RESUMO
Pulmonary fibrosis is a lethal interstitial lung disease, for which current treatments are inadequate in halting its progression. A significant factor contributing to the development of fibrosis is insufficient autophagy, which leads to increased fibroblast proliferation and collagen deposition. However, treatments aimed at upregulating autophagy often cause further lung pathology due to the disruption of epithelial cell balance. In response, we have developed a novel macrophage delivery system loaded with an epithelial-to-mesenchymal transition inhibitor, hyperoside (HYP), and an autophagy inducer, rapamycin (RAP). This system targets the fibrotic areas of the lung through chemotaxis, releases liposomes via macrophage extracellular traps, and effectively inhibits fibroblast proliferation while restoring the alveolar structure through the combined effects of RAP and HYP, ultimately reducing lung pathology without causing systemic toxicity. Our findings not only highlight a promising method to enhance autophagy-based treatments for pulmonary fibrosis but also demonstrate the potential of macrophages as effective nanocarriers for drug delivery.
Assuntos
Autofagia , Transição Epitelial-Mesenquimal , Macrófagos , Fibrose Pulmonar , Quercetina , Sirolimo , Autofagia/efeitos dos fármacos , Sirolimo/farmacologia , Sirolimo/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Animais , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Quercetina/química , Quercetina/farmacologia , Quercetina/análogos & derivados , Humanos , Regulação para Cima/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Sistemas de Liberação de Medicamentos , Células RAW 264.7RESUMO
Purpose: Renal cell carcinoma (RCC) is the most common and lethal type of urogenital cancer, with one-third of new cases presenting as metastatic RCC (mRCC), which, being the seventh most common cancer in men and the ninth in women, poses a significant challenge. For patients with poor prognosis, temsirolimus (TEM) has been approved for first-line therapy, possessing pharmacodynamic activities that block cancer cell growth and inhibit proliferation-associated proteins. However, TEM suffers from poor water solubility, low bioavailability, and systemic side effects. This study aims to develop a novel drug formulation for the treatment of RCC. Methods: In this study, amphiphilic block copolymer (poly(ethylene glycol) monomethyl ether-poly(beta-amino ester)) (mPEG-PBAE) was utilized as a drug delivery vehicle and TEM-loaded micelles were prepared by thin-film hydration method by loading TEM inside the nanoparticles. Then, the molecular weight of mPEG-PBAE was controlled to make it realize hydrophobic-hydrophilic transition in the corresponding pH range thereby constructing pH-responsive TEM-loaded micelles. Characterization of pH-responsive TEM-loaded nanomicelles particle size, potential and micromorphology while its determination of drug-loading properties, in vitro release properties. Finally, pharmacodynamics and hepatorenal toxicity were further evaluated. Results: TEM loading in mPEG-PBAE increased the solubility of TEM in water from 2.6 µg/mL to more than 5 mg/mL. The pH-responsive TEM-loaded nanomicelles were in the form of spheres or spheroidal shapes with an average particle size of 43.83 nm and a Zeta potential of 1.79 mV. The entrapment efficiency (EE) of pH-responsive TEM nanomicelles with 12.5% drug loading reached 95.27%. Under the environment of pH 6.7, the TEM was released rapidly within 12 h, and the release rate could reach 73.12% with significant pH-dependent characteristics. In vitro experiments showed that mPEG-PBAE preparation of TEM-loaded micelles had non-hemolytic properties and had significant inhibitory effects on cancer cells. In vivo experiments demonstrated that pH-responsive TEM-loaded micelles had excellent antitumor effects with significantly reduced liver and kidney toxicity. Conclusion: In conclusion, we successfully prepared pH-responsive TEM-loaded micelles. The results showed that pH-responsive TEM-loaded micelles can achieve passive tumor targeting of TEM, and take advantage of the acidic conditions in tumor tissues to achieve rapid drug release.
