RESUMEN
Poly(lactide) (PLA) and poly(ethylene glycol) (PEG)-based hydrogels were prepared by mixing phosphate buffer saline (PBS, pH 7.4) solutions of four-arm (PEG-PLA)2-R-(PLA-PEG)2 enantiomerically pure copolymers having the opposite chirality of the poly(lactide) blocks. Dynamic Light Scattering, rheology measurements, and fluorescence spectroscopy suggested that, depending on the nature of the linker R, the gelation process followed rather different mechanisms. In all cases, mixing of equimolar amounts of the enantiomeric copolymers led to micellar aggregates with a stereocomplexed PLA core and a hydrophilic PEG corona. Yet, when R was an aliphatic heptamethylene unit, temperature-dependent reversible gelation was mainly induced by entanglements of PEG chains at concentrations higher than 5 wt.%. When R was a linker containing cationic amine groups, thermo-irreversible hydrogels were promptly generated at concentrations higher than 20 wt.%. In the latter case, stereocomplexation of the PLA blocks randomly distributed in micellar aggregates is proposed as the major determinant of the gelation process.
Asunto(s)
Polietilenglicoles , Polímeros , Polímeros/química , Polietilenglicoles/química , Poliésteres/química , Micelas , Hidrogeles/químicaRESUMEN
Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu+-independent strain-promoted alkyne-azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.
Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Inmunoconjugados/química , Cadenas Pesadas de Inmunoglobulina/química , Nanopartículas/química , Polietilenglicoles/química , Anticuerpos de Dominio Único/química , Alquinos/química , Aminoaciltransferasas/metabolismo , Animales , Azidas/química , Proteínas Bacterianas/metabolismo , Química Clic/métodos , Reacción de Cicloadición/métodos , Cisteína Endopeptidasas/metabolismo , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Polietilenglicoles/metabolismo , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismoRESUMEN
In this paper we describe a straightforward supramolecular strategy to encapsulate silicon phthalocyanine (SiPc) photosensitizers (PS) in polymeric micelles made of poly(ε-caprolactone)-b-methoxypoly(ethylene glycol) (PCL-PEG) block copolymers. While PCL-PEG micelles are promising nanocarriers based on their biocompatibility and biodegradability, the design of our new PS favors their encapsulation. In particular, they combine two axial benzoyl substituents, each of them carrying either three hydrophilic methoxy(triethylenoxy) chains (1), three hydrophobic dodecyloxy chains (3), or both kinds of chains (2). The SiPc derivatives 1 and 2 are therefore amphiphilic, with the SiPc unit contributing to the hydrophobic core, while lipophilicity increases along the series, making it possible to correlate the loading efficacy in PCL-PEG micelles with the hydrophobic/hydrophilic balance of the PS structure. This has led to a new kind of third-generation nano-PS that efficiently photogenerates 1O2, while preliminary in vitro experiments demonstrate an excellent cellular uptake and a promising PDT activity.
Asunto(s)
Indoles/química , Compuestos de Organosilicio/química , Fármacos Fotosensibilizantes/química , Portadores de Fármacos/química , Micelas , Poliésteres/química , Polietilenglicoles/químicaRESUMEN
Selective elimination of macrophages by photodynamic therapy (PDT) is a new and promising therapeutic modality for the reduction of atherosclerotic plaques. m-Tetra(hydroxyphenyl)chlorin (mTHPC, or Temoporfin) may be suitable as photosensitizer for this application, as it is currently used in the clinic for cancer PDT. In the present study, mTHPC was encapsulated in polymeric micelles based on benzyl-poly(ε-caprolactone)-b-methoxy poly(ethylene glycol) (Ben-PCL-mPEG) using a film hydration method, with loading capacity of 17%. Because of higher lipase activity in RAW264.7 macrophages than in C166 endothelial cells, the former cells degraded the polymers faster, resulting in faster photosensitizer release and higher in vitro photocytotoxicity of mTHPC-loaded micelles in those macrophages. However, we observed release of mTHPC from the micelles in 30min in blood plasma in vitro which explains the observed similar in vivo pharmacokinetics of the mTHPC micellar formulation and free mTHPC. Therefore, we could not translate the beneficial macrophage selectivity from in vitro to in vivo. Nevertheless, we observed accumulation of mTHPC in atherosclerotic lesions of mice aorta's which is probably the result of binding to lipoproteins upon release from the micelles. Therefore, future experiments will be dedicated to increase the stability and thus allow accumulation of intact mTHPC-loaded Ben-PCL-mPEG micelles to macrophages of atherosclerotic lesions.
Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Mesoporfirinas/administración & dosificación , Micelas , Fármacos Fotosensibilizantes/administración & dosificación , Animales , Enfermedades Cardiovasculares/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/efectos de la radiación , Femenino , Luz , Mesoporfirinas/sangre , Mesoporfirinas/farmacocinética , Mesoporfirinas/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Fotoquimioterapia , Fármacos Fotosensibilizantes/sangre , Fármacos Fotosensibilizantes/farmacocinética , Fármacos Fotosensibilizantes/uso terapéutico , Poliésteres/administración & dosificación , Poliésteres/farmacocinética , Poliésteres/uso terapéutico , Células RAW 264.7 , Oxígeno Singlete/química , Distribución TisularRESUMEN
In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells.
Asunto(s)
Materiales Biocompatibles , Plaquetas/química , Cartílago/crecimiento & desarrollo , Condrogénesis/fisiología , Dextranos , Fenómenos Biomecánicos , Plaquetas/fisiología , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Condrocitos/efectos de los fármacos , Condrocitos/fisiología , Condrogénesis/efectos de los fármacos , Técnicas de Cocultivo , Sustancias de Crecimiento/administración & dosificación , Humanos , Hidrogeles , Ensayo de Materiales , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Microscopía Electrónica de Rastreo , Osteoartritis/patología , Osteoartritis/fisiopatología , Osteoartritis/terapia , Ingeniería de Tejidos , TiraminaRESUMEN
Tubular scaffolds (internal diameter approximately 3 mm and wall thickness approximately 0.8 mm) with a porosity of approximately 83% and an average pore size of 116 µm were prepared from flexible poly(trimethylene carbonate) (PTMC) polymer by dip-coating and particulate leaching methods. PTMC is a flexible and biocompatible polymer that crosslinks upon irradiation; porous network structures were obtained by irradiating the specimens in vacuum at 25 kGy before leaching soluble salt particles. To assess the suitability of these scaffolds in dynamic cell culturing for cardiovascular tissue engineering, the scaffolds were coated with a thin (0.1 to 0.2 mm) non-porous PTMC layer and its performance was evaluated in a closed pulsatile flow system (PFS). For this, the PFS was operated at physiological conditions at liquid flows of 1.56 ml/s with pressures varying from 80-120 mmHg at a frequency of 70 pulsations per minute. The mechanical properties of these coated porous PTMC scaffolds were not significantly different than non-coated scaffolds. Typical tensile strengths in the radial direction were 0.15 MPa, initial stiffness values were close to 1.4 MPa. Their creep resistance in cyclic deformation experiments was excellent. In the pulsatile flow setup, the distention rates of these flexible and elastic scaffolds were approximately 0.10% per mmHg, which is comparable to that of a porcine carotid artery (0.11% per mmHg). The compliance and stiffness index values were close to those of natural arteries.?In long-term deformation studies, where the scaffolds were subjected to physiological pulsatile pressures for one week, the morphology and mechanical properties of the PTMC scaffolds did not change. This suggests their suitability for application in a dynamic cell culture bioreactor.
Asunto(s)
Reactores Biológicos , Prótesis Vascular , Materiales Biocompatibles Revestidos , Dioxanos/química , Polímeros/química , Flujo Pulsátil , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Arterias Carótidas/fisiología , Adaptabilidad , Dioxanos/efectos de la radiación , Humanos , Arteria Ilíaca/fisiología , Bombas de Infusión , Arterias Mamarias/fisiología , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Polímeros/efectos de la radiación , Porosidad , Presión , Vena Safena/fisiología , Propiedades de Superficie , Porcinos , Resistencia a la Tracción , Factores de Tiempo , Microtomografía por Rayos XRESUMEN
Porous, tubular, flexible, and elastic poly(trimethylene carbonate) (PTMC) scaffolds (length 8 cm and inner diameter 3 mm) for vascular tissue engineering were prepared by means of a dip-coating and particulate leaching procedure. Using NaCl as porogen, scaffolds with an average pore size of 110 µm and a porosity of 85% were obtained. Before leaching the salt, the structures were made creep-resistant by means of crosslinking at 25 kGy gamma irradiation. To increase the efficiency of cell seeding, the scaffolds were provided with a microporous outer layer of 0.2 mm with an average pore size of 28 µm and a porosity of 65% (total wall thickness 1 mm). Human smooth muscle cells (SMCs) were seeded in these scaffolds with an efficiency of 43%, as determined after 24 h cell adhesion. SMCs were cultured in the scaffolds up to 14 days under stationary conditions or under pulsatile flow conditions in a bioreactor (pressure 70-130 mmHg, 69 pulsations/min, and average wall shear rate 320 s(-1)). Although SMCs proliferated under both conditions, cell numbers were three to five times higher in case of dynamic culturing. This was qualitatively confirmed by means of histology. Also, in terms of mechanical properties, the dynamically cultured constructs performed better than the statically cultured constructs. After culturing for 14 days, the maximum tensile strengths of the constructs, determined in the radial direction, had increased from 0.16 MPa (unseeded scaffold) to 0.48 MPa (dynamic culturing) and 0.38 MPa (static culturing). The results of this study indicate that a potentially useful medial layer for tissue-engineered vascular grafts can be prepared by dynamic culturing of human SMCs seeded in porous tubular poly(trimethylene carbonate) scaffolds.