RESUMO
A range of novel BODIPY derivatives with a tripodal aromatic core was synthesized and characterized spectroscopically. These new fluorophores showed promising features as probes for inâ vitro assays in live cells and offer strategic routes for further functionalization towards hybrid nanomaterials. Incorporation of biotin tags facilitated proof-of-concept access to targeted bioconjugates as molecular probes. Computational explorations using DFT and TD-DFT calculations identified the most stable tripodal linker conformations and predicted their absorption and emission behavior. The uptake and speciation of these molecules in living prostate cancer cells was imaged by single- and two-photon excitation techniques coupled with two-photon fluorescence lifetime imaging (2P FLIM).
RESUMO
5-Aminolevulinic acid-based photodynamic therapy (ALA-PDT) was first implemented over three decades ago and has since been mainly part of clinical practice for the management of pre-cancerous and cancerous skin lesions. Photodynamic therapy relies on the combination of a photosensitizer, light and oxygen to cause photo-oxidative damage of cellular components. 5-Aminolevulinic acid (ALA) is a natural precursor of the heme biosynthetic pathway, which when exogenously administered leads to the accumulation of the photoactivatable protoporphyrin IX. Although, effective and providing excellent cosmetic outcomes, its use has been restricted by the burning, stinging, and prickling sensation associated with treatment, as well as cutaneous adverse reactions that may be induced. Despite intense research in the realm of drug delivery, pain moderation, and light delivery, a novel protocol design using sunlight has led to some of the best results in terms of treatment response and patient satisfaction. Daylight PDT is the protocol of choice for the management of treatment of multiple or confluent actinic keratoses (AK) skin lesions. This review aims to revisit the photophysical, physicochemical and biological characteristics of ALA-PDT, and the underlying mechanisms resulting in daylight PDT efficiency and limitations.
Assuntos
Ceratose Actínica , Fotoquimioterapia , Ácido Aminolevulínico/uso terapêutico , Humanos , Ceratose Actínica/tratamento farmacológico , Fotoquimioterapia/métodos , Luz Solar , Resultado do TratamentoRESUMO
Over the past decade, porphyrin derivatives have emerged as invaluable synthetic building blocks and theranostic kits for the delivery of cellular fluorescence imaging and photodynamic therapy. Tetraphenylporphyrin (TPP), its metal complexes, and related derivatives have been investigated for their use as dyes in histology and as components of multimodal imaging probes. The photophysical properties of porphyrin-metal complexes featuring radiometals have been a focus of our attention for the realization of fluorescence imaging probes coupled with radioimaging capabilities and therapeutic potential having "true" theranostic promise. We report hereby on the synthesis, radiochemistry, structural investigations, and preliminary in vitro and in vivo uptake studies on a range of functionalized porphyrin-based derivatives. In pursuit of developing new porphyrin-based probes for multimodality imaging applications, we report new functionalized neutral, polycationic, and polyanionic porphyrins incorporating nitroimidazole and sulfonamide moieties, which were used as targeting groups to improve the notoriously poor pharmacokinetics of porphyrin tags. The resulting functional metalloporphyrin species were stable under serum challenges and the nitroimidazole and sulfonamide derivatives remained fluorescent, allowing in vitro confocal studies and visualization of the lysosomal uptake in a gallium(III) sulfonamide derivative. The molecular structures of selected porphyrin derivatives were determined by single crystal X-ray diffraction using synchrotron radiation. We also investigated the nature of the emission/excitation behavior of model functional porphyrins using in silico approaches such as TD DFT in simple solvation models. The conjugation of porphyrins with the [7-13] and [7-14] fragments of bombesin was also achieved, to provide targeting of the gastrin releasing peptide receptor (GRPR). Depending on the metal, probe conjugates of relevance for single photon emission computed tomography (SPECT) or positron emission tomography (PET) probes have been designed and tested hereby, using TPP and related functional free base porphyrins as the bifunctional chelator synthetic scaffold and 111In[In] or 68Ga[Ga], respectively, as the central metal ions. Interestingly, for simple porphyrin conjugates good radiochemical incorporation was obtained for both radiometals, but the presence of peptides significantly diminished the radio-incorporation yields. Although the gallium-68 radiochemistry of the bombesin conjugates did not show radiochemical incorporation suitable for in vivo studies, likely because the presence of the peptide changed the behavior of the TPP-NH2 synthon taken alone, the optical imaging assays indicated that the conjugated peptide tags do mediate uptake of the porphyrin units into cells.