Assuntos
Antineoplásicos , Carcinoma de Células Renais , Neoplasias Renais , Micelas , Polietilenoglicóis , Sirolimo , Sirolimo/administração & dosagem , Sirolimo/química , Sirolimo/farmacocinética , Sirolimo/farmacologia , Sirolimo/análogos & derivados , Humanos , Polietilenoglicóis/química , Concentração de Íons de Hidrogênio , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Tamanho da Partícula , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Camundongos , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Feminino , MasculinoRESUMO
In the realm of arterial disease interventions, drug-eluting stents (DES) have become a vital therapeutic choice in preventing atherosclerotic plaque formation and restenosis and facilitating vessel healing. Sirolimus-encapsulated poly Lactic-co-Glycolic acid (PLGA) Microparticles (MPs) were developed using solvent evaporation. MPs were freeze-dried with a cryoprotectant and coated on the stent surface using an efficient and reproducible nitrogen-assisted spray coating technique. The MPs displayed a uniform distribution particle size of 4.38 ± 1.1 µm, span value of 0.88 ± 0.02, coating mass transfer efficiency of 13.45 ± 1.1 % on the stent, and a coating time of ≤ 2 min per stent. Post sterilization, the particle size and morphology of the coated stents remained unchanged. Accelerated in vitro drug release profiles were evaluated under different conditions, indicating significant influences based on dissolution methods ranging from 28.2 %±4.3 %, 42.5 %±5.3 %, 76.6 %±4.7 %, and 84.25 %±3.1 % for dialysis bag (DB), vessel simulating flow-through cell (vFTC), flow-through cell (FTC), and sample and separate (SS) technique respectively for 48 h. The drug release mechanism from the coated stents is governed by the combination of the Korsmeyer Peppas and Higuchi models. The developed dissolution method exhibited discriminative effectiveness when evaluated with critical formulation attributes and process parameter variations. The 48 h accelerated drug release studies correlated well with the 6-month real-time release rate with an R2 value of 0.9142 and Pearson's R2 of 0.9561. Ex-vivo studies demonstrated the permeation of MPs into artery tissues. Stability studies confirmed that MPs coated stents maintained desired properties at 4 °C and 30 °C/65 % RH for 6 months. Overall, these findings contribute to advancing stent technology, suggesting the potential for improvement of arterial interventions and enhanced patient outcomes.
Assuntos
Liberação Controlada de Fármacos , Stents Farmacológicos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Sirolimo , Sirolimo/administração & dosagem , Sirolimo/farmacocinética , Sirolimo/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Tamanho da PartículaRESUMO
Purpose: Ovarian cancer has the highest mortality rate and lowest survival rate among female reproductive system malignancies. There are treatment options of surgery and chemotherapy, but both are limited. In this study, we developed and evaluated micelles composed of D-α-tocopheryl polyethylene-glycol (PEG) 1000 succinate (TPGS) and Soluplus® (SOL) loaded with olaparib (OLA), a poly(ADP-ribose)polymerase (PARP) inhibitor, and rapamycin (RAPA), a mammalian target of rapamycin (mTOR) inhibitor in ovarian cancer. Methods: We prepared micelles containing different molar ratios of OLA and RAPA embedded in different weight ratios of TPGS and SOL (OLA/RAPA-TPGS/SOL) were prepared and physicochemical characterized. Furthermore, we performed in vitro cytotoxicity experiments of OLA, RAPA, and OLA/RAPA-TPGS/SOL. In vivo toxicity and antitumor efficacy assays were also performed to assess the efficacy of the mixed micellar system. Results: OLA/RAPA-TPGS/SOL containing a 4:1 TPGS:SOL weight ratio and a 2:3 OLA:RAPA molar ratio showed synergistic effects and were optimized. The drug encapsulation efficiency of this formulation was >65%, and the physicochemical properties were sustained for 180 days. Moreover, the formulation had a high cell uptake rate and significantly inhibited cell migration (**p < 0.01). In the in vivo toxicity test, no toxicity was observed, with the exception of the high dose group. Furthermore, OLA/RAPA-TPGS/SOL markedly inhibited tumor spheroid and tumor growth in vivo. Conclusion: Compared to the control, OLA/RAPA-TPGS/SOL showed significant tumor inhibition. These findings lay a foundation for the use of TPGS/SOL mixed micelles loaded with OLA and RAPA in the treatment of ovarian cancer.
