RESUMO
Two-dimension graphene oxide (GO) nanosheets with high and low serum protein binding profiles (high/low hard-bound protein corona/HChigh/low) are used in this study as model materials and screening tools to investigate the underlying roles of the protein corona on nanomaterial toxicities in vivo. We proposed that the in vivo biocompatibility/nanotoxicity of GO is protein corona-dependent and host immunity-dependent. The hypothesis was tested by injecting HChigh/low GO nanosheets in immunocompetent ICR/CD1 and immunodeficient NOD-scid II2rγnull mice and performed histopathological and hematological evaluation studies on days 1 and 14 post-injection. HClow GO induced more severe acute lung injury compared to HChigh GO in both immunocompetent and immunodeficient mice, with the effect being particularly pronounced in immunocompetent animals. Additionally, HClow GO caused more significant liver injury in both types of mice, with immunodeficient mice being more susceptible to its hepatotoxic effects. Moreover, administration of HClow GO resulted in increased hematological toxicity and elevated levels of serum pro-inflammatory cytokines in immunocompromised and immunocompetent mice, respectively. Correlation studies were conducted to explore the impact of distinct protein corona compositions on resulting toxicities in both immunocompetent and immunodeficient mice. This facilitated the identification of consistent patterns, aligning with those observed in vitro, thus indicating a robust in vitro-in vivo correlation. This research will advance our comprehension of how hard corona proteins interact with immune cells, leading to toxicity, and will facilitate the development of improved immune-modulating nanomaterials for therapeutic purposes.
Assuntos
Grafite , Camundongos Endogâmicos ICR , Nanoestruturas , Coroa de Proteína , Animais , Grafite/química , Grafite/toxicidade , Camundongos , Coroa de Proteína/química , Coroa de Proteína/imunologia , Nanoestruturas/química , Nanoestruturas/toxicidade , Camundongos SCID , Camundongos Endogâmicos NODRESUMO
Immunotherapy is currently a standard of care in the treatment of many malignancies. However, predictable side effects caused by systemic administration of highly immunostimulatory molecules have been a serious concern within this field. Intratumoural expression or silencing of immunogenic and immunoinhibitory molecules using nucleic acid-based approaches such as plasmid DNA (pDNA) and small interfering RNA (siRNA), respectively, could represent a next generation of cancer immunotherapy. Here, we employed lipid nanoparticles (LNPs) to deliver either non-specific pDNA and siRNA, or constructs targeting two prominent immunotherapeutic targets OX40L and indoleamine 2,3-dioxygenase-1 (IDO), to tumours in vivo. In the B16F10 mouse model, intratumoural delivery of LNP-formulated non-specific pDNA and siRNA led to strong local immune activation and tumour growth inhibition even at low doses due to the pDNA immunogenic nature. Replacement of these non-specific constructs by pOX40L and siIDO resulted in more prominent immune activation as evidenced by increased immune cell infiltration in tumours and tumour-draining lymph nodes. Consistently, pOX40L alone or in combination with siIDO could prolong overall survival, resulting in complete tumour regression and the formation of immunological memory in tumour rechallenge models. Our results suggest that intratumoural administration of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy.
