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Extracellular vesicles (EVs) are nanoparticles secreted by various organisms. Methods for modifying EVs functionally have garnered attention for developing EV-based therapeutic systems. However, most technologies used to integrate these functions are limited to mammalian-derived EVs and a promising modification method for bacteria-derived EVs has not yet been developed. In this study, we propose a novel method for the versatile functionalization of immunostimulatory probiotic Bifidobacteria-derived EVs (B-EVs) using amino acid metabolic labeling and azide-alkyne click reaction. Azide D-alanine (ADA), a similar molecule to D-alanine in bacteria cell-wall peptidoglycan, was selected as an azide group-functionalized amino acid. Azide-modified B-EVs were isolated from Bifidobacteria incubated with ADA. The physicochemical and compositional characteristics, as well as adjuvanticity of B-EVs against immune cells were not affected by azide loading, demonstrating that this functionalization approach can retain the endogenous usefulness of B-EVs. By using the fluorescent B-EVs obtained by this method, the intracellular trafficking of B-EVs after uptake by immune cells was successfully observed. Furthermore, this method enabled the formulation of B-EVs for hydrogelation and enhanced adjuvanticity in the host. Our findings will be helpful for further development of EV-based immunotherapy.
Assuntos
Azidas , Bifidobacterium , Química Click , Vesículas Extracelulares , Imunoterapia , Vesículas Extracelulares/metabolismo , Bifidobacterium/metabolismo , Azidas/química , Animais , Imunoterapia/métodos , Alanina/química , Probióticos/administração & dosagem , Camundongos , Aminoácidos/química , Aminoácidos/metabolismo , Humanos , Células RAW 264.7RESUMO
The olfactory and trigeminal pathways are direct delivery pathways between the nose and brain. To determine the effect of direct delivery on drug distribution in the brain, two model drugs with different physical properties, antipyrine (ANP), with high membrane permeability, and ranitidine (RNT), with low membrane permeability, were selected. For ANP, direct delivery from the nose to the brain was observed only in the olfactory bulb beside the nasal cavity, with a direct transport percentage (DTP) of approximately 45â¯%, whereas in the frontal and occipital brains, the contribution from the systemic circulation to the brain was observed as the primary route of brain distribution. No significant variations were observed in the pharmacokinetics of ANP in the left and right brain, whereas RNT was distributed in all brain regions with a DTP of > 95â¯%. The closer the brain region is to the nasal cavity, the higher the DTP. Furthermore, the left brain, the same nostril site (left nostril) of administration, had a larger level of drug delivery than the right brain. These findings imply that the influence of the administered nostril site differs based on the physicochemical properties and amount of the drug.
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Extracellular vesicles (EVs) are cell-derived nanoparticles that can be used as novel biomaterials. In the development of EVs-based therapeutic systems, it is essential to understand the in vivo fate of exogenously administered EVs and subsequent biological responses mediated by EVs. Although probiotics and microorganisms that modulate the host immune system also secrete EVs, their tissue distribution and biological reactions after administration to the host have not been sufficiently elucidated. In this study, we characterized EVs released from the probiotics Bifidobacterium longum (B-EVs) and Lactobacillus plantarum WCFS1 (L-EVs) in terms of tissue distribution and immune-activating capacity after intravenous and subcutaneous administration in mice. B-EVs and L-EVs exhibited particle sizes of approximately 100-160 nm and negative zeta potentials. These EVs contained peptidoglycan, DNA, and RNA as their cargoes. Intravenously administered B-EVs and L-EVs mainly accumulated in the liver and spleen. Furthermore, liver F4/80 and splenic CD169 macrophages took up the intravenously administered EVs. Subcutaneously administered B-EVs and L-EVs accumulated in the lymph nodes and were mainly located in the B-lymphocyte zone, indicating that exogenously administered probiotic-derived EVs showed a similar biodistribution, irrespective of the EVs-secreting cell type. Evaluation of EVs-mediated immune reactions demonstrated that intravenously administered EVs showed little activation potency. In contrast, subcutaneously administered B-EVs strongly increased the expression of inflammatory cytokine (TNF-α) and co-stimulatory molecules (CD40 and CD80) than L-EVs. These findings indicate that the subcutaneous administration of B-EVs is a useful strategy for the development of novel EVs-based immunotherapies.
