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Advances in polymer chemistry over the last decade have enabled the synthesis of molecularly precise polymer networks that exhibit homogeneous structure. These precise polymer gels create the opportunity to establish true multiscale, molecular to macroscopic, relationships that define their elastic and failure properties. In this work, a theory of network fracture that accounts for loop defects is developed by drawing on recent advances in network elasticity. This loop-modified Lake-Thomas theory is tested against both molecular dynamics (MD) simulations and experimental fracture measurements on model gels, and good agreement between theory, which does not use an enhancement factor, and measurement is observed. Insight into the local and global contributions to energy dissipated during network failure and their relation to the bond dissociation energy is also provided. These findings enable a priori estimates of fracture energy in swollen gels where chain scission becomes an important failure mechanism.
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Bottlebrush networks (BBNs) are an exciting new class of materials with interesting physical properties derived from their unique architecture. While great strides have been made in our fundamental understanding of bottlebrush polymers and networks, an interdisciplinary approach is necessary for the field to accelerate advancements. This review aims to act as a primer to BBN chemistry and physics for both new and current members of the community. In addition to providing an overview of contemporary BBN synthetic methods, we developed a workflow and desktop application (LengthScale), enabling bottlebrush physics to be more approachable. We conclude by addressing several topical issues and asking a series of pointed questions to stimulate conversation within the community.
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There is significant potential in exploiting antibody specificity to develop new therapeutic treatments. However, intracellular protein delivery is a paramount challenge because of the difficulty in transporting large, polar molecules across cell membranes. Cell-penetrating peptide mimics (CPPMs) are synthetic polymers that are versatile materials for intracellular delivery of biological molecules, including nucleic acids and proteins, with superior performance compared to their natural counterparts and commercially available peptide-based reagents. Studies have demonstrated that noncovalent complexation with these synthetic carriers is necessary for the delivery of proteins, but the fundamental interactions dominating CPPM-protein complexation are not well understood. Beyond these interactions, the mechanism of release for many noncovalent carriers is not well established. Herein, interactions expected to be critical in CPPM-protein binding and unbinding were explored, including hydrogen bonding, electrostatics, and hydrophobic interactions. Despite the guanidinium-rich functionality of these polymeric carriers, hydrogen bonding was shown not to be a dominant interaction in CPPM-protein binding. Fluorescence quenching assays were used to decouple the effect of electrostatic and hydrophobic interactions between amphiphilic CPPMs and proteins. Furthermore, by conducting competition assays with other proteins, unbinding of protein cargoes from CPPM-protein complexes was demonstrated and provided insight into mechanisms of protein release. This work offers understanding toward the role of carrier and cargo binding and unbinding in intracellular outcomes. In turn, an improved fundamental understanding of noncovalent polymer-protein complexation will enable more effective methods for intracellular protein delivery.
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Péptidos de Penetración Celular , Péptidos de Penetración Celular/química , Guanidina/química , Interacciones Hidrofóbicas e Hidrofílicas , Polímeros/química , Unión ProteicaRESUMEN
Reconfigurable polymer networks are gaining interest for their potential applications as self-healing, recyclable, and stimuli-responsive smart materials. Relating the bond strength of dynamic interactions to material properties including stress relaxation time and modulus is crucial for smart material design. In this work, in situ crosslinked transition metal-terpyridine reconfigurable networks were utilized to modulate the characteristic network stress relaxation time, τR. The use of stress relaxation experiments rather than oscillatory frequency sweeps allowed for the measurement of network bond dynamics across a wider dynamic range than has been previously reported. The stress relaxation time was shown to be tunable by metal center, counterion, and crosslink density. Remarkably, the network crosslinked with covalent-like ruthenium chloride-terpyridine interaction, while having a longer τR, was qualitatively similar to the other metal-ligand networks. Furthermore, the relaxation time was independent of crosslink density in strongly bonded networks, allowing for independent tunability of modulus and τR. In contrast, increasing crosslink density reduced τR in networks crosslinked with weaker interactions.
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Rapid expansion of soft solids subjected to a negative hydrostatic stress can occur through cavitation or fracture. Understanding how these two mechanisms relate to a material's molecular structure is important to applications in materials characterization, adhesive design, and tissue damage. Here, a recently improved needle-induced cavitation (NIC) protocol is applied to a set of model end-linked PEG gels with quantitatively linked elastic and fracture properties. This quantitative link between molecular scale structure and macroscopic properties is exploited to experimentally probe the relationship between cavitation, fracture, and molecular scale damage. This work indicates that rational tuning of the elastofracture length relative to the crack geometry can be used to alter the expansion mechanism from cavitation to fracture during NIC.
