RESUMO
Pancreatic cancer is a unique cancer in that up to 90% of its tumour mass is composed of a hypovascular and fibrotic stroma. This makes it extremely difficult for chemotherapies to be delivered into the core of the cancer mass. We tissue-engineered a biomimetic 3D pancreatic cancer ("tumouroid") model comprised of a central artificial cancer mass (ACM), containing MIA Paca-2 cells, surrounded by a fibrotic stromal compartment. This stromal compartment had a higher concentration of collagen type I, fibronectin, laminin, and hyaluronic acid (HA) than the ACM. The incorporation of HA was validated with alcian blue staining. Response to paclitaxel was determined in 2D MIA Paca-2 cell cultures, the ACMs alone, and in simple and complex tumouroids, in order to demonstrate drug sensitivity within pancreatic tumouroids of increasing complexity. The results showed that MIA Paca-2 cells grew into the complex stroma and invaded as cell clusters with a maximum distance of 363.7 µm by day 21. In terms of drug response, the IC50 for paclitaxel for MIA Paca-2 cells increased from 0.819 nM in 2D to 3.02 nM in ACMs and to 5.87 nM and 3.803 nM in simple and complex tumouroids respectively, indicating that drug penetration may be significantly reduced in the latter. The results demonstrate the need for biomimetic models during initial drug testing and evaluation.
Assuntos
Paclitaxel/farmacologia , Neoplasias Pancreáticas/patologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Engenharia Tecidual , Microambiente Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Imunofluorescência , Humanos , Imuno-Histoquímica , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Esferoides Celulares , Células Estromais/patologia , Células Tumorais CultivadasRESUMO
Parenteral chemotherapy is usually administered intravenously, although patient preference and health economics suggest the subcutaneous (sc) route could be an attractive alternative. However, due to the low aqueous solubility of hydrophobic drugs and injection volume limitations, the total amount of drug that can be administered in a single sc injection is frequently insufficient. We have developed hyaluronidase coated nanoparticles (NPs) that efficiently encapsulate such drugs, thus addressing both issues and allowing sufficient amounts of hydrophobic drug to be administered and absorbed effectively. CUDC-101, a poorly water-soluble multitargeted anticancer drug that simultaneously inhibits the receptor tyrosine kinases (RTKs) EGFR and HER2, as well as histone deacetylase (HDAC), was encapsulated in polymeric Molecular Envelope Technology (MET) NPs. The role of polymer chemistry, formulation parameters, and coating with hyaluronidase (HYD) on MET-CUDC-101 NP formulations was examined and optimized to yield high drug loading and colloidal stability, and, after freeze-drying, stable storage at room temperature for up to 90 days. The pharmacokinetic studies in healthy rats showed that plasma AUC0-24h after sc administration correlates tightly with formulation physical chemistry, specifically in vitro colloidal stability. Compared to uncoated NPs, the HYD-coating doubled the drug plasma exposure. In a murine A431 xenograft model, the coated HYD-MET-CUDC-101 NPs at a dose equivalent to 90 mg kg-1 CUDC-101 increased the survival time from 15 days (control animals treated with hyaluronidase alone) to 43 days. Polymer MET nanoparticles coated with hyaluronidase enabled the subcutaneous delivery of a hydrophobic drug with favorable therapeutic outcomes.
Assuntos
Antineoplásicos/administração & dosagem , Carcinoma de Células Escamosas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Inibidores de Histona Desacetilases/farmacologia , Hialuronoglucosaminidase/química , Ácidos Hidroxâmicos/administração & dosagem , Nanopartículas/química , Polímeros/química , Quinazolinas/administração & dosagem , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Quitosana/análogos & derivados , Quitosana/química , Portadores de Fármacos/química , Feminino , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/sangue , Histona Desacetilases , Histonas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ácidos Hidroxâmicos/sangue , Ácidos Hidroxâmicos/farmacocinética , Camundongos , Camundongos Nus , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Tamanho da Partícula , Quinazolinas/sangue , Quinazolinas/farmacocinética , Ratos , Solubilidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The global prevalence of neurologic disorders is rising, and yet we are still unable to deliver most drug molecules, in therapeutic quantities, to the brain. The blood brain barrier consists of a tight layer of endothelial cells surrounded by astrocyte foot processes, and these anatomic features constitute a significant barrier to drug transport from the blood to the brain. One way to bypass the blood brain barrier and thus treat diseases of the brain is to use the nasal route of administration and deposit drugs at the olfactory region of the nares, from where they travel to the brain via mechanisms that are still not clearly understood, with travel across nerve fibers and travel via a perivascular pathway both being hypothesized. The nose-to-brain route has been demonstrated repeatedly in preclinical models, with both solution and particulate formulations. The nose-to-brain route has also been demonstrated in human studies with solution and particle formulations. The entry of device manufacturers into the arena will enable the benefits of this delivery route to become translated into approved products. The key factors that determine the efficacy of delivery via this route include the following: delivery to the olfactory area of the nares as opposed to the respiratory region, a longer retention time at the nasal mucosal surface, penetration enhancement of the active through the nasal epithelia, and a reduction in drug metabolism in the nasal cavity. Indications where nose-to-brain products are likely to emerge first include the following: neurodegeneration, post-traumatic stress disorder, pain, and glioblastoma.