Assuntos
Vesículas Extracelulares , Probióticos , Camundongos , Animais , Distribuição Tecidual , Adjuvantes Imunológicos/farmacologia , Macrófagos , Vesículas Extracelulares/metabolismoRESUMO
The yeast strain Saccharomyces cerevisiae is an eukaryotic organism that has been widely used for the production of fermented foods. Most cells secrete extracellular vesicles (EVs), small particles composed of lipid membranes. Elucidating the role of EVs as a new intercellular communication system and developing novel EV-based therapies have attracted the increased attention of researchers. Although recent studies have reported the secretion of EVs from S. cerevisiae, their in vivo fate and subsequent EV-mediated biological responses in the host are unclear. In this study, we characterized both the biodistribution of locally (intradermally and subcutaneously) administered Saccharomyces cerevisiae-derived EVs (S-EVs) and the EV-mediated immune responses to evaluate their potential use as biocompatible vaccine adjuvants. S-EVs were round but heterogeneous in size and contained glucan, DNA, and RNA. Their mean particle sizes and zeta potentials were approximately 177.5 nm and -14.6 mV, respectively. We provided evidence that locally administered S-EVs were delivered to the lymph nodes, mainly reaching the B-cell zone. Measurement of host immune reactions revealed that administration of S-EVs increased the expression of cytokine (tumor necrosis factor (TNF)-α) and costimulatory molecules (CD40, CD80, CD86), which are indicators of immune activation. Especially, subcutaneously injected S-EVs showed potent adjuvanticity, indicating that subcutaneous administration of S-EVs is the desirable approach for achieving effective immune stimulation. These findings will facilitate the development of novel EV-based immunotherapies.
Assuntos
Vesículas Extracelulares , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Adjuvantes de Vacinas , Distribuição Tecidual , Citocinas/metabolismo , Vesículas Extracelulares/metabolismoRESUMO
Extracellular vesicles (EV) are nanoparticles secreted from cells that are involved in biological functions by transferring their cargo to target cells. Novel disease diagnostic and therapeutic methods may be developed utilizing EV derived from specific cells. In particular, mesenchymal stem cell-derived EV have several useful effects, including tissue repair. Several clinical trials are currently underway. Recent studies have demonstrated that EV secretion is not limited to mammals but also occurs in microorganisms. Since EV from microorganisms contain various bioactive molecules, elucidation of their effects on the host and their practical use is of great interest. On the other hand, for EV utilization, it is necessary to clarify their basic characteristics, such as physical properties and effects on target cells, and to develop a drug delivery system that can manipulate and utilize EV functions. However, the current state of knowledge on EV derived from microorganisms is very limited compared to that of mammalian cell-derived EV. Therefore, we focused on probiotics, microorganisms that have beneficial effects on living organisms. Since probiotics are widely used as pharmaceuticals and functional foods, the utilization of EV secreted from probiotics is expected to benefit clinical fields. In this review, we describe our research on elucidating the effects of probiotic-derived EV on the innate immune response of the host and evaluating their availability as a novel adjuvant.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Probióticos , Animais , Cicatrização , Imunidade Inata , MamíferosRESUMO
Kidney-targeted drug delivery is vital in treating kidney diseases by improving therapeutic efficacy and safety. However, targeting drugs to the kidney is challenging, as drug nano-carriers are usually trapped by the reticuloendothelial system in the liver and spleen. Recently, we reported that serine-modified polyamidoamine (Ser-PAMAM) dendrimer functions as a highly potent kidney-targeting drug carrier. Further, we demonstrated that Ser-PAMAM predominantly accumulated in the kidney, especially in proximal tubules, a pattern associated with the pathogenesis of chronic kidney diseases and renal carcinoma cells. Furthermore, captopril was successfully targeted to the kidney using Ser-PAMAM, and cysteine- or S-nitrosothiol (source of nitric oxide)-loaded Ser-PAMAM effectively suppressed the occurrence of renal injury following renal ischemia/reperfusion. In this review, we summarized recent challenges in developing a kidney-targeted drug delivery system and discussed the utility of our serine modification-based improvements to this system for the efficient treatment of kidney diseases.
