RESUMO
NaCT mediates citrate uptake in the liver cell line HepG2. When these cells were exposed to iron (Fe3+), citrate uptake/binding as monitored by the association of [14C]-citrate with cells increased. However, there was no change in NaCT expression and function, indicating that NaCT was not responsible for this Fe3+-induced citrate uptake/binding. Interestingly however, the process exhibited substrate selectivity and saturability as if the process was mediated by a transporter. Notwithstanding these features, subsequent studies demonstrated that the iron-induced citrate uptake/binding did not involve citrate entry into cells; instead, the increase was due to the formation of citrate-Fe3+ chelate that adsorbed to the cell surface. Surprisingly, the same phenomenon was observed in culture wells without HepG2 cells, indicating the adsorption of the citrate-Fe3+ chelate to the plastic surface of culture wells. We used this interesting phenomenon as a simple screening technique for new iron chelators with the logic that if another iron chelator is present in the assay system, it would compete with citrate for binding to Fe3+ and prevent the formation and adsorption of citrate-Fe3+ to the culture well. This technique was validated with the known iron chelators deferiprone and deferoxamine, and with the bacterial siderophore 2,3-dihydroxybenzoic acid and the catechol carbidopa.
Assuntos
Artefatos , Ácido Cítrico , Ácido Cítrico/farmacologia , Desferroxamina/farmacologia , Compostos Férricos/farmacologia , Ferro/metabolismo , Quelantes de Ferro/farmacologia , PlásticosRESUMO
SLC13A5/NaCT is a sodium-coupled citrate transporter expressed in the plasma membrane of the liver, testis, and brain. In these tissues, SLC13A5 has important functions in the synthesis of fatty acids, cholesterol, and neurotransmitters. In recent years, patients homozygous for recessive mutations in SLC13A5, known as SLC13A5 deficiency [early infantile epileptic encephalopathy-25 (EIEE-25)], exhibit severe global developmental delay, early-onset intractable seizures, spasticity, and amelogenesis imperfecta affecting tooth development. Although the pathogenesis of SLC13A5 deficiency remains not clearly understood, cytoplasmic citrate deficits, decreased energy status in neurons, and citrate-zinc chelation are hypothesized to explain the neurological deficits. However, no study has examined the possibility of specific pharmacological drugs and/or lifestyle changes synergizing with heterozygosity of SLC13A5 deficiency to increase the risk of EIEE-25 clinical phenotype. Here, we report on a heterozygous SLC13A5-deficient patient who demonstrated evidence of pharmaco-synergistic heterozygosity upon administration of metformin, valproic acid, and starvation. The report illustrates the importance of careful consideration of the potential adverse effects of specific pharmacological treatments in patients with heterozygosity for disease-causing recessive mutations in SLC13A5.
Assuntos
Epilepsia/genética , Metformina/efeitos adversos , Simportadores/deficiência , Ácido Valproico/efeitos adversos , Adulto , Substituição de Aminoácidos , Amônia/sangue , Animais , Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/uso terapêutico , Transtorno Autístico/genética , Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/genética , Citratos/sangue , Transportadores de Ácidos Dicarboxílicos/fisiologia , Proteínas de Drosophila/fisiologia , Epilepsia/sangue , Epilepsia/induzido quimicamente , Epilepsia/etiologia , Feminino , Privação de Alimentos , Heterozigoto , Humanos , Lactatos/sangue , Longevidade/genética , Metformina/uso terapêutico , Camundongos , Mutação de Sentido Incorreto , Mutação Puntual , Transtornos Psicóticos/tratamento farmacológico , Transtornos Psicóticos/genética , Psicotrópicos/uso terapêutico , Piruvatos/sangue , Recidiva , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Simportadores/genética , Simportadores/fisiologia , Anormalidades Dentárias/genética , Ácido Valproico/uso terapêuticoRESUMO
Background: SLC6A14 (ATB0,+), a Na+/Cl-coupled transporter for neutral/cationic amino acids, is overexpressed in many cancers; It has been investigated as a target for improved liposomal drug delivery to treat liver cancer.Research design and methods: Here we explored the mechanism of ATB0,+-mediated entry of such liposomes. As ATB0,+ is highly expressed in pancreatic cancer, we also examined the therapeutic utility of ATB0,+-targeted liposomal drug delivery to treat this cancer.Results: The uptake of lysine-conjugated liposomes (LYS-LPs) was greater in ATB0,+-positive MCF7 cells. The uptake process consisted of two steps: binding and internalization. The binding of LYS-LPs to MCF7 cells was higher than that of bare liposomes, and the process was dependent on Na+ and Cl-, and inhibitable by ATB0,+ substrates or blocker. In contrast, the internalization step was independent of lysine. The cellular entry of LYS-LPs facilitated by ATB0,+ occurred via endocytosis with transient endosomal degradation of ATB0,+ protein with subsequent recovery. Moreover, LYS-LPs also enhanced the uptake and cytotoxicity of gemcitabine in these cells in an ATB0,+-dependent manner.Conclusions: We conclude that ATB0,+ could be exploited for targeted drug delivery in the form of lysine-conjugated liposomes and that the approach represents a novel strategy for enhanced pancreatic cancer therapy.