Assuntos
Metaloporfirinas/química , Radioisótopos/química , Ânions , Cátions , Linhagem Celular Tumoral , Teoria da Densidade Funcional , Humanos , Estrutura Molecular , Estudo de Prova de Conceito , Análise Espectral/métodosRESUMO
Iron is an essential nutrient for virtually all microbes and limiting the concentration of available iron is a potential strategy to be used as an alternative to antibiotic treatment. In this study we analysed the antimicrobial activity of two chelators, specifically 3-hydroxy-1,2-dimethyl-4(1H)-pyridone (deferiprone, DFP), which is clinically approved for the treatment of iron overload disorders, and its 1,2-diethyl homologue, CP94. Both compounds showed moderate activity towards planktonically growing P. aeruginosa cells, and the mechanism of action of these chelators was indeed by limiting the amount of free iron. Surprisingly, the compounds behaved very differently when the cells were grown in biofilms. DFP also showed inhibitory effects on biofilm formation but in contrast, CP94 stimulated this process, in particular at high concentrations. We hypothesised that CP94 behaves as an iron carrier, which was confirmed by our observation that it had antimicrobial synergy with the toxic metals, gallium and copper. This suggests that P. aeruginosa produces a biofilm-specific transport protein that recognises CP94 but not the closely related compound DFP.
Assuntos
Antibacterianos/farmacologia , Quelantes de Ferro/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Piridonas/farmacologia , Biofilmes/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
Heme plays a vital role in cell biology and dysregulation of heme levels is implicated in a wide range of diseases. However, monitoring heme levels in biological systems is currently not straightforward. A short synthetic peptide probe containing 7-azatryptophan is shown to bind hemin in vitro with quenching of the azatryptophan fluorescence. This chemical tool can be used to detect the change in free heme induced in human skin cells upon exposure to UVA irradiation.
Assuntos
Corantes Fluorescentes/química , Heme/análise , Peptídeos/química , Linhagem Celular , Corantes Fluorescentes/síntese química , Humanos , Conformação Molecular , Peptídeos/síntese química , Espectrometria de Fluorescência , Raios UltravioletaRESUMO
The administration of 5-aminolevulinic acid (ALA) to generate enhanced intracellular levels of endogenous porphyrins is currently one of the most important approaches for photodynamic therapy (PDT) and photodiagnosis (PDD). Despite the great promise of ALA-based PDT, the physicochemical behaviour and chemical reactivity of ALA are problematic, and a variety of chemical approaches have been brought to bear to improve cellular delivery, enhance porphyrin production, and generate ALA prodrugs that have appropriate stability for convenient clinical use, as well as selectivity for cancerous tissues. While there has been considerable success, there are still a number of challenges to be addressed and opportunities to be exploited through application of chemical insight in this area.