Assuntos
Administração Intranasal/métodos , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Nariz , Animais , Humanos , Cavidade Nasal/metabolismo , Mucosa Nasal/metabolismoRESUMO
PURPOSE: The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression. METHODS: Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg(-1)) or ethanolic lomustine (6.5 mg kg(-1)) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg(-1)) or ethanolic lomustine (daily 1.2 mg kg(-1) - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated. RESULTS: The MET formulation resulted in modest brain targeting (brain/ bone AUC0-4h ratios for MET and ethanolic lomustine = 0.90 and 0.53 respectively and brain/ liver AUC0-4h ratios for MET and ethanolic lomustine = 0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes. CONCLUSIONS: Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.
Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Lomustina/administração & dosagem , Animais , Antineoplásicos Alquilantes/efeitos adversos , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/uso terapêutico , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Lomustina/efeitos adversos , Lomustina/farmacocinética , Lomustina/uso terapêutico , Masculino , Camundongos , Nanopartículas/químicaRESUMO
There are very few drug delivery systems that target key organs via the oral route, as oral delivery advances normally address gastrointestinal drug dissolution, permeation, and stability. Here we introduce a nanomedicine in which nanoparticles, while also protecting the drug from gastric degradation, are taken up by the gastrointestinal epithelia and transported to the lung, liver, and spleen, thus selectively enhancing drug bioavailability in these target organs and diminishing kidney exposure (relevant to nephrotoxic drugs). Our work demonstrates, for the first time, that oral particle uptake and translocation to specific organs may be used to achieve a beneficial therapeutic response. We have illustrated this using amphotericin B, a nephrotoxic drug encapsulated within N-palmitoyl-N-methyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycol chitosan (GCPQ) nanoparticles, and have evidenced our approach in three separate disease states (visceral leishmaniasis, candidiasis, and aspergillosis) using industry standard models of the disease in small animals. The oral bioavailability of AmB-GCPQ nanoparticles is 24%. In all disease models, AmB-GCPQ nanoparticles show comparable efficacy to parenteral liposomal AmB (AmBisome). Our work thus paves the way for others to use nanoparticles to achieve a specific targeted delivery of drug to key organs via the oral route. This is especially important for drugs with a narrow therapeutic index.
Assuntos
Anfotericina B/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Administração Oral , Animais , Antifúngicos/farmacocinética , Antiprotozoários/farmacocinética , Estabilidade de Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NanomedicinaRESUMO
Neurological diseases such as neurodegeneration, pain, psychiatric disorders, stroke, and brain cancers would greatly benefit from the use of highly potent and specific peptide pharmaceuticals. Peptides are especially desirable because of their low inherent toxicity. The presence of the blood brain barrier (BBB), their short duration of action, and their need for parenteral administration limits their clinical use. However, over the past decade there have been significant advances in delivering peptides to the central nervous system. Angiopep peptides developed by Angiochem (Montreal, Canada), transferrin antibodies developed by ArmaGen (Santa Monica, USA), and cell penetrating peptides have all shown promise in delivering therapeutic peptides across the BBB after intravenous administration. Noninvasive methods of delivering peptides to the brain include the use of chitosan amphiphile nanoparticles for oral delivery and nose to brain strategies. The uptake of the chitosan amphiphile nanoparticles by the gastrointestinal epithelium is important for oral peptide delivery. Finally protecting peptides from plasma degradation is integral to the success of most of these peptide delivery strategies.