Assuntos
Dendrímeros , Nefropatias , Humanos , Serina , Sistemas de Liberação de Medicamentos , Portadores de Fármacos , Rim , Nefropatias/tratamento farmacológicoRESUMO
Extracellular vesicles (EVs) encapsulate various bioactive molecules, and much effort has been directed towards developing a novel EV-based therapy. Although recent studies reported the secretion of EVs from probiotics baker's yeast Saccharomyces cerevisiae (S. cerevisiae), their properties and functions remain obscure. The aim of this study was to clarify the usefulness of EVs from S. cerevisiae (S-EVs) as a novel vaccine material by defining their physicochemical properties and biological functions. The collected S-EVs contained ß-D-glucan and showed particle sizes and zeta potentials approximately 128.8 nm and -7.39 mV, respectively. S-EVs were positive for heat shock protein 70 kDa (HSP70). These S-EVs considerably enhanced the production of proinflammatory tumor necrosis factor-α and interleukin 6 from RAW264.7 cells (mouse macrophage-like cells) and DC2.4 cells (mouse dendritic cells). The expression of maturation markers CD40, CD80 and CD86 on the surface of these immune cells incubated with S-EVs was remarkably upregulated. Immune cells endocytosed S-EVs, and toll like receptor 2 on immune cells was involved in immune activation by S-EVs. These results indicate that extracellular vesicles derived from baker's yeast Saccharomyces cerevisiae are an attractive source as a novel vaccine material for immune cells maturation.
Assuntos
Vesículas Extracelulares , Saccharomyces cerevisiae , Animais , Camundongos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Diferenciação Celular , Vesículas Extracelulares/metabolismo , Interleucina-6/metabolismo , Células RAW 264.7RESUMO
In the present study, L-serine (Ser)-modified poly-L-lysine (PLL) was synthesized to develop a biodegradable, kidney-targeted drug carrier for efficient radionuclide therapy in renal cell carcinoma (RCC). Ser-PLL was labeled with 111In/90Y via diethylenetriaminepentaacetic acid (DTPA) chelation for biodistribution analysis/radionuclide therapy. In mice, approximately 91% of the total dose accumulated in the kidney 3 h after intravenous injection of 111In-labeled Ser-PLL. Single-photon emission computed tomography/computed tomography (SPECT/CT) imaging showed that 111In-labeled Ser-PLL accumulated in the renal cortex following intravenous injection. An intrarenal distribution study showed that fluorescein isothiocyanate (FITC)-labeled Ser-PLL accumulated mainly in the renal proximal tubules. This pattern was associated with RCC pathogenesis. Moreover, 111In-labeled Ser-PLL rapidly degraded and was eluted along with the low-molecular-weight fractions of the renal homogenate in gel filtration chromatography. Continuous Ser-PLL administration over five days had no significant effect on plasma creatinine, blood urea nitrogen (BUN), or renal histology. In a murine RCC model, kidney tumor growth was significantly inhibited by the administration of the beta-emitter 90Y combined with Ser-PLL. The foregoing results indicate that Ser-PLL is promising as a biodegradable drug carrier for kidney-targeted drug delivery and efficient radionuclide therapy in RCC.