Assuntos
Ácido Aminolevulínico/farmacologia , Antineoplásicos/farmacologia , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Ácido Aminolevulínico/administração & dosagem , Ácido Aminolevulínico/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Humanos , Neoplasias/patologia , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/químicaRESUMO
The interaction of Tat-conjugated PEGylated CdSe/ZnS quantum dots (QD) with the amphiphilic disulfonated aluminium phthalocyanine photosensitiser is investigated in aqueous solution and in a human breast cancer cell line. In aqueous solution, the QDs and phthalocyanine form stable nanocomposites. Using steady-state and time-resolved fluorescence measurements combined with singlet oxygen detection, efficient Förster resonance energy transfer (FRET) is observed with the QDs acting as donors, and the phthalocyanine photosensitiser, which mediates production of singlet oxygen, as acceptors. In cells, the Tat-conjugated QDs localise in lysosomes and the QD fluorescence lifetimes are close to values observed in aqueous solution. Strong FRET-induced quenching of the QD lifetime is observed in cells incubated with the nanocomposites using fluorescence lifetime imaging microscopy (FLIM). Using excitation of the QDs at wavelengths where phthalocyanine absorption is negligible, FRET-induced release of QDs from endo/lysosomes is confirmed using confocal imaging and FLIM, which is attributed to photooxidative damage to the endo/lysosomal membranes mediated by the phthalocyanine acceptor.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Indóis/farmacologia , Espaço Intracelular/metabolismo , Nanopartículas/química , Imagem Óptica/métodos , Fármacos Fotossensibilizantes/farmacologia , Pontos Quânticos/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Isoindóis , Células MCF-7 , Microscopia de FluorescênciaRESUMO
The chitinase-like proteins YKL-39 (chitinase 3-like-2) and YKL-40 (chitinase 3-like-1) are highly expressed in a number of human cells independent of their origin (mesenchymal, epithelial or haemapoietic). Elevated serum levels of YKL-40 have been associated with a negative outcome in a number of diseases ranging from cancer to inflammation and asthma. YKL-39 expression has been associated with osteoarthritis. However, despite the reported association with disease, the physiological or pathological role of these proteins is still very poorly understood. Although YKL-39 is homologous to the two family 18 chitinases in the human genome, it has been reported to lack any chitinase activity. In the present study, we show that human YKL-39 possesses a chitinase-like fold, but lacks key active-site residues required for catalysis. A glycan screen identified oligomers of N-acetylglucosamine as preferred binding partners. YKL-39 binds chitooligosaccharides and a newly synthesized derivative of the bisdionin chitinase-inhibitor class with micromolar affinity, through a number of conserved tryptophan residues. Strikingly, the chitinase activity of YKL-39 was recovered by reverting two non-conservative substitutions in the active site to those found in the active enzymes, suggesting that YKL-39 is a pseudo-chitinase with retention of chitinase-like ligand-binding properties.
Assuntos
Adipocinas/química , Adipocinas/metabolismo , Lectinas/química , Lectinas/metabolismo , Acetilglucosamina/metabolismo , Adipocinas/antagonistas & inibidores , Adipocinas/genética , Substituição de Aminoácidos , Domínio Catalítico , Quitina/metabolismo , Proteína 1 Semelhante à Quitinase-3 , Quitinases/química , Quitinases/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Lectinas/antagonistas & inibidores , Lectinas/genética , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Mutação Puntual , Conformação Proteica , Dobramento de Proteína , Triptofano/metabolismoRESUMO
Given that cell-penetrating peptides (CPP) are cationic and often amphipathic, similar to membrane-active antimicrobial peptides, it may be possible to use CPP conjugation to improve the delivery of photosensitisers for antimicrobial photodynamic therapy (antimicrobial PDT). We investigated the possibility of using a Tat peptide to deliver the photosensitiser, tetrakis(phenyl)porphyrin (TPP) and kill bacteria. The Tat peptide is a positively-charged mammalian cell-penetrating peptide with potent antimicrobial activity but no haemolytic activity. Fluorescence spectroscopy revealed that the bioconjugate can bind to and/or be incorporated into all bacterial species tested. All species were susceptible to the Tat-porphyrin, with the bactericidal effect being dependent on both the concentration and the light dose. Using the highest light dose, treatment with the Tat-porphyrin achieved reductions of 6.6 log(10) and 6.37 log(10) in the viable counts of Staphylococcus aureus and Streptococcus pyogenes, and reductions of 5.74 log(10) and 6.6 log(10) in the viable counts of Pseudomonas aeruginosa and Escherichia coli. Moreover, the Tat moiety appears to confer antimicrobial properties to the conjugate, particularly for the Gram positive strains, based on the observation of dark toxicity using 1 µM of Tat-porphyrin. Finally, the conjugate induced membrane destabilization by synergistic action of the peptide and PDT, resulting in carboxyfluorescein leakage from bacterial membrane-mimicking liposomes. These findings demonstrate that the use of CPP to deliver a photosensitiser is an effective way of improving the uptake and the treatment efficacy of antimicrobial PDT.
