RESUMEN

Chitosan, a natural polysaccharide derived from chitin, possesses biocompatibility, biodegradability, and mucoadhesive characteristics, making it an attractive material for the delivery of mRNA payloads to the nasal mucosa and promoting their uptake by target cells such as epithelial and immune cells (e.g., dendritic cells and macrophages). In this project, we aimed at developing novel lipid-based nanoformulations for mRNA delivery to counteract the pandemic caused by SARS-CoV-2 virus. The formulations achieved a mRNA encapsulation efficiency of ~80.2% with chitosan-lipid nanoparticles, as measured by the RiboGreen assay. Furthermore, the evaluation of SARS-CoV-2 Spike (S) receptor-binding domain (RBD) expression via ELISA for our vaccine formulations showed transfection levels in human embryonic kidney cells (HEK 293), lung carcinoma cells (A549), and dendritic cells (DC 2.4) equal to 9.9 ± 0.1 ng/mL (174.7 ± 1.1 fold change from untreated cells (UT)), 7.0 ± 0.2 ng/mL (128.1 ± 4.9 fold change from UT), and 0.9 ± 0.0 ng/mL (18.0 ± 0.1 fold change from UT), respectively. Our most promising vaccine formulation was also demonstrated to be amenable to lyophilization with minimal degradation of loaded mRNA, paving the way towards a more accessible and stable vaccine. Preliminary in vivo studies in mice were performed to assess the systemic and local immune responses. Nasal bronchoalveolar lavage fluid (BALF) wash showed that utilizing the optimized formulation resulted in local antibody concentrations and did not trigger any systemic antibody response. However, if further improved and developed, it could potentially contribute to the management of COVID-19 through nasopharyngeal immunization strategies.

RESUMEN

Understanding the stability of mRNA loaded lipid nanoparticles (mRNA-LNPs) is imperative for their clinical development. Herein, we propose the use of size-exclusion chromatography coupled with dual-angle light scattering (SEC-MALS) as a new approach to assessing mRNA-LNP stability in pure human serum and plasma. By applying a dual-column configuration to attenuate interference from plasma components, SEC-MALS was able to elucidate the degradation kinetics and physical property changes of mRNA-LNPs, which have not been observed accurately by conventional dynamic light scattering techniques. Interestingly, both serum and plasma had significantly different impacts on the molecular weight and radius of gyration of mRNA-LNPs, suggesting the involvement of clotting factors in desorption of lipids from mRNA-LNPs. We also discovered that a trace impurity (~1 %) in ALC-0315, identified as its O-tert-butyloxycarbonyl-protected form, greatly diminished mRNA-LNP stability in serum. These results demonstrated the potential utility of SEC-MALS for optimization and quality control of LNP formulations.

Asunto(s)

Cromatografía en Gel , Lípidos , Nanopartículas , ARN Mensajero , Humanos , ARN Mensajero/genética , ARN Mensajero/sangre , Nanopartículas/química , Lípidos/química , Dispersión Dinámica de Luz , Plasma/química , Luz , Dispersión de Radiación , Suero/química , Estabilidad del ARN , Liposomas

RESUMEN

The advent of SARS-CoV-2 variants with defined mutations that augment pathogenicity and/or increase immune evasiveness continues to stimulate global efforts to improve vaccine formulation and efficacy. The extraordinary advantages of lipid nanoparticles (LNPs), including versatile design, scalability, and reproducibility, make them ideal candidates for developing next-generation mRNA vaccines against circulating SARS-CoV-2 variants. Here, we assess the efficacy of LNP-encapsulated mRNA booster vaccines encoding the spike protein of SARS-CoV-2 for variants of concern (Delta, Omicron) and using a predecessor (YN2016C isolated from bats) strain spike protein to elicit durable cross-protective neutralizing antibody responses. The mRNA-LNP vaccines have desirable physicochemical characteristics, such as small size (~78 nm), low polydispersity index (<0.13), and high encapsulation efficiency (>90%). We employ in vivo bioluminescence imaging to illustrate the capacity of our LNPs to induce robust mRNA expression in secondary lymphoid organs. In a BALB/c mouse model, a three-dose subcutaneous immunization of mRNA-LNPs vaccines achieved remarkably high levels of cross-neutralization against the Omicron B1.1.529 and BA.2 variants for extended periods of time (28 weeks) with good safety profiles for all constructs when used in a booster regime, including the YN2016C bat virus sequences. These findings have important implications for the design of mRNA-LNP vaccines that aim to trigger durable cross-protective immunity against the current and newly emerging variants.