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Agujas , GelesRESUMEN
Over the past decade, extensive optimization of polymeric cell-penetrating peptide (CPP) mimics (CPPMs) by our group has generated a substantial library of broadly effective carriers which circumvent the need for covalent conjugation often required by CPPs. In this study, design rules learned from CPPM development were applied to reverse-engineer the first library of simple amphiphilic block copolypeptides for non-covalent protein delivery, namely, poly(alanine-block-arginine), poly(phenylalanine-block-arginine), and poly(tryptophan-block-arginine). This new CPP library was screened for enhanced green fluorescent protein and Cre recombinase delivery alongside a library of CPPMs featuring equivalent side-chain configurations. Due to the added hydrophobicity imparted by the polymer backbone as compared to the polypeptide backbone, side-chain functionality was not a universal predictor of carrier performance. Rather, overall carrier hydrophobicity predicted the top performers for both internalization and activity of protein cargoes, regardless of backbone identity. Furthermore, comparison of protein uptake and function revealed carriers which facilitated high gene recombination despite remarkably low Cre internalization, leading us to formalize the concept of intracellular availability (IA) of the delivered cargo. IA, a measure of cargo activity per quantity of cargo internalized, provides valuable insight into the physical relationship between cellular internalization and bioavailability, which can be affected by bottlenecks such as endosomal escape and cargo release. Importantly, carriers with maximal IA existed within a narrow hydrophobicity window, more hydrophilic than those exhibiting maximal cargo uptake. Hydrophobicity may be used as a scaffold-independent predictor of protein uptake, function, and IA, enabling identification of new, effective carriers which would be overlooked by uptake-based screening methods.
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Péptidos de Penetración Celular , Transporte Biológico , Péptidos de Penetración Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Polímeros , Transporte de ProteínasRESUMEN
T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype.
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Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo L/metabolismo , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/genética , Proteína Quinasa C-theta/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Factores de Transcripción Forkhead/genética , Regiones Promotoras Genéticas , Unión Proteica , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Estabilidad Proteica , Transducción de SeñalRESUMEN
Regulatory T cells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory T cells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the T cell-specific kinase, protein kinase C theta, which promotes T cell effector functions and represses regulatory T cell differentiation, augments regulatory T cell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 T cells. When differentiated ex vivo into induced regulatory T cells, treated cells expressed elevated levels of the regulatory T cell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory T cells. The in vitro suppressive capacity of these induced regulatory T cells was 10-fold greater than that of T cells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory T cells were superior to non-treated T cells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory T cells as a cell-based therapy for immune modulation.
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Traslado Adoptivo/métodos , Anticuerpos/inmunología , Anticuerpos/farmacología , Péptidos de Penetración Celular , Enfermedad Injerto contra Huésped/terapia , Tolerancia Inmunológica/efectos de los fármacos , Líquido Intracelular/inmunología , Proteína Quinasa C-theta/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/metabolismo , Enfermedad Injerto contra Huésped/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptor de Muerte Celular Programada 1/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Resultado del TratamientoRESUMEN
Characterizing the high-strain-rate and high-strain mechanics of soft materials is critical to understanding the complex behavior of polymers and various dynamic injury mechanisms, including traumatic brain injury. However, their dynamic mechanical deformation under extreme conditions is technically difficult to quantify and often includes irreversible damage. To address such challenges, we investigate an experimental method, which allows quantification of the extreme mechanical properties of soft materials using ultrafast stroboscopic imaging of highly reproducible laser-induced cavitation events. As a reference material, we characterize variably cross-linked polydimethylsiloxane specimens using this method. The consistency of the laser-induced cavitation is achieved through the introduction of laser absorbing seed microspheres. Based on a simplified viscoelastic model, representative high-strain-rate shear moduli and viscosities of the soft specimens are quantified across different degrees of crosslinking. The quantified rheological parameters align well with the time-temperature superposition prediction of dynamic mechanical analysis. The presented method offers significant advantages with regard to quantifying high-strain rate, irreversible mechanical properties of soft materials and tissues, compared to other methods that rely upon the cyclic dynamics of cavitation. These advances are anticipated to aid in the understanding of how damage and injury develop in soft materials and tissues.
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Acute graft-versus-host disease is a frequent complication associated with allogeneic hematopoietic stem cell transplantation. Patients that become refractory to initial steroid treatment have a poor prognosis. apceth-201 consists of human allogeneic mesenchymal stromal cells, engineered by lentiviral transduction to express the protease inhibitor alpha-1 antitrypsin, to augment the anti-inflammatory potential of the mesenchymal stromal cells. We show that apceth-201 mesenchymal stromal cells efficiently suppress T cell proliferation and polarize macrophages to an anti-inflammatory M2 type, in vitro. To assess the in vivo efficacy of apceth-201, it was tested in two different mouse models of acute graft-versus-host disease. Control animals in a humanized model succumbed quickly to disease, whereas median survival was doubled in apceth-201-treated animals. The product was also tested in a graft-versus-host disease model system that closely mimics haploidentical hematopoietic stem cell transplantation, an approach that is now being evaluated for use in the clinic. Control animals succumbed quickly to disease, whereas treatment with apceth-201 resulted in long-term survival of 57% of the animals. Within 25 days after the second injection, clinical scores returned to baseline in responding animals, indicating complete resolution of graft-versus-host disease. These promising data have led to planning of a phase I study using apceth-201.
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Expresión Génica , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , alfa 1-Antitripsina/genética , Animales , Quimiotaxis de Leucocito/inmunología , Citocinas/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Orden Génico , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/terapia , Xenoinjertos , Mediadores de Inflamación/metabolismo , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Ratones , Especificidad de Órganos/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Trasplante Homólogo , Resultado del Tratamiento , alfa 1-Antitripsina/metabolismoRESUMEN
Delivering peptides and proteins with intracellular function represents a promising avenue for therapeutics, but remains a challenge due to the selective permeability of the plasma membrane. The successful delivery of cytosolically active proteins would enable many opportunities, including improved vaccine development through major histocompatibility complex (MHC) class I antigen display. Extended research using cell-penetrating peptides (CPPs) has aimed to facilitate intracellular delivery of exogenous proteins with some success. A new class of polymer-based mimics termed protein transduction domain mimics (PTDMs), which maintain the positive charge and amphiphilic nature displayed by many CPPs, was developed using a poly-norbornene-based backbone. Herein, we use a previously characterized PTDM to investigate delivery of the model antigen SIINFEKL into leukocytes. Peptide delivery into over 90% of CD14+ monocytes was detected in less than 15 min with nominal inflammatory cytokine response and high cell viability. The co-delivery of a TLR9 agonist and antigen using the PTDM into antigen-presenting cells in vitro showed presentation of SIINFEKL in association with MHC class I molecules, in addition to upregulation of classical differentiation markers revealing the ability of the PTDM to successfully deliver cargo intracellularly and show application in the field of immunotherapy.
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Monocitos/metabolismo , Presentación de Antígeno/fisiología , Supervivencia Celular/fisiología , Péptidos de Penetración Celular/metabolismo , Células Dendríticas/metabolismo , Citometría de Flujo , Voluntarios Sanos , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Receptores de Lipopolisacáridos/metabolismo , Células THP-1 , Receptor Toll-Like 9/metabolismoRESUMEN
As the field of anion exchange membranes (AEMs) employs an increasing variety of cations, a critical understanding of cation properties must be obtained, especially as they relate to membrane ion conductivity. Here, to elucidate such properties, metal cation-based AEMs, featuring bis(norbornene) nickel, ruthenium, or cobalt complexes, were synthesized and characterized. In addition, isothermal titration calorimetry (ITC) was used to probe counterion exchange thermodynamics in order to understand previously reported differences in conductivity. The ion conductivity data reported here further demonstrated that nickel-complex cations had higher conductivity as compared to their ruthenium and cobalt counterparts. Surprisingly, bulk hydration number, ion concentration, ion exchange capacity, and activation energy were not sufficient to explain differences in conductivity, so the thermodynamics of metal cation-counterion association were explored using ITC. Specifically, for the nickel cation as compared to the other two metal-based cations, a larger thermodynamic driving force for chloride counterion release was observed, shown through a smaller Δ Htot for counterion exchange, which indicated weaker cation-counterion association. The use of ITC to study cation-counterion association was further exemplified by characterizing more traditional AEM cations, such as quaternary ammoniums and an imidazolium cation, which demonstrated small variances in their enthalpic response, but an overall Δ Htot similar to that of the nickel-based cation. The cation hydration, rather than its hydration shell or the bulk hydration of the membrane, likely played the key role in determining the strength of the initial cation-counterion pair. This report identifies for the first time how ITC can be used to experimentally determine thermodynamic quantities that are key parameters for understanding and predicting conductivity in AEMs.