Assuntos
Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Peptídeos/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/farmacologia , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/síntese química , Porfirinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Espectrometria de Fluorescência , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pyogenes/efeitos dos fármacosRESUMO
Epithionitriles represent a previously unrecognized class of cancer chemopreventive phytochemical generated from alkenyl glucosinolates in cruciferous vegetables. In rat liver RL-34 epithelial cells, 1-cyano-2,3-epithiopropane (CETP), 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent) were shown to induce cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione (GSH) S-transferase A3 and the glutamate-cysteine ligase modifier subunit; CETP was more potent in this regard than were either CETB or CETPent, with 50 microM CETP eliciting a remarkable approximately 10-fold induction of NQO1. Furthermore, 50 microM CETP stimulated a 2.0-fold overproduction of GSH in RL-34 cells. Transfection experiments demonstrated that epithionitriles induced gene expression through an antioxidant response element (ARE) and that transactivation of an Nqo1-luciferase reporter plasmid was dependent on NF-E2 p45-related factor 2 (Nrf2), a cap'n'collar basic region leucine zipper transcription factor. Evidence is presented that CETP affected Nrf2-mediated induction of ARE-driven transcription by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase substrate adaptor that negatively regulates Nrf2. We found that Nqo1 was expressed constitutively at high levels in Keap1(-/-) mouse embryonic fibroblasts (MEFs) and it was not further induced by CETP. However, knock-in of mouse Keap1 or zebrafish Keap1a into Keap1(-/-) MEFs repressed Nqo1-luciferase reporter gene activity, but repression by the murine or zebrafish proteins was antagonized by CETP. Pre-treatment of Nrf2(+/+) MEFs, but not Nrf2(-/-) MEFs, with 15 microM CETP for 24 h conferred 2.4-fold resistance against subsequent exposure to the alpha,beta-unsaturated aldehyde acrolein, indicating that the phytochemical exerts chemopreventive properties against genotoxic xenobiotics.
Assuntos
Acroleína/antagonistas & inibidores , Nitrilas/farmacologia , Extratos Vegetais/farmacologia , Propano/análogos & derivados , Compostos de Sulfidrila/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Glucosinolatos/metabolismo , Glutationa/metabolismo , Fígado/citologia , Fígado/fisiologia , Luciferases/genética , Camundongos , Nitrilas/metabolismo , Propano/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RatosRESUMO
Sulforaphane can stimulate cellular adaptation to redox stressors through transcription factor Nrf2. Using mouse embryonic fibroblasts (MEFs) as a model, we show herein that the normal homeostatic level of glutathione in Nrf2(-/-) MEFs was only 20% of that in their wild-type counterparts. Furthermore, the rate of glutathione synthesis following its acute depletion upon treatment with 3 micromol/l sulforaphane was very substantially lower in Nrf2(-/-) MEFs than in wild-type cells, and the rebound leading to a approximately 1.9-fold increase in glutathione that occurred 12-24 h after Nrf2(+/+) MEFs were treated with sulforaphane was not observed in Nrf2(-/-) fibroblasts. Wild-type MEFs that had been pre-treated for 24 h with 3 micromol/l sulforaphane exhibited between 1.4- and 3.2-fold resistance against thiol-reactive electrophiles, including isothiocyanates, alpha,beta-unsaturated carbonyl compounds (e.g. acrolein), aryl halides and alkene epoxides. Pre-treatment of Nrf2(+/+) MEFs with sulforaphane also protected against hydroperoxides (e.g. cumene hydroperoxide, CuOOH), free radical-generating compounds (e.g. menadione), and genotoxic electrophiles (e.g. chlorambucil). By