Assuntos
DNA , Imunoterapia , Camundongos Endogâmicos C57BL , Nanopartículas , Plasmídeos , RNA Interferente Pequeno , Animais , Imunoterapia/métodos , RNA Interferente Pequeno/administração & dosagem , Nanopartículas/administração & dosagem , Nanopartículas/química , Plasmídeos/administração & dosagem , DNA/administração & dosagem , DNA/imunologia , Camundongos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Feminino , Linhagem Celular Tumoral , Melanoma Experimental/terapia , Melanoma Experimental/imunologia , Lipídeos/química , Lipídeos/administração & dosagem , Portadores de Fármacos/químicaRESUMO
Extracellular vesicles (EVs) derived from mesenchymal stem cells are promising nanotherapeutics in liver diseases due to their regenerative and immunomodulatory properties. Nevertheless, a concern has been raised regarding the rapid clearance of exogenous EVs by phagocytic cells. Here we explore the impact of protein corona on EVs derived from two culturing conditions in which specific proteins acquired from media were simultaneously adsorbed on the EV surface. Additionally, by incubating EVs with serum, simulating protein corona formation upon systemic delivery, further resolved protein corona-EV complex patterns were investigated. Our findings reveal the potential influences of corona composition on EVs under in vitro conditions and their in vivo kinetics. Our data suggest that bound albumin creates an EV signature that can retarget EVs from hepatic macrophages. This results in markedly improved cellular uptake by hepatocytes, liver sinusoidal endothelial cells and hepatic stellate cells. This phenomenon can be applied as a camouflage strategy by precoating EVs with albumin to fabricate the albumin-enriched protein corona-EV complex, enhancing non-phagocytic uptake in the liver. This work addresses a critical challenge facing intravenously administered EVs for liver therapy by tailoring the protein corona-EV complex for liver cell targeting and immune evasion.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Coroa de Proteína , Vesículas Extracelulares/metabolismo , Coroa de Proteína/metabolismo , Coroa de Proteína/química , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Humanos , Camundongos , Hepatócitos/metabolismo , Hepatócitos/citologia , Fígado/metabolismo , Macrófagos/metabolismo , Macrófagos/citologiaRESUMO
The clinical application of EDV, a potent antioxidant drug approved for amyotrophic lateral sclerosis (ALS), is limited by its short biological half-life and poor water solubility necessitating hospitalization during intravenous infusion. Nanotechnology-based drug delivery constitutes a powerful tool through inferring drug stability and targeted drug delivery improving drug bioavailability at the diseased site. Nose-to-brain drug delivery offers direct access to the brain bypassing the blood brain barrier and reducing systemic biodistribution. In this study, we designed EDV-loaded poly(lactic-co-glycolic acid) (PLGA)-based polymeric nanoparticles (NP-EDV) for intranasal administration. NPs were formulated by the nanoprecipitation method. Morphology, EDV loading, physicochemical properties, shelf-life stability, in vitro release and pharmacokinetic assessment in mice were conducted. EDV was efficiently loaded into â¼90 nm NPs, stable up to 30 days of storage, at â¼3% drug loading. NP-EDV reduced H2O2-induced oxidative stress toxicity in mouse microglial cell line BV-2. Optical imaging and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) showed that intranasal delivery of NP-EDV offered higher and more sustained brain uptake of EDV compared to intravenous administration. This study is the first of its kind to develop an ALS drug in a nanoparticulate formulation for nose-to-brain delivery raising hope to ALS patients where currently treatment options are limited to two clinically approved drugs only.
Assuntos
Esclerose Lateral Amiotrófica , Nanopartículas , Camundongos , Animais , Administração Intranasal , Edaravone/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/metabolismo , Portadores de Fármacos/química , Disponibilidade Biológica , Distribuição Tecidual , Cromatografia Líquida , Peróxido de Hidrogênio/metabolismo , Espectrometria de Massas em Tandem , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Tamanho da PartículaRESUMO
Therapeutic nucleic acids (TNAs) comprise an alternative to conventional drugs for cancer therapy. Recently, stable nucleic acid lipid particles (SNALPs) have been explored to deliver TNA efficiently and safely both in vitro and in vivo. Small interfering RNA (siRNA) and messenger RNA (mRNA) based drugs have been suggested for a wide range of pathologies, and their respective lipid nanoparticle (LNP) formulations have been optimised using a Design of Experiments (DoE) approach. However, it is uncertain as to whether data obtained from DoE using simple experimental outputs can be used to generate a general heuristic for delivery of diverse TNA both in vitro and in vivo. Using plasmid DNA (pDNA), for which limited DoE optimisation has been performed, and siRNA to represent the two extremities of the TNA spectrum in terms of size and biological requirements, we performed a comparative DoE for both molecules and assessed the predictive qualities of the model both in vitro and in vivo. By producing a minimum run of 24 SNALP formulations with different lipid compositions incorporating either pDNA or siRNA, DoE models were successfully established for predicting the effect of individual lipid composition on particle size, TNA encapsulation and transfection both in vitro and in vivo. The results showed that the particle size, and in vitro and in vivo transfection efficiency of both pDNA and siRNA SNALP formulations were affected by lipid compositions. The encapsulation efficiency of pDNA SNALPs but not siRNA SNALPs was affected by the lipid composition. Notably, the optimal lipid compositions of SNALPs for pDNA/siRNA delivery were not identical. Furthermore, in vitro transfection efficiency could not be used to predict promising LNP candidates in vivo. The DoE approach described in this study may provide a method for comprehensive optimisation of LNPs for various applications. The model and optimal formulation described in this study can serve as a foundation from which to develop other novel NA containing LNPs for multiple applications such as NA based vaccines, cancer immunotherapies and other TNA therapies.