Assuntos
Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Peptídeos/administração & dosagem , Sequência de Aminoácidos , Animais , Barreira Hematoencefálica , Humanos , Dados de Sequência Molecular , Nanopartículas/administração & dosagem , Peptídeos/farmacocinéticaRESUMO
Glioblastoma multiforme (GBM) is a fast-growing and aggressive brain tumour, which remains largely resistant to treatment; the prognosis for patients is poor, with a median survival time of about 12-18 months, post diagnosis. In an effort to bring more efficacious treatments to patients, we targeted the down regulation of ITCH, an E3 ligase that is overexpressed in a variety of cancers, and which inhibits P73, a tumour suppressor gene. 6-O-glycolchitosan (GC) was used to deliver siRNA ITCH (GC60-siRNA-ITCH) and gemcitabine via the nose to brain route in CD-1 nude mice which had previously been implanted intracranially with U87-MG-luc2 cells. Prior to this in vivo study, an in vitro study established the synergistic effect of siRNA-ITCH in combination with a chemotherapy drug-gemcitabine. A downregulation of ITCH, an upregulation of p73 and enhanced apoptosis were observed in vitro in U87-MG cells, using qPCR, Western blot analysis, confocal laser scanning microscopy, flow cytometry and cytotoxicity assays. When GC60-siRNA-ITCH was combined with gemcitabine, there was a resultant decrease in cell proliferation in vitro. In CD1 mice, the administration of siRNA-ITCH (7 doses of 0.081 mg/kg) alone did not significantly affect animal survival (increasing mean survival from 29 to 33 days when compared to untreated animals), whereas intranasal gemcitabine had a significant effect on survival (increasing survival from 29 to 45 days when compared to untreated animals, p < 0.01). The most significant effect was seen with combination therapy (GC60-siRNA-ITCH plus gemcitabine), where survival increased by 89%, increasing from 29 to 54 days (p < 0.01). Our data demonstrate that siRNA chemosensitises brain tumours to gemcitabine and that the nose-to-brain delivery route may be a viable route for the treatment of intracranial tumours.
RESUMO
Respiratory diseases, including influenza, infectious pneumonia, and severe acute respiratory syndrome (SARS), are a leading cause of morbidity and mortality worldwide. The recent COVID-19 pandemic claimed over 6.9 million lives globally. With the possibility of future pandemics, the creation of affordable antimicrobial meshes for protective gear, such as facemasks, is essential. Electrospinning has been a focus for much of this research, but most approaches are complex and expensive, often wasting raw materials by distributing antiviral agents throughout the mesh despite the fact they can only be active if at the fibre surface. Here, we report a low cost and efficient one-step method to produce nanofibre meshes with antimicrobial activity, including against SARS-CoV-2. Cetrimonium bromide (CTAB) was deposited directly onto the surface of polycaprolactone (PCL) fibres by coaxial electrospinning. The CTAB-coated samples have denser meshes with finer nanofibres than non-coated PCL fibres (mean diameter: â¼300 nm versus â¼900 nm, with mean pore size: â¼300 nm versus > 600 nm). The formulations have > 90% coating efficiency and exhibit a burst release of CTAB upon coming into contact with aqueous media. The CTAB-coated materials have strong antibacterial activity against Staphylococcus aureus (ca. 100%) and Pseudomonas aeruginosa (96.5 ± 4.1%) bacteria, as well as potent antiviral activity with over 99.9% efficacy against both respiratory syncytial virus and SARS-CoV-2. The CTAB-coated nanofibre mesh thus has great potential to form a mask material for preventing both bacterial and viral respiratory infections.