RESUMO
The aim of this study was to develop a polyethylene glycol (PEG)-conjugated third-generation polyamidoamine dendrimer (PAMAM) with phosphorylated serine as an osteoid surface-targeting drug carrier for the treatment of bone diseases. We conjugated PAMAM backbones to l-serine and obtained Ser-PAMAM. Then, phosphoric acid and PEG were covalently bound to the Ser-PAMAM to generate PEGylated phosphorylated Ser-PAMAM (PEG-phosSer-PAMAM). Using osteoblast-like cells (MC3T3-E1 cells) cultured in 3D collagen gels, we showed that phosSer-PAMAM adsorbed both the hydroxyapatite and type I collagen components of the bone matrix. Fourier transform infrared spectroscopy analysis indicated that the phosphoryl side chains of phosSer-PAMAM formed electrostatic interactions and hydrogen bonds with the anionic amino acid residues of type I collagen. Mice were intravenously injected with the foregoing molecules, and a tissue distribution study disclosed that the lower limb bone took up about twice as much 111In-labeled PEG-phosSer-PAMAM as 111In-labeled nonphosphorylated PEG-Ser-PAMAM or unmodified PAMAM. An intrabone distribution experiment showed that fluorescein isothiocyanate (FITC)-labeled PEG-phosSer-PAMAM accumulated on the osteoid surfaces, which is associated with bone pathogenesis such as skeletal dysplasias and osteoporosis to a far greater extent than nonphosphorylated PEG-Ser-PAMAM. Our findings indicated that PEG-phosSer-PAMAM is a promising carrier for efficient drug targeting to osteoid surfaces.
Assuntos
Dendrímeros , Portadores de Fármacos , Animais , Matriz Óssea , Colágeno Tipo I , Dendrímeros/química , Portadores de Fármacos/química , Camundongos , Poliaminas , Polietilenoglicóis/química , SerinaRESUMO
The surface properties of nanoparticles (NPs) affect their stability and formation of the protein corona, which influence their targeting abilities. We evaluated these properties using bone (hydroxyapatite; HAP) targeting peptide on tamoxifen (TAM)-loaded stereocomplexformed polylactide-polyethyleneglycol (SC-PLA-PEG) NPs. Octaaspartic acid-octaglycine-cysteine (D8G8C) anionic derivative (Ani. pep.) and octa-aspartic acid-octa lysine-cysteine (D8K8C), a zwitterionic derivative (Zwi. pep.) were conjugated with SC-PLA-PEG NPs as HAP-targeting peptides. The addition of hydrophobic PLA homopolymers increased the surface PEG density on the NPs. Denser PEG chains on NPs decreased their specific surface area, reducing protein adsorption on the NPs and TAM release from NPs. NPs with dense PEG chains and Zwi. pep. showed superior shelf stability and lower protein adsorption than NPs with dense PEG chains and Ani. pep. in murine serum. Furthermore, the HAP-binding ability of NPs with Zwi. pep. was significantly higher than that of NPs with Ani. pep. These results indicate that decreasing the specific surface area and zwitterionization of HAP-targeting peptides on NPs are promising approaches to improve the serum compatibility and stability of NPs.
Assuntos
Nanopartículas , Coroa de Proteína , Animais , Ácido Aspártico , Cisteína , Hidroxiapatitas , Lisina , Camundongos , Nanopartículas/química , Peptídeos , Poliésteres/química , Polietilenoglicóis/química , TamoxifenoRESUMO
Since probiotic-derived extracellular vesicles (EVs) are capable of activating innate immunity, they are expected to be useful as novel adjuvants. To elucidate the mechanisms underlying the immunostimulatory effects of EVs released from probiotic cells, we newly investigated the role of Toll-like receptor 2 (TLR2) and immune cell downstream signaling in the generation of proinflammatory cytokines. Isolated Bifidobacterium- and Lactobacillus-derived EVs expressed peptidoglycan, one of the major pathogen-associated molecular patterns. EVs particle diameter were approximately 110-120 nm with a negative-zeta potential. The generation of proinflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in TLR2-expressing mouse macrophage-like RAW264.7 cells and mouse dendritic DC2.4 cells treated with Bifidobacterium- and Lactobacillus-derived EVs decreased after the addition of T2.5, a TLR2 inhibitory antibody. Furthermore, we showed that the signaling pathways of c-Jun-NH2-terminal kinase (JNK)/mitogen-activated protein kinases (MAPK) and nuclear factor-kappaB (NF-κB) were also involved in the production of proinflammatory cytokines from EV-treated immune cells. These results provide valuable information for understanding of the host biological function induced by probiotic-derived EVs, which is helpful for developing an EV-based immunotherapeutic system.