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Delivering proteins into the intracellular environment is a critical step toward probing vital cellular processes for the purposes of ultimately developing new therapeutics. Polymeric carriers are widely used to facilitate protein delivery with guanidinium-rich macromolecules leading the way within this category. Although binding interactions between natural proteins and synthetic polymers have been studied extensively, the relationship between polymer-protein binding and intracellular delivery is seldom explored. Elucidating the role of cargo binding in delivery is a promising direction that is expected to provide new insights that further optimize intracellular protein delivery. Herein, model polymeric carriers called protein transduction domain mimics (PTDMs) were studied for their ability to bind to a variety of protein cargoes, including an antibody, where the proteins encompassed a range of sizes (â¼16-151 kDa) and isoelectric points (4.7-11.4). The PTDM-protein complexes were also delivered into Jurkat T cells in an attempt to establish a general correlation between binding ability and delivery outcomes. Binding assays resulted in a vast range of dissociation constants (Kd), which spanned from 3.5 to 4820 nM and indicated a variety of binding strengths between PTDM and protein. More significantly, PTDMs preferentially bound certain types of proteins over others, such as the antibody fragment over the whole antibody. Furthermore, increased PTDM-protein binding affinity did not correlate with protein delivery, suggesting that the successful internalization of complexes is independent of binding equilibrium. Although binding did not correlate with internalization here, the potential for binding affinity to impact other aspects of delivery, like cargo functionality inside the cell, remains an open possibility.
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Polímeros/química , Proteínas/administración & dosificación , Avidina/química , Citoplasma/metabolismo , Sistemas de Liberación de Medicamentos , Endocitosis , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/administración & dosificación , Proteínas Fluorescentes Verdes/metabolismo , Guanidina/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/metabolismo , Células Jurkat , Muramidasa/química , Unión Proteica , Proteínas/metabolismo , Estreptavidina/químicaRESUMEN
Elucidating the predominant cellular entry mechanism for protein transduction domains (PTDs) and their synthetic mimics (PTDMs) is a complicated problem that continues to be a significant source of debate in the literature. The PTDMs reported here provide a well-controlled platform to vary molecular composition for structure activity relationship studies to further our understanding of PTDs, their non-covalent association with cargo, and their cellular internalization pathways. Specifically, several guanidine rich homopolymers, along with an amphiphilic block copolymer were used to investigate the relationship between structure and internalization activity in HeLa cells, both alone and non-covalently complexed with EGFP by flow cytometery and confocal imaging. The findings indicate that while changing the amount of positive charge on our PTDMs does not seem to affect the endosomal uptake, the presence of hydrophobicity appears to be a critical factor for the polymers to enter cells either alone, or with associated cargo.
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Endocitosis/fisiología , Endosomas/metabolismo , Guanidinas/metabolismo , Peptidomiméticos/metabolismo , Polímeros/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Guanidinas/síntesis química , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Peptidomiméticos/síntesis química , Polímeros/síntesis química , Estructura Terciaria de Proteína , Transporte de Proteínas , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Cell-penetrating peptides are an important class of molecules with promising applications in bioactive cargo delivery. A diverse series of guanidinium-containing polymeric cell-penetrating peptide mimics (CPPMs) with varying backbone chemistries was synthesized and assessed for delivery of both GFP and fluorescently tagged siRNA. Specifically, we examined CPPMs based on norbornene, methacrylate, and styrene backbones to determine how backbone structure impacted internalization of these two cargoes. Either charge content or degree of polymerization was held constant at 20, with diguanidinium norbornene molecules being polymerized to both 10 and 20 repeat units. Generally, homopolymer CPPMs delivered low amounts of siRNA into Jurkatâ T cells, with no apparent backbone dependence; however, by adding a short hydrophobic methyl methacrylate block to the guanidinium-rich methacrylate polymer, siRNA delivery to nearly the entire cell population was achieved. Protein internalization yielded similar results for most of the CPPMs, though the block polymer was unable to deliver proteins. In contrast, the styrene-based CPPM yielded the highest internalization for GFP (≈40 % of cells affected), showing that indeed backbone chemistry impacts protein delivery, specifically through the incorporation of an aromatic group. These results demonstrate that an understanding of how polymer structure affects cargo-dependent internalization is critical to designing new, more effective CPPMs.