Assuntos
Micelas , Neoplasias Ovarianas , Ftalazinas , Piperazinas , Polietilenoglicóis , Polivinil , Sirolimo , Vitamina E , Feminino , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Piperazinas/química , Piperazinas/farmacologia , Polietilenoglicóis/química , Humanos , Animais , Linhagem Celular Tumoral , Vitamina E/química , Vitamina E/farmacologia , Sirolimo/química , Sirolimo/farmacologia , Sirolimo/administração & dosagem , Sirolimo/farmacocinética , Ftalazinas/química , Ftalazinas/farmacologia , Ftalazinas/administração & dosagem , Ftalazinas/farmacocinética , Polivinil/química , Polivinil/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagem , Camundongos , Portadores de Fármacos/química , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Camundongos Endogâmicos BALB C , Sobrevivência Celular/efeitos dos fármacosRESUMO
Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events.
Assuntos
Multimerização Proteica , Proteínas Recombinantes de Fusão , Sirolimo , Proteínas de Ligação a Tacrolimo , Sirolimo/química , Sirolimo/farmacologia , Sirolimo/metabolismo , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Humanos , Cristalografia por Raios X , Modelos Moleculares , Domínios Proteicos , Ligação ProteicaRESUMO
Heart transplantation offers life-saving treatment for patients with end-stage heart failure; however, ischemia-reperfusion injury (IRI) and subsequent immune responses remain significant challenges. Current therapies primarily target adaptive immunity, with limited options available for addressing IRI and innate immune activation. Although plant-derived vesicle-like nanoparticles show promise in managing diseases, their application in organ transplantation complications is unexplored. Here, this work develops a novel reactive oxygen species (ROS)-responsive multifunctional fusion extracellular nanovesicles carrying rapamycin (FNVs@RAPA) to address early IRI and Ly6C+Ly6G- inflammatory macrophage-mediated rejection in heart transplantation. The FNVs comprise Exocarpium Citri grandis-derived extracellular nanovesicles with anti-inflammatory and antioxidant properties, and mesenchymal stem cell membrane-derived nanovesicles expressing calreticulin with macrophage-targeting ability. A novel ROS-responsive bio-orthogonal chemistry approach facilitates the active targeting delivery of FNVs@RAPA to the heart graft site, effectively alleviating IRI and promoting the polarization of Ly6C+Ly6G- inflammatory macrophages toward an anti-inflammatory phenotype. Hence, FNVs@RAPA represents a promising therapeutic approach for mitigating early transplantation complications and immune rejection. The fusion-targeted delivery strategy offers superior heart graft site enrichment and macrophage-specific targeting, promising improved transplant outcomes.
Assuntos
Rejeição de Enxerto , Transplante de Coração , Macrófagos , Espécies Reativas de Oxigênio , Sirolimo , Espécies Reativas de Oxigênio/metabolismo , Animais , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/prevenção & controle , Sirolimo/química , Sirolimo/farmacologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Nanopartículas/química , Calreticulina/metabolismo , Calreticulina/química , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismoRESUMO
Sirolimus (SR) is a macrolide with antifungal and antitumor immunosuppressant properties, classified as a selective inhibitor of mammalian target of rapamycin (mTOR). In this study, an ionic in situ gel of SR (SR-SUS-ISG) was formulated using gellan gum, exhibiting stability regardless of temperature and pH variations, causing minimal irritation. Harnessing the physiological conditions of the eye, SR-SUS-ISG underwent gelation upon contact with ions, increasing drug viscosity and prolonging retention on the ocular surface. Concurrently, SR-SUS-ISG displayed favorable shear dilution properties, reducing viscosity at ambient temperature, enhancing fluidity, and facilitating convenient packaging and transport. Biocompatibility assessments on both human corneal epithelial cells and rabbit eyes demonstrated that SR-SUS-ISG could well be tolerated. Pharmacokinetic investigations in rabbit ocular aqueous humor revealed sustained release, improved corneal penetration, and enhanced bioavailability. Additionally, in a rat corneal alkali burn model, SR-SUS-ISG exhibited inhibitory effects on corneal neovascularization, associated with decreased levels of the inflammatory factors VEGF and MMPs. These findings suggested that SR-SUS-ISG held promise as an effective ocular drug delivery system.