RESUMO
There are numerous pharmaceutical, food, and consumer product applications requiring the incorporation of hydrophobic solutes within aqueous media. Often amphiphiles and/or polymers are used to produce encapsulating nanostructures. Because the encapsulation efficiencies of these nanostructures directly impact on the process or product, it is often desirable to optimize this parameter. To produce these advanced functional materials, we hypothesized that an amphiphile with a claw shape would favor polymer aggregation into nanoparticles and hydrophobic compound encapsulation. Claw amphiphiles were prepared by attaching one end of comb-shaped chitosan amphiphile chains [N,N,N-trimethyl, N,N-dimethyl, N-monomethyl, N-palmitoyl, N-acetyl, 6-O-glycol chitosan (GCPQA)] to a central dendrimer core [generation 3 diaminobutane poly(propylenimine) dendrimer (DAB)] to give DAB-GCPQA. The linear chitosan amphiphile (GCPQA) forms the digits of the claw. These claw amphiphiles were very stable and had a high encapsulating efficiency. DAB-GCPQAs (Mn = 30 and 70 kDa) had extremely low critical micelle concentrations [CMCs = 0.43 µg mL(-1) (13 nM) and 0.093 µg mL(-1) (0.9 nM), respectively], and their CMCs were lower than that of linear GCPQA [Mn = 14 kDa, CMC = 0.77 µg mL(-1) (38 nM)]. The claw amphiphile CMCs decreased linearly with the number of digits (r(2) = 0.98), and drug encapsulation (hydrophobic drug propofol) in 4 mg mL(-1) dispersions of the amphiphiles increased linearly (r(2) = 0.94) with the number of digits. DAB-GCPQA70 (4 mg mL(-1), 0.058 mM) encapsulated propofol (7.3 mg mL(-1), 40 mM). Finally, despite their stability, claw amphiphile nanoparticles are able to release the encapsulated drug in vivo, as shown with the claw amphiphile-propofol formulations in a murine loss of righting reflex model.
Assuntos
Dendrímeros/química , Composição de Medicamentos , Nanopartículas/química , Tensoativos/química , Dendrímeros/síntese química , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Tensoativos/síntese químicaRESUMO
Disulfiram (DS) is an anti-alcoholism drug capable of acting against important and hard-to-treat cancers. The drug's relative instability and variable absorption/distribution have led to its variable pharmacokinetics and suboptimal exposure. Hence, it was hypothesised that a nano-enabled form of DS might be able to overcome such limitations. Encapsulation of the labile DS was achieved with quaternary ammonium palmitoyl glycol chitosan (GCPQ) to form a high-capacity, soybean oil-based DS-GCPQ nanoemulsion. DS-GCPQ showed capability of oil-loading up to 50% v/v for a stable entrapment of high drug content. With increasing oil content (10 to 50% v/v), the mean particle size and polydispersity index were also increased (166 to 351 nm and 0.14 to 0.22, respectively) for a given amount of GCPQ. Formulations showed a highly positive particle surface charge (50.9 ± 1.3 mV), contributing to the colloidal stability of the individual particles. DS-GCPQ showed marked cytotoxicity against pancreatic cancer cell lines with enhanced activity in the presence of copper. An intravenous pharmacokinetic study of DS-GCPQ in vivo showed improved plasma drug stability with a DS half-life of 17 min. Prolonged survival was seen in tumour-bearing animals treated with DS-GCPQ supplemented with copper. In conclusion, DS-GCPQ nanoemulsion has the potential to be developed further for cancer therapeutic purposes.
Assuntos
Quitosana , Dissulfiram , Nanopartículas , Animais , Cobre , Composição de Medicamentos , Emulsões , Micelas , Tamanho da Partícula , Quitosana/química , Sistemas de Liberação de Fármacos por NanopartículasRESUMO
The use of nucleic acids to treat various brain diseases could offer new therapeutic modalities, providing the nucleic acids may be effectively delivered to areas of the brain using non-toxic vectors. In this study, we present evidence that genes may be successfully delivered in a dose-dependent manner via the nose, primarily to the cerebral cortex using a 6-O-glycolchitosan (GC) formulation of plasmid DNA. Positively charged (zeta potential = +13 - + 25 mV) GC-DNA nanoparticles of 100-500 nm in diameter with favourable cell biocompatibility were shown to deliver the reporter Green Fluorescent Protein (GFP) plasmid to the U87MG cell line and the resulting protein expression was not significantly different from that obtained with Lipofectamine 2000. On intranasal delivery of GC-luciferase-plasmid nanoparticles to Balb/ C mice at 4 doses, ranging from 0.02 to 0.1 mg/ kg, luciferase activity was observed qualitatively in intact mouse brains, 48 h after intranasal, using the IV-VIS visualisation. In further confirmation of brain delivery, dose-dependent protein expression was quantified in multiple brain areas 48 h after dosing; with protein expression seen mainly in the cerebral cortex and striatum and following expression levels: cerebral cortex = olfactory bulb > striatum > brain stem > mid brain = cerebellum. No protein expression was observed in the liver and lungs of dosed animals. GC-DNA protein expression was not significantly different to that observed with Lipofectamine 2000. These results demonstrate that GC-DNA nanoparticles are able to deliver genes preferably to specific brain regions such as the cerebral cortex and striatum; offering the possibility of using genes to treat a range of neurological disorders using a non-invasive method of dosing.
Assuntos
Encefalopatias , Ácidos Nucleicos , Animais , Camundongos , Nariz , Encéfalo , Córtex CerebralRESUMO
A new class of anticancer prodrugs was designed by combining the cytotoxicity of platinum(IV) complexes and the drug carrier properties of glycol chitosan polymers: Unsymmetrically carboxylated platinum(IV) analogues of cisplatin, carboplatin and oxaliplatin, namely (OC-6-44)-acetatodiammine(3-carboxypropanoato)dichloridoplatinum(IV), (OC-6-44)-acetaodiammine(3-carboxypropanoato)(cyclobutane-1,1-dicarboxylato)platinum(IV) and (OC-6-44)-acetato(3-carboxypropanoato)(1R,2R-cyclohexane-1,2-diamine)oxalatoplatinum(IV) were synthesised and conjugated via amide bonding to degraded glycol chitosan (dGC) polymers with different chain lengths (5, 10, 18 kDa). The 15 conjugates were investigated with 1H and 195Pt NMR spectroscopy, and average amounts of platinum(IV) units per dGC polymer molecule with ICP-MS, revealing a range of 1.3-22.8 platinum(IV) units per dGC molecule. Cytotoxicity was tested with MTT assays in the cancer cell lines A549, CH1/PA-1, SW480 (human) and 4T1 (murine). IC50 values in the low micromolar to nanomolar range were obtained, and higher antiproliferative activity (up to 72 times) was detected with dGC-platinum(IV) conjugates in comparison to platinum(IV) counterparts. The highest cytotoxicity (IC50 of 0.036 ± 0.005 µM) was determined in CH1/PA-1 ovarian teratocarcinoma cells with a cisplatin(IV)-dGC conjugate, which is hence 33 times more potent than the corresponding platinum(IV) complex and twice more potent than cisplatin. Biodistribution studies of an oxaliplatin(IV)-dGC conjugate in non-tumour-bearing Balb/C mice showed an increased accumulation in the lung compared to the unloaded oxaliplatin(IV) analogue, arguing for further activity studies.
RESUMO
Quaternary ammonium palmitoyl glycol chitosan (GCPQ) has already shown beneficial drug delivery properties and has been studied as a carrier for anticancer agents. Consequently, we synthesised cytotoxic platinum(IV) conjugates of cisplatin, carboplatin and oxaliplatin by coupling via amide bonds to five GCPQ polymers differing in their degree of palmitoylation and quaternisation. The conjugates were characterised by 1H and 195Pt NMR spectroscopy as well as inductively coupled plasma mass spectrometry (ICP-MS), the latter to determine the amount of platinum(IV) units per GCPQ polymer. Cytotoxicity was evaluated by the MTT assay in three human cancer cell lines (A549, non-small-cell lung carcinoma; CH1/PA-1, ovarian teratocarcinoma; SW480, colon adenocarcinoma). All conjugates displayed a high increase in their cytotoxic activity by factors of up to 286 times compared to their corresponding platinum(IV) complexes and mostly outperformed the respective platinum(II) counterparts by factors of up to 20 times, also taking into account the respective loading of platinum(IV) units per GCPQ polymer. Finally, a biodistribution experiment was performed with an oxaliplatin-based GCPQ conjugate in non-tumour-bearing BALB/c mice revealing an increased accumulation in lung tissue. These findings open promising opportunities for further tumouricidal activity studies especially focusing on lung tissue.
RESUMO
The oral use of neuropeptides to treat brain disease is currently not possible because of a combination of poor oral absorption, short plasma half-lives and the blood-brain barrier. Here we demonstrate a strategy for neuropeptide brain delivery via the (a) oral and (b) intravenous routes. The strategy is exemplified by a palmitic ester prodrug of the model drug leucine(5)-enkephalin, encapsulated within chitosan amphiphile nanoparticles. Via the oral route the nanoparticle-prodrug formulation increased the brain drug levels by 67% and significantly increased leucine(5)-enkephalin's antinociceptive activity. The nanoparticles facilitate oral absorption and the prodrug prevents plasma degradation, enabling brain delivery. Via the intravenous route, the nanoparticle-prodrug increases the peptide brain levels by 50% and confers antinociceptive activity on leucine(5)-enkephalin. The nanoparticle-prodrug enables brain delivery by stabilizing the peptide in the plasma although the chitosan amphiphile particles are not transported across the blood-brain barrier per se, and are excreted in the urine.
