RESUMEN

Pancreatic cancer is a more aggressive and refractory malignancy. Resistance and toxicity limit drug efficacy. Herein, we report a lower toxic and higher effective miriplatin (MPt)-loaded liposome, LMPt, exhibiting totally different anti-cancer mechanism from previously reported platinum agents. Both in gemcitabine (GEM)-resistant/sensitive (GEM-R/S) pancreatic cancer cells, LMPt exhibits prominent anti-cancer activity, led by faster cellular entry-induced larger accumulation of MPt. The level of caveolin-1 (Cav-1) determines entry rate and switch of entry pathways of LMPt, indicating a novel role of Cav-1 in nanoparticle entry. After endosome-lysosome processing, in unchanged metabolite, MPt is released and targets mitochondria to enhance binding of mitochondria protease LONP1 with POLG and TFAM, to degrade POLG and TFAM. Then, via PINK1-Parkin axis, mitophagy is induced by POLG and TFAM degradation-initiated mitochondrial DNA (mtDNA) replication blocking. Additionally, POLG and TFAM are identified as novel prognostic markers of pancreatic cancer, and mtDNA replication-induced mitophagy blocking mediates their pro-cancer activity. Our findings reveal that the target of this liposomal platinum agent is mitochondria but not DNA (target of most platinum agents), and totally distinct mechanism of MPt and other formulations of MPt. Self-assembly offers LMPt special efficacy and mechanisms. Prominent action and characteristic mechanism make LMPt a promising cancer candidate.

RESUMEN

Gene therapy based on miRNAs has broad application prospects in the treatment of tumors. However, due to degradation and ineffective release during intracellular transport, current gene delivery vectors used for miRNAs limited their actual transfection efficiency. This study develops a novel nonviral vector PEI-SPDP-Man (PSM) that can simultaneously target cellular uptake pathways and intracellular responsive release for miR-34a. PSM is synthesized by connected mannitol (Man) to branched polyethylenimine (PEI) using a disulfide bond. The prepared PSM/miR-34a gene delivery system can induce and enter to tumor cells through caveolae-mediated endocytosis to reduce the degradation of miR-34a in lysosomes. The disulfide bond is sensed at high concentration of glutathione (GSH) in the tumor cells and miR-34a is released, thereby reducing the expression of Bcl-2 and CD44 to suppress the proliferation and invasion of tumor cells. In vitro and in vivo experiments show that through the targeted cellular uptake and the efficient release of miR-34a, an effective antitumor and antimetastasis profiles for the treatment of orthotopic triple negative breast cancer (TNBC) are achieved. This strategy of controlling intracellular transport pathways by targeting cellular uptake pathways in the gene therapy is an approach that could be developed for highly effective cancer therapy.

Asunto(s)

MicroARNs , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/terapia , Neoplasias de la Mama Triple Negativas/patología , Línea Celular Tumoral , Polímeros , Caveolas/metabolismo , Caveolas/patología , MicroARNs/metabolismo , Técnicas de Transferencia de Gen , Endocitosis , Disulfuros , Proliferación Celular

RESUMEN

Signal-amplified imaging of microRNAs (miRNAs) is a promising strategy at the single-cell level because liquid biopsy fails to reflect real-time dynamic miRNA levels. However, the internalization pathways for available conventional vectors predominantly involve endo-lysosomes, showing nonideal cytoplasmic delivery efficiency. In this study, size-controlled 9-tile nanoarrays are designed and constructed by integrating catalytic hairpin assembly (CHA) with DNA tile self-assembly technology to achieve caveolae-mediated endocytosis for the amplified imaging of miRNAs in a complex intracellular environment. Compared with classical CHA, the 9-tile nanoarrays possess high sensitivity and specificity for miRNAs, achieve excellent internalization efficiency by caveolar endocytosis, bypassing lysosomal traps, and exhibit more powerful signal-amplified imaging of intracellular miRNAs. Because of their excellent safety, physiological stability, and highly efficient cytoplasmic delivery, the 9-tile nanoarrays can realize real-time amplified monitoring of miRNAs in various tumor and identical cells of different periods, and imaging effects are consistent with the actual expression levels of miRNAs, ultimately demonstrating their feasibility and capacity. This strategy provides a high-potential delivery pathway for cell imaging and targeted delivery, simultaneously offering a meaningful reference for the application of DNA tile self-assembly technology in relevant fundamental research and medical diagnostics.