Assuntos
Vesículas Extracelulares , Probióticos , Animais , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismoRESUMO
To optimize the characteristics of stereocomplex polylactide-b-polyethylene glycol nanoparticles (SC-PEG NPs) in terms of pharmacokinetics (PK), we chose continuous anti-solvent precipitation with a T-junction as a preparation method and investigated the effect of using solvents containing an ion excipient (lithium bromide, LiBr) on the characteristics of SC-PEG NPs by changing the processing temperature and total flow rate (TFR). Processing temperatures above the melting temperature (Tm) of the PEG domain produced a sharper polydispersity and denser surface PEG densities of SC-PEG NPs than those produced by processing temperatures below the Tm of the PEG domains. Response surface analysis revealed that a higher LiBr concentration and slower TFR resulted in larger and denser hydrodynamic diameters (Dh) and surface PEG densities, respectively. However, a high concentration (300 mM) of LiBr resulted in a decreased drug loading content (DLC). 14C-tamoxifen-loaded 111In-SC-PEG NPs with larger Dh and denser surface PEG densities showed a prolonged plasma retention and low tissue distribution after intravenous injection in mice. These results indicate that the novel strategy of using solvents containing LiBr at different processing temperatures and TFR can broadly control characteristics of SC-PEG NPs, such as Dh, surface PEG densities, and DLC, which alter the PK profiles and tissue distributions.
RESUMO
The dissolution behaviors of base excipients from sustained-release formulations have been investigated using various methodologies. However, the dissolution of polymers has not been fully evaluated because differences between formulations are still verified only by the release of active pharmaceutical ingredients (APIs). In our previous study, we proposed a quick and simultaneous analysis of dissolved APIs and water-soluble polymers by ultra HPLC using charged aerosol and photodiode array detectors. The purpose of this study was to verify whether the analysis system could be adapted to other water-soluble polymers. Dissolution tests were conducted using matrix model tablets prepared from three polymers and three APIs (propranolol, ranitidine, and cilostazol) with different solubilities. The dissolution profiles of the polymers and APIs were determined using the proposed analysis system and compared. The results clarified differences in the dissolution behaviors of the APIs and polymers. The polymers, especially hydroxypropyl cellulose, exhibited the dissolution properties characteristic of each model formulation. Propranolol and ranitidine showed the diffusion type, while cilostazol showed the erosion type release mechanism due to their different solubilities. The release of cilostazol was delayed in all models compared to the polymer, which may be due to the aggregation of cilostazol in the gel layer. This analytical method can be used to study the dissolution behavior (diffusion or erosion) of APIs from matrix tablets containing various polymers. This method will provide useful information on release control, which will make it easier and more efficient to design appropriate formulations and analyze the release mechanisms.