contrast, Nrf2(-/-) MEFs were typically approximately 50% less tolerant of these agents than wild-type fibroblasts, and sulforaphane pre-treatment did not protect the mutant cells against xenobiotics. To test whether Nrf2-mediated up-regulation of glutathione represents the major cytoprotective mechanism stimulated by sulforaphane, 5 micromol/l buthionine sulfoximine (BSO) was used to inhibit glutathione synthesis. In Nrf2(+/+) MEFs pre-treated with sulforaphane, BSO diminished intrinsic resistance and abolished inducible resistance to acrolein, CuOOH and chlorambucil, but not menadione. Thus Nrf2-dependent up-regulation of GSH is the principal mechanism by which sulforaphane pre-treatment induced resistance to acrolein, CuOOH and chlorambucil, but not menadione.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/fisiologia , Fibroblastos/fisiologia , Fator 2 Relacionado a NF-E2/fisiologia , Peróxidos/toxicidade , Tiocianatos/toxicidade , Animais , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Radicais Livres/toxicidade , Glutationa/metabolismo , Isotiocianatos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Oxirredução/efeitos dos fármacos , Sulfóxidos , Xenobióticos/toxicidadeRESUMO
Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides, and antiasthmatics. Argifin, a natural product cyclopentapeptide, competitively inhibits family 18 chitinases in the nanomolar to micromolar range and shows extensive substrate mimicry. In an attempt to map the active fragments of this large natural product, the cyclopentapeptide was progressively dissected down to four linear peptides and dimethylguanylurea, synthesized using a combination of solution and solid phase peptide synthesis. The peptide fragments inhibit chitinase B1 from Aspergillus fumigatus (AfChiB1), the human chitotriosidase, and chitinase activity in lung homogenates from a murine model of chronic asthma, with potencies ranging from high nanomolar to high micromolar inhibition. X-ray crystallographic analysis of the chitinase-inhibitor complexes revealed that the conformations of the linear peptides were remarkably similar to that of the natural product. Strikingly, the dimethylguanylurea fragment, representing only a quarter of the natural product mass, was found to harbor all significant interactions with the protein and binds with unusually high efficiency. The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.
Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Quitinases/antagonistas & inibidores , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Arginina/análogos & derivados , Arginina/metabolismo , Arginina/farmacologia , Aspergillus fumigatus/enzimologia , Produtos Biológicos/síntese química , Produtos Biológicos/metabolismo , Quitinases/química , Quitinases/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/metabolismo , Relação Estrutura-Atividade , Ureia/análogos & derivados , Ureia/metabolismo , Ureia/farmacologiaRESUMO
A new, highly efficient, all-solid-phase synthesis of argifin, a natural product cyclic pentapeptide chitinase inhibitor, is reported. The synthesis features attachment of an orthogonally protected Asp residue to the solid support and assembly of the linear peptide chain by Fmoc SPPS prior to cyclisation and side-chain manipulation on-resin. Introduction of the key N-methyl carbamoyl-substituted Arg side chain is achieved via derivatisation of a selectively protected Orn residue, prior to cleavage from the resin and side-chain deprotection. A severe aspartimide side-reaction observed upon final deprotection is circumvented by the use of a novel aqueous acidolysis procedure. The flexibility of the synthesis is demonstrated by the preparation of a series of argifin analogues designed from the X-ray structure of the natural product in complex with a representative family 18 chitinase.