Asunto(s)

MicroARNs , Nanoestructuras , MicroARNs/genética , Caveolas , ADN/química , Nanoestructuras/química , Endocitosis

RESUMEN

Pancreatic cancer is a more aggressive and refractory malignancy. Resistance and toxicity limit drug efficacy. Herein, we report a lower toxic and higher effective miriplatin (MPt)-loaded liposome, LMPt, exhibiting totally different anti-cancer mechanism from previously reported platinum agents. Both in gemcitabine (GEM)-resistant/sensitive (GEM-R/S) pancreatic cancer cells, LMPt exhibits prominent anti-cancer activity, led by faster cellular entry-induced larger accumulation of MPt. The level of caveolin-1 (Cav-1) determines entry rate and switch of entry pathways of LMPt, indicating a novel role of Cav-1 in nanoparticle entry. After endosome-lysosome processing, in unchanged metabolite, MPt is released and targets mitochondria to enhance binding of mitochondria protease LONP1 with POLG and TFAM, to degrade POLG and TFAM. Then, via PINK1-Parkin axis, mitophagy is induced by POLG and TFAM degradation-initiated mitochondrial DNA (mtDNA) replication blocking. Additionally, POLG and TFAM are identified as novel prognostic markers of pancreatic cancer, and mtDNA replication-induced mitophagy blocking mediates their pro-cancer activity. Our findings reveal that the target of this liposomal platinum agent is mitochondria but not DNA (target of most platinum agents), and totally distinct mechanism of MPt and other formulations of MPt. Self-assembly offers LMPt special efficacy and mechanisms. Prominent action and characteristic mechanism make LMPt a promising cancer candidate.

RESUMEN

It is well recognized that redox imbalance, nitric oxide (NO), and vitamin D deficiencies increase risk of cardiovascular, metabolic, and infectious diseases. However, clinical studies assessing efficacy of NO and vitamin D supplementation have failed to produce unambiguous efficacy outcomes suggesting that the understanding of the pharmacologies involved is incomplete. This raises the need for using systems pharmacology tools to better understand cause-effect relationships at biological systems levels. We describe the use of spectral clustering methodology to analyze protein network interactions affected by a complex nutraceutical, Cardio Miracle (CM), that contains arginine, citrulline, vitamin D, and antioxidants. This examination revealed that interactions between protein networks affected by these substances modulate functions of a network of protein complexes regulating caveolae-mediated endocytosis (CME), TGF beta activity, vitamin D efficacy and host defense systems. Identification of this regulatory scheme and the working of embedded reciprocal feedback loops has significant implications for treatment of vitamin D deficiencies, atherosclerosis, metabolic and infectious diseases such as COVID-19.

RESUMEN

(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NH4Cl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-ß-cyclodextrin (MßCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.

Asunto(s)

Caveolas , Internalización del Virus , Animales , Caveolas/metabolismo , Línea Celular , Colesterol/metabolismo , Clatrina/metabolismo , Deltacoronavirus , Dinaminas/metabolismo , Endocitosis , Porcinos

RESUMEN

Pas2r12 is comprised of a repeat of the penetration-accelerating sequence (Pas) (Pas2: FFLIG-FFLIG) and D-form dodeca-arginine (r12), a cell-penetrating peptide. Pas2r12 significantly enhances cytosolic delivery of cargo proteins, including enhanced green fluorescent protein and immunoglobulin G. Simply incubating Pas2r12 with cargo leads to their cytosolic tranlsocation. Cytosolic delivery of cargo by Pas2r12 involves caveolae-mediated endocytosis. In this chapter, we describe methods of cytosolic delivery of cargo using Pas2r12 and provide methods for investigating the cellular uptake pathway of cargo by Pas2r12.

Asunto(s)

Péptidos de Penetración Celular/análisis , Arginina , Citosol , Endocitosis , Secuencias Repetidas en Tándem

RESUMEN

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Asunto(s)

Caveolas/efectos de los fármacos , Colesterol/química , Células Endoteliales/efectos de los fármacos , Liposomas/química , Microdominios de Membrana/efectos de los fármacos , Transfección/métodos , Animales , Caveolas/química , Caveolas/metabolismo , Caveolina 1/antagonistas & inhibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Línea Celular Transformada , Colesterol/metabolismo , Clatrina/metabolismo , ADN/química , ADN/metabolismo , Endocitosis/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Filipina/química , Filipina/farmacología , Expresión Génica , Liposomas/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Nistatina/química , Nistatina/farmacología , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacología , Pinocitosis/efectos de los fármacos , Plásmidos/química , Plásmidos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas

RESUMEN

Growing evidence demonstrated that bioaccumulation of polystyrene nanoplastics (PS-NPs) in various organisms including human beings caused destructive effects on health. Nanoplastics may adversely affect fetal development potentially since they can pass through the placental barrier. However, very little has been known about the embryonic toxicity of polystyrene nanoplastics, especially in embryonic neurulation, the early developmental stage of the fetus, as well as the corresponding mechanisms. In this study, we first observed that 60- or 900-nm PS-NPs (especially 60-nm PS-NPs) could cross mouse placentas and affect developing mice fetuses. To avoid the indirect adverse effects derived from the restricted placenta, we employed early chick embryos as a developmental model to evaluate direct adverse effects of PS-NPs on embryo/fetal development, revealing suppressive effects on embryo development and an increased frequency of congenital abnormalities (especially in the nervous system), including neural tube defects. Thus, we focused on the potential negative effects of PS-NPs on neurulation, the earliest stage of nervous system development. Using caveolin-1 immunofluorescent staining of SH-SY5Y cells exposed to PS-NPs-GFP, we demonstrated that PS-NPs were internalized by SH-SY5Y cells via caveolae-mediated endocytosis. Transmission electron microscopy; LC3B immunofluorescent staining; and Atg7, Atg5, p62 and LC3B western blot results revealed that autophagy was activated in SH-SY5Y cells exposed to PS-NPs. However, PS-NPs were not degraded by the autophagic-lysosomal system given the lack of LAMP1 changes and minimal PS-NPs-GFP and LAMP1 colocalization. Furthermore, the cytoplasmic accumulation of PS-NPs caused faulty apoptotic cell death in SH-SY5Y cells and the developing neural tube as revealed by c-caspase3 immunofluorescent staining. Thus, defective neural tube morphogenesis, as demonstrated by neural tube defects, occurred during embryogenesis in the context of PS-NP exposure.

Asunto(s)

Nanopartículas , Contaminantes Químicos del Agua , Animales , Apoptosis , Caveolas , Embrión de Pollo , Endocitosis , Femenino , Ratones , Microplásticos , Tubo Neural , Neurulación , Placenta , Poliestirenos/toxicidad , Embarazo

RESUMEN

Cell membrane acts as a barrier to the entry of impermeable drugs into cells. Recent studies have suggested that using magnetic fields can enable molecules to overcome the cell membrane barrier. However, the mechanism of membrane permeabilization remains unclear. Therefore, we evaluated the increases in bleomycin (CT) uptake, a non-permanent chemotherapy agent, using high-pulsed magnetic fields and investigated whether endocytosis was involved in the process. This study exposed MCF-7 cells to magnetic fields (2.2 T strength, different number of 28 and 56 pulses, and frequency of 1 and 10 Hz) in order to investigate whether this approach could promote the cell-killing efficiency of bleomycin. The involvement of endocytosis as a possible mechanism was tested by exposing cells to three endocytosis inhibitors, namely chlorpromazine, genistein, and amiloride. Our results illustrated that magnetic fields, depending on their conditions, could induce different endocytosis pathways. In such conditions as 10 Hz-28 pulses, 10 Hz-56 pulses, and 1 Hz-56 pulse, clathrin-mediated endocytosis was observed. Moreover, macropinocytosis was induced by the 10 Hz magnetic field and caveolae-mediated endocytosis occurred in all the magnetic field conditions. The findings imply that high-pulsed magnetic fields generate different endocytosis pathways in the MCF-7 cells, thus increasing the efficiency of chemotherapy agents.

Asunto(s)

Caveolas , Clatrina , Membrana Celular , Endocitosis , Campos Magnéticos

RESUMEN

Members of the Polyomaviridae family differ in their host range, pathogenesis, and disease severity. To date, some of the most studied polyomaviruses include human JC, BK, and Merkel cell polyomavirus and non-human subspecies murine and simian virus 40 (SV40) polyomavirus. Although dichotomies in host range and pathogenesis exist, overlapping features of the infectious cycle illuminate the similarities within this virus family. Of particular interest to human health, JC, BK, and Merkel cell polyomavirus have all been linked to critical, often fatal, illnesses, emphasizing the importance of understanding the underlying viral infections that result in the onset of these diseases. As there are significant overlaps in the capacity of polyomaviruses to cause disease in their respective hosts, recent advancements in characterizing the infectious life cycle of non-human murine and SV40 polyomaviruses are key to understanding diseases caused by their human counterparts. This review focuses on the molecular mechanisms by which different polyomaviruses hijack cellular processes to attach to host cells, internalize, traffic within the cytoplasm, and disassemble within the endoplasmic reticulum (ER), prior to delivery to the nucleus for viral replication. Unraveling the fundamental processes that facilitate polyomavirus infection provides deeper insight into the conserved mechanisms of the infectious process shared within this virus family, while also highlighting critical unique viral features.

Asunto(s)

Interacciones Microbiota-Huesped/genética , Poliomavirus/genética , Internalización del Virus , Replicación Viral , Animales , Núcleo Celular/virología , Especificidad del Huésped , Humanos , Poliomavirus/patogenicidad , Infecciones por Polyomavirus/virología

RESUMEN

Biodegradable polymers from renewable resources have attracted much attention in recent years within the biomedical field. Lately, poly(δ-decalactone) based copolymer micelles have emerged as a potential drug delivery carrier material as a sustainable alternative to fossil-based polymers. However, their intracellular drug delivery potential is not yet investigated and therefore, in this work, we report on the synthesis and cellular uptake efficiency of poly(δ-decalactone) based micelles with or without a targeting ligand. Folic acid was chosen as a model targeting ligand and Rhodamine B as a fluorescent tracer to demonstrate the straightforward functionalisation aspect of copolymers. The synthesis of block copolymers was accomplished by a combination of facile ring-opening polymerisation and click chemistry to retain the structure uniformity. The presence of folic acid on the surface of micelles with diameter ~150 nm upsurge the uptake efficiency by 1.6 fold on folate receptor overexpressing MDA-MB-231 cells indicating the attainment of targeting using ligand functionality. The drug delivery capability of these carriers was ascertained by using docetaxel as a model drug, whereby the in vitro cytotoxicity of the drug was significantly increased after incorporation in micelles 48 h post incubation. We have also investigated the possible endocytosis route of non-targeted micelles and found that caveolae-mediated endocytosis was the preferred route of uptake. This work strengthens the prospect of using novel bio-based poly(δ-decalactone) micelles as efficient multifunctional drug delivery nanocarriers towards medical applications.

RESUMEN

Multidisciplinary research efforts in the field of drug delivery have led to the development of a variety of drug delivery systems (DDS) designed for site-specific delivery of diagnostic and therapeutic agents. Since efficient uptake of drug carriers into target cells is central to effective drug delivery, a comprehensive understanding of the biological pathways for cellular internalization of DDS can facilitate the development of DDS capable of precise tissue targeting and enhanced therapeutic outcomes. Diverse methods have been applied to study the internalization mechanisms responsible for endocytotic uptake of extracellular materials, which are also the principal pathways exploited by many DDS. Chemical inhibitors remain the most commonly used method to explore endocytotic internalization mechanisms, although genetic methods are increasingly accessible and may constitute more specific approaches. This review highlights the molecular basis of internalization pathways most relevant to internalization of DDS, and the principal methods used to study each route. This review also showcases examples of DDS that are internalized by each route, and reviews the general effects of biophysical properties of DDS on the internalization efficiency. Finally, options for intracellular trafficking and targeting of internalized DDS are briefly reviewed, representing an additional opportunity for multi-level targeting to achieve further specificity and therapeutic efficacy.

Asunto(s)

Membrana Celular/metabolismo , Sistemas de Liberación de Medicamentos , Endocitosis/fisiología , Animales , Transporte Biológico/fisiología , Portadores de Fármacos/química , Humanos

RESUMEN

Integration of multiple agent therapy (MAT) into one probe is promising for improving therapeutic efficiency for cancer treatment. However, MAT probe, if entering the cell as a whole, may not be optimal for each therapeutic agent (with different physicochemical properties), to achieve their best performance, hindering strategy optimization. A peptide-conjugated-AIEgen (FC-PyTPA) is presented: upon loading with siRNA, it self-assembles into FCsiRNA -PyTPA. When approaching the region near tumor cells, FCsiRNA -PyTPA responds to extracellular MMP-2 and is cleaved into FCsiRNA and PyTPA. The former enters cells mainly by macropinocytosis and the latter is internalized into cells mainly through caveolae-mediated endocytosis. This two-part strategy greatly improves the internalization efficiency of each individual therapeutic agent. Inside the cell, self-assembly of nanofiber precursor F, gene interference of CsiRNA , and ROS production of PyTPA are activated to inhibit tumor growth.

Asunto(s)

Antineoplásicos/farmacología , Endocitosis/efectos de los fármacos , Sondas Moleculares/química , Neoplasias/terapia , Antineoplásicos/uso terapéutico , Humanos , Neoplasias/patología , ARN Interferente Pequeño/administración & dosificación

RESUMEN

Caveolae are membrane organelles formed by submicron invaginations in the plasma membrane, and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally-responsive elastin-like polypeptides (ELPs) can trigger formation of 'genetically engineered protein microdomains (GEPMs)' functionalized with either Clathrin-light chain or the epidermal growth factor receptor. This manuscript is the first report of this strategy to modulate caveolin-1 (CAV1). By attaching different ELP sequences to CAV1, mild heating can be used to self-assemble CAV1-ELP microdomains inside of cells. The temperature of self-assembly can be controlled by tuning the ELP sequence. The formation of CAV1-ELP microdomains internalizes Cholera Toxin Subunit B, a commonly used marker of caveolae mediated endocytosis. CAV1-ELPs also colocalize with Cavin 1, an essential component of functional caveolae biogenesis. With the emerging significance of caveolae in health and disease and the lack of specific probes to rapidly and reversibly affect caveolar function, CAV1-ELP microdomains are a new tool to rapidly probe caveolae associated processes in endocytosis, cell signaling, and mechanosensing.

Asunto(s)

Caveolas , Caveolina 1 , Caveolas/metabolismo , Caveolina 1/genética , Elastina , Endocitosis , Temperatura

RESUMEN

Protein therapeutics is playing an increasingly critical role in treatment of human diseases. However, current vectors are captured by the digestive endo-lysosomal system, which results in an extremely low fraction (<2%) of protein being released in the cytoplasm. This paper reports a drug-delivering-drug platform (HA-PNPplex, 200 nm) for potent intracellular delivery of protein and combined treatment of cancer. Methods: The platform was prepared by loading functional protein on pure drug nanoparticles (PNPs) followed by hyaluronic acid coating and was characterized by dynamic light scattering, transmission electron microscopy, and gel electrophoresis. In vitro, cellular uptake, trafficking, and cytotoxicity were evaluated by flow cytometry and confocal laser microscopy. Protein expression was assayed by western blot. In vivo, blood circulation and biodistribution were studied using a fluorescence imaging system, antitumor efficacy was assessed in a caspase 3-deficient tumor model, and biocompatibility was determined by comparison of hemolytic activity and proinflammatory cytokines and tissue histology. Results: HA-PNPplex delivered the functional protein, caspase 3, to cells via bypassing endo-lysosomes and raised the caspase-3 level 6.5-fold in caspase 3-deficient cells. Promoted tumor accumulation (1.5-fold) and penetration were exhibited, demonstrating a high tumor-targeting ability of HA-PNPplex. HA-PNPplex rendered a 7-fold increase in caspase 3 in tumor and allowed for a 100% tumor growth inhibition and >60% apoptosis, implying significant antitumor activities. Conclusions: This platform gains cellular entry without entrapment in the endo-lysosomes and enables efficient intracellular protein delivery and resultant profound cancer treatment. This platform, with extremely high drug-loading, is a valuable platform for combined cancer therapy with small-molecule drugs and proteins. More importantly, this work offers a robust and safe approach for protein therapeutics and intracellular delivery of other functional peptides, as well as gene-based therapy.

Asunto(s)

Antineoplásicos/farmacocinética , Productos Biológicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Caspasa 3/farmacocinética , Sistemas de Liberación de Medicamentos , Nanopartículas/metabolismo , Animales , Antineoplásicos/administración & dosificación , Disponibilidad Biológica , Productos Biológicos/administración & dosificación , Western Blotting , Caspasa 3/administración & dosificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Ratones Endogámicos BALB C , Microscopía Confocal , Modelos Biológicos , Imagen Óptica , Ratas , Resultado del Tratamiento

RESUMEN

Rationale: Non-invasive tracking of transplanted cells is critical in evaluating delivery, migration and prognosis of cell therapies. Methods: We formulated a nano-contrast agent consisting of a perfluorooctylbromide (PFOB) core within a shell of poly (lactic-co-glycolic acid) (PLGA) followed by a coat of polystyrene sulfonate (PSS) for 19F MRI. The nano-contrast agent (PSS-NP) was characterised by DLS and the uptake efficiency of the nano-contrast agent (PSS-NP) was tested using flow cytometry, in vitro MRI and confocal microscopy. In vitro and in vivo assays of labelled cells were tested for their ability to provide an MRI signal while retaining their osteoblastic differentiation capabilities. Results: PSS-NPs were internalised via caveolae-mediated endocytosis in mesenchymal stromal/stem cells without affecting cell proliferation and differentiation in osteoblasts, both in vitro and in vivo. Furthermore, labelled cells were monitored by 19F MRI for up to 2 months after transplantation in mice. In particular, PSS-NP-labelled cells can be used to monitor the enhanced immune rejection of grafted human cells in normal BALB/c mice compared to immune-compromised NOD/SCID mice. One week after transplantation, 40% of the 19F MRI signal was lost in normal mice, whereas only 10% was lost in immune-compromised mice. Conclusion: Overall, these results show that PSS-NPs can label MSCs effectively, and be employed in vivo as a novel nano-contrast agent for non-invasive cell tracking using clinically relevant 19F MRI techniques.

Asunto(s)

Caveolas/metabolismo , Medios de Contraste/química , Imagen por Resonancia Magnética/métodos , Células Madre Mesenquimatosas/metabolismo , Nanopartículas/química , Animales , Línea Celular , Medios de Contraste/farmacocinética , Endocitosis , Fluorocarburos/química , Humanos , Hidrocarburos Bromados , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Nanopartículas/metabolismo , Osteogénesis , Poliestirenos/química

RESUMEN

Previously developed Asn-Gly-Arg (NGR) peptide-modified multifunctional poly(ethyleneimine)-poly(ethylene glycol) (PEI-PEG)-based nanoparticles (TPIC) have been considered to be promising carriers for the co-delivery of DNA and doxorubicin (DOX). As a continued effort, the aim of the present study was to further evaluate the interaction between TPIC and human umbilical vein endothelial cells (HUVEC) to better understand the cellular entry mechanism. In the present investigation, experiments relevant to co-localization, endocytosis inhibitors and factors influencing the internalization were performed. Without any treatment, there was no co-localization between aminopeptidase N/CD13 (APN/CD13) and caveolin 1 (CAV1). However, co-localization between CD13 and CAV1 was observed when cells were incubated with an anti-CD13 antibody or TPIC. As compared with antibody treatment, TPIC accelerated the speed and enhanced the degree of co-localization. TPIC entered HUVEC not only together with CD13 but also together with CAV1. However, this internalization was not dependent on the enzyme activity of CD13 but could be inhibited by methyl-ß-eyclodextfin (MßCD), further identifying the involvement of caveolae-mediated endocytosis (CvME). This conclusion was also verified by endocytosis inhibitor experiments.

RESUMEN

α-Lactalbumin (α-LA) can bind oleic acid (OA) to form the anti-tumor α-LA-OA complex. Previous studies suggested α-LA-OA induced apoptosis or autophagy in an independent way. Furthermore, as a large molecule, α-LA-OA could enter tumor cells and accumulated in the nucleus, which was speculated as the basis of its anti-tumor activity. In this study, we evaluated the internalization property of α-LA-OA with and without endocytosis inhibitors using flow cytometry and laser scanning confocal microscopy. It was shown α-LA-OA transported from the cell membrane to the cytoplasm, then accumulated around the nucleus, which consequently began to shrink and condense. The α-LA component only located in the membrane whereas the OA component entered cytoplasm. When pre-treated cells with these inhibitors, the internalization of OA would all decrease while the interaction of α-LA with membrane did not influence. As for the complex, the internalization of α-LA-OA was completely blocked at 4°C and significantly decreased in the presence of cytochalasin D, an inhibitor of phagocytosis (p<0.01). In conclusion, the anti-tumor activity of α-LA-OA was mainly dependent on the OA component whereas the internalization mechanism was related to the α-LA component to be temperature-dependent and have a close relationship with the phagocytosis pathway.

Asunto(s)

Antineoplásicos/química , Antineoplásicos/metabolismo , Lactalbúmina/química , Lactalbúmina/metabolismo , Ácido Oléico/química , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Transporte Biológico , Bovinos , Endocitosis/efectos de los fármacos , Células HeLa , Humanos , Lactalbúmina/farmacología

RESUMEN

Previously developed Asn-Gly-Arg (NGR) peptide-modified multifunctional poly(ethyleneimine)-poly(ethylene glycol) (PEI-PEG)-based nanoparticles (TPIC) have been considered to be promising carriers for the co-delivery of DNA and doxorubicin (DOX). As a continued effort, the aim of the present study was to further evaluate the interaction between TPIC and human umbilical vein endothelial cells (HUVEC) to better understand the cellular entry mechanism. In the present investigation, experiments relevant to co-localization, endocytosis inhibitors and factors influencing the internalization were performed. Without any treatment, there was no co-localization between aminopeptidase N/CD13 (APN/CD13) and caveolin 1 (CAV1). However, co-localization between CD13 and CAV1 was observed when cells were incubated with an anti-CD13 antibody or TPIC. As compared with antibody treatment, TPIC accelerated the speed and enhanced the degree of co-localization. TPIC entered HUVEC not only together with CD13 but also together with CAV1. However, this internalization was not dependent on the enzyme activity of CD13 but could be inhibited by methyl--eyclodextfin (MCD), further identifying the involvement of caveolae-mediated endocytosis (CvME). This conclusion was also verified by endocytosis inhibitor experiments.