Assuntos
Preparações Farmacêuticas/análise , Polímeros/análise , Cromatografia Líquida de Alta Pressão , Composição de Medicamentos , Liberação Controlada de Fármacos , Solubilidade , Água/químicaRESUMO
The objective of the study is to develop a quick and simultaneous analysis system for the dissolution of the active pharmaceutical ingredient (API) and the formulation excipient in samples from the dissolution test by UHPLC using the charged aerosol and PDA detectors. The combination of two columns for size-exclusion chromatography (SEC) and the equipment of the charged aerosol detector allowed the quick determination of various water-soluble polymers. Three model sustained-release tablets, each containing a different API of different water solubility (propranolol (soluble), ranitidine (very soluble), and cilostazol (practically insoluble)), were prepared from polyethylene oxide (PEO) matrix to verify the applicability and utility of the analysis system. The dissolution of propranolol was the same as that of PEO, indicating that the diffusion rate of propranolol was consistent with the erosion rate of the PEO and that the dissolution of PRO was based on diffusion. Ranitidine was released faster than PEO, suggesting that ranitidine was diffused through the gel layer of PEO early upon contact with the dissolution medium and before PEO gel erosion. Cilostazol was released slower as compared to PEO, indicating that cilostazol dissolution was based on the polymer's erosion. These results suggested that the analysis system developed in this study is a precise and valid tool to study the dissolution behavior of both APIs and excipients. Optimization of the SEC column for the appropriate separation of APIs and excipients makes the analysis system more efficient and convenient to study the drug release mechanisms and to design formulations.
Assuntos
Química Farmacêutica , Excipientes , Aerossóis , Cromatografia Líquida de Alta Pressão , Preparações de Ação Retardada , Solubilidade , ComprimidosRESUMO
To optimize prolonged and sustained delivery of polylactide-block-polyethyleneglycol polymeric nanoparticles (PLA-PEG NPs), in terms of the PLA isomer and molecular weight, we performed orthogonal physicochemical characterization and evaluated the pharmacokinetics of tamoxifen (TAM)-loaded PLA-PEG NPs. DL-lactide- (DL-PEG NP), L-lactide- (L-PEG NPs), and stereocomplex-based (SC-PEG NPs) PLA-PEGs, with two different PLA to PEG ratios (12k-5k and 5k-5k Da) were synthesized, and NPs were prepared by anti-solvent precipitation. Size exclusion chromatography, multi-angle light scattering, dynamic light scattering, and 1H nuclear magnetic resonance studies revealed that SC-PEG NPs (12k-5k) had a compact structure and the highest PEG density, followed by L-PEG NPs (12k-5k), DL-PEG NPs (12k-5k), and all PLA-PEG NPs (5k-5k). Additionally, solid-phase extraction indicated that SC-PEG NPs (12k-5k) had the highest drug loading content and the lowest surface TAM adsorption, of the PLA-PEGs evaluated. These results were explained by the crystallinity of the PLA core, which was analyzed by X-ray diffraction. In the pharmacokinetic studies, 14C-TAM-loaded 111In-SC-PEG NPs (12k-5k) exhibited the highest area under the plasma concentration-time curve, followed by L-PEG NPs (12k-5k) and DL-PEG NPs (12k-5k), after intravenous injection in mice. These results indicate that SC-PEG NPs (12k-5k) are promising drug carriers for the sustained and prolonged delivery of TAM.
Assuntos
Nanopartículas , Polietilenoglicóis , Animais , Portadores de Fármacos , Camundongos , Tamanho da Partícula , PoliésteresRESUMO
Extracellular vesicles (EVs) secreted from probiotics, defined as live microorganisms with beneficial effects on the host, are expected to be new nanomaterials for EV-based therapy. To clarify the usability of probiotic-derived EVs in terms of EV-based therapy, we systematically evaluated their characteristics, including the yield, physicochemical properties, the cellular uptake mechanism, and biological functions, using three different types of probiotics: Bifidobacterium longum, Clostridium butyricum, and Lactobacillus plantarum WCFS1. C. butyricum secreted the largest amounts of EVs, whereas all the EVs showed comparable particle sizes and zeta potentials, ranging from 100 to 150 nm and -8 to -10 mV, respectively. The silkworm larvae plasma assay indicated that these EVs contain peptidoglycan that activates the host's immune response. Moreover, a cellular uptake study of probiotic-derived EVs in RAW264.7 cells (mouse macrophage-like cells) and DC2.4 cells (mouse dendritic cells) in the presence of inhibitors (cytochalasin B, chlorpromazine, and methyl-ß-cyclodextrin) revealed that probiotic-derived EVs were mainly taken up by these immune cells via clathrin-mediated endocytosis and macropinocytosis. Furthermore, all the probiotic-derived EVs stimulated the innate immune system through the production of inflammatory cytokines (TNF-α and IL-6) from these immune cells, clarifying their utility as a novel adjuvant formulation. These findings on probiotic-derived EVs are valuable for understanding the biological significance of probiotic-derived EVs and the development of EV-based immunotherapy.