Assuntos
Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Peptídeos Cíclicos/síntese química , Cristalografia por Raios X , Ciclização , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (ALA) has been widely used in photodynamic therapy for a range of malignant and nonmalignant lesions. However, ALA is relatively hydrophilic and lacks stability at physiologic pH, limiting its bioavailability. We have investigated more lipophilic, uncharged ALA-peptide prodrugs based on phenylalanyl-ALA conjugates, which are water soluble and chemically stable for improving ALA delivery. Pharmacokinetics of the induced protoporphyrin IX (PpIX) were studied in transformed PAM212 keratinocyte cells and pig skin explants. The intracellular porphyrin production was substantially increased with Ac-L-Phe-ALA-Me (compound 1) and Ac-L-Phe-ALA (compound 3) compared with equimolar ALA: after 6-h incubation, the PpIX fluorescence measured using 0.01 mmol/L of compound 1 was enhanced by a factor of 5 compared with ALA. Phototoxicity results showed good correlation with PpIX levels, giving a LD(50) (2.5 J/cm(2)) of 25 micromol/L for ALA, 6 micromol/L for 5-aminolaevulinic hexyl ester, and 2.6 micromol/L for compound 1, which exhibited the highest phototoxicity. However, these results were stereospecific because the corresponding D-enantiomer, Ac-D-Phe-ALA-Me (compound 2), induced neither porphyrin synthesis nor phototoxicity. PpIX levels were considerably reduced when cells were incubated with compound 1 at low temperatures, consistent with active transport. In pig skin explants, compound 1 induced higher porphyrin fluorescence than ALA by a factor of 3. These results show that water-soluble peptide prodrugs of ALA can greatly increase its cellular uptake, generating more intracellular PpIX and improved tumor cell photosensitization. The derivatives are comparable in efficacy with 5-aminolaevulinic hexyl ester but less toxic and more stable at physiologic pH.
Assuntos
Ácido Aminolevulínico/farmacologia , Peptídeos/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Pró-Fármacos/farmacologia , Ácido Aminolevulínico/química , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Endocitose/efeitos dos fármacos , Esterases/metabolismo , Feminino , Fluoresceína/metabolismo , Fluorescência , Técnicas In Vitro , Cinética , Camundongos , Peptídeos/química , Protoporfirinas/metabolismo , Pele/efeitos dos fármacos , Pele/patologia , SuínosRESUMO
The development of a reliable sensitive method for the HPLC determination of 5-aminolaevulinic acid (ALA) and ALA esters in cell lysates is described. The method relies on the quantification of a fluorescent derivative of ALA following its derivatisation with acetylacetone and formaldehyde. Following this procedure it is possible to quantify ALA in cell lysates with no need for pre-purification of the sample. The method has been validated in two ranges of concentration (0.6-65 microM, 0.1-10 microg/mL, and 30-600 microM, 5-100 microg/mL), and has also been extended and validated for the determination of ALA released from ALA prodrugs after acidic hydrolysis.
Assuntos
Ácido Aminolevulínico/análise , Cromatografia Líquida de Alta Pressão/métodos , Queratinócitos/química , Ácido Aminolevulínico/metabolismo , Análise de Variância , Animais , Linhagem Celular , Esterificação , Ésteres/análise , Queratinócitos/metabolismo , Modelos Lineares , Camundongos , Pró-Fármacos/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). While TNF-α/IFN-γ treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes.
Assuntos
Mucosa Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Células CACO-2 , Claudina-2/genética , Claudina-2/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Cadeias Leves de Miosina/metabolismo , Permeabilidade/efeitos dos fármacos , Ratos , Junções Íntimas/metabolismoRESUMO
Tight junction (TJ) structures restrict the movement of solutes between adjacent epithelial cells to maintain homeostatic conditions. A peptide, termed PIP 640, with the capacity to regulate the transient opening of intestinal TJ structures through an endogenous mechanism involving the induction of myosin light chain (MLC) phosphorylation at serine 19 (MLC-pS19) has provided a promising new method to enhance the in vivo oral bioavailability of peptide therapeutics. PIP 640 is a decapeptide composed of all D-amino acids (rrdykvevrr-NH2) that contains a central sequence designed to emulates a specific domain of C-kinase potentiated protein phosphatase-1 inhibitor-17â¯kDa (CPI-17) surrounded by positively-charged amino acids that provide a cell penetrating peptide (CPP)-like character. Here, we examine compositional requirements of PIP 640 with regard to its actions on MLC phosphorylation, its intracellular localization to TJ structures, and its interactions with MLC phosphatase (MLCP) elements that correlate with enhanced solute uptake. These studies showed that a glutamic acid and tyrosine within this peptide are critical for PIP 640 to retain its ability to increase MLC-pS19 levels and enhance the permeability of