Assuntos
Géis , Sirolimo , Animais , Coelhos , Sirolimo/farmacologia , Sirolimo/farmacocinética , Sirolimo/química , Humanos , Géis/química , Córnea/efeitos dos fármacos , Córnea/metabolismo , Ratos , Masculino , Polissacarídeos Bacterianos/química , Nanopartículas/química , Administração Oftálmica , Neovascularização da Córnea/tratamento farmacológico , Ratos Sprague-Dawley , Viscosidade , Sistemas de Liberação de Medicamentos , Soluções Oftálmicas/química , Soluções Oftálmicas/farmacologia , Linhagem Celular , Disponibilidade BiológicaRESUMO
Drug delivery systems based on biomimetic peptide nanoparticles are steadily gaining prominence in the treatment of diverse medical conditions. This study focused on the development of peptides that depend on ligand-receptor interactions to load rapamycin (RAPA). Furthermore, a multifunctional peptide was engineered to target oxidized low-density lipoprotein (oxLDL) within atherosclerotic plaques, facilitating the localized delivery of RAPA. The interactions between peptides and RAPA/oxLDL were analyzed by simulations and experimental approaches. Results show that the main amino acid residues on the mammalian target of rapamycin that bind to RAPA are constructed as peptides (P1 and P2), which have specific interactions with RAPA and can effectively improve the loading efficiency of RAPA. The encapsulation and drug loading efficiencies of P1/P2 were 68.0/47.9% and 48.3/36.5%, respectively. In addition, the interaction force of the multifunctional peptide (P3) and oxLDL surpassed that of their interaction with human umbilical vein endothelial cells by a factor of 3.6, conclusively establishing the specific targeting of oxLDL by these nanoparticles. The encapsulation and drug loading efficiencies of P3 for RAPA were determined to be 60.2% and 41.5%. P3 can effectively load RAPA and target oxLDL within the plaque, suggesting that P3 has potential as a therapeutic agent for atherosclerotic disease.
Assuntos
Células Endoteliais da Veia Umbilical Humana , Lipoproteínas LDL , Nanopartículas , Peptídeos , Placa Aterosclerótica , Sirolimo , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Sirolimo/administração & dosagem , Sirolimo/química , Sirolimo/farmacologia , Humanos , Placa Aterosclerótica/tratamento farmacológico , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/administração & dosagem , Nanopartículas/química , Nanopartículas/administração & dosagem , Sistemas de Liberação de Medicamentos , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Portadores de Fármacos/químicaRESUMO
Immune rejection remains the major cause of corneal graft failure. Immunosuppressants (such as rapamycin; RAPA) adjunctive to antibiotics (such as levofloxacin hydrochloride; Lev) are a clinical mainstay after corneal grafts but suffer from poor ocular bioavailability associated with severe side effects. In this study, we fabricated a Lev@RAPA micelle loaded cationic peptide-based hydrogel (NapFFKK) as a dual-drug delivery system by integrating RAPA micelles with Lev into a cationic NapFFKK hydrogel to potentially reduced the risk of corneal graft rejection. The properties of the resulting hydrogels were characterized using transmission electronmicroscopy and rheometer. Lev@RAPA micelles loaded NapFFKK hydrogel provided sustained in vitro drug release without compromising their inherent pharmacological activities. Topical instillation of Lev@RAPA micelles loaded NapFFKK hydrogel resulted in the great ocular tolerance and extended precorneal retention over 60 min, thus significantly enhancing the ocular bioavailability of both Lev and RAPA. Overall, such dual-drug delivery system might be a promising formulation for the suppression of corneal graft failure.
Assuntos
Transplante de Córnea , Sistemas de Liberação de Medicamentos , Rejeição de Enxerto , Hidrogéis , Micelas , Nanopartículas , Rejeição de Enxerto/prevenção & controle , Hidrogéis/química , Animais , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Transplante de Córnea/métodos , Coelhos , Liberação Controlada de Fármacos , Sirolimo/administração & dosagem , Sirolimo/farmacocinética , Sirolimo/química , Levofloxacino/administração & dosagem , Levofloxacino/farmacocinética , Levofloxacino/química , Imunossupressores/administração & dosagem , Imunossupressores/farmacocinética , Imunossupressores/química , Disponibilidade Biológica , Masculino , Córnea/efeitos dos fármacos , Córnea/metabolismo , Portadores de Fármacos/químicaRESUMO
In the context of arteriovenous fistula (AVF) failure, local delivery enables the release of higher concentrations of drugs that can suppress neointimal hyperplasia (NIH) while reducing systemic adverse effects. However, the radiolucency of polymeric delivery systems hinders long-term in vivo surveillance of safety and efficacy. We hypothesize that using a radiopaque perivascular wrap to deliver anti-NIH drugs could enhance AVF maturation. Through electrospinning, we fabricated multifunctional perivascular polycaprolactone (PCL) wraps loaded with bismuth nanoparticles (BiNPs) for enhanced radiologic visibility and drugs that can attenuate NIHârosuvastatin (Rosu) and rapamycin (Rapa). The following groups were tested on the AVFs of a total of 24 Sprague-Dawley rats with induced chronic kidney disease: control (i.e., without wrap), PCL-Bi (i.e., wrap with BiNPs), PCL-Bi-Rosu, and PCL-Bi-Rapa. We found that BiNPs significantly improved the wraps' radiopacity without affecting biocompatibility. The drug release profiles of Rosu (hydrophilic drug) and Rapa (hydrophobic drug) differed significantly. Rosu demonstrated a burst release followed by gradual tapering over 8 weeks, while Rapa demonstrated a gradual release similar to that of the hydrophobic BiNPs. In vivo investigations revealed that both drug-loaded wraps can reduce vascular stenosis on ultrasonography and histomorphometry, as well as reduce [18F]Fluorodeoxyglucose uptake on positron emission tomography. Immunohistochemical studies revealed that PCL-Bi-Rosu primarily attenuated endothelial dysfunction and hypoxia in the neointimal layer, while PCL-Bi-Rapa modulated hypoxia, inflammation, and cellular proliferation across the whole outflow vein. In summary, the controlled delivery of drugs with different properties and mechanisms of action against NIH through a multifunctional, radiopaque perivascular wrap can improve imaging and histologic parameters of AVF maturation.
Assuntos
Bismuto , Ratos Sprague-Dawley , Rosuvastatina Cálcica , Sirolimo , Animais , Ratos , Sirolimo/química , Sirolimo/farmacologia , Rosuvastatina Cálcica/química , Rosuvastatina Cálcica/farmacologia , Rosuvastatina Cálcica/farmacocinética , Bismuto/química , Bismuto/farmacologia , Poliésteres/química , Masculino , Fístula Arteriovenosa/patologia , Nanopartículas Metálicas/química , Neointima/patologia , Nanopartículas/química , Humanos , Liberação Controlada de FármacosAssuntos
Estabilidade de Medicamentos , Sirolimo , Solubilidade , Água , Sirolimo/farmacologia , Sirolimo/química , Água/químicaRESUMO
Stroke is a leading cause of global mortality and severe disability. However, current strategies used for treating ischemic stroke lack specific targeting capabilities, exhibit poor immune escape ability, and have limited drug release control. Herein, we developed an ROS-responsive nanocarrier for targeted delivery of the neuroprotective agent rapamycin (RAPA) to mitigate ischemic brain damage. The nanocarrier consisted of a sulfated chitosan (SCS) polymer core modified with a ROS-responsive boronic ester enveloped by a red blood cell membrane shell incorporating a stroke homing peptide. When encountering high levels of intracellular ROS in ischemic brain tissues, the release of SCS combined with RAPA from nanoparticle disintegration facilitates effective microglia polarization and, in turn, maintains blood-brain barrier integrity, reduces cerebral infarction, and promotes cerebral neurovascular remodeling in a mouse stroke model involving transient middle cerebral artery occlusion (tMCAO). This work offers a promising strategy to treat ischemic stroke therapy.
Assuntos
Barreira Hematoencefálica , Quitosana , Portadores de Fármacos , AVC Isquêmico , Nanopartículas , Sirolimo , Animais , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/patologia , Camundongos , Quitosana/química , Portadores de Fármacos/química , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Sirolimo/farmacologia , Sirolimo/química , Sirolimo/uso terapêutico , Nanopartículas/química , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Polissacarídeos/química , Polissacarídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sulfatos/química , Sulfatos/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismoRESUMO
A complete and detailed characterization of Rapamycin (1) and Prolylrapamycin (2) has been conducted by homo- and hetero-nuclear NMR experiments in DMSO-d6 along with HRMS and FT-IR spectra and DSCs analyses. The NMR experiments allowed the assignment of every single proton and carbon atom belonging to the two structures and the definitive confirm of the presence of a pyrrolidine ring in Prolylrapamycin (2) in place of the piperidine ring that characterizes the structure of Sirolimus.
Assuntos
Espectroscopia de Ressonância Magnética , Sirolimo , Sirolimo/química , Pirrolidinas/química , Espectroscopia de Infravermelho com Transformada de Fourier , Estrutura MolecularRESUMO
Rapamycin (RAP) is currently being developed as potential antibreast cancer drug. However, its poor solubility completely limits its use. The aim of this study was to develop polyethylene glycol-poly(lactide-co-glycolide) (PEG-PLGA)-based nanoparticles (NPs) to load RAP via microfluidics with an appropriate polyethylene glycol (PEG) content to enhance the bioavailability of RAP. Polydimethylsiloxane (PDMS) chips with a Y-shaped channel were designed to obtain RAP-loaded PEG-PLGA NPs (RAP-PEG-PLGA). The entrapment efficiency (EE) and drug loading (DL) as well as release profile of RAP-PEG-PLGA were evaluated, and their resistance to plasma albumin adsorption of NPs with different PEG contents was evaluated and compared. RAW264.7 and 4T1 cells were used to assess the antiphagocytic and anticancer cells effect of NPs, respectively. RAP-PEG-PLGA of around 124 nm in size were successfully prepared with the EE of 82.0% and DL of 12.3%, and sustained release for around 40 d. A PEG relative content of 10% within the PEG-PLGA molecule was shown superior in resisting protein adsorption. RAP-PEG-PLGA inhibited the growth of breast cancer cells when the concentration was over 10 µg/mL, and the inhibition efficiency was significantly higher than free RAP. Hence, the current RAP-PEG-PLGA could be a potential therapeutic system for breast cancer treatment.
Assuntos
Portadores de Fármacos , Nanopartículas , Polietilenoglicóis , Sirolimo , Sirolimo/química , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Sirolimo/farmacocinética , Polietilenoglicóis/química , Animais , Nanopartículas/química , Camundongos , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Linhagem Celular Tumoral , Células RAW 264.7 , Tamanho da Partícula , Precipitação Química , Adsorção , Humanos , PoliésteresRESUMO
Biodegradable drug-eluting stents (DESs) are gaining importance owing to their attractive features, such as complete drug release to the target site. Magnesium (Mg) alloys are promising materials for future biodegradable DESs. However, there are few explorations using biodegradable Mg for cardiovascular stent application. In this present study, sirolimus-loaded poly D, L-lactic-co-glycolic acid (PLGA)-coated/ sirolimus-fixed/AZ91 Mg alloy-based substrate was developed via a layer-by-layer approach for cardiovascular stent application. The AZ91 Mg alloy was prepared through the squeeze casting technique. The casted AZ91 Mg alloy (Mg) was alkali-treated to provide macroporous networks to hold the sirolimus and PLGA layers. The systematic characterization was investigated via electrochemical, optical, physicochemical, and in-vitro biological characteristics. The presence of the Mg17 Al12 phase in the Mg sample was found in the x-ray diffraction system (XRD) spectrum which influences the corrosion behavior of the developed substrate. The alkali treatment increases the substrate's hydrophilicity which was confirmed through static contact angle measurement. The anti-corrosion characteristic of casted-AZ91 Mg alloy (Mg) was slightly less than the sirolimus-loaded PLGA-coated alkali-treated AZ91 Mg alloy (Mg/Na/S/P) substrate. However, dissolution rates for both substrates were found to be controlled at cell culture conditions. Radiographic densities of AZ91 Mg alloy substrates (Mg, Mg/Na, and Mg/Na/S/P) were measured to be 0.795 ± 0.015, 0.742 ± 0.01, and 0.712 ± 0.017, respectively. The star-shaped structure of 12% sirolimus/PLGA ensures the bioavailability of the drugs. Sirolimus release kinetic was fitted up to 80% with the "Higuchi model" for Mg samples, whereas Mg/Na/S/P showed 45% fitting with a zero-order mechanism. The Mg/Na/S/P substrate showed a 70% antithrombotic effect compared to control. Further, alkali treatment enhances the antibacterial characteristic of AZ91 Mg alloy. Also, the alkali-treated sirolimus-loaded substrates (Mg/Na/S and Mg/Na/S/P) inhibit the valvular interstitial cell's growth significantly in in-vitro. Hence, the results imply that sirolimus-loaded PLGA-coated AZ91 Mg alloy-based substrate can be a potential candidate for cardiovascular stent application.
Assuntos
Glicolatos , Magnésio , Sirolimo , Sirolimo/farmacologia , Sirolimo/química , Magnésio/farmacologia , Magnésio/química , Glicóis , Ligas/química , Materiais Revestidos Biocompatíveis/farmacologia , Materiais Revestidos Biocompatíveis/química , Ácido Láctico/química , Álcalis , CorrosãoRESUMO
Restenosis remains the main reason for treatment failure of arterial disease. Sirolimus (SIR) as a potent anti-proliferative agent is believed to prevent the phenomenon. The application of exosomes provides an extended-release delivery platform for SIR intramural administration. Herein, SIR was loaded into fibroblast-derived exosomes isolated by ultracentrifugation. Different parameters affecting drug loading were optimized, and exosome samples were characterized regarding physicochemical, pharmaceutical, and biological properties. Cytotoxicity, scratch wound assays, and quantitative real-time PCR for inflammation- and migration-associated genes were performed. Restenosis was induced by carotid injury in a rat carotid model and then exosomes were locally administered. After 14 days, animals were investigated by computed tomography (CT) angiography, morphometric, and immunohistochemical analyses. Western blotting confirmed the presence of specific protein markers in exosomes. Characterization of empty and SIR-loaded exosomes verified round and nanoscale structure of vesicles. Among prepared formulations, desired entrapment efficiency (EE) of 76% was achieved by protein:drug proportion of 2:1 and simple incubation for 30 min at 37 °C. Also, the optimal formulation released about 30% of the drug content during the first 24 h, followed by a prolonged release for several days. In vitro studies revealed the uptake and functional efficacy of the optimized formulation. In vivo studies revealed that %restenosis was in the following order: saline > empty exosomes > SIR-loaded exosomes. Furthermore, Ki67, alpha smooth muscle actin (α-SMA), and matrix metalloproteinase (MMP) markers were less expressed in the SIR-exosomes-treated arteries. These findings confirmed that exosomal SIR could be a hopeful strategy for the prevention of restenosis.
Assuntos
Exossomos , Sirolimo , Ratos , Animais , Sirolimo/química , AngioplastiaRESUMO
Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.
Assuntos
Acústica , DNA , Proteínas , Análise Espectral , Análise Espectral/métodos , DNA/química , Proteínas/química , Mapas de Interação de Proteínas , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Sirolimo/química , Sirolimo/metabolismo , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
Imaging protein-protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.
Assuntos
Sondas Moleculares , Proteínas de Ligação a Tacrolimo , Ligação Proteica , Luciferases/genética , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Sirolimo/química , Sirolimo/metabolismoRESUMO
Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by â¼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.
Assuntos
Neoplasias da Mama , Hydra , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Portadores de Fármacos/química , Elastina/química , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Hydra/metabolismo , Peptídeos/química , Sirolimo/química , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/uso terapêuticoRESUMO
Loading and eluting drugs on self-expandable metallic stents (SEMSs) can be challenging in terms of fabrication, mechanical stability, and therapeutic effects. In this study, a flexible 3D nanonetworked silica film (NSF) capable of withstanding mechanical stress during dynamic expansion is constructed to function as a drug delivery platform on an entire SEMS surface. Despite covering a broad curved area, the synthesized NSF is defect-free and thin enough to increase the stent strut diameter (110 µm) by only 0.4 percent (110.45 µm). The hydrophobic modification of the surface enables loading of 4.7 times the sirolimus (SRL) concentration in NSF than Cypher, polymer-coated commercial stent, which is based on the same thickness of coating layer. Furthermore, SRL-loaded NSF exhibits a twofold delay in release compared to the control group without NSF. The SRL-loaded NSF SEMS significantly suppresses stent-induced tissue hyperplasia than the control SEMS in the rat esophagus (all variables, p < 0.05). Thus, the developed NSF is a promising polymer-free drug delivery platform to efficiently treat esophageal stricture.