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vesículas Extracelulares/metabolismo , Probióticos/metabolismo , Animais , Células Cultivadas , Clorpromazina/farmacologia , Citocalasina B/farmacologia , Citocinas/imunologia , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Fatores Imunológicos/metabolismo , Imunoterapia/métodos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Camundongos , Células RAW 264.7 , beta-Ciclodextrinas/farmacologiaRESUMO
To establish a system for assessing drug permeation and irritation of the skin, the permeation of benzoic acid and isosorbide dinitrate, which are listed in the Pharmacopoeia, and the chemical irritation were evaluated using skin generated from human induced pluripotent stem cells (iPSCs). Multilayer structures and cellular markers (keratin 14 and 10, which are in basal and suprabasal epidermal layers) were clearly detected in our iPSC-based skin. Transepidermal water loss (TEWL) decreased after iPSC-derived keratinocytes were cultured on collagen gels from human primary fibroblasts. These results indicate that the barrier function was partly increased by formation of the living epidermis. The cumulative amount of benzoic acid and isosorbide dinitrate across human iPSC-based skin gradually increased after an initial lag time. Moreover, the irritancy of various chemicals (non-irritants: ultrapure water, allyl phenoxy-acetate, isopropanol, and hexyl salicylate and irritants: 5% sodium dodecyl sulfate (SDS), heptanal, potassium hydroxide (5% aq.) and cyclamen aldehyde) to iPSC-based skin was almost met the irritation criteria of the Organisation for Economic Co-operation and Development (OECD) guideline. The results of our iPSC-based skin evaluation provide useful basic information for developing an assessment system to predict the permeation and safety of new transdermal drugs in human skin.
Assuntos
Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Irritantes/metabolismo , Absorção Cutânea/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Administração Cutânea , Animais , Células Cultivadas , Prepúcio do Pênis/citologia , Prepúcio do Pênis/efeitos dos fármacos , Prepúcio do Pênis/metabolismo , Humanos , Recém-Nascido , Irritantes/administração & dosagem , Masculino , Ratos Wistar , Pele/citologia , Absorção Cutânea/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) have a tumor-homing ability-they accumulate inside tumors after systemic injection, and may thus be useful as carriers for tumor-targeting therapy. To use MSCs effectively as an anti-cancer therapy, they must first be functionalized with a large amount of anti-cancer drugs without causing any significant changes to their tumor-tropism. In the present study, we attempted to modify the cell surface of MSCs with doxorubicin-loaded liposomes (DOX-Lips), using the avidin-biotin complex method, and evaluated delivery efficiency and anti-tumor efficacy of DOX-Lip-modified MSCs. The amount of DOX in DOX-Lip-modified C3H10T1/2 cells, a murine mesenchymal stem cell line, was approximately 21.5 pg per cell, with no significant changes to the tumor-tropism of C3H10T1/2 cells. Notably, DOX-Lip-modified C3H10T1/2 cells significantly suppressed the proliferation of firefly luciferase-expressing murine colon adenocarcinoma colon26/fluc cells, compared to DOX-Lips alone. Fluorescent DOX accumulated at the cell contact surface and inside green fluorescence protein-expressing colon26 (colon26/GFP) in co-cultures of DOX-Lip-modified C3H10T1/2 and colon26/GFP cells. This localized distribution was not observed when only DOX-Lips was added to colon26/GFP cells. These results suggest that DOX-Lips are efficiently delivered from DOX-Lip-modified C3H10T1/2 cells to the neighboring colon26 cells. Furthermore, DOX-Lip-modified C3H10T1/2 cells suppressed tumor growth in subcutaneous tumor-bearing mice, and in a lung metastasis mouse model. Taken together, these results indicate that the intercellular delivery of DOX may be enhanced using DOX-Lip-modified MSCs as an efficient carrier system for targeted tumor therapy.
