RESUMEN
In this work, we exploit computational fluid dynamics (CFD) to evaluate stirred tank reactor (STR) process engineer parameters (PEP) and design a scale-down system (SDS) to be representative of the formulation and filling process steps for an Aluminum adjuvanted vaccine drug product (DP). To study the shear history in the SDS we used the concept of number of passages, combined with an appropriate stirring speed down scale strategy comprising of either (i) tip speed equivalence, widely used as a scale-up criterion for a shear-sensitive product, or (ii) rotating shear, a shear metric introduced by Metz and Otto in 1957 but never used as scaling criterion. The outcome of the CFD simulations shows that the tip equivalence generates a worst-case SDS in terms of shear, whereas the rotating shear scaling approach could be used to design a more representative SDS. We monitored the trend over time for "In Vitro Relative Potency" as DP Critical Quality Attribute for both scaling approaches, which highlighted the crucial role of choosing the appropriate scaling-down approach to be representative of the manufacturing scale during process characterization studies.
Asunto(s)
Hidrodinámica , Vacunas , Simulación por Computador , Adyuvantes Inmunológicos/química , Química Farmacéutica/métodos , Tecnología Farmacéutica/métodosRESUMEN
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.
Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Ácidos Nucleicos , Vacunas , Ratones , Animales , Células Dendríticas , Ovalbúmina , Células Presentadoras de Antígenos , Activación de Linfocitos , Presentación de Antígeno , Linfocitos T , Ácidos Nucleicos/metabolismo , Ratones Endogámicos C57BL , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/metabolismo , Neoplasias/terapia , Neoplasias/metabolismoRESUMEN
Carbohydrate-based materials are increasingly investigated for a range of applications spanning from healthcare to advanced functional materials. Synthetic glycopolymers are particularly attractive as they possess low toxicity and immunogenicity and can be used as multivalent ligands to target sugar-binding proteins (lectins). Here, we utilised RAFT polymerisation to synthesize two families of novel diblock copolymers consisting of a glycopolymers block containing either mannopyranose or galactopyranose pendant units, which was elongated with sodium 2-acrylamido-2-methyl-1-propanesulfonate (AMPS) to generate a polyanionic block. The latter enabled complexation of cationic aminoglycoside antibiotic tobramycin through electrostatic interactions (loading efficiency in the 0.5-6.3 wt% range, depending on the copolymer). The resulting drug vectors were characterized by dynamic light scattering, zeta-potential, and transmission electron microscopy. Tobramycin-loaded complexes were tested for their ability to prevent clustering or disrupt biofilm of the Pseudomonas aeruginosa Gram-negative bacterium responsible for a large proportion of nosocomial infection, especially in immunocompromised patients. P. aeruginosa possesses two specific tetrameric carbohydrate-binding adhesins, LecA (PA-IL, galactose/N-acetyl-D-galactosamine-binding) and LecB (PA-IIL, fucose/mannose-binding), and the cell-associated and extracellular adhesin CdrA (Psl/mannose-binding) thus ideally suited for targeted drug delivery using sugar-decorated tobramycin-loaded complexes here developed. Both aliphatic and aromatic linkers were utilised to link the sugar pendant units to the polyacrylamide polymer backbone to assess the effect of the nature of such linkers on bactericidal/bacteriostatic properties of the complexes. Results showed that tobramycin-loaded complexes efficiently suppressed (40 to 60% of inhibition) in vitro biofilm formation in PAO1-L P. aeruginosa and that preferential targeting of PAO1-L biofilm can be achieved using mannosylated glycopolymer-b-AMPSm.
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Pseudomonas aeruginosa , Tobramicina , Antibacterianos/química , Antibacterianos/farmacología , Biopelículas , Humanos , Manosa , Tobramicina/químicaRESUMEN
Despite significant advances in chemotherapy, the overall prognosis of hepatocellular carcinoma (HCC) remains extremely poor. HCC targeting strategies were combined with the tumor cell cytotoxicity of oncolytic viruses (OVs) to develop a more efficient and selective therapeutic system. OVs were coated with a polygalactosyl-b-agmatyl diblock copolymer (Gal32-b-Agm29), with high affinity for the asialoglycoprotein receptor (ASGPR) expressed on the liver cell surface, exploiting the electrostatic interaction of the positively charged agmatine block with the negatively charged adenoviral capsid surface. The polymer coating altered the viral particle diameter (from 192 to 287 nm) and zeta-potential (from -24.7 to 23.3 mV) while hiding the peculiar icosahedral symmetrical OV structure, as observed by TEM. Coated OVs showed high potential therapeutic value on the human hepatoma cell line HepG2 (cytotoxicity of 72.4% ± 4.96), expressing a high level of ASGPRs, while a lower effect was attained with ASPGR-negative A549 cell line (cytotoxicity of 54.4% ± 1.59). Conversely, naked OVs showed very similar effects in both tested cell lines. Gal32-b-Agm29 OV coating enhanced the infectivity and immunogenic cell death program in HepG2 cells as compared to the naked OV. This strategy provides a rationale for future studies utilizing oncolytic viruses complexed with polymers toward effective treatment of hepatocellular carcinoma.
RESUMEN
A library of amphiphilic monomethoxypolyethylene glycol (mPEG) terminating polyaminoacid co-polymers able to self-assemble into colloidal systems was screened for the delivery and controlled release of doxorubicin (Doxo). mPEG-Glu/Leu random co-polymers were generated by Ring Opening Polymerization from 5 kDa mPEG-NH2 macroinitiator using 16:0:1, 8:8:1, 6:10:1, 4:12:1 γ-benzyl glutamic acid carboxy anhydride monomer/leucine N-carboxy anhydride monomer/PEG molar ratios. Glutamic acid was selected for chemical conjugation of Doxo, while leucine units were introduced in the composition of the polyaminoacid block as spacer between adjacent glutamic repeating units to minimize the steric hindrance that could impede the Doxo conjugation and to promote the polymer self-assembly by virtue of the aminoacid hydrophobicity. The benzyl ester protecting the γ-carboxyl group of glutamic acid was quantitatively displaced with hydrazine to yield mPEG5kDa-b-(hydGlum-r-Leun). Doxo was conjugated to the diblock co-polymers through pH-sensitive hydrazone bond. The Doxo derivatized co-polymers obtained with a 16:0:1, 8:8:1, 6:10:1 Glu/Leu/PEG ratios self-assembled into 30-40 nm spherical nanoparticles with neutral zeta-potential and CMC in the range of 4-7 µM. At pH 5.5, mimicking endosome environment, the carriers containing leucine showed a faster Doxo release than at pH 7.4, mimicking the blood conditions. Doxo-loaded colloidal formulations showed a dose dependent cytotoxicity on two cancer cell lines, CT26 murine colorectal carcinoma and 4T1 murine mammary carcinoma with IC50 slightly higher than those of free Doxo. The carrier assembled with the polymer containing 6:10:1 hydGlu/Leu/PEG molar ratio {mPEG5kDa-b-[(Doxo-hydGlu)6-r-Leu10]} was selected for subsequent in vitro and in vivo investigations. Confocal imaging on CT26 cell line showed that intracellular fate of the carrier involves a lysosomal trafficking pathway. The intratumor or intravenous injection to CT26 and 4T1 subcutaneous tumor bearing mice yielded higher antitumor activity compared to free Doxo. Furthermore, mPEG5kDa-b-[(Doxo-hydGlu)6-r-Leu10] displayed a better safety profile when compared to commercially available Caelyx®.
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Portadores de Fármacos , Micelas , Animales , Preparaciones de Acción Retardada , Doxorrubicina , Concentración de Iones de Hidrógeno , Ratones , Polietilenglicoles , PolímerosRESUMEN
The biorecognition-based control of attachment/detachment of MCF-7 cancer cells from polymer-coated surfaces is demonstrated. A glass surface is coated with a thermoresponsive statistical copolymer of poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAm-co-Am)], which is end-capped with the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, and the hydrophilic polymer poly(ethylene glycol) (PEG). Below the lower critical solution temperature (LCST) of p(NIPAm-co-Am) (38 °C), the copolymers are in the extended conformation, allowing for accessibility of the GRGDS peptides to membrane-associated integrins thus enabling cell attachment. Above the LCST, the p(NIPAm-co-Am) polymers collapse into globular conformations, resulting in the shielding of the GRGDS peptides into the PEG brush with consequent inaccessibility to cell-surface integrins, causing cell detachment. The surface coating is carried out by a multi-step procedure that included: glass surface amination with 3-aminopropyltriethoxysilane; reaction of mPEG5kDa -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am)15.1kDa -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am)15.1kDa -COOH. A range of spectrophotometric, surface, and microscopy assays confirmed the identity of the polymer-coated substrates. Competition studies prove that MCF-7 cancer cells are attached via peptide recognition at the coated surfaces according to the mPEG5kDa /p(NIPam-co-Am)15.1kDa -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release.
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Temperatura , Resinas Acrílicas/síntesis química , Resinas Acrílicas/química , Adhesión Celular , Recuento de Células , Fluorescencia , Vidrio/química , Humanos , Células MCF-7 , Microscopía de Fuerza Atómica , Oligopéptidos/química , Espectroscopía de Fotoelectrones , Polietilenglicoles/química , Propilaminas/química , Silanos/química , Succinimidas/química , Propiedades de SuperficieRESUMEN
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.
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Dexametasona/química , Liposomas/química , Microfluídica/métodos , Acetatos , Compuestos de Calcio , Línea Celular , Química Farmacéutica/métodos , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Humanos , Tamaño de la PartículaRESUMEN
Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG5kDa-cholane and mPEG5kDa-cholesterol, respectively. Microcalorimetric analyses showed that mPEG5kDa-cholane and mPEG5kDa-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 104 and 1.14 105 M-1, respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of α-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG5kDa-cholesterol has superior stabilizing effect of the protein conformation than mPEG5kDa-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG5kDa-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG5kDa-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG5kDa-cholane and mPEG5kDa-cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation.
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Colanos/química , Colesterol/análogos & derivados , Excipientes/química , Insulina/química , Polietilenglicoles/química , Colesterol/química , Coloides , Composición de Medicamentos , Liberación de Fármacos , Estabilidad de Medicamentos , Cinética , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Estabilidad Proteica , Solubilidad , TemperaturaRESUMEN
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.
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Colanos/administración & dosificación , Colesterol/administración & dosificación , Portadores de Fármacos/farmacocinética , Fosfatidiletanolaminas/administración & dosificación , Polietilenglicoles/administración & dosificación , Animales , Disponibilidad Biológica , Colanos/química , Colanos/farmacocinética , Colesterol/química , Colesterol/farmacocinética , Composición de Medicamentos/métodos , Estabilidad de Medicamentos , Células HeLa , Humanos , Lípidos , Liposomas , Ratones , Ratones Endogámicos BALB C , Simulación de Dinámica Molecular , Peso Molecular , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacocinética , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Propiedades de SuperficieRESUMEN
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.