its highest level after 24 h of culture and was efficiently exocytosed until it was no longer detectable after 4 days. When tested in vitro, these particles induced a substantial increase in the presentation of the immunodominant ovalbumin-derived peptide SIINFEKL in both macrophages and dendritic cells. In addition, vaccination with these particles generated a cytotoxic T-lymphocyte response that was superior to both free ovalbumin and particles made from an analogous but slower-degrading acid-labile polyurethane polymer. Overall, we present a fully degradable polymer system with nontoxic byproducts, which may find use in various biomedical applications including protein-based vaccines.


Assuntos
Portadores de Fármacos/administração & dosagem , Nanopartículas/administração & dosagem , Poliuretanos/metabolismo , Proteínas/química , Vacinas/administração & dosagem , Ácidos/metabolismo , Animais , Apresentação do Antígeno/imunologia , Células Cultivadas , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Epitopos Imunodominantes/imunologia , Luz , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Nanopartículas/química , Ovalbumina/química , Ovalbumina/imunologia , Tamanho da Partícula , Poliuretanos/química , Poliuretanos/farmacocinética , Proteínas/imunologia , Espalhamento de Radiação , Linfócitos T Citotóxicos/imunologia , Vacinas/química , Vacinas/imunologia
15.
J Am Chem Soc ; 130(32): 10494-5, 2008 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-18630909

RESUMO

Dextran, a biocompatible, water-soluble polysaccharide, was modified at its hydroxyls with acetal moieties such that it became insoluble in water but freely soluble in common organic solvents enabling its use in the facile preparation of acid-sensitive microparticles. These particles degrade in a pH-dependent manner: FITC-dextran was released with a half-life at 37 degrees C of 10 h at pH 5.0 compared to a half-life of approximately 15 days at pH 7.4. Both hydrophobic and hydrophilic cargoes were successfully loaded into these particles using single and double emulsion techniques, respectively. When used in a model vaccine application, particles loaded with the protein ovalbumin (OVA) increased the presentation of OVA-derived peptides to CD8+ T-cells 16-fold relative to OVA alone. Additionally, this dextran derivative was found to be nontoxic in preliminary in vitro cytotoxicity assays. Owing to its ease of preparation, processability, pH-sensitivity, and biocompatibility, this type of modified dextran should find use in numerous drug delivery applications.


Assuntos
Materiais Biocompatíveis/síntese química , Dextranos/síntese química , Acetais/química , Ácidos/química , Animais , Apresentação do Antígeno , Materiais Biocompatíveis/química , Materiais Biocompatíveis/uso terapêutico , Materiais Biocompatíveis/toxicidade , Biodegradação Ambiental , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular , Dextranos/química , Dextranos/uso terapêutico , Dextranos/toxicidade , Sistemas de Liberação de Medicamentos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Concentração de Íons de Hidrogênio , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Teste de Materiais , Camundongos , Ovalbumina/imunologia , Vacinas/química , Vacinas/imunologia
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