Assuntos
Antineoplásicos , Neoplasias do Colo , Neoplasias Pulmonares , Células-Tronco Mesenquimais , Animais , Antineoplásicos/uso terapêutico , Avidina/uso terapêutico , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Doxorrubicina/uso terapêutico , CamundongosRESUMO
PURPOSE: We have previously reported that Capryol 90 improves the intestinal absorption of insulin, a peptide drug, without causing serious damage to the intestinal epithelium. However, the effects of Capryol 90 and its related formulations on the intestinal absorption of other drugs, and their absorption-enhancing mechanisms are still unclear. The aim of this study is to evaluate the effects of Capryol 90 and its related formulations on the intestinal absorption of drugs and elucidate their absorption-enhancing mechanisms. METHODS: The intestinal absorption of 5(6)-carboxyfluorescein, fluorescein isothiocyanate-dextrans, and alendronate was evaluated using an in situ closed loop method. Brush border membrane vesicles (BBMVs) were labeled with fluorescent probes, and the fluidity of membrane was evaluated by a fluorescence depolarization method. The expression levels of tight junction (TJ) proteins were measured using a Western blot method and immunofluorescence staining. RESULTS: Among the tested excipients, Capryol 90 significantly improved the small and large intestinal absorption of drugs. In mechanistic studies, Capryol 90 increased the membrane fluidity of lipid bilayers in BBMVs. Additionally, Capryol 90 decreased the expression levels of TJ-associated proteins, namely claudin-4, occludin, and ZO-1. CONCLUSIONS: Capryol 90 is an effective absorption enhancer for improving the intestinal absorption of poorly absorbed drugs via both transcellular and paracellular pathways.
Assuntos
Alendronato/metabolismo , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Polímeros/farmacologia , Propilenoglicóis/farmacologia , Animais , Células CACO-2 , Claudina-4/metabolismo , Dextranos/metabolismo , Impedância Elétrica , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceínas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Masculino , Fluidez de Membrana/efeitos dos fármacos , Ocludina/metabolismo , Ratos Wistar , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Bone-drug targeting therapies using nanoparticles based on targeting ligands remain challenging due to their uptake clearance at non-target sites such as the liver, kidney, and spleen. Furthermore, the distribution sites of nanoparticles in bones have not been fully investigated, thus halting the development of more effective bone metastasis treatment strategies. In this study, we developed nanoparticles self-assembled from cholesterol-terminated, polyethylene glycol-conjugated, aspartic acid (Asp)-modified polyamidoamine dendrimer (Asp-PAMAM-Micelles) with targeting to active bone turnover sites associated with bone metastasis pathogenesis. On analysis through whole-body single photon emission computed tomography/computed tomography (SPECT/CT) imaging, 111In-Asp-PAMAM-Micelles showed high specificity to active bone turnover sites (especially the joints in the lower limbs, shoulder, and pelvis) after intravenous injection in mice. The lower limb bone uptake clearance for 111In-Asp-PAMAM-Micelles encapsulating paclitaxel (PTX) was 3.5-fold higher than that for 111In-unmodified PAMAM-Micelles (PTX). 3H-PTX encapsulated Asp-PAMAM-Micelles effectively accumulated in the lower limb bones in a similar manner as the 111In-Asp-PAMAM-Micelles (PTX). In a bone metastatic tumor mouse model, the tumor growth in the lower limb bones was significantly inhibited by injection of Asp-PAMAM-Micelles (PTX) compared to unmodified PAMAM-Micelles (PTX). Our results demonstrate that Asp-PAMAM-Micelles are sophisticated drug delivery systems for highly potent targeting to active bone turnover sites.