macromolecular solutes of the size range of therapeutic peptides without detectable cytotoxicity. On the other hand, exchange of the aspartic acid for alanine and then arginine resulted in an increasingly greater bias toward protein phosphatase-1 (PP1) relative to MLCP inhibition, an outcome that resulted in increased paracellular permeability for solutes in the size range of therapeutic peptides, but with a significant increase in cytotoxicity. Together, these data further our understanding of the composition requirements of PIP 640 with respect to the desired goal of transiently altering the intestinal epithelial cell paracellular barrier properties through an endogenous mechanism, providing a novel approach to enhance the oral bioavailability of poorly absorbed therapeutic agents of < ~ 5â¯kDa.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Mucosa Intestinal/metabolismo , Cadeias Leves de Miosina/metabolismo , Administração Oral , Aminoácidos/química , Animais , Disponibilidade Biológica , Células CACO-2 , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacocinética , Humanos , Masculino , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Permeabilidade , Fosforilação/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Endosomal entrapment is a key issue for the intracellular delivery of many nano-sized biotherapeutics to their cytosolic or nuclear targets. Photochemical internalisation (PCI) is a novel light-based solution that can be used to trigger the endosomal escape of a range of bioactive agents into the cytosol leading to improved efficacy in pre-clinical and clinical studies. PCI typically depends upon the endolysosomal colocalisation of the bioactive agent with a suitable photosensitiser that is administered separately. In this study we demonstrate that both these components may be combined for codelivery via a novel multifunctional liposomal nanocarrier, with a corresponding increase in the biological efficacy of the encapsulated agent. As proof of concept, we show here that the cytotoxicity of the 30 kDa protein toxin, saporin, in MC28 fibrosarcoma cells is significantly enhanced when delivered via a cell penetrating peptide (CPP)-modified liposome, with the CPP additionally functionalised with a photosensitiser that is targeted to endolysosomal membranes. This innovation opens the way for the efficient delivery of a range of biotherapeutics by the PCI approach, incorporating a clinically proven liposome delivery platform and using bioorthogonal ligation chemistries to append photosensitisers and peptides of choice.
Assuntos
Citosol/metabolismo , Citotoxinas/metabolismo , Lipossomos/química , Fármacos Fotossensibilizantes/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Citotoxinas/química , Citotoxinas/farmacologia , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Luz , Lipossomos/síntese química , Maleimidas/química , Microscopia Confocal , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Porfirinas/metabolismo , Ratos , Saporinas/química , Saporinas/metabolismoRESUMO
A major problem with many promising nano-sized biotherapeutics including macromolecules is that owing to their size they are subject to cellular uptake via endocytosis, and become entrapped and then degraded within endolysosomes, which can significantly impair their therapeutic efficacy. Photochemical internalisation (PCI) is a technique for inducing cytosolic release of the entrapped agents that harnesses sub-lethal photodynamic therapy (PDT) using a photosensitiser that localises in endolysosomal membranes. Using light to trigger reactive oxygen species-mediated rupture of the photosensitised endolysosomal membranes, the spatio-temporal selectivity of PCI then enables cytosolic release of the agents at the selected time after administration so that they can reach their intracellular targets. However, conventional photosensitisers used clinically for PDT are ineffective for photochemical internalisation owing to their sub-optimal intracellular localisation. In this work we demonstrate that such a photosensitiser, chlorin e6, can be repurposed for PCI by conjugating the chlorin to a cell penetrating peptide, using bioorthogonal ligation chemistry. The peptide conjugation enables targeting of endosomal membranes so that light-triggered cytosolic release of an entrapped nano-sized cytotoxin can be achieved with consequent improvement in cytotoxicity. The photoproperties of the chlorin moiety are also conserved, with comparable singlet oxygen quantum yields found to the free chlorin.
Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Porfirinas/farmacologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Humanos , Estrutura Molecular , Processos Fotoquímicos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Transporte Proteico , RatosRESUMO
The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein-protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4kDa fluorescent dextran but not 70kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3-4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier.