RESUMO
The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives (IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound (IP-002H-Rho) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R+) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002x-Rho association with A431-CCK2-R+ cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002M-Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002G-Rho and IP-002L-Rho, respectively. Unexpectedly, IP-002H-Rho showed a similar cell association to that of IP-002L-Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R+ cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho, respectively, proving IP-002M-Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R+ cells incubated with IP-002M-Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002M-Rho by A431-CCK2-R+ cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.
Assuntos
Receptor de Colecistocinina B , Humanos , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/metabolismo , Linhagem Celular Tumoral , Trissacarídeos/química , Lactose/análogos & derivados , Lactose/química , Glucose/metabolismoRESUMO
Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium responsible for severe nosocomial infections and is considered a critical pulmonary pathogen for both immunocompromised and cystic fibrosis patients. Planktonic cells of P. aeruginosa possess intrinsic and acquired resistances, inactivating several classes of conventional antibiotics. Additionally, this bacterium can grow, forming biofilms, and complex structures, further hampering the action of multiple antibiotics. Here, we report the biological properties of D-Q53 CecB, an all-D enantiomer of the silkworm natural peptide Q53 CecB. Compared to the L-variant, D-Q53 CecB was resistant to in vitro degradation by humans and P. aeruginosa elastases and showed an enhanced bactericidal activity against P. aeruginosa planktonic bacteria. D-Q53 CecB was thermostable and maintained its antimicrobial activity at high salt concentrations and in the presence of divalent cations or fetal-bovine serum, although at reduced levels. Against different types of human cells, D-Q53 CecB showed cytotoxic phenomena at concentrations several folds higher compared to those active against P. aeruginosa. When L- and D-Q53 CecB were compared for their antibiofilm properties, both peptides were active in inhibiting biofilm formation. However, the D-enantiomer was extremely effective in inducing biofilm degradation, suggesting this peptide as a favorable candidate in an anti-Pseudomonas therapy.
Assuntos
Cecropinas , Infecções por Pseudomonas , Animais , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Bombyx , Cecropinas/farmacologia , Cecropinas/uso terapêutico , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologiaRESUMO
The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer-CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.
Assuntos
Receptor de Manose , Lectinas de Ligação a Manose , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Lectinas/química , Macrófagos/metabolismo , Lectinas Tipo C/metabolismo , Manose/químicaRESUMO
Immunotherapy is deemed one of the most powerful therapeutic approaches to treat cancer. However, limited response and tumor specificity are still major challenges to address. Herein, mannosylated polycations targeting mannose receptor- are developed as vectors for plasmid DNA (pDNA)-based vaccines to improve selective delivery of genetic material to antigen-presenting cells and enhance immune cell activation. Three diblock glycopolycations (M15A12, M29A25, and M58A45) and two triblock copolymers (M29A29B9 and M62A52B32) are generated by using mannose (M), agmatine (A), and butyl (B) derivatives to target CD206, complex nucleic acids, and favor the endosomal escape, respectively. All glycopolycations efficiently complex pDNA at N/P ratios <5, protecting the pDNA from degradation in a physiological milieu. M58A45 and M62A52B32 complexed with plasmid encoding for antigenic ovalbumin (pOVA) trigger the immune activation of cultured dendritic cells, which present the SIINFEKL antigenic peptide via specific major histocompatibility complex-I. Importantly, administration of M58A45/pOVA elicits SIINFEKL-specific T-cell response in C56BL/6 mice bearing the melanoma tumor model B16-OVA, well in line with a reduction in tumor growth. These results qualify mannosylation as an efficient strategy to target immune cells in cancer vaccination and emphasize the potential of these glycopolycations as effective delivery vehicles for nucleic acids.
Assuntos
Vacinas Anticâncer , Neoplasias , Ácidos Nucleicos , Vacinas , Camundongos , Animais , Células Dendríticas , Ovalbumina , Células Apresentadoras de Antígenos , Ativação Linfocitária , Apresentação de Antígeno , Linfócitos T , Ácidos Nucleicos/metabolismo , Camundongos Endogâmicos C57BL , Vacinas Anticâncer/genética , Vacinas Anticâncer/metabolismo , Neoplasias/terapia , Neoplasias/metabolismoRESUMO
Dendritic cells serve as the main immune cells that trigger the immune response. We developed a simple and cost-effective nanovaccine platform based on the α1',2-mannobiose derivative for dendritic cell targeting. In previous work, we have formulated the α1,2-mannobiose-based nanovaccine platform with plasmid DNA and tested it in cattle against BoHV-1 infection. There, we have shown that the dendritic cell targeting using this nanovaccine platform in vivo can boost the immunogenicity, resulting in a long-lasting immunity. In this work, we aim to characterize the α1',2-mannobiose derivative, which is key in the nanovaccine platform. This DC-targeting strategy takes advantage of the specific receptor known as DC-SIGN and exploits its capacity to bind α1,2-mannobiose that is present at terminal ends of oligosaccharides in certain viruses, bacteria, and other pathogens. The oxidative conjugation of α1',2-mannobiose to NH2-PEG2kDa-DSPE allowed us to preserve the chemical structure of the non-reducing mannose of the disaccharide and the OH groups and the stereochemistry of all carbons of the reducing mannose involved in the binding to DC-SIGN. Here, we show specific targeting to DC-SIGN of decorated micelles incubated with the Raji/DC-SIGN cell line and uptake of targeted liposomes that took place in human, bovine, mouse, and teleost fish DCs in vitro, by flow cytometry. Specific targeting was found in all cultures, demonstrating a species-non-specific avidity for this ligand, which opens up the possibility of using this nanoplatform to develop new vaccines for various species, including humans.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Linfoma/imunologia , Manose/química , Receptores de Superfície Celular/imunologia , Vacinas/imunologia , Animais , Bovinos , Feminino , Peixes , Humanos , Linfoma/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da Espécie , Vacinas/administração & dosagemRESUMO
OBJECTIVES: The aim of this study was to investigate the relationship between the degree of crystal phagocytosis and the magnitude of the local inflammatory process using fresh synovial fluid (SF) collected from patients with crystal-induced arthritis. In parallel, an in vitro model of crystal-induced inflammation was used to assess the effect of cell priming on crystal phagocytosis and IL-1ß production. METHODS: SF was collected from 20 patients with gout and 20 with pyrophosphate crystal-induced arthritis and examined under ordinary and polarised light microscopy for total and differential white blood cell (WBC) count and crystal search. The total phagocytosis index was determined in SF along with IL-1ß, IL-8, IL-10, and TGFß levels. The in vitro studies were performed using primed or unprimed THP-1 cells stimulated with calcium pyrophosphate (CPP) crystals, monosodium urate (MSU) crystals and/or cytochalasin D. RESULTS: We demonstrated that the phagocytosis index calculated on the total number of cells was independent from the inflammatory local indices such as WBC and the percentage of polymorphonuclear cells but showed a positive correlation with pro-inflammatory cytokines. By contrast, the local inflammatory indices (WBC and IL-1ß) showed a strong positive correlation with the percentage of polymorphonuclear cells with crystals internalised and a negative correlation with the percentage of mononuclear cells with crystals internalised. The in vitro study showed that phagocytosis represents a fundamental step in the induction of the inflammatory response to MSU and CPP crystals, but it also occurs in absence of cell priming. CONCLUSIONS: The results of this study indicate a possible role of non-inflammatory phagocytosis in limiting the acute attack of crystal-induced arthritis.