RESUMEN

Acute myeloid leukemia carrying FMS-like tyrosine kinase receptor-3 (FLT3) mutations is a fatal blood cancer with a poor prognosis. Although the FLT3 inhibitor gilteritinib has recently been approved, it still suffers from limited efficacy and relatively high nonresponse rates. In this study, we report the potentiation of gilteritinib efficacy using nanocomplexation with a hyaluronic acid-epigallocatechin gallate conjugate. The self-assembly, colloidal stability, and gilteritinib loading capacity of the nanocomplex were characterized by reversed-phase high-performance liquid chromatography and dynamic light scattering technique. Flow cytometric analysis revealed that the nanocomplex efficiently internalized into FLT3-mutated leukemic cells via specific interactions between the surface-exposed hyaluronic acid and CD44 receptor overexpressed on the cells. Moreover, this nanocomplex was found to induce an eradication of the leukemic cells in a synergistic manner by elevating the levels of reactive oxygen species and caspase-3/7 activities more effectively than free gilteritinib. This study may provide a useful strategy to design nanomedicines capable of augmenting the therapeutic efficacy of FLT3 inhibitors for effective leukemia therapy.

RESUMEN

BACKGROUND: Overproduction of reactive oxygen species (ROS) is known to delay wound healing by causing oxidative tissue damage and inflammation. The green tea catechin, (-)-Epigallocatechin-3-O-gallate (EGCG), has drawn a great deal of interest due to its strong ROS scavenging and anti-inflammatory activities. In this study, we developed EGCG-grafted silk fibroin hydrogels as a potential wound dressing material. METHODS: The introduction of EGCG to water-soluble silk fibroin (SF-WS) was accomplished by the nucleophilic addition reaction between lysine residues in silk proteins and EGCG quinone at mild basic pH. The resulting SF-EGCG conjugate was co-crosslinked with tyramine-substituted SF (SF-T) via horseradish peroxidase (HRP)/H2O2 mediated enzymatic reaction to form SF-T/SF-EGCG hydrogels with series of composition ratios. RESULTS: Interestingly, SF-T70/SF-EGCG30 hydrogels exhibited rapid in situ gelation (< 30 s), similar storage modulus to human skin (≈ 1000 Pa) and superior wound healing performance over SF-T hydrogels and a commercial DuoDERM® gel dressings in a rat model of full thickness skin defect. CONCLUSION: This study will provide useful insights into a rational design of ROS scavenging biomaterials for wound healing applications.

RESUMEN

BACKGROUND: Currently available anti-leukemia drugs have shown limited success in the treatment of acute myeloid leukemia (AML) due to their poor access to bone marrow niche supporting leukemic cell proliferation. RESULTS: Herein, we report a bone marrow-targetable green tea catechin-based micellar nanocomplex for synergistic AML therapy. The nanocomplex was found to synergistically amplify the anti-leukemic potency of sorafenib via selective disruption of pro-survival mTOR signaling. In vivo biodistribution study demonstrated about 11-fold greater bone marrow accumulation of the nanocomplex compared to free sorafenib. In AML patient-derived xenograft (AML-PDX) mouse model, administration of the nanocomplex effectively eradicated bone marrow-residing leukemic blasts and improved survival rates without noticeable off-target toxicity. CONCLUSION: This study may provide insights into the rational design of nanomedicine platforms enabling bone marrow-targeted delivery of therapeutic agents for the treatment of AML and other bone marrow diseases.

Asunto(s)

Catequina , Leucemia Mieloide Aguda , Ratones , Animales , Humanos , Médula Ósea , Catequina/farmacología , Micelas , Sorafenib , Distribución Tisular , Leucemia Mieloide Aguda/tratamiento farmacológico , Modelos Animales de Enfermedad , Té

RESUMEN

(-)-Epigallocatechin-3-O-gallate (EGCG), the most bioactive catechin in green tea, has drawn significant interest as a potent antioxidant and anti-inflammatory compound. However, the application of EGCG has been limited by its rapid autoxidation at physiological pH, which generates cytotoxic levels of reactive oxygen species (ROS). Herein, we report the synthesis of poly(acrylic acid)-EGCG conjugates with tunable degrees of substitution and their spontaneous self-assembly into micellar nanoparticles with enhanced resistance against autoxidation. These nanoparticles not only exhibited superior oxidative stability and cytocompatibility over native EGCG, but also showed excellent ROS-scavenging and anti-inflammatory effects. This work presents a potential strategy to overcome the stability and cytotoxicity issues of EGCG, making it one step closer toward its widespread application.

Asunto(s)

Catequina , Nanopartículas , Resinas Acrílicas , Antiinflamatorios/farmacología , Catequina/análogos & derivados , Catequina/farmacología , Micelas , Especies Reactivas de Oxígeno , Té/química

RESUMEN

Chemoresistance is one of the major challenges for the treatment of acute myeloid leukemia. Epigallocatechin gallate (EGCG), a bioactive polyphenol from green tea, has attracted immense interest as a potential chemosensitizer, but its application is limited due to the need for effective formulations capable of co-delivering EGCG and anti-leukemic drugs. Herein, we describe the formation and characterization of a micellar nanocomplex self-assembled from EGCG and daunorubicin, an anthracycline drug for the first-line treatment of acute myeloid leukemia. This nanocomplex was highly stable at pH 7.4 but stimulated to release the incorporated daunorubicin at pH 5.5, mimicking an acidic endosomal environment. More importantly, the nanocomplex exhibited superior cytotoxic efficacy against multidrug-resistant human leukemia cells over free daunorubicin by achieving a strong synergism, as supported by median-effect plot analysis. The observed chemosensitizing effect was in association with enhanced nucleus accumulation of daunorubicin, elevation of intracellular reactive oxygen species and caspase-mediated apoptosis induction. Our study presents a promising strategy for circumventing chemoresistance for more effective leukemia therapy.

Asunto(s)

Catequina , Leucemia Mieloide Aguda , Humanos , Daunorrubicina/farmacología , Apoptosis , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/tratamiento farmacológico , Catequina/farmacología , Té/química

RESUMEN

Fibroblast-like synoviocytes are a key effector cell type involved in the pathogenesis of rheumatoid arthritis. The major green tea catechin, epigallocatechin-3-O-gallate (EGCG), has attracted significant interest for rheumatoid arthritis therapy because of its ability to suppress the proliferation and interleukin-6 secretion of synoviocytes. However, therapeutic efficacy of EGCG has been limited by a lack of target cell specificity. Herein we report hyaluronic acid-EGCG (HA-EGCG) conjugates as an anti-arthritic agent that is capable of targeting fibroblast-like synoviocytes via HA-CD44 interactions. These conjugates exhibited superior anti-proliferative and anti-inflammatory activities compared with EGCG under simulated physiological conditions. Near-infrared fluorescence imaging revealed preferential accumulation of the conjugates at inflamed joints in a collagen-induced arthritis rat model, and their anti-arthritic efficacy was investigated by measuring a change in the edema and histopathological scores. Our findings suggest the potential of HA-EGCG conjugates as an anti-arthritic agent for the treatment of rheumatoid arthritis.

