RESUMEN
Cascade-reaction containers generating reactive oxygen species (ROS) as an alternative for antibiotic-based strategies for bacterial infection control, require endogenous oxygen-sources and ROS-generation close to or preferably inside target bacteria. Here, this is achieved by cetyltrimethylammonium-chloride (CTAC) assisted in situ metabolic labeling and incorporation of mesoporous SiO2-nanoparticles, dual-loaded with glucose-oxidase and Fe3O4-nanoparticles as cascade-reaction containers, inside bacterial cell walls. First, azide-functionalized d-alanine (D-Ala-N3) was inserted in cell wall peptidoglycan layers of growing Gram-positive pathogens. In Gram-negatives, this could only be achieved after outer lipid-membrane permeabilization, using a low concentration of CTAC. Low concentrations of CTAC had no adverse effect on in vitro blood clotting or hemolysis nor on the health of mice when blood-injected. Next, dibenzocyclooctyne-polyethylene-glycol modified, SiO2-nanoparticles were in situ click-reacted with d-Ala-N3 in bacterial cell wall peptidoglycan layers. Herewith, a two-step cascade-reaction is facilitated inside bacteria, in which glucose-oxidase generates H2O2 at endogenously-available glucose concentrations, while subsequently Fe3O4-nanoparticles catalyze generation of â¢OH from the H2O2 generated. Generation of â¢OH inside bacterial cell walls by dual-loaded mesoporous SiO2-nanoparticles yielded more effective in vitro killing of both planktonic Gram-positive and Gram-negative bacteria suspended in 10 % plasma than SiO2-nanoparticles solely loaded with glucose-oxidase. Gram-positive or Gram-negative bacterially induced sepsis in mice could be effectively treated by in situ pre-treatment with tail-vein injected CTAC and d-Ala-N3, followed by injection of dual-loaded cascade-reaction containers without using antibiotics. This makes in situ metabolic incorporation of cascade-reaction containers as described attractive for further investigation with respect to the control of other types of infections comprising planktonic bacteria. STATEMENT OF SIGNIFICANCE: In situ metabolic-incorporation of cascade-reaction-containers loaded with glucose-oxidase and Fe3O4 nanoparticles into bacterial cell-wall peptidoglycan is described, yielding ROS-generation from endogenous glucose, non-antibiotically killing bacteria before ROS inactivates. Hitherto, only Gram-positives could be metabolically-labeled, because Gram-negatives possess two lipid-membranes. The outer membrane impedes direct access to the peptidoglycan. This problem was solved by outer-membrane permeabilization using a quaternary-ammonium compound. Several studies on metabolic-labeling perform crucial labeling steps during bacterial-culturing that in real-life should be part of a treatment. In situ metabolic-incorporation as described, can be applied in well-plates during in vitro experiments or in the body as during in vivo animal experiments. Surprisingly, metabolic-incorporation proceeded unhampered in blood and a murine, bacterially-induced sepsis could be well treated.
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Peptidoglicano , Especies Reactivas de Oxígeno , Sepsis , Animales , Especies Reactivas de Oxígeno/metabolismo , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Ratones , Nanopartículas/química , Dióxido de Silicio/química , Bacterias Grampositivas/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacosRESUMEN
It is well known that the physicochemical properties of nanocarriers, which are closely related to the surface modification of nanoparticles, have crucial impacts on their biological effects. Herein, the interaction between functionalized degradable dendritic mesoporous silica nanoparticles (DDMSNs) and bovine serum albumin (BSA) was investigated for probing into the nanocarriers' potential toxicity using multi-spectroscopy such as ultraviolet/visible (UV/Vis), synchronous fluorescence, Raman and circular dichroism (CD) spectroscopy. BSA, owing to its structural homology and high sequence similarity with HSA, was employed as the model protein to study the interactions with DDMSNs, amino-modified DDMSNs (DDMSNs-NH2) and hyaluronic acid (HA) coated nanoparticles (DDMSNs-NH2-HA). It was found that the static quenching behavior of DDMSNs-NH2-HA to BSA was accompanied by an endothermic and hydrophobic force-driven thermodynamic process, which was confirmed by fluorescence quenching spectroscopic studies and thermodynamic analysis. Furthermore, the conformational variations of BSA upon interaction with nanocarriers were observed by combination of UV/Vis, synchronous fluorescence, Raman and CD spectroscopy. The microstructure of amino residues in BSA changed due to the existence of nanoparticles, for example, the amino residues and hydrophobic groups exposed to microenvironment and the alpha helix (α-helix) content of BSA decreased. Specially, through thermodynamic analysis, the diverse binding modes and driving forces between nanoparticles and BSA were discovered because of different surface modifications on DDMSNs, DDMSNs-NH2 and DDMSNs-NH2-HA. We believe that this work can promote the interpretation of mutual impact between nanoparticles and biomolecules, which will be in favor of predicting the biological toxicity of nano-DDS and engineering functionalized nanocarriers.
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Nanopartículas , Albúmina Sérica Bovina , Albúmina Sérica Bovina/química , Dicroismo Circular , Espectrometría de Fluorescencia/métodos , Termodinámica , Unión Proteica , Sitios de Unión , Espectrofotometría Ultravioleta/métodosRESUMEN
Despite unprecedented successes of antibody-based cancer immunotherapy, the serious side effects and rapid clearance following systemic administration remain big challenges to realize its full potential. At the same time, combination immunotherapy using multiple antibodies has shown particularly promising in cancer treatment. It is noticed that the working mechanisms of natural holdase and foldase chaperone are desirable to overcome the limitations of therapeutic antibodies. Holdase chaperone stabilizes unfolded client and prevents it from activation and degradation, while foldase chaperone assists unfolded client to its native state to function. Here a holdase/foldase mimetic nanochaperone (H/F-nChap) to co-delivery two types of monoclonal antibodies (mAbs), αCD16 and αPDL1, and resiquimod (R848) is developed, which significantly improves cancer immunotherapy. The H/F-nChap presents holdase activity in blood and normal tissues that hides and protects mAbs from unnecessary targeted activation and degradation, thereby prolonging blood circulation and reducing immunotoxicity in vivo. Furthermore, H/F-nChap switches to foldase activity in the tumor microenvironment that exposes mAbs and releases R848 to enhance the engagement between NK cells and tumor cells and promote immune activation, respectively. The H/F-nChap represents a strategy for safe and spatiotemporal delivery of multiple mAbs, providing a promising platform for improved cancer immunotherapy.
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Anticuerpos Monoclonales , Neoplasias , Humanos , Anticuerpos Monoclonales/uso terapéutico , Inmunoterapia , Neoplasias/terapiaRESUMEN
Cascade-reaction chemistry can generate reactive-oxygen-species that can be used for the eradication of infectious biofilms. However, suitable and sufficient oxygen sources are not always available near an infection site, while the reactive-oxygen-species generated are short-lived. Therefore, we developed a magnetic cascade-reaction container composed of mesoporous Fe3O4@SiO2 nanoparticles containing glucose-oxidase and l-arginine for generation of reactive-oxygen-species. Glucose-oxidase was conjugated with APTES facilitating coupling to Fe3O4@SiO2 nanoparticles and generation of H2O2 from glucose. l-arginine was loaded into the nanoparticles to generate NO from the H2O2 generated. Using an externally-applied magnetic field, cascade-reaction containers could be homogeneously distributed across the depth of an infectious biofilm. Cascade-reaction containers with coupled glucose-oxidase were effective in killing planktonic, Gram-positive and Gram-negative bacteria. Additional efficacy of the l-arginine based second cascade-reaction was only observed when H2O2 as well as NO were generated in-biofilm. In vivo accumulation of cascade-reaction containers inside abdominal Staphylococcus aureus biofilms upon magnetic targeting was observed real-time in living mice through an implanted, intra-vital window. Moreover, vancomycin-resistant, abdominal S. aureus biofilms could be eradicated consuming solely endogenous glucose, without any glucose addition. Herewith, a new, non-antibiotic-based infection-control strategy has been provided, constituting a welcome addendum to the shrinking clinical armamentarium to control antibiotic-resistant bacterial infections.
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Diabetic foot ulcers infected with antibiotic-resistant bacteria form a severe complication of diabetes. Antimicrobial-loaded hydrogels are used as a dressing for infected wounds, but the ongoing rise in the number of antimicrobial-resistant infections necessitates new, nonantibiotic based designs. Here, a guanosine-quadruplex (G4 )-hydrogel composed of guanosine, 2-formylphenylboronic acid, and putrescine is designed and used as a cascade-reaction container. The G4 -hydrogel is loaded with glucose-oxidase and hemin. The first cascade-reaction, initiated by glucose-oxidase, transforms glucose and O2 into gluconic acid and H2 O2 . In vitro, this reaction is most influential on killing Staphylococcus aureus or Pseudomonas aeruginosa in suspension, but showed limited killing of bacteria in biofilm-modes of growth. The second cascade-reaction, however, transforming H2 O2 into reactive-oxygen-species (ROS), also enhances killing of biofilm bacteria due to hemin penetration into biofilms and interaction with eDNA G-quadruplexes in the biofilm matrix. Therewith, the second cascade-reaction generates ROS close to the target bacteria, facilitating killing despite the short life-time of ROS. Healing of infected wounds in diabetic mice proceeds faster upon coverage by these G4 -hydrogels than by clinically common ciprofloxacin irrigation. Moreover, local glucose concentrations around infected wounds decrease. Concluding, a G4 -hydrogel loaded with glucose-oxidase and hemin is a good candidate for infected wound dressings, particularly in diabetic patients.
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Diabetes Mellitus Experimental , Infección de Heridas , Animales , Glucosa , Guanosina/farmacología , Humanos , Hidrogeles , Ratones , Infección de Heridas/tratamiento farmacológicoRESUMEN
Nanochaperones have been designed and used for regulating the (re)folding of proteins, treating protein misfolding-related diseases, and, more recently, in drug delivery. Despite various successes, a complete understanding of the working mechanisms remains elusive, which represents a challenge for the realization of their full potential. Here, we thoroughly investigated the functioning of differently charged nanochaperones that regulate the refolding of thermally denatured lysozyme. We found that the balance between the capture and release of lysozyme clients that are controlled by nanochaperones plays a key role in regulating refolding. More importantly, the findings could be applied to other enzymes with various physicochemical properties. On the basis of these results, we could recover the activity of enzymes with high efficiency either after 20â days of storage at 40 °C or heating at high temperatures for 30-60â min. Our results provide important new design strategies for nanochaperone systems to improve their properties and expand their applications.
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Chaperonas Moleculares/química , Muramidasa/química , Temperatura , Muramidasa/metabolismo , Tamaño de la Partícula , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
Extracellular polymeric substances (EPS) hold infectious biofilms together and limit antimicrobial penetration and clinical infection control. Here, we present zwitterionic micelles as a previously unexplored, synthetic self-targeting dispersant. First, a pH-responsive poly(ε-caprolactone)-block-poly(quaternary-amino-ester) was synthesized and self-assembled with poly(ethylene glycol)-block-poly(ε-caprolactone) to form zwitterionic, mixed-shell polymeric micelles (ZW-MSPMs). In the acidic environment of staphylococcal biofilms, ZW-MSPMs became positively charged because of conversion of the zwitterionic poly(quaternary-amino-ester) to a cationic lactone ring. This allowed ZW-MSPMs to self-target, penetrate, and accumulate in staphylococcal biofilms in vitro. In vivo biofilm targeting by ZW-MSPMs was confirmed for staphylococcal biofilms grown underneath an implanted abdominal imaging window through direct imaging in living mice. ZW-MSPMs interacted strongly with important EPS components such as eDNA and protein to disperse biofilm and enhance ciprofloxacin efficacy toward remaining biofilm, both in vitro and in vivo. Zwitterionic micellar dispersants may aid infection control and enhance efficacy of existing antibiotics against remaining biofilm.
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Antibacterianos , Micelas , Animales , Antibacterianos/farmacología , Biopelículas , Microscopía Intravital , Ratones , PolímerosRESUMEN
Insulin would undergo unfolding and fibrillation under stressed conditions, which may cause serious biotechnological and medical problems. Herein, by mimicking the structure and functions of natural chaperones HSP70s, self-assembled polymeric micelles are used as nanochaperones for the delivery of insulin. The confined hydrophobic domains on the surface of nanochaperones adsorb partially unfolded insulin, inhibiting the aggregation and fibrillation and enhancing the stability of insulin. The bioactivity of insulin is well-reserved after incubation with the nanochaperones at 37 °C for 7 d or heating at 70 °C for 1 h. The stealthy poly(ethylene glycol) chains around the confined domains protect the adsorbed insulin from enzymatic degradation and prolong the circulation time. More importantly, the excellent glucose sensitivity of the hydrophobic domains enables the nanochaperones to release and refold insulin in native form in response to hyperglycemia. This kind of nanochaperone may offer a hopeful strategy for the protection and delivery of insulin.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Insulina , Chaperonas Moleculares , Nanoestructuras , Animales , Diabetes Mellitus Experimental/metabolismo , Insulina/química , Insulina/farmacocinética , Insulina/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Chaperonas Moleculares/química , Chaperonas Moleculares/farmacocinética , Chaperonas Moleculares/farmacología , Células 3T3 NIH , Nanoestructuras/química , Nanoestructuras/uso terapéuticoRESUMEN
Internalization of Staphylococcus aureus by macrophages can inactivate bacterial killing mechanisms, allowing intracellular residence and dissemination of infection. Concurrently, these staphylococci can evade antibiotics that are frequently unable to pass mammalian cell membranes. A binary, amphiphilic conjugate composed of triclosan and ciprofloxacin is synthesized that self-assemble through micelle formation into antimicrobial nanoparticles (ANPs). These novel ANPs are stabilized through encapsulation in macrophage membranes, providing membrane-encapsulated, antimicrobial-conjugated NPs (Me-ANPs) with similar protein activity, Toll-like receptor expression and negative surface charge as their precursor murine macrophage/human monocyte cell lines. The combination of Toll-like receptors and negative surface charge allows uptake of Me-ANPs by infected macrophages/monocytes through positively charged, lysozyme-rich membrane scars created during staphylococcal engulfment. Me-ANPs are not engulfed by more negatively charged sterile cells possessing less lysozyme at their surface. The Me-ANPs kill staphylococci internalized in macrophages in vitro. Me-ANPs likewise kill staphylococci more effectively than ANPs without membrane-encapsulation or clinically used ciprofloxacin in a mouse peritoneal infection model. Similarly, organ infections in mice created by dissemination of infected macrophages through circulation in the blood are better eradicated by Me-ANPs than by ciprofloxacin. These unique antimicrobial properties of macrophage-monocyte Me-ANPs provide a promising direction for human clinical application to combat persistent infections.
RESUMEN
In the past decades, insulin delivery systems have been widely developed for diabetes treatment. Though a few works have investigated polymeric micelles with glucose and H2O2 dual-responsiveness for the delivery of insulin, great efforts should still be devoted to enhancing the therapeutic efficacy. Herein, glucose/H2O2 dual-responsive polymeric micelles were fabricated for the self-regulated insulin delivery. The polymeric micelles were self-assembled by poly(ethylene glycol)-block-poly(amino phenylboronic ester) (PEG-b-PAPBE), where the hydrophilic PEG offered the shell and the hydrophobic PAPBE endowed the polymeric micelles with the dual-sensibility to glucose and H2O2. The built-in phenylboronic ester (PBE) could be not only broken by glucose but also hydrolyzed by H2O2, thus resulting in the disintegration of the polymeric micelles. The glucose-responsive release of insulin was achieved and could be further facilitated by the coencapsulation of glucose oxidase (GOx) in the micelles, which would produce H2O2 by catalytic oxidation of glucose and thus lead to the hydrolysis of the phenylboronic ester by H2O2. Compared with free insulin or micelles that carried insulin alone, a subcutaneous injection of the insulin/GOx-coloaded polymeric micelles to the diabetic mice presented a superior hypoglycemic effect in vivo. This kind of polymeric micelle with glucose and H2O2 dual-responsiveness provides a promising approach for diabetes therapy.
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Tumor heterogeneity has been one of the most important factors leading to the failure of conventional cancer therapies due to the accumulation of genetically distinct tumor-cell subpopulations during the tumor development process. Due to the diversity of genetic mutations during tumor growth, combining the use of multiple drugs has only achieved limited success in combating heterogeneous tumors. Herein, we report a novel antitumor strategy that effectively addresses tumor heterogeneity by using a CRISPR/Cas9-based nanoRNP carrying a combination of sgRNAs. Such nanoRNP is synthesized from Cas9 ribonucleoprotein, any combinations of required sgRNAs, and a rationally designed responsive polymer that endows nanoRNP with high circulating stability, enhanced tumor accumulation, and the efficient gene editing in targeted tumor cells eventually. By carrying a combination of sgRNAs that targets STAT3 and RUNX1, the nanoRNP exhibited efficient gene expression disruptions on a heterogeneous tumor model with two subsets of cells whose proliferations were sensitive to the reduced expression of STAT3 and RUNX1, respectively, leading to the effective growth inhibition of the heterogeneous tumor. Considering the close relationship between tumor heterogeneity and cancer progression, resistance to therapy, and recurrences, nanoRNP provides a feasible strategy to overcome tumor heterogeneity in the development of more advanced cancer therapy against malignant tumors.
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Sistemas CRISPR-Cas , Edición Génica/métodos , Neoplasias/terapia , Animales , Línea Celular Tumoral , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Terapia Genética/métodos , Humanos , Ratones , Ratones Desnudos , Nanomedicina/métodos , Neoplasias/genética , Neoplasias/patología , Factor de Transcripción STAT3/genéticaRESUMEN
Bacterial infection is becoming the biggest threat to human health. The scenario is partly due to the ineffectiveness of the conventional antibiotic treatments against the emergence of multidrug-resistant bacteria and partly due to the bacteria living in biofilms or cells. Adaptive biomaterials can change their physicochemical properties in the microenvironment of bacterial infection, thereby facilitating either their interactions with bacteria or drug release. The trends in treating bacterial infections using adaptive biomaterials-based systems are flourishing and generate innumerous possibility to design novel antimicrobial therapeutics. This feature article aims to summarize the recent developments in the formulations, mechanisms, and advances of adaptive materials in bacterial infection diagnosis, contact killing of bacteria, and antimicrobial drug delivery. Also, the challenges and limitations of current antimicrobial treatments based on adaptive materials and their clinical and industrial future prospects are discussed.
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Antiinfecciosos/síntesis química , Bacterias/efectos de los fármacos , Biopelículas/efectos de los fármacos , Técnicas Biosensibles , Interacciones Huésped-Patógeno , Nanoestructuras/química , Antiinfecciosos/farmacología , Aptámeros de Nucleótidos/química , Aptámeros de Péptidos/química , Bacterias/crecimiento & desarrollo , Bacterias/patogenicidad , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Biopelículas/crecimiento & desarrollo , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Colorantes Fluorescentes/síntesis química , Humanos , Concentración de Iones de Hidrógeno , Hidrolasas , Indoles/química , Indoles/farmacología , Plancton/efectos de los fármacos , Plancton/crecimiento & desarrollo , Plancton/patogenicidad , Polímeros/química , Polímeros/farmacologíaRESUMEN
Diabetes is a chronic metabolic disorder disease characterized by high blood glucose levels and has become one of the most serious threats to human health. In recent decades, a number of insulin delivery systems, including bulk gels, nanogels, and polymeric micelles, have been developed for the treatment of diabetes. Herein, a kind of glucose and H2O2 dual-responsive polymeric nanogel was designed for enhanced glucose-responsive insulin delivery. The polymeric nanogels composed of poly(ethylene glycol) and poly(cyclic phenylboronic ester) (glucose and H2O2 dual-sensitive groups) were synthesized by a one-pot thiol-ene click chemistry approach. The nanogels displayed glucose-responsive release of insulin and the release rate could be promoted by the incorporation of glucose oxidase (GOx), which generated H2O2 at high glucose levels and H2O2 further oxidizes and hydrolyzes the phenylboronic ester group. The nanogels have characteristics of long blood circulation time, a fast response to glucose, and excellent biocompatibility. Moreover, subcutaneous delivery of insulin to diabetic mice with the insulin/GOx-loaded nanogels presented an effective hypoglycemic effect compared to that of injection of insulin or insulin-loaded nanogels. This kind of nanogel would be a promising candidate for the delivery of insulin in the future.