Assuntos
Gota , Líquido Sinovial , Humanos , Inflamação , Fagocitose , Ácido ÚricoRESUMO
The cholecystokinin-2 receptor (CCK-2R) is overexpressed in several human cancers but displays limited expression in normal tissues. For this reason, it is a suitable target for developing specific radiotracers. In this study, a nastorazepide-based ligand functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator (IP-001) was synthesized and labelled with indium-111. The radiolabeling process yielded >95% with a molar activity of 10 MBq/nmol and a radiochemical purity of >98%. Stability studies have shown a remarkable resistance to degradation (>93%) within 120 h of incubation in human blood. The in vitro uptake of [111In]In-IP-001 was assessed for up to 24 h on a high CCK-2R-expressing tumor cell line (A549) showing maximal accumulation after 4 h of incubation. Biodistribution and single photon emission tomography (SPECT)/CT imaging were evaluated on BALB/c nude mice bearing A549 xenograft tumors. Implanted tumors could be clearly visualized after only 4 h post injection (2.36 ± 0.26% ID/cc), although a high amount of radiotracer was also found in the liver, kidneys, and spleen (8.25 ± 2.21%, 6.99 ± 0.97%, and 3.88 ± 0.36% ID/cc, respectively). Clearance was slow by both hepatobiliary and renal excretion. Tumor retention persisted for up to 24 h, with the tumor to organs ratio increasing over-time and ending with a tumor uptake (1.52 ± 0.71% ID/cc) comparable to liver and kidneys.
Assuntos
Benzodiazepinas/química , Radioisótopos de Índio/farmacocinética , Neoplasias Pulmonares/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Radioisótopos de Índio/administração & dosagem , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Compostos Radiofarmacêuticos/administração & dosagem , Receptor de Colecistocinina B/metabolismo , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The colloidal stability, in vitro toxicity, cell association, and in vivo pharmacokinetic behavior of liposomes decorated with monomethoxy-poly(ethylene glycol)-lipids (mPEG-lipids) with different chemical features were comparatively investigated. Structural differences of the mPEG-lipids used in the study included: (a) surface-anchoring moiety [1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), cholesterol (Chol), and cholane (Chln)]; (b) mPEG molecular weight (2 kDa mPEG45 and 5 kDa mPEG114); and (c) mPEG shape (linear and branched PEG). In vitro results demonstrated that branched (mPEG114)2-DSPE confers the highest stealth properties to liposomes (â¼31-fold lower cell association than naked liposomes) with respect to all PEGylating agents tested. However, the pharmacokinetic studies showed that the use of cholesterol as anchoring group yields PEGylated liposomes with longer permeance in the circulation and higher systemic bioavailability among the tested formulations. Liposomes decorated with mPEG114-Chol had 3.2- and â¼2.1-fold higher area under curve (AUC) than naked liposomes and branched (mPEG114)2-DSPE-coated liposomes, respectively, which reflects the high stability of this coating agent. By comparing the PEGylating agents with same size, namely, linear 5 kDa PEG derivatives, linear mPEG114-DSPE yielded coated liposomes with the best in vitro stealth performance. Nevertheless, the in vivo AUC of liposomes decorated with linear mPEG114-DSPE was lower than that obtained with liposomes decorated with linear mPEG114-Chol. Computational molecular dynamics modeling provided additional insights that complement the experimental results.
Assuntos
Colanos/administração & dosagem , Colesterol/administração & dosagem , Portadores de Fármacos/farmacocinética , Fosfatidiletanolaminas/administração & dosagem , Polietilenoglicóis/administração & dosagem , Animais , Disponibilidade Biológica , Colanos/química , Colanos/farmacocinética , Colesterol/química , Colesterol/farmacocinética , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Células HeLa , Humanos , Lipídeos , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Dinâmica Molecular , Peso Molecular , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacocinética , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Propriedades de SuperfícieRESUMO
Liposomes have been one of the most exploited drug delivery systems in recent decades. However, their large-scale production with low batch-to-batch differences is a challenge for industry, which ultimately delays the clinical translation of new products. We have investigated the effects of formulation parameters on the colloidal and biopharmaceutical properties of liposomes generated with a thin-film hydration approach and microfluidic procedure. Dexamethasone hemisuccinate was remotely loaded into liposomes using a calcium acetate gradient. The liposomes produced by microfluidic techniques showed a unilamellar structure, while the liposomes produced by thin-film hydration were multilamellar. Under the same remote loading conditions, a higher loading capacity and efficiency were observed for the liposomes obtained by microfluidics, with low batch-to-batch differences. Both formulations released the drug for almost one month with the liposomes prepared by microfluidics showing a slightly higher drug release in the first two days. This behavior was ascribed to the different structure of the two liposome formulations. In vitro studies showed that both formulations are non-toxic, associate to human Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells, and efficiently reduce inflammation, with the liposomes obtained by the microfluidic technique slightly outperforming. The results demonstrated that the microfluidic technique offers advantages to generate liposomal formulations for drug-controlled release with an enhanced biopharmaceutical profile and with scalability.