RESUMEN

Acute myeloid leukemia (AML) is an aggressive malignancy that leads to a poor prognosis even with intensive chemotherapy. As the key feature of AML is the blockade of hematopoietic cell maturation, considerable attention has been paid to 'differentiation therapy' aimed at transforming AML cells into more mature, benign phenotypes using pharmacological agents. Here we report a hyaluronic acid-(-)-epigallocatechin-3-O-gallate (HA-EGCG) conjugate as a unique anti-leukemic agent, capable of selectively killing AML cells as well as promoting their terminal differentiation into monocytes and granulocytes. This 'two-pronged' effect of the HA-EGCG conjugate was demonstrated in two different AML cell lines (NB4 and HL60), but absent in a physical mixture (HA + EGCG), highlighting the importance of HA conjugation for targeting of EGCG moieties to AML cells. Moreover, administration of the HA-EGCG conjugate not only suppressed AML progression, but also prolonged survival in the HL60 xenograft mouse model. Our study suggests new opportunities for designing two-pronged anti-leukemic agents for more effective AML treatment.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Catequina/análogos & derivados , Ácido Hialurónico/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Catequina/administración & dosificación , Catequina/química , Catequina/farmacología , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HEK293 , Células HL-60 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Masculino , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Although a few nanomedicines have been approved for clinical use in cancer treatment, that recognizes improved patient safety through targeted delivery, their improved efficacy over conventional drugs has remained marginal. One of the typical drawbacks of nanocarriers for cancer therapy is a low drug-loading capacity that leads to insufficient efficacy and requires an increase in dosage and/or frequency of administration, which in turn increases carrier toxicity. In contrast, elevating drug-loading would cause the risk of nanocarrier instability, resulting in low efficacy and off-target toxicity. This intractable drug-to-carrier ratio has imposed constraints on the design and development of nanocarriers. However, if the nanocarrier has intrinsic therapeutic effects, the efficacy would be synergistically augmented with less concern for the drug-to-carrier ratio. Sunitinib-loaded micellar nanocomplex (SU-MNC) was formed using poly(ethylene glycol)-conjugated epigallocatechin-3-O-gallate (PEG-EGCG) as such a carrier. SU-MNC specifically inhibited the vascular endothelial growth factor-induced proliferation of endothelial cells, exhibiting minimal cytotoxicity to normal renal cells. SU-MNC showed enhanced anticancer effects and less toxicity than SU administered orally/intravenously on human renal cell carcinoma-xenografted mice, demonstrating more efficient effects on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors. In comparison, a conventional nanocarrier, SU-loaded polymeric micelle (SU-PM) comprised of PEG-b-poly(lactic acid) (PEG-PLA) copolymer, only reduced toxicity with no elevated efficacy, despite comparable drug-loading and tumor-targeting efficiency to SU-MNC. Improved efficacy of SU-MNC was ascribed to the carrier-drug synergies with the high-performance carrier of PEG-EGCG besides tumor-targeted delivery.

Asunto(s)

Antineoplásicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Neoplasias Renales/tratamiento farmacológico , Nanopartículas/química , Sunitinib/farmacología , Té/química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Catequina/análogos & derivados , Catequina/química , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Portadores de Fármacos/química , Femenino , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , Micelas , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Tamaño de la Partícula , Polietilenglicoles/química , Sunitinib/administración & dosificación , Sunitinib/química , Propiedades de Superficie , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Enzymatic crosslinking chemistry using horseradish peroxidase (HRP) has been widely utilized as an effective approach to fabricating injectable hydrogels with high efficiency under mild reaction conditions. However, their clinical applications are limited by the immunogenicity of the plant-derived enzyme. Herein we report the design, synthesis and characterization of HRP-immobilized porous silica particles (HRP-particles) and their use for in situ formation of HRP-free hydrogels. HRP was immobilized on aminopropyl-modified porous silica particles of 70-140 µm in diameter via poly(ethylene glycol) spacers of different molecular weights by reductive amination reaction. Two different HRP-free hydrogels based on dextran-tyramine and gelatin-hydroxyphenylpropionic acid (GHPA) conjugates were produced by passing a solution containing the conjugates and H2O2 through a syringe packed with HRP-particles. The storage modulus and gelation rate of both hydrogels were tunable by varying the contact time between the polymer solution and HRP-particles. Our in vitro study revealed that HRP-free GHPA hydrogel was less stimulatory to activated mouse macrophages than HRP-containing GHPA hydrogel with the same stiffness. Furthermore, HRP-free GHPA hydrogel exhibited remarkably lower levels of local and systemic inflammation than HRP-containing one upon subcutaneous injection in immunocompetent C57BL/6J mice. The attenuated immunogenicity of HRP-free GHPA hydrogels makes them an attractive platform for tissue engineering applications. STATEMENT OF SIGNIFICANCE: The immunogenicity of HRP is a significant issue for clinical application of HRP-catalyzed in situ forming hydrogels. HRP-particles are developed to overcome the safety concerns by fabricating HRP-free hydrogels. The porosity of silica particles and molecular weight of poly(ethylene glycol) spacers are discovered as important factors determining the catalytic ability of HRP-particles to induce the in situ crosslinking of polymer-phenol conjugates. Although several articles speculate the potential of HRP to trigger immune responses when administered as a part of hydrogel formulation, no literature has attempted to investigate the immunogenicity of HRP-containing hydrogels in comparison with HRP-free hydrogels. Our findings suggest that the immunogenicity issue should be carefully considered before clinical translation of HRP-containing hydrogels.

Asunto(s)

Hidrogeles , Ingeniería de Tejidos , Animales , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/inmunología , Enzimas Inmovilizadas/farmacología , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/inmunología , Peroxidasa de Rábano Silvestre/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hidrogeles/química , Hidrogeles/farmacología , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/inmunología , Peróxido de Hidrógeno/farmacología , Masculino , Ratones , Porosidad , Células RAW 264.7

RESUMEN

The green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has gained significant attention as a potent adjuvant to enhance the antitumor efficacy of cisplatin while mitigating its harmful side effects. Herein we report the development of a fail-safe cisplatin nanomedicine constructed with hyaluronic acid-EGCG conjugate for ovarian cancer therapy. A simple mixing of this conjugate and cisplatin induces spontaneous self-assembly of micellar nanocomplexes having a spherical core-shell structure. The surface-exposed hyaluronic acid enables efficient delivery of cisplatin into CD44-overexpressing cancer cells via receptor-mediated endocytosis whereas the internally packed EGCG moieties offer an environment favorable for the encapsulation of cisplatin. In addition, the antioxidant effect of EGCG moieties ensures fail-safe protection against off-target organ toxicity originating from cisplatin-evoked oxidative stress. Pharmacokinetic and biodistribution studies reveal the prolonged blood circulation and preferential tumor accumulation of intravenously administered nanocomplexes. Moreover, the nanocomplexes exhibit superior antitumor efficacy over free cisplatin while displaying no toxicity in both a subcutaneous xenograft model and peritoneal metastatic model of human ovarian cancer. Our findings demonstrate proof of concept for the feasibility of green tea catechin-based micellar nanocomplexes as a safe and effective cisplatin nanomedicine for ovarian cancer treatment.

Asunto(s)

Antineoplásicos/farmacología , Catequina/análogos & derivados , Cisplatino/química , Ácido Hialurónico/farmacología , Nanoconjugados/química , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Catequina/química , Catequina/metabolismo , Catequina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Liberación de Fármacos , Femenino , Humanos , Ácido Hialurónico/química , Ratones SCID , Micelas , Estrés Oxidativo/efectos de los fármacos , Tamaño de la Partícula , Propiedades de Superficie , Té/química , Distribución Tisular

RESUMEN

Hyaluronic acid (HA)-based biomaterials have demonstrated only limited in vivo stability as a result of rapid degradation by hyaluronidase and reactive oxidative species. The green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has received considerable attention because of its powerful antioxidant and enzyme-inhibitory activities. We describe here the synthesis of HA-EGCG conjugate using a thiol-mediated reaction and its use for the preparation of a long-lasting injectable hydrogel. HA-EGCG conjugates with tunable degrees of substitution were synthesized by the nucleophilic addition reaction between EGCG quinone and thiolated HA under mild conditions. Contrary to unmodified HA, the conjugates exhibited free radical scavenging and hyaluronidase-inhibitory activities. Peroxidase-catalyzed coupling reaction between EGCG moieties was employed to produce in situ forming HA-EGCG hydrogel with surprisingly high resistance to hyaluronidase-mediated degradation. When injected subcutaneously in mice, HA-EGCG hydrogel was retained much longer than HA-tyramine hydrogel with minimal inflammation.

Asunto(s)

Catequina/análogos & derivados , Depuradores de Radicales Libres/química , Ácido Hialurónico/química , Hidrogeles/química , Animales , Catequina/química , Línea Celular , Femenino , Depuradores de Radicales Libres/efectos adversos , Depuradores de Radicales Libres/síntesis química , Depuradores de Radicales Libres/farmacocinética , Hidrogeles/efectos adversos , Hidrogeles/síntesis química , Hidrogeles/farmacocinética , Macrófagos/efectos de los fármacos , Ratones , Compuestos de Sulfhidrilo/química , Distribución Tisular

RESUMEN

Hydrogels have evolved into indispensable biomaterials in the fields of drug delivery and regenerative medicine. This minireview aims to highlight the recent advances in the hydrogel design for controlled release of bioactive proteins. The latest developments of enzyme-responsive and externally regulated drug delivery systems are summarized. The design strategies and applications of phase-separated hydrogel systems are also described. We expect that these emerging approaches will enable expanded use of hydrogels in biomedicine and healthcare.