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Liberação de Medicamentos , Endocitose , Neoplasias Pancreáticas/tratamento farmacológico , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Lipossomos , Células MCF-7RESUMO
Targeted nanocarriers have shown great promise in drug delivery because of optimized drug behavior and improved therapeutic efficacy. How to improve the targeting efficiency of nanocarriers for the maximum possible drug delivery is a critical issue. Here we developed L-carnitine-conjugated nanoparticles targeting the carnitine transporter OCTN2 on enterocytes for improved oral absorption. As a variable, we introduced various lengths of the polyethylene glycol linker (0, 500, 1000, and 2000) between the nanoparticle surface and the ligand (CNP, C5NP, C10NP and C20NP) to improve the ligand flexibility, and consequently for more efficient interaction with the transporter, to enhance the oral delivery of the cargo load into cells. An increased absorption was observed in cellular uptake in vitro and in intestinal perfusion assay in situ when the polyethylene glycol was introduced to link L-carnitine to the nanoparticles; the highest absorption was achieved with C10NP. In contrast, the linker decreased the absorption efficiency in vivo. As the presence or absence of the mucus layer was the primary difference between in vitro/in situ versus in vivo, the presence of this layer was the likely reason for this differential effect. In summary, the size of the polyethylene glycol linker improved the absorption in vitro and in situ, but interfered with the absorption in vivo. Even though this strategy of increasing the ligand flexibility with the variable size of the polyethylene glycol failed to increase oral absorption in vivo, this approach is likely to be useful for enhanced cellular uptake following intravenous administration of the nanocarriers.
Assuntos
Carnitina/farmacologia , Portadores de Fármacos/química , Nanopartículas/química , Paclitaxel/farmacologia , Membro 5 da Família 22 de Carreadores de Soluto/efeitos dos fármacos , Administração Oral , Animais , Células CACO-2 , Carnitina/administração & dosagem , Carnitina/farmacocinética , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Enterócitos , Humanos , Masculino , Paclitaxel/administração & dosagem , Paclitaxel/farmacocinética , Tamanho da Partícula , Polietilenoglicóis/química , Ratos , Ratos Sprague-DawleyRESUMO
l-Carnitine, obligatory for oxidation of fatty acids, is transported into cells by the Na+-coupled transporter OCTN2 and the Na+/Cl--coupled transporter ATB0,+. Here we investigated the potential of L-carnitine-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (LC-PLGA NPs) to deliver chemotherapeutic drugs into cancer cells by targeting the nanoparticles to both OCTN2 and ATB0,+. The cellular uptake of LC-PLGA NPs in the breast cancer cell line MCF7 and the colon cancer cell line Caco-2 was increased compared to unmodified nanoparticles, but decreased in the absence of co-transporting ions (Na+ and/or Cl-) or in the presence of competitive substrates for the two transporters. Studies with fluorescently labeled nanoparticles showed their colocalization with both OCTN2 and ATB0,+, confirming the involvement of both transporters in the cellular uptake of LC-PLGA NPs. As the expression levels of OCTN2 and ATB0,+ are higher in colon cancer cells than in normal colon cells, LC-PLGA NPs can be used to deliver chemotherapeutic drugs selectively into cancer cells for colon cancer therapy. With 5-fluorouracil-loaded LC-PLGA NPs, we were able to demonstrate significant increases in the uptake efficiency and cytotoxicity in colon cancer cells that were positive for OCTN2 and ATB0,+. In a 3D spheroid model of tumor growth, LC-PLGA NPs showed increased uptake and enhanced antitumor efficacy. These findings indicate that dual-targeting LC-PLGA NPs to OCTN2 and ATB0,+ has great potential to deliver chemotherapeutic drugs for colon cancer therapy. Dual targeting LC-PLGA NPs to OCTN2 and ATB0,+ can selectively deliver chemotherapeutics to colon cancer cells where both transporters are overexpressed, preventing targeting to normal cells and thus avoiding off-target side effects.
Assuntos
Nanopartículas , Sistema ASC de Transporte de Aminoácidos , Células CACO-2 , Carnitina , Neoplasias do Colo , Humanos , Ácido Láctico , Antígenos de Histocompatibilidade Menor , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Membro 5 da Família 22 de Carreadores de SolutoRESUMO
OCTN2 (SLC22A5) is a Na+ -coupled absorption transporter for l-carnitine in small intestine. This study tests the potential of this transporter for oral delivery of therapeutic drugs encapsulated in l-carnitine-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (LC-PLGA NPs) and discloses the molecular mechanism for cellular endocytosis of transporter-targeting nanoparticles. Conjugation of l-carnitine to a surface of PLGA-NPs enhances the cellular uptake and intestinal absorption of encapsulated drug. In both cases, the uptake process is dependent on cotransporting ion Na+ . Computational OCTN2 docking analysis shows that the presence of Na+ is important for the formation of the energetically stable intermediate complex of transporter-Na+ -LC-PLGA NPs, which is also the first step in cellular endocytosis of nanoparticles. The transporter-mediated intestinal absorption of LC-PLGA NPs occurs via endocytosis/transcytosis rather than via the traditional transmembrane transport. The portal blood versus the lymphatic route is evaluated by the plasma appearance of the drug in the control and lymph duct-ligated rats. Absorption via the lymphatic system is the predominant route in the oral delivery of the NPs. In summary, LC-PLGA NPs can effectively target OCTN2 on the enterocytes for enhancing oral delivery of drugs and the critical role of cotransporting ions should be noticed in designing transporter-targeting nanoparticles.
Assuntos
Carnitina/química , Endocitose , Nanopartículas/química , Paclitaxel/administração & dosagem , Paclitaxel/uso terapêutico , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , Administração Oral , Disponibilidade Biológica , Células CACO-2 , Carnitina/síntese química , Humanos , Absorção Intestinal , Íons , Ácido Láctico/química , Sistema Linfático/efeitos dos fármacos , Simulação de Acoplamento Molecular , Nanopartículas/ultraestrutura , Paclitaxel/farmacocinética , Espectroscopia Fotoeletrônica , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sódio/química , Membro 5 da Família 22 de Carreadores de Soluto/genéticaRESUMO
Rapidly proliferating tumor cells upregulate specific amino acid transporters, which hold great potential for tumor-selective drug delivery. Published reports have focused primarily on blocking these transporters as a means of starving the tumor cells of amino acids, but their potential in drug delivery remains understudied. In the present study, we developed liposomes functionalized with lysine and polyoxyethylene stearate conjugate (LPS) to interact with ATB0,+, an amino acid transporter overexpressed in hepatocarcinoma and the liver cancer cell line HepG2. The LPS modified liposomes (LPS-Lips) were ~100nm in size and exhibited high drug encapsulation efficiency as 94.7%. The uptake of LPS-Lips in HepG2 cells was dependent on Na+ and Cl-. Molecular dynamic simulation showed that a sustained occluded state of the transporter upon binding to co-transported ions was formed and LPS-Lips triggered the cellular internalization of liposomes. We loaded these LPS-Lips with docetaxel and evaluated the potential of ATB0,+-mediated endocytosis of the drug-loaded LPS-Lips in HepG2 cells in vitro and in syngeneic mouse transplants in vivo. Compared with unmodified liposomes, which did not interact with ATB0,+, LPS-Lips exhibited the ability to deliver docetaxel more efficiently into tumor cells with consequent greater antitumor efficacy and less systemic toxicity. These studies provide first evidences that ATB0,+ can be used as a novel and effective target for drug delivery system in tumor cells using chemically modified liposomes for loading with chemotherapeutics and targeting them for the transporter-mediated endocytosis. As ATB0,+ is highly upregulated in several cancers, this approach holds potential for tumor-selective delivery of drugs to treat these cancer types.