Assuntos
Dexametasona/química , Lipossomos/química , Microfluídica/métodos , Acetatos , Compostos de Cálcio , Linhagem Celular , Química Farmacêutica/métodos , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Tamanho da PartículaRESUMO
BACKGROUND: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway. METHODS: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG5kDa-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice. RESULTS: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition. CONCLUSIONS: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.
Assuntos
Cromonas/farmacologia , Glioma/tratamento farmacológico , Glioma/metabolismo , Metabolômica , Animais , Proliferação de Células/efeitos dos fármacos , Glioma/diagnóstico , Glicólise/efeitos dos fármacos , Humanos , Masculino , Camundongos , Neoplasias Experimentais/diagnóstico , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
A novel unconventional supramolecular oligo-cationic structure (Agm6-M-PEG-OCH3) has been synthesized to yield high efficiency therapeutic oligonucleotide (ON) delivery. Agm6-M-PEG-OCH3 was obtained by a multistep protocol that included the conjugation of agmatine (Agm) moieties to maltotriose (M), which was further derivatized with one poly(ethylene glycol) (PEG) chain. Gel electrophoresis analysis showed that the 19 base pairs dsDNA model ON completely associates with Agm6-M-PEG-OCH3 at 3 N/P molar ratio, which is in agreement with the in silico molecular predictions. Isothermal titration calorimetry (ITC) analyses showed that the Agm6-M-PEG-OCH3/ON association occurs through a combination of mechanisms depending on the N/P ratios resulting in different nanostructures. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the Agm6-M-PEG-OCH3/ON polyplexes have rod-shape structure with a mean diameter of 50-75 nm and aspect ratio depending on the N/P ratio. The polyplexes were stable over time in buffer, while a slight size increase was observed in the presence of serum proteins. Cell culture studies showed that neither Agm6-M-PEG-OCH3 nor polyplexes displayed cytotoxic effects. Cellular uptake depended on the cell line and polyplex composition: cellular internalization was higher in the case of MCF-7 and KB cells compared to MC3T3-E1 cells and polyplexes with smaller aspect ratio were taken-up by cells more efficiently than polyplexes with higher aspect ratio. Finally, preliminary studies showed that our novel carrier efficiently delivered ONs into cells providing gene silencing.
Assuntos
Portadores de Fármacos/química , Guanidina/química , Nanoestruturas/química , Oligonucleotídeos/química , Polietilenoglicóis/química , Polímeros/química , Proliferação de Células , Humanos , Nanoestruturas/administração & dosagem , Neoplasias/genética , Neoplasias/terapia , Oligonucleotídeos/administração & dosagem , Polímeros/administração & dosagem , Células Tumorais CultivadasRESUMO
Ligand-mediated targeting and internalization of plasma membrane receptors is central to cellular function. These types of receptors have accordingly been investigated as targets to facilitate entry of diagnostic and therapeutic constructs into cells. However, there remains a need to characterize how receptor targeting agents on nanoparticles interact at surface receptors and whether it is possible to control these interactions via exogenous stimuli. Here, we describe the switchable display of the iron-transporting protein, transferrin (Tf), at the surface of thermoresponsive polymer-coated gold nanoparticles and show that internalization of the coated nanoparticles into target cells changes across temperature ranges over which transferrin is expected to be sterically "hidden" by an extended polymer chain and then "revealed" by polymer chain collapse. The switching process is dependent on the numbers of transferrin molecules and thermoresponsive polymer chains attached and whether the assay temperature is above or below the transition temperatures of the responsive polymers at the nanoparticle surfaces. Significantly, however, the control of internalization is critically reliant on overall nanoparticle colloidal stability while the thermoresponsive component of the surface undergoes conformational change. The data show that the cell entry function of complex and large biomolecule ligands can be modulated by polymer-induced accessibility change but that a simple "hide and reveal" mechanism for ligand display following polymer chain collapse is insufficient to account for nanoparticle uptake and subsequent intracellular trafficking.
Assuntos
Endocitose/efeitos dos fármacos , Substâncias Macromoleculares/química , Nanopartículas Metálicas/química , Polímeros/química , Polímeros/farmacologia , Sítios de Ligação , Entropia , Ouro/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas/química , Espectrofotometria Ultravioleta , Temperatura , Transferrina/químicaRESUMO
Peptide therapeutics have the potential to self-associate, leading to aggregation and fibrillation. Noncovalent PEGylation offers a strategy to improve their physical stability; an understanding of the behavior of the resulting polymer/peptide complexes is, however, required. In this study, we have performed a set of experiments with additional mechanistic insight provided by in silico simulations to characterize the molecular organization of these complexes. We used palmitoylated vasoactive intestinal peptide (VIP-palm) stabilized by methoxy-poly(ethylene glycol)5kDa-cholane (PEG-cholane) as our model system. Homogeneous supramolecular assemblies were found only when complexes of PEG-cholane/VIP-palm exceeded a molar ratio of 2:1; at and above this ratio, the simulations showed minimal exposure of VIP-palm to the solvent. Supramolecular assemblies formed, composed of, on average, 9-11 PEG-cholane/VIP-palm complexes with 2:1 stoichiometry. Our in silico results showed the structural content of the helical conformation in VIP-palm increases when it is complexed with the PEG-cholane molecule; this behavior becomes yet more pronounced when these complexes assemble into larger supramolecular assemblies. Our experimental results support this: the extent to which VIP-palm loses helical structure as a result of thermal denaturation was inversely related to the PEG-cholane:VIP-palm molar ratio. The addition of divalent buffer species and increasing the ionic strength of the solution both accelerate the formation of VIP-palm fibrils, which was partially and fully suppressed by 2 and >4 mol equivalents of PEG-cholane, respectively. We conclude that the relative freedom of the VIP-palm backbone to adopt nonhelical conformations is a key step in the aggregation pathway.
Assuntos
Colanos/química , Ácido Palmítico/química , Polietilenoglicóis/química , Polímeros/química , Peptídeo Intestinal Vasoativo/química , Humanos , Lipoilação , Conformação ProteicaRESUMO
Polymer-protein conjugates can be engineered to self-assemble into discrete and well-defined drug delivery systems, which combine the advantages of receptor targeting and controlled drug release. We designed specific conjugates of the iron-binding and transport protein, transferrin (Tf), to combine the advantages of this serum-stable protein as a targeting agent for cancer cells with self-assembling polymers to act as carriers of cytotoxic drugs. Tf variants were expressed with cysteine residues at sites spanning different regions of the protein surface, and the polymer conjugates grown from these variants were compared with polymer conjugates grown from nonselectively derivatized sites on native Tf. The resulting synthetic biopolymer hybrids were evaluated for self-assembly properties, size and topology, ability to carry an anticancer drug (paclitaxel), and cytotoxicity with and without a drug payload in a representative human colon cancer cell line. The results demonstrated that the engineered Tf variant polymer conjugates formed better-defined self-assembled nanoparticles than the nonselectively derivatized conjugates and showed greater efficacy in paclitaxel delivery. A polymer conjugate grown from a specific Tf variant, S415C was found to be taken up rapidly into cancer cells expressing the Tf-receptor, and, while tolerated well by cells in the absence of drugs, was as cytotoxic as free paclitaxel, when loaded with the drug. Importantly, the S415C conjugate polymer was not the most active variant in Tf-receptor binding, suggesting that the nanoscale self-assembly of the polymer-protein hybrid is also a key factor in delivery efficacy. The data overall suggest new design rules for polymer-biopolymer hybrids and therapeutic delivery systems, which include engineering specific residues for conjugation that mediate nanoscale assembly as well as control of ligand-receptor interactions to target specific cell types.
Assuntos
Nanoconjugados/química , Transferrina/química , Antineoplásicos/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Células HCT116 , Humanos , Células MCF-7 , Nanoconjugados/efeitos adversos , Paclitaxel/administração & dosagemRESUMO
A small library of thermoresponsive amphiphilic copolymers based on polylactide-block-poly((2-(2-methoxyethoxy)ethyl methacrylate)-co-(oligoethylene glycol methacrylate)) (PLA-b-P(DEGMA)-co-(OEGMA)), was synthesised by copper-mediated controlled radical polymerisation (CRP) with increasing ratios of OEGMA : DEGMA. These polymers were combined in two ways to form nanoparticles with controllable thermal transition temperatures as measured by particle aggregation. The first technique involved the blending of two (PLA-b-P(DEGMA)-co-(OEGMA)) polymers together prior to assembling nanoparticles (NPs). The second method involved mixing pre-formed nanoparticles of single (PLA-b-P(DEGMA)-co-(OEGMA)) polymers. The observed critical aggregation temperature Tt did not change in a linear relationship with the ratios of each copolymer either in the nanoparticles blended from different copolymers or in the mixtures of pre-formed nanoparticles. However, where co-polymer mixtures were based on (OEG)9MA ratios within 5-10 mole%, a linear relationship between (OEG)9MA composition in the blends and Tt was obtained. The data suggest that OEGMA-based copolymers are tunable over a wide temperature range given suitable co-monomer content in the linear polymers or nanoparticles. Moreover, the thermal transitions of the nanoparticles were reversible and repeatable, with the cloud point curves being essentially invariant across at least three heating and cooling cycles, and a selected nanoparticle formulation was found to be readily endocytosed in representative cancer cells and fibroblasts.
Assuntos
Materiais Biocompatíveis/química , Nanopartículas/química , Polímeros/química , Temperatura , Materiais Biocompatíveis/toxicidade , Humanos , Células MCF-7 , Polímeros/toxicidadeRESUMO
Triple-negative breast cancer (TNBC) is considered one of the most incurable malignancies due to its clinical characteristics, including high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Therefore, it remains a critical unmet medical need. On the other hand, poor delivery efficiency continues to reduce the efficacy of anti-cancer therapeutics developed against solid tumours using various strategies, such as genetically engineered oncolytic vectors used as nanocarriers. The study was designed to evaluate the anti-tumour efficacy of a novel combinatorial therapy based on oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with an anti-PD-1 (pembrolizumab) and paclitaxel (PTX). Here, we first tested the antineoplastic effect in two-dimensional (2D) and three-dimensional (3D) breast cancer models in MDA-MB-231, MDA-MB-468 and MCF-7 cells. Then, to further evaluate the efficacy of combinatorial therapy, including immunological aspects, we established a three-dimensional (3D) co-culture model based on MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) to create an integrated system that more closely mimics the complexity of the tumour microenvironment and interacts with the immune system. Treatment with OV as a priming agent, followed by pembrolizumab and then paclitaxel, was the most effective in reducing the tumour volume in TNBC co-cultured spheroids. Further, T-cell phenotyping analyses revealed significantly increased infiltration of CD8+, CD4+ T and Tregs cells. Moreover, the observed anti-tumour effects positively correlated with the level of CD4+ T cell infiltrates, suggesting the development of anti-cancer immunity. Our study demonstrated that combining different immunotherapeutic agents (virus, pembrolizumab) with PTX reduced the tumour volume of the TNBC co-cultured spheroids compared to relevant controls. Importantly, sequential administration of the investigational agents (priming with the vector) further enhanced the anti-cancer efficacy in 3D culture over other groups tested. Taken together, these results support further evaluation of the virus in combination with anti-PD-1 and PTX for the treatment of triple-negative breast cancer patients. Importantly, further studies with in vivo models should be conducted to better understand the translational aspects of tested therapy.
Assuntos
Antineoplásicos , Terapia Viral Oncolítica , Paclitaxel , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/terapia , Adenoviridae , Paclitaxel/farmacologia , Paclitaxel/toxicidade , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Técnicas de Cultura de Células em Três Dimensões , Leucócitos Mononucleares/imunologia , Terapia Combinada , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Morte Celular , Microambiente Tumoral , Inibidores de Checkpoint Imunológico/farmacologia , Antineoplásicos Imunológicos/farmacologia , Humanos , Linfócitos T/imunologiaRESUMO
The aim of the present study consists of the evaluation of the biodistribution of a novel 68Ga-labeled radiopharmaceutical, [68Ga]Ga-NODAGA-Z360, injected into Balb/c nude mice through histopathological analysis on bioptic samples and radiomics analysis of positron emission tomography/computed tomography (PET/CT) images. The 68Ga-labeled radiopharmaceutical was designed to specifically bind to the cholecystokinin receptor (CCK2R). This receptor, naturally present in healthy tissues such as the stomach, is a biomarker for numerous tumors when overexpressed. In this experiment, Balb/c nude mice were xenografted with a human epidermoid carcinoma A431 cell line (A431 WT) and overexpressing CCK2R (A431 CCK2R+), while controls received a wild-type cell line. PET images were processed, segmented after atlas-based co-registration and, consequently, 112 radiomics features were extracted for each investigated organ / tissue. To confirm the histopathology at the tissue level and correlate it with the degree of PET uptake, the studies were supported by digital pathology. As a result of the analyses, the differences in radiomics features in different body districts confirmed the correct targeting of the radiopharmaceutical. In preclinical imaging, the methodology confirms the importance of a decision-support system based on artificial intelligence algorithms for the assessment of radiopharmaceutical biodistribution.
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Despite substantial improvements in breast cancer (BC) treatment there is still an urgent need to find alternative treatment options to improve the outcomes for patients with advanced-stage disease. Photodynamic therapy (PDT) is gaining a lot of attention as a BC therapeutic option because of its selectivity and low off-target effects. However, the hydrophobicity of photosensitizers (PSs) impairs their solubility and limits the circulation in the bloodstream, thus representing a major challenge. The use of polymeric nanoparticles (NPs) to encapsulate the PS may represent a valuable strategy to overcome these issues. Herein, we developed a novel biomimetic PDT nanoplatform (NPs) based on a polymeric core of poly(lactic-co-glycolic)acid (PLGA) loaded with the PS meso-tetraphenylchlorin disulfonate (TPCS2a). TPCS2a@NPs of 98.89 ± 18.56 nm with an encapsulation efficiency percentage (EE%) of 81.9 ± 7.92% were obtained and coated with mesenchymal stem cells-derived plasma membranes (mMSCs) (mMSC-TPCS2a@NPs, size of 139.31 ± 12.94 nm). The mMSC coating armed NPs with biomimetic features to impart long circulation times and tumor-homing capabilities. In vitro, biomimetic mMSC-TPCS2a@NPs showed a decrease in macrophage uptake of 54% to 70%, depending on the conditions applied, as compared to uncoated TPCS2a@NPs. Both NP formulations efficiently accumulated in MCF7 and MDA-MB-231 BC cells, while the uptake was significantly lower in normal breast epithelial MCF10A cells with respect to tumor cells. Moreover, encapsulation of TPCS2a in mMSC-TPCS2a@NPs effectively prevents its aggregation, ensuring efficient singlet oxygen (1O2) production after red light irradiation, which resulted in a considerable in vitro anticancer effect in both BC cell monolayers (IC50 < 0.15 µM) and three-dimensional spheroids.
RESUMO
Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.