Assuntos
Encéfalo/metabolismo , Encefalinas/administração & dosagem , Encefalinas/química , Nanopartículas/administração & dosagem , Peptídeos/administração & dosagem , Peptídeos/química , Pró-Fármacos/administração & dosagem , Administração Oral , Animais , Barreira Hematoencefálica/metabolismo , Encefalinas/farmacocinética , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Camundongos , Nanopartículas/química , Peptídeos/síntese química , Pró-Fármacos/síntese química , Pró-Fármacos/química , Ratos , Ratos WistarRESUMO
Gold nanoparticles (AuNPs) are continuing to gain popularity in the field of nanotechnology. New methods are continuously being developed to tune the particles' physicochemical properties, resulting in control over their biological fate and applicability to in vivo diagnostics and therapy. This review focuses on the effects of varying particle size on optical properties, opsonization, cellular internalization, renal clearance, biodistribution, tumor accumulation, and toxicity. We review the common methods of synthesizing ultrasmall AuNPs, as well as the emerging constructs termed ultrasmall-in-nano-an approach which promises to provide the desirable properties from both ends of the AuNP size range. We review the various applications and outcomes of ultrasmall-in-nano constructs in vitro and in vivo.
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PURPOSE: Chronic infections of Candida albicans are characterised by the embedding of budding and entwined filamentous fungal cells into biofilms. The biofilms are refractory to many drugs and Candida biofilms are associated with ocular fungal infections. The objective was to test the activity of nanoparticulate amphotericin B (AmB) against Candida biofilms. METHODS: AmB was encapsulated in the Molecular Envelope Technology (MET, N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan) nanoparticles and tested against Candida biofilms in vitro. Confocal laser scanning microscopy (CLSM) imaging of MET nanoparticles' penetration into experimental biofilms was carried out and a MET-AmB eye drop formulation was tested for its stability. RESULTS: MET-AmB formulations demonstrated superior activity towards C. albicans biofilms in vitro with the EC50 being ~30 times lower than AmB alone (EC50 MET-AmB = 1.176 µg mL-1, EC50 AmB alone = 29.09 µg mL-1). A similar superior activity was found for Candida glabrata biofilms, where the EC50 was ~10× lower than AmB alone (EC50 MET-AmB = 0.0253 µg mL-1, EC50 AmB alone = 0.289 µg mL-1). CLSM imaging revealed that MET nanoparticles penetrated through the C. albicans biofilm matrix and bound to fungal cells. The activity of MET-AmB was no different from the activity of AmB alone against C. albicans cells in suspension (MET-AmB MIC90 = 0.125 µg mL-1, AmB alone MIC90 = 0.250 µg mL-1). MET-AmB eye drops were stable at room temperature for at least 28 days. CONCLUSIONS: These biofilm activity findings raise the possibility that MET-loaded nanoparticles may be used to tackle Candida biofilm infections, such as refractory ocular fungal infections.
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Therapeutic gene silencing in the brain is usually achieved using highly invasive intracranial administration methods and/or comparatively toxic vectors. In this work, we use a relatively biocompatible vector: poly(ethylene glycol) star-shaped polymer capped with amine groups (4APPA) via the nose to brain route. 4APPA complexes anti- itchy E3 ubiquitin protein ligase (anti-ITCH) siRNA to form positively charged (zeta potential +15 ± 5 mV) 150 nm nanoparticles. The siRNA-4APPA polyplexes demonstrated low cellular toxicity (IC50 = 13.92 ± 6 mg mL-1) in the A431 cell line and were three orders of magnitude less toxic than Lipofectamine 2000 (IC50 = 0.033 ± 0.04 mg mL-1) in this cell line. Cell association and uptake of fluorescently labelled siRNA bound to siRNA-4APPA nanoparticles was demonstrated using fluorescent activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), respectively. Gene silencing of the ITCH gene was observed in vitro in the A431 cell line (65% down regulation when compared to the use of anti-ITCH siRNA alone). On intranasal dosing with fluorescently labelled siRNA-4APPA polyplexes, fluorescence was seen in the cells of the olfactory bulb, cerebral cortex and mid-brain regions. Finally, down regulation of ITCH was seen in the brain cells (54 ± 13% ITCH remaining compared to untreated controls) in a healthy rat model, following intranasal dosing of siRNA-4APPA nanoparticles (0.15 mg kg-1 siRNA twice daily for 3 days). Gene silencing in the brain may be achieved by intranasal administration of siRNA- poly(ethylene glycol) based polyplexes.
