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1.
Mol Pharm ; 20(7): 3356-3366, 2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-36952227

RESUMO

Despite the success of mRNA-based vaccines against infectious diseases (including COVID-19), safety concerns have been raised relating to the lipid nanoparticles (LNPs) used to deliver the mRNA cargo. Antibodies against the polyethylene glycol (PEG) coating on these non-viral vectors are present in the general population and can in some instances induce allergic reactions. Furthermore, treatment with PEGylated therapeutics may increase the plasma concentration of such anti-PEG antibodies. The widespread use of PEGylated nanoparticles for mRNA vaccines concerns researchers and clinicians about a potential rise in future cases of allergic reactions against mRNA vaccines and cross-reactions with other PEGylated therapeutics. To determine if vaccination with Comirnaty increased the plasma concentration of antibodies against LNPs, we investigated the blood plasma concentration of anti-LNP antibodies in healthy individuals before and after vaccination with the mRNA-based COVID-19 vaccine Comirnaty (BNT162b2). Blood samples were acquired from 21 healthy adults before vaccination, 3-4 weeks after the first vaccination dose but before the second dose, and 2-6 months after the second (booster) dose. The blood plasma concentration of antibodies recognizing the LNPs was analyzed using a microscopy-based assay capable of measuring antibody-binding to individual authentic LNPs. No significant increase in anti-LNP antibodies was observed after two doses of Comirnaty. The LNPs used for intramuscular delivery of mRNA in the vaccine against COVID-19, Comirnaty, do, therefore, not seem to induce the generation of anti-vector antibodies.


Assuntos
COVID-19 , Hipersensibilidade , Nanopartículas , Adulto , Humanos , Vacinas contra COVID-19 , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas de mRNA , Vacinação , Anticorpos
2.
Small ; 18(14): e2106529, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35187804

RESUMO

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non-opsonized liposomes co-existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.


Assuntos
Lipossomos , Proteínas Opsonizantes , Anticorpos , Proteínas do Sistema Complemento/metabolismo , Lipossomos/química , Proteínas Opsonizantes/metabolismo , Opsonização , Polietilenoglicóis/química
3.
Cytometry A ; 95(8): 917-924, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31120635

RESUMO

The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles. © 2019 International Society for Advancement of Cytometry.


Assuntos
Calibragem , Citometria de Fluxo/métodos , Corantes Fluorescentes/farmacologia , Nanopartículas/química , Vesículas Extracelulares/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Lipossomos/química , Lipossomos/farmacologia , Nanopartículas/ultraestrutura
4.
Biophys J ; 113(6): 1269-1279, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28738989

RESUMO

Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and thereby synergistically regulate the trafficking and sorting of lipidated proteins.


Assuntos
Genes ras , Lipossomos/química , Modelos Moleculares , Fosfatidilcolinas/química , Pressão , Ligação Proteica , Propriedades de Superfície
5.
J Colloid Interface Sci ; 674: 139-144, 2024 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-38925059

RESUMO

Nucleic acid-based therapies are transforming medicine, but rely on an efficient delivery vehicle such as lipid nanoparticles (LNPs). Concerns exists in the nanomedicine field, that a large fraction of the LNPs in the ensemble does not contain any nucleic acid cargo and thus exert no functional effect. Nevertheless, how LNP lipid formulation, the LNP preparation method employed and nucleic acid cargo size correlates with the proportion of empty LNPs remains largely unexplored. Here we employ a well-established single particle based method to study nucleic acid loading heterogeneity in LNPs. We find that only a minor fraction of LNPs are "empty", both for LNPs loaded with siRNA, mRNA and plasmids. For clinically relevant LNPs for mRNA delivery, we never detected more than 16% empty nanoparticles in the ensemble. Thus employing standard LNP lipid-cargo combinations and preparation schemes results in LNPs with the potential to serve their biomedical function.


Assuntos
Lipídeos , Nanopartículas , RNA Interferente Pequeno , Nanopartículas/química , Lipídeos/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/administração & dosagem , Tamanho da Partícula , RNA Mensageiro/genética , Plasmídeos/química , Humanos , Ácidos Nucleicos/química , Ácidos Nucleicos/administração & dosagem , Propriedades de Superfície , Lipossomos
6.
J Colloid Interface Sci ; 669: 198-210, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38713958

RESUMO

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.


Assuntos
Calcitonina , Lipossomas Unilamelares , Calcitonina/química , Calcitonina/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Animais , Fluoresceínas/química , Membrana Celular/metabolismo , Membrana Celular/química
7.
Mol Ther Methods Clin Dev ; 29: 450-459, 2023 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-37251983

RESUMO

Following the recent approval of both siRNA- and mRNA-based therapeutics, nucleic acid therapies are considered a game changer in medicine. Their envisioned widespread use for many therapeutic applications with an array of cellular target sites means that various administration routes will be employed. Concerns exist regarding adverse reactions against the lipid nanoparticles (LNPs) used for mRNA delivery, as PEG coatings on nanoparticles can induce severe antibody-mediated immune reactions, potentially being boosted by the inherently immunogenic nucleic acid cargo. While exhaustive information is available on how physicochemical features of nanoparticles affects immunogenicity, it remains unexplored how the fundamental choice of administration route regulates anti-particle immunity. Here, we directly compared antibody generation against PEGylated mRNA-carrying LNPs administered by the intravenous, intramuscular, or subcutaneous route, using a novel sophisticated assay capable of measuring antibody binding to authentic LNP surfaces with single-particle resolution. Intramuscular injections in mice were found to generate overall low and dose-independent levels of anti-LNP antibodies, while both intravenous and subcutaneous LNP injections generated substantial and highly dose-dependent levels. These findings demonstrate that before LNP-based mRNA medicines can be safely applied to new therapeutic applications, it will be crucial to carefully consider the choice of administration route.

8.
FASEB Bioadv ; 5(9): 355-366, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37674540

RESUMO

Extracellular vesicle (EV) secretion rate is stimulated by hypoxia that causes increased reactive oxygen species (ROS) production by the mitochondrial electron transport chain (ETC) and hypoxia-induced factor (HIF)-1 signaling; however, their contribution to the increased EV secretion rate is unknown. We found that the EV marker secretion rate in our EV reporter cell line CD9truc-EGFP was unaffected by the HIF-1α stabilizer roxadustat; yet, ETC stimulation by dichloroacetic acid (DCA) significantly increased EV secretion. The DCA-induced EV secretion was blocked by the antioxidant TEMPO and rotenone, an inhibitor of the ETC's Complex I. Under hypoxic conditions, the limited oxygen reduction impedes the ETC's Complex III. To mimic this, we inhibited Complex III with antimycin A, which increased ROS-dependent EV secretion. The electron transport between Complex I and III is accomplished by coenzyme Q created by the mevalonate pathway and tyrosine metabolites. Blocking an early step in the mevalonate pathway using pitavastatin augmented the DCA-induced EV secretion, and 4-nitrobenzoate-an inhibitor of the condensation of the mevalonate pathway with tyrosine metabolites-increased ROS-dependent EV secretion. Our findings indicate that hypoxia-mimetics targeting the ETC modify EV secretion and that ROS produced by the ETC is a potent stimulus for EV secretion.

9.
Small Methods ; 6(3): e2101364, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34994103

RESUMO

Flow cytometry (FCM) is a high-throughput fluorescence-based technique for multiparameter analysis of individual particles, including cells and nanoparticles. Currently, however, FCM does in many cases not permit proper counting of fluorophore-tagged markers on individual particles, due to a lack of tools for translating FCM output intensities into accurate numbers of fluorophores. This lack hinders derivation of detailed biologic information and comparison of data between experiments with FCM. To address this technological void, the authors here use DNA nanotechnology to design and construct barrel-shaped DNA-origami nanobeads for fluorescence/antigen quantification in FCM. Each bead contains a specific number of calibrator fluorophores and a fluorescent trigger domain with an alternative fluorophore for proper detection in FCM. Using electron microscopy, single-particle fluorescence microscopy, and FCM, the design of each particle is verified. To validate that the DNA bead-based FCM calibration enabled the authors to determine the number of antigens on a biological particle, the uniform and well-characterized murine leukemia virus (MLV) is studied. 48 ± 11 envelope surface protein (Env) trimers per MLV is obtained, which is consistent with reported numbers that relied on low-throughput imaging. Thus, the authors' DNA-beads should accelerate quantitative studies of the biology of individual particles with FCM.


Assuntos
DNA , Corantes Fluorescentes , Animais , Antígenos , Calibragem , Citometria de Fluxo/métodos , Ionóforos , Camundongos , Nanotecnologia
10.
Biochim Biophys Acta Biomembr ; 1864(2): 183820, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34813768

RESUMO

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provide detailed information but often rely on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000-2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Citometria de Fluxo/métodos , Fluoresceínas/metabolismo , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Lipossomas Unilamelares/metabolismo , Fluorescência
11.
Acta Biomater ; 144: 96-108, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35314364

RESUMO

Stimulation of monocytes with immunomodulating agents can harness the immune system to treat a long range of diseases, including cancers, infections and autoimmune diseases. To this end we aimed to develop a monocyte-targeting delivery platform based on cationic liposomes, which can be utilized to deliver immunomodulators and thus induce monocyte-mediated immune responses while avoiding off-target side-effects. The cationic liposome design is based on functionalizing the liposomal membrane with a cholesterol-anchored tri-arginine peptide (TriArg). We demonstrate that TriArg liposomes can target monocytes with high specificity in both human and murine blood and that this targeting is dependent on the content of TriArg in the liposomal membrane. In addition, we show that the mechanism of selective monocyte targeting involves the CD14 co-receptor, and selectivity is compromised when the TriArg content is increased, resulting in complement-mediated off-target uptake in granulocytes. The presented mechanistic findings of uptake by peripheral blood leukocytes may guide the design of future drug delivery systems utilized for immunotherapy. STATEMENT OF SIGNIFICANCE: Monocytes are attractive targets for immunotherapies of cancers, infections and autoimmune diseases. Specific delivery of immunostimulatory drugs to monocytes is typically achieved using ligand-targeted drug delivery systems, but a simpler approach is to target monocytes using cationic liposomes. To achieve this, however, a deep understanding of the mechanisms governing the interactions of cationic liposomes with monocytes and other leukocytes is required. We here investigate these interactions using liposomes incorporating a cationic arginine-rich lipopeptide. We demonstrate that monocyte targeting can be achieved by fine-tuning the lipopeptide content in the liposomes. Additionally, we reveal that the CD14 receptor is involved in the targeting process, whereas the complement system is not. These mechanistic findings are critical for future design of monocyte-targeting liposomal therapies.


Assuntos
Doenças Autoimunes , Neoplasias , Animais , Arginina/farmacologia , Cátions , Humanos , Lipopeptídeos/farmacologia , Receptores de Lipopolissacarídeos , Lipossomos/química , Camundongos , Monócitos
12.
Front Bioeng Biotechnol ; 10: 965200, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36159696

RESUMO

Unsuccessful clinical translation of orally delivered biological drugs remains a challenge in pharmaceutical development and has been linked to insufficient mechanistic understanding of intestinal drug transport. Live cell imaging could provide such mechanistic insights by directly tracking drug transport across intestinal barriers at subcellular resolution, however traditional intestinal in vitro models are not compatible with the necessary live cell imaging modalities. Here, we employed a novel microfluidic platform to develop an in vitro intestinal epithelial barrier compatible with advanced widefield- and confocal microscopy. We established a quantitative, multiplexed and high-temporal resolution imaging assay for investigating the cellular uptake and cross-barrier transport of biologics while simultaneously monitoring barrier integrity. As a proof-of-principle, we use the generic model to monitor the transport of co-administrated cell penetrating peptide (TAT) and insulin. We show that while TAT displayed a concentration dependent difference in its transport mechanism and efficiency, insulin displayed cellular internalization, but was restricted from transport across the barrier. This illustrates how such a sophisticated imaging based barrier model can facilitate mechanistic studies of drug transport across intestinal barriers and aid in vivo and clinical translation in drug development.

13.
ACS Omega ; 6(1): 871-880, 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33458538

RESUMO

Biomimetic high-density lipoproteins (b-HDL) have in the past two decades been applied for various drug delivery applications. As b-HDL inherently have relatively long circulation half-life and high tumor accumulation, this has inspired researchers to use b-HDL to selectively deliver drugs to tumors. PEGylation of the b-HDL has been pursued to increase the circulation half-life and therapeutic efficacy even further. The b-HDL consist of lipids stabilized by a protein/peptide scaffold, and while PEGylation of the scaffold has been shown to greatly increase the circulation half-life of the scaffold, the effect of PEGylation of the lipids is much less significant. Still, it remains to be evaluated how the biological fate, including cellular uptake, biodistribution, and circulation half-life, of the b-HDL lipids is affected by PEGylation of the b-HDL scaffold. We studied this with apolipoprotein A-I (apoA-I)-based b-HDL and mono-PEGylated b-HDL (PEG b-HDL) both in vitro and in vivo. We found that PEGylation of the b-HDL scaffold only seemed to have minimal effect on the biological fate of the lipids. Both b-HDL and PEG b-HDL overall shared similar biological fates, which includes cellular uptake through the scavenger receptor class B type 1 (SR-BI) and relatively high tumor accumulation. This highlights that b-HDL are dynamic particles, and the biological fates of the b-HDL components (lipids and scaffold) can differ. A phenomenon that may also apply for other multicomponent nanoparticles.

14.
Int J Pharm ; 588: 119715, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32750439

RESUMO

Targeting nanocarrier drug delivery systems, that deliver drug payloads to the site of disease action, are frequently viewed as the future of nanocarrier based therapies but have struggled to breakthrough to the clinic in comparison to non-targeting counterparts. Using unilamellar liposomes as model nanocarriers, we show that cell targeting strategy (electrostatic, ligand and antigen) influences both the intracellular fate of the liposomes and the corresponding efficacy of the loaded drug, doxorubicin, in endothelial cells. We show that increased liposome uptake by cells does not translate to improved efficacy in this scenario but that liposome intracellular trafficking, particularly distribution between recycling endosomes and lysosomes, influences in vitro efficacy. Choosing targeting strategies that promote desired nanocarrier intracellular trafficking may be a viable strategy to enhance the in vivo efficacy of drug delivery systems.


Assuntos
Doxorrubicina/metabolismo , Células Endoteliais/metabolismo , Lipídeos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Doxorrubicina/química , Doxorrubicina/farmacologia , Composição de Medicamentos , Liberação Controlada de Fármacos , Endocitose , Endossomos/metabolismo , Células Endoteliais/efeitos dos fármacos , Humanos , Cinética , Lipossomos , Lisossomos/metabolismo
15.
ACS Cent Sci ; 6(7): 1159-1168, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32724850

RESUMO

Biological membranes have distinct geometries that confer specific functions. However, the molecular mechanisms underlying the phenomenological geometry/function correlations remain elusive. We studied the effect of membrane geometry on the localization of membrane-bound proteins. Quantitative comparative experiments between the two most abundant cellular membrane geometries, spherical and cylindrical, revealed that geometry regulates the spatial segregation of proteins. The measured geometry-driven segregation reached 50-fold for membranes of the same mean curvature, demonstrating a crucial and hitherto unaccounted contribution by Gaussian curvature. Molecular-field theory calculations elucidated the underlying physical and molecular mechanisms. Our results reveal that distinct membrane geometries have specific physicochemical properties and thus establish a ubiquitous mechanistic foundation for unravelling the conserved correlations between biological function and membrane polymorphism.

16.
Cell Rep ; 23(7): 2056-2069, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29768204

RESUMO

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , Drosophila melanogaster/metabolismo , Insulina/metabolismo , Secreção de Insulina , Lipossomos , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
17.
ACS Appl Mater Interfaces ; 9(19): 15907-15921, 2017 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-28117959

RESUMO

Cell organelles are subcellular structures entrapping a set of enzymes to achieve a specific functionality. The incorporation of artificial organelles into cells is a novel medical paradigm which might contribute to the treatment of various cell disorders by replacing malfunctioning organelles. In particular, artificial organelles are expected to be a powerful solution in the context of enzyme replacement therapy since enzymatic malfunction is the primary cause of organelle dysfunction. Although several attempts have been made to encapsulate enzymes within a carrier vehicle, only few intracellularly active artificial organelles have been reported to date and they all consist of single-compartment carriers. However, it is noted that biological organelles consist of multicompartment architectures where enzymatic reactions are executed within distinct subcompartments. Compartmentalization allows for multiple processes to take place in close vicinity and in a parallel manner without the risk of interference or degradation. Here, we report on a subcompartmentalized and intracellularly active carrier, a crucial step for advancing artificial organelles. In particular, we develop and characterize a novel capsosome system, which consists of multiple liposomes and fluorescent gold nanoclusters embedded within a polymer carrier capsule. We subsequently demonstrate that encapsulated enzymes preserve their activity intracellularly, allowing for controlled enzymatic cascade reaction within a host cell.


Assuntos
Células Artificiais , Cápsulas , Ouro , Lipossomos , Polímeros
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