Asunto(s)

Preparaciones de Acción Retardada/química , Sistemas de Liberación de Medicamentos , Hidrogeles/química , Proteínas/química , Animales , Materiales Biocompatibles/química , Enzimas/química , Humanos , Polietilenglicoles/química

RESUMEN

Nanosized polyelectrolyte complexes are attractive delivery vehicles for the transfer of therapeutic genes to diseased cells. Here we report the application of self-assembled ternary complexes constructed with plasmid DNA, branched polyethylenimine and hyaluronic acid-green tea catechin conjugates for targeted gene delivery. These conjugates not only stabilize plasmid DNA/polyethylenimine complexes via the strong DNA-binding affinity of green tea catechin, but also facilitate their transport into CD44-overexpressing cells via receptor-mediated endocytosis. The hydrodynamic size, surface charge and physical stability of the complexes are characterized. We demonstrate that the stabilized ternary complexes display enhanced resistance to nuclease attack and polyanion-induced dissociation. Moreover, the ternary complexes can efficiently transfect the difficult-to-transfect HCT-116 colon cancer cell line even in serum-supplemented media due to their enhanced stability and CD44-targeting ability. Confocal microscopic analysis demonstrates that the stabilized ternary complexes are able to promote the nuclear transport of plasmid DNA more effectively than binary complexes and hyaluronic acid-coated ternary complexes. The present study suggests that the ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates can be widely utilized for CD44-targeted delivery of nucleic acid-based therapeutics.

Asunto(s)

Catequina/análogos & derivados , ADN/administración & dosificación , Ácido Hialurónico/metabolismo , Plásmidos/administración & dosificación , Transfección/métodos , Catequina/química , Catequina/metabolismo , ADN/genética , Endocitosis , Proteínas Fluorescentes Verdes/genética , Células HCT116 , Células HEK293 , Humanos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/química , Plásmidos/genética , Polietileneimina/química , Polietileneimina/metabolismo , Té/química , Té/metabolismo

RESUMEN

Immunotherapy including interferon-alpha (IFN-α) is one of the treatment options for metastatic renal cell carcinoma (mRCC) patients. Despite clinical benefits for the selected patients, IFN-α therapy has some problems, such as poor tolerability and dose-limiting adverse effects. In addition, the frequent injections reduce a patient's quality of life and compliance. Recently, an injectable and biodegradable hydrogel system to prolong drug release is reported. In this study, we investigated the anticancer effect of IFN-α (Sumiferon®)-incorporated hyaluronic acid-tyramine (HA-Tyr) hydrogels in human RCC-xenografted in nude mice. We also evaluated the synergistic efficacy of IFN-α-incorporated HA-Tyr hydrogels+sorafenib in this model. IFN-α-incorporated HA-Tyr hydrogels+sorafenib most effectively inhibited tumor growth on human RCC cells xenografted in nude mice. In addition, IFN-α-incorporated HA-Tyr hydrogels+sorafenib inhibited the proliferation of tumor in nude mice by inducing apoptosis and the suppression of angiogenesis. Our results suggest a possibility that HA-Tyr hydrogel drug delivery system prolongs the biological half-life of natural human IFN-α and enhances its anticancer effects on human RCC cells. STATEMENT OF SIGNIFICANCE: The scope of this study is to provide an alternative approach to improve the anticancer efficacy in renal cell carcinoma (RCC) treatment by using hyaluronic acid-tyramine (HA-Tyr) hydrogel drug delivery system. We investigated the anticancer effect of natural interferon-α (IFN-α)-incorporated HA-Tyr hydrogels in RCC cells. We also evaluated the synergistic efficacy of natural human IFN-α-incorporated HA-Tyr hydrogels+sorafenib. We demonstrated that HA-Tyr hydrogel system is able to release natural human IFN-α in sustained manner and enhances its anticancer effects on human RCC cells. In addition, we suggested that IFN-α-incorporated HA-Tyr hydrogels+sorafenib exhibited most effectively anticancer effects. Hence, we believe that this approach could be applied to treatment with RCC in the future.