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Gene delivery to the cerebral cortex is challenging due to the blood brain barrier and the labile and macromolecular nature of DNA. Here we report gene delivery to the cortex using a glycol chitosan-DNA polyplex (GCP). In vitro, GCPs carrying a reporter plasmid DNA showed approximately 60% of the transfection efficiency shown by Lipofectamine lipoplexes (LX) in the U87 glioma cell line. Aiming to maximise penetration through the brain extracellular space, GCPs were coated with hyaluronidase (HYD) to form hyaluronidase-coated polyplexes (GCPH). The GCPH formulation retained approximately 50% of the in vitro hyaluronic acid (HA) digestion potential but lost its transfection potential in two-dimensional U87 cell lines. However, intranasally administered GCPH (0.067 mg kg-1 DNA) showed high levels of gene expression (IVIS imaging of protein expression) in the brain regions. In a separate experiment, involving GCP, LX and naked DNA, the intranasal administration of the GCP formulation (0.2 mg kg-1 DNA) resulted in protein expression predominantly in the cerebral cortex, while a similar dose of intranasal naked DNA led to protein expression in the cerebellum. Intranasal LX formulations did not show any evidence of protein expression. GCPs may provide a means to target protein expression to the cerebral cortex via the intranasal route.
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Treatment of posterior eye diseases with intravitreal injections of drugs, while effective, is invasive and associated with side effects such as retinal detachment and endophthalmitis. In this work, we have formulated a model compound, rapamycin (RAP), in nanoparticle-based eye drops and evaluated the delivery of RAP to the posterior eye tissues in a healthy rabbit. We have also studied the formulation in experimental autoimmune uveitis (EAU) mouse model with retinal inflammation. Aqueous RAP eye drops were prepared using N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (Molecular Envelope Technology - MET) containing 0.23 ± 0.001% w/v RAP with viscosity, osmolarity, and pH within the ocular comfort range, and the formulation (MET-RAP) was stable in terms of drug content at both refrigeration and room temperature for one month. The MET-RAP eye drops delivered RAP to the choroid-retina with a Cmax of 145 ± 49 ng/g (tmax = 1 h). The topical application of the MET-RAP eye drops to the EAU mouse model resulted in significant disease suppression compared to controls, with activity similar to dexamethasone eye drops. The MET-RAP eye drops also resulted in a reduction of RORγt and an increase in both Foxp3 expression and IL-10 secretion, indicating a mechanism involving the inhibition of Th17 cells and the up-regulation of T-reg cells. The MET-RAP formulation delivers RAP to the posterior eye segments, and the formulation is active in EAU.
Assuntos
Segmento Posterior do Olho , Uveíte , Animais , Camundongos , Soluções Oftálmicas/farmacologia , Coelhos , Retina , Sirolimo/farmacologia , Uveíte/tratamento farmacológicoRESUMO
Levodopa (L-DOPA) is an oral Parkinson's Disease drug that generates the active metabolite - dopamine (DA) in vivo. However, oral L-DOPA exhibits low oral bioavailability, limited brain uptake, peripheral DA-mediated side effects and its poor brain bioavailability can lead to long-term complications. Here we show that L-DOPA forms stable (for at least 5 months) 300 nm nanoparticles when encapsulated within N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ). A nano-in-microparticle GCPQ-L-DOPA formulation (D50 = 7.2 µm), prepared by spray-drying, was stable for one month when stored at room and refrigeration temperatures and was capable of producing the original GCPQ-L-DOPA nanoparticles upon aqueous reconstitution. Nasal administration of reconstituted GCPQ-L-DOPA nanoparticles to rats resulted in significantly higher DA levels in the brain (Cmax of 94 ng g-1 above baseline levels 2 h post-dosing) when compared to nasal administration of L-DOPA alone, with DA being undetectable in the brain with the latter. Furthermore, nasal GCPQ-L-DOPA resulted in higher levels of L-DOPA in the plasma (a 17-fold increase in the Cmax, when compared to L-DOPA alone) with DA undetectable in the plasma from both formulations. These data provide evidence of effective delivery of DA to the brain with the GCPQ-L-DOPA formulation.