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Materiales Biocompatibles/síntesis química , Guanidina/química , Polímeros/química , Materiales Biocompatibles/química , Péptidos de Penetración Celular/química , Citometría de Flujo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Células Jurkat , Metilmetacrilato/química , Norbornanos/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Transfección/métodosRESUMEN
The use of proteins as biological tools and therapeutic agents is limited due to the fact that proteins do not effectively cross the plasma membrane of cells. Here, we report a novel class of protein transporter molecules based on protein transduction domain mimics (PTDMs) synthesized via ring opening metathesis polymerization (ROMP). The PTDMs reported here were specifically inspired by amphiphilic peptides known to deliver functional proteins into cells via noncovalent interactions between the peptide and the cargo. This contrasts with peptides like TAT, penetratin, and R9, which often require covalent fusion to their cargoes. Using the easily tunable synthetic ROMP platform, the importance of a longer hydrophobic segment with cationic guanidinium groups was established through the delivery of EGFP into Jurkat T cells. The most efficient of these protein transporters was used to deliver functional Cre Recombinase with â¼80% knockdown efficiency into hard to transfect human T cells. Additionally, a C-terminally deleted form of the transcription factor Runx1 (Runx1.d190) was delivered into primary murine splenocytes, producing a 2-fold increase in c-Myc mRNA production, showcasing the versatility of this platform to deliver biologically active proteins into hard to transfect cell types.
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Biomimética , Transporte de Proteínas , Proteínas/química , Transfección , Animales , Sistemas de Liberación de Medicamentos , Proteínas Fluorescentes Verdes/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Integrasas/química , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Polimerizacion , Bazo/citología , Bazo/efectos de los fármacos , Linfocitos T/efectos de los fármacosRESUMEN
Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Péptidos de Penetración Celular/síntesis química , Enfermedad Injerto contra Huésped/inmunología , Isoenzimas/antagonistas & inhibidores , Leucocitos Mononucleares/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacología , Diferenciación Celular , Proliferación Celular , Péptidos de Penetración Celular/química , Humanos , Inmunomodulación , Leucocitos Mononucleares/inmunología , Activación de Linfocitos , Ratones , Fosforilación/efectos de los fármacos , Proteína Quinasa C-theta , Transducción de Señal/efectos de los fármacos , Células TH1/inmunologíaRESUMEN
The mechanism(s) by which certain small peptides and peptide mimics carry large cargoes across membranes through exclusively non-covalent interactions has been difficult to resolve. Here, we use the droplet-interface bilayer as a platform to characterize distinct mechanistic differences between two such carriers: Pep-1 and a guanidinium-rich peptide mimic we call D9. While both Pep-1 and D9 can carry an enzyme, horseradish peroxidase (HRP) across a lipid bilayer, we found that they do so by different mechanisms. Specifically, Pep-1 requires voltage or membrane asymmetry while D9 does not. In addition, D9 can facilitate HRP transport without pre-forming a complex with HRP. By contrast, complex formation is required by Pep-1. Both carriers are capable of forming pores in membranes but our data hints that these pores are not responsible for cargo transport. Overall, D9 appears to be a more potent and versatile transporter when compared with Pep-1 because D9 does not require an applied voltage or other forces to drive transport. Thus, D9 might be used to deliver cargo across membranes under conditions where Pep-1 would be ineffective.
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Membrana Celular/metabolismo , Cisteamina/análogos & derivados , Membrana Dobles de Lípidos/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/química , Membrana Celular/química , Cisteamina/química , Cisteamina/metabolismo , Guanidina/química , Peroxidasa de Rábano Silvestre/metabolismo , Membrana Dobles de Lípidos/química , Potenciales de la Membrana , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Molecular , Péptidos/química , Transporte de ProteínasRESUMEN
Many biophysical studies of protein transduction domains (PTDs) and their synthetic mimics (PTDMs) focus on the interaction between the polycationic PTD(M) and anionic phospholipid surfaces. Most, but not all, of these studies suggest that these cation-anion interactions are vital for membrane activity. In this study, the effect of anionic lipid content on PTDM performance was examined for three ring-opening metathesis (ROMP)-based PTDMs with varying hydrophobicity. Using a series of dye-loaded vesicles with gradually increasing anionic lipid content, we saw that increased anionic lipid content inhibited dye release caused by these PTDMs. This result is the opposite of what was found in studies with poly- and oligo-arginine. While the effect is reduced for more hydrophobic PTDMs, it is observable even with the most hydrophobic PTDMs of our test panel. Additional experiments included dynamic light scattering and zeta potential measurements to measure size as a function of vesicle surface charge in the presence of increasing PTDM concentration and surface plasmon resonance spectroscopy to quantify binding between PTDMs and surface-bound lipid layers with varying anion content. The results from these measurements suggested that PTDM hydrophobicity, not cation-anion interactions, is the main driving force of the interaction between our PTDMs and the model membranes investigated. This suggests a model of interaction where surface association and membrane insertion are driven by PTDM hydrophobicity, while anionic lipid content serves primarily to "pin" the PTDM to the membrane surface and limit insertion.