Asunto(s)

Carcinoma de Células Renales/tratamiento farmacológico , Ácido Hialurónico , Hidrogeles , Interferón-alfa , Neoplasias Renales/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Tiramina , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Hidrogeles/química , Hidrogeles/farmacología , Interferón-alfa/química , Interferón-alfa/farmacología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Ratones Desnudos , Niacinamida/química , Niacinamida/farmacología , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Sorafenib , Tiramina/química , Tiramina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Hydrogels are widely used as reservoirs in drug delivery and scaffolds for tissue engineering. In particular, injectable hydrogel systems, which are formed by physical, chemical, or enzyme-mediated crosslinking reactions in situ, offer the advantages of minimal invasiveness, ease of application, and void-filling property. Examples of these hydrogels are provided in the first part of this paper. In the second part, hydrogels that are formed by the enzymatic activity of horseradish peroxidase (HRP) are highlighted. HRP catalyzes the crosslinking reaction of polymer-phenol conjugates in the presence of hydrogen peroxide (H2O2), resulting in hydrogels with tunable gelation rate and crosslinking density. The catalytic mechanism of the HRP-mediated crosslinking reaction is discussed in detail, and the recent biomedical applications of the HRP-crosslinked hydrogels are described. Lastly, the concerns associated with HRP-mediated crosslinking and the future outlook of HRP-crosslinked hydrogels are addressed.

Asunto(s)

Materiales Biocompatibles/síntesis química , Reactivos de Enlaces Cruzados/química , Peroxidasa de Rábano Silvestre/química , Hidrogeles/química , Trasplante de Células Madre/métodos , Ingeniería de Tejidos/métodos , Materiales Biocompatibles/administración & dosificación , Activación Enzimática , Enzimas Inmovilizadas/química , Hidrogeles/administración & dosificación , Inyecciones/métodos , Viscosidad

RESUMEN

The propagation of human embryonic stem cells (hESCs) in three-dimensional (3D) scaffolds facilitates the cell expansion process and supplies pluripotent cells of high quality for broad-spectrum applications in regenerative medicine. Herein, we report an enzyme-mediated hyaluronic acid-tyramine (HA-Tyr) hydrogel that encapsulated and propagated hESCs in 3D. HA-Tyr hydrogels were formed by crosslinking the tyramine moieties with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentration, we prepared HA-Tyr hydrogels of different mechanical strength and studied the self-renewal properties of hESCs in these scaffolds. We observed that both the chemical composition and mechanical strength of substrates were important factors affecting cell proliferation and pluripotency. The HA-Tyr hydrogel with a compressive modulus of ∼350Pa supported the proliferation of hESCs at the pluripotent state in both mTeSR1 medium and mouse embryonic fibroblast (MEF)-conditioned medium. Immunohistochemical analyses revealed that hESCs proliferated well and formed spheroid structures in 3D, without undergoing apoptosis. The hESCs cultured in HA-Tyr hydrogels showed high expression of CD44 and pluripotency markers. These cells exhibited the capability to form cell derivatives of all three embryonic germ layers in vitro and in vivo. In addition, the genetic integrity of the hESCs was unaffected in the 3D cultivation system. STATEMENT OF SIGNIFICANCE: The scope of this study is to provide a stable 3D cultivation system for the expansion of human embryonic stem cells (hESCs) towards clinical applications. We report an enzyme mediated hyaluronic acid-tyramine (HA-Tyr) hydrogel that encapsulated and propagated hESCs in 3D. Unlike other HA-based photo-crosslinked hydrogel systems reported, we investigated the effects of mechanical strength of hydrogels on the self-renewal properties of hESCs in 3D. Then, we characterized hESCs cultured in hydrogels with lower mechanical strength that best supported the self-renewal of hESCs. Hence, we demonstrated a reliable approach for the controlled propagation of hESCs in 3D. We believe that such an approach would facilitate the development of stem cell-based therapy towards clinical applications.

Asunto(s)

Células Madre Embrionarias Humanas/metabolismo , Ácido Hialurónico/química , Hidrogeles/química , Ensayo de Materiales , Andamios del Tejido/química , Tiramina/química , Animales , Peroxidasa de Rábano Silvestre/química , Células Madre Embrionarias Humanas/citología , Humanos , Peróxido de Hidrógeno/química , Ratones