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Glucosa Oxidasa/química , Glucosa/metabolismo , Peróxido de Hidrógeno/metabolismo , Hipoglucemiantes/metabolismo , Insulina/metabolismo , Polietilenglicoles/química , Polietileneimina/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Supervivencia Celular/efectos de los fármacos , Química Clic , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/tratamiento farmacológico , Portadores de Fármacos/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Glucosa/química , Glucosa Oxidasa/metabolismo , Prueba de Tolerancia a la Glucosa , Peróxido de Hidrógeno/química , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Insulina/química , Insulina/uso terapéutico , Ratones , Células 3T3 NIH , Nanogeles , Polietilenglicoles/toxicidad , Polietileneimina/toxicidadRESUMEN
Large amounts of insulin-loaded glucose-responsive micelles based on poly(amino acid)s have been developed for diabetes treatment over last decades, but most of them could not effectively protect insulin from enzymatic degradation in vivo because the micellar core was biodegradable and lacked protective structure for insulin, which would lower the efficacy of insulin to a large extent. In this study, we fabricated a new type of insulin-loaded glucose-responsive complex micelles (CMs), which were self-assembled by a phenylboronic acid (PBA)-modified block copolymer PEG-b-P(Asp-co-AspPBA) and a glucosamine (GA)/nitrilotriacetic acid (NTA)-functionalized block copolymer PNIPAM-b-P(Asp-co-AspGA-co-AspNTA), for self-regulated delivery of insulin with effective protection of insulin and enhanced hypoglycemic activity in vivo. The CMs possessed mixed shell of PEG/PNIPAM and cross-linked core of PBA/GA complex, which could be disintegrated under the condition of high glucose concentration (5 g/L) while maintaining stable at low glucose concentration (1 g/L). The NTA groups of CMs greatly improved the loading content of insulin by specifically bind insulin via the chelated zinc ions. More importantly, PNIPAM chains in the mixed shell would collapse under 37 °C and form hydrophobic domains around the micellar core, which could significantly protect the micellar core as well as the encapsulated insulin from attacking by external proteases. In a murine model of type 1 diabetes, the CMs with insulin chelated by NTA showed a long hypoglycemic effect, which is superior to insulin-loaded simple micelles without PNIPAM and insulin in PBS buffer (pH 7.4). Therefore, this kind of CMs could be a potential candidate for insulin delivery in diabetes therapy.
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Sistemas de Liberación de Medicamentos , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Insulina/administración & dosificación , Micelas , Animales , Glucemia/metabolismo , Preparaciones de Acción Retardada , Dispersión Dinámica de Luz , Endopeptidasa K/metabolismo , Fluorescencia , Masculino , Ratones Endogámicos BALB C , Polímeros/síntesis química , Polímeros/química , Proteolisis/efectos de los fármacosRESUMEN
Because of their abnormal vasculature and the dense tumor extra-cellular matrix, solid tumors prevent the deep and uniform penetration of nanocarriers. Numerous studies have shown that nanocarriers with a positively charged surface exhibit enhanced tumor penetration. Therefore, a hypoxia responsive nanocarrier [responsive micelles (RMs)] was developed, which can gradually increase the positive surface charge by responding to hypoxia gradients, and eventually achieve deep penetration in tumors. The nanocarrier was composed of a poly(caprolactone) core and a mixed shell of poly(ethylene glycol) (PEG) and 4-nitrobenzyl chloroformate (NBCF)-modified polylysine (PLL). During the blood circulation, the NBCF-modified PLL was shielded by the PEG, which gave it the ability to inhibit its rapid removal by the immune system. After reaching the tumor, the hypoxia microenvironment triggered partial NBCF degradation that recovered the amine groups of PLL, leading to a remarkable change in the surface to a positively charged one that enabled the penetration of the nanocarrier into the tumor. As the nanocarrier penetrated into the interior of the tumor, the decrease in oxygen concentration led to the further degradation of the NBCF-modified PLL, resulting in the increase of the positive surface charge which further facilitated the deep penetration. The subsequent in vitro and in vivo experiments certified that RM/doxorubicin had a better penetration ability and improved inhibition efficacy on tumor tissues, which demonstrated its potential application in cancer therapy.
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Antineoplásicos/química , Antineoplásicos/farmacología , Portadores de Fármacos/química , Nanopartículas/química , Hipoxia Tumoral/efectos de los fármacos , Animales , Antineoplásicos/metabolismo , Transporte Biológico , Línea Celular Tumoral , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacocinética , Liberación de Fármacos , Humanos , Concentración de Iones de Hidrógeno , Ratones , Micelas , Oxígeno/metabolismo , Tamaño de la Partícula , Polímeros/química , Distribución TisularRESUMEN
CRISPR/dCas9 systems can precisely control endogenous gene expression without interrupting host genomic sequence and have provided a novel and feasible strategy for the treatment of cancers at the transcriptional level. However, development of CRISPR/dCas9-based anti-cancer therapeutics remains challenging due to the conflicting requirements for the design of the delivery system: a cationic and membrane-binding surface facilitates the tumor accumulation and cellular uptake of the CRISPR/dCas9 system, but hinders the circulating stability in vivo. Here, a multistage delivery nanoparticle (MDNP) that can achieve tumor-targeted delivery of CRISPR/dCas9 systems and restore endogenous microRNA (miRNA) expression in vivo is described. MDNP is designed as a core-shell structure in which the shell is made of a responsive polymer that endows MDNP with the capability to present different surface properties in response to its surrounding microenvironment, allowing the MNDP overcoming multiple physiological barriers and delivering the payload to tumor tissues with an optimal efficiency. Systemic administration of MDNP/dCas9-miR-524 to tumor-bearing mice achieved effective upregulation of miR-524 in tumors, leading to the simultaneous interferences of multiple signal pathways related to cancer cell proliferation and presenting remarkable tumor growth retardation, suggesting the feasibility of utilizing MDNP to achieve tumor-targeting delivery of CRISPR/dCas9 with sufficient levels to realize its therapeutic effects.
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Novel artificial enzymes are highly desired to overcome the shortcomings of natural enzymes during industrial or biological applications. Here we designed and prepared nanogel-based artificial enzymes (NAEs) to mimic natural horseradish peroxidase (HRP) using a facile one-pot, scalable method. The poly(N-isopropylacrylamide) (PNIPAM) matrix provided a temperature-responsive and size-controllable scaffold for the NAEs, and 1-vinylimidazole (Vim) moieties stabilized the enzymatic centers (Hemin) through coordination interaction. The feeding ratios of the components to prepare NAEs were subsequently studied and optimized to ensure the NAEs possess the highest catalytic activity and stability. The optimized NAEs were quite stable and can maintain their catalytic activities over a broad range of heat or pH treatments, and a long storage period as well. The NAEs are active to catalytic oxidation of several azo compounds and their activities can easily be switched on/off by changing the surrounding temperature. Taken together, these easily made, highly stable, efficient and activity-switchable NAEs could mimic natural HRP while overcoming their shortcomings and have a potential in wastewater treatment and controllable catalysis.
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Resinas Acrílicas/química , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Hemina/química , Peroxidasa de Rábano Silvestre/química , Nanopartículas/química , Polietileneimina/química , Catálisis , Peroxidasa de Rábano Silvestre/metabolismo , Oxidación-Reducción , Polietilenglicoles/química , TemperaturaRESUMEN
Alzheimer's disease (AD) is a progressive and irreversible brain disorder. Recent studies revealed the pivotal role of ß-amyloid (Aß) in AD. However, there is no conclusive indication that the existing therapeutic strategies exerted any effect on the mitigation of Aß-induced neurotoxicity and the elimination of Aß aggregates simultaneously in vivo. Herein, we developed a novel nanocomposite that can eliminate toxic Aß aggregates and mitigate Aß-induced neurotoxicity in AD mice. This nanocomposite was designed to be a small-sized particle (14 ± 4 nm) with Aß-binding peptides (KLVFF) integrated on the surface. The nanocomposite was prepared by wrapping a protein molecule with a cross-linked KLVFF-containing polymer layer synthesized by in situ polymerization. The presence of the nanocomposite remarkably changed the morphology of Aß aggregates, which led to the formation of Aß/nanocomposite coassembled nanoclusters instead of Aß oligomers. With the reduction of the pathological Aß oligomers, the nanocomposites attenuated the Aß-induced neuron damages, regained endocranial microglia's capability to phagocytose Aß, and eventually protected hippocampal neurons against apoptosis. Thus, we anticipate that the small-sized nanocomposite will potentially offer a feasible strategy in the development of novel AD treatments.
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Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Nanocompuestos/uso terapéutico , Nanomedicina/métodos , Péptidos/uso terapéutico , Agregación Patológica de Proteínas/terapia , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Péptidos beta-Amiloides/aislamiento & purificación , Animales , Modelos Animales de Enfermedad , Ratones , Modelos Moleculares , Nanocompuestos/química , Nanocompuestos/ultraestructura , Péptidos/química , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patologíaRESUMEN
Biofilms that contribute to the persistent bacterial infections pose serious threats to human health, due in part to the extracellular polymeric substances (EPS) matrix of biofilm block the diffusion of intact antimicrobials. The poor penetration of antimicrobials into biofilm greatly reduces their bacterial killing efficacy. Here, we have demonstrated a nanocapsule PMPC-CS synthesized by encapsulating a chitosan nanoparticle with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC). Such PMPC-based surface exhibited low EPS-adsorption, allowing enhanced penetration of PMPC-CS. Additionally, PMPC-CS showed effective targeting toward negatively charged bacterial cell surfaces and pH-responsive drug release mediated by the swelling of chitosan core under the acidic environment of biofilm. These unique features ensured targeted delivery of antimicrobials throughout the depth of a biofilm. Delivery of triclosan with PMPC-CS outperformed direct application of free triclosan in inhibiting the growth of bacteria in biofilm, suggesting the potential of PMPC-CS as an effective delivery system for the treatment of bacterial infections.
RESUMEN
BACKGROUND: Successful implementation of gene therapy heavily relies on efficiently delivering genetic materials and specific targeting into cells. Oncogene-driven endocytosis stimulates nutrient uptake and also develops an endocytosis-mediated defense against therapeutic agents. Cell-penetrating peptides, typically HIV-Tat, are well known for efficient delivery of nucleic acid drugs but lack targeting specificity. Various passive targeting strategies were pursued to enhance the tumor targeting efficiency; however, they are still limited by complicated cellular endocytosis routes and the heterogeneity of cancer types. METHODS: Tat/pDNA complexes were noncovalently compacted and their physiochemical properties were determined. The siRNA pool and pLV-RNAi-GFP lentivirus were used to knock down dbl oncogene (originally isolated from diffuse B-cell lymphoma) expression, and its overexpression was performed by plasmid transient transfection. The cellular uptake of fluorescent ligands was quantified by confocal imaging and flow cytometry analysis. The transgene efficiency was determined by the Luciferase expression assay. Rho GTPase activation was checked by the GST-Rho GTPase-binding domain pull-down assay. RESULTS: pGL3 plasmid DNA was noncovalently compacted with the Tat peptide into nano-size complexes at high N/P ratios. Macropinocytosis, a clathrin- and caveolin-independent endocytosis process, was shown to contribute to the uptake of middle-sized (â¼600 nm) Tat/pGL3 complexes. Cell-type-specific variation in macropinocytosis was essentially controlled by the action of the Dbl oncogene. Onco-Dbl presentation constantly induced a high level of macropinocytosis activity in ovarian cancer cells. Onco-Dbl overexpression hyperstimulated macropinocytosis enhancement in cells mainly through actin cytoskeleton reorganization mediated by the PH domain and Rac1 activation. The Dbl-driven Rho GTPase signaling collectively determined the cell-type-specific macropinocytosis phenotype. CONCLUSION: Such an aspect can be exploited to selectively confer targeted delivery of Tat/pDNA nano-complexes into ovarian cancer cells. Our work provides a novel alternative for targeted delivery of cell-penetrating peptide-based nucleic acid drugs into certain tumor types if specific endocytosis pathways are used.