Assuntos
Nanopartículas , Lipossomos , DNA , RNA Interferente Pequeno , RNA Mensageiro , LipídeosRESUMO
Intranasal administration is becoming increasingly more attractive as a fast delivery route to the brain for therapeutics circumventing the blood-brain barrier (BBB). Gold nanorods (AuNRs) demonstrate unique optical and biological properties compared to other gold nanostructures due to their high aspect ratio. In this study, we investigated for the first time the brain region-specific distribution of AuNRs and their potential as a drug delivery platform for central nervous system (CNS) therapy following intranasal administration to mice using a battery of analytical and imaging techniques. AuNRs were functionalized with a fluorescent dye (Cyanine5, Cy5) or a metal chelator (diethylenetriaminepentaacetic dianhydride, DTPA anhydride) to complex with Indium-111 via a PEG spacer for optical and nuclear imaging, respectively. Direct quantification of gold was achieved by inductively coupled plasma mass spectrometry. Rapid AuNRs uptake in mice brains was observed within 10 min following intranasal administration which gradually reduced over time. This was confirmed by the 3 imaging/analytical techniques. Autoradiography of sagittal brain sections suggested entry to the brain via the olfactory bulb followed by diffusion to other brain regions within 1 h of administration. The presence of AuNR in glioblastoma (GBM) tumors following intranasal administration was also proven which opens doors for AuNRs applications, as nose-to-brain drug delivery carriers, for treatment of a range of CNS diseases.
Assuntos
Glioblastoma , Nanotubos , Camundongos , Animais , Administração Intranasal , Ouro/química , Encéfalo , Nanotubos/químicaRESUMO
In situ vaccination with immunostimulatory compounds is a demonstrated means to treat tumors preclinically. While these therapeutic effects have been attributed to the actions of T cells or innate immune activation, characterisation of the humoral immune response is seldom performed. This study aims to identify whether the injection of immunoadjuvants, Addavax (Adda) and cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG), intratumorally can influence the antibody response. Specifically, whether intratumoral injection of immunoadjuvants can alter the tumor-specific antibody target, titre and isotype. Following this, the study aimed to investigate whether serum obtained from in situ vaccinated mice could neutralise circulating tumor cells. Serum was obtained from mice bearing B16F10-OVA-Luc-GFP tumors treated with immunoadjuvants. Antibody targets' titre and isotype were assessed by indirect ELISA. The ability of serum to neutralise circulating cancer cells was evaluated in a B16F10 pseudo-metastatic model. It was observed that tumor-bearing mice mount a specific anti-tumor antibody response. Antibody titre and target were unaffected by in situ vaccination with immunoadjuvants; however, a higher amount of IgG2c was produced in mice receiving Adda plus CpG. Serum from in situ vaccinated mice was unable to neutralise circulating B16F10 cells. Thus, this study has demonstrated that anti-tumor antibody isotype may be modified using in situ vaccination; however, this alone is not sufficient to neutralise circulating cancer cells.
Assuntos
Adjuvantes Imunológicos , Neoplasias , Camundongos , Animais , AnticorposRESUMO
Tumour-targeted near-infrared (NIR) optical imaging is an emerging tool for the detection of malignant tissues. This modality can be useful in both diagnosis and intraoperative visualisation, to help defining tumour margins and allow a more precise removal of all the cancerous mass during surgery. In this context, we have developed a series of NIR fluorescent probes that target the prostate-specific membrane antigen (PSMA), an established biomarker overexpressed in prostate cancer. Four new NIR imaging agents were prepared by conjugating the well-known urea-based PSMA targeting module to the NIR fluorophore Cy7.5, with linkers of 7, 10, 17 and 24 atoms. The affinity of each probe for PSMA was assessed through competitive binding and IC50 measurement in prostate cancer cells, using a previously reported PSMA-targeted NIR probe (i.e. PSMA-IRDye800CW) as reference. The NIR probe PSMA-Cy7.5_2 demonstrated a high affinity for PSMA (i.e. IC50 = 58.8 nM) and was further studied in mouse xenograft models of prostate cancer, to assess its ability to image PSMA positive tumour tissues. While PSMA-Cy7.5_2 out-performed PSMA-IRDye800CW in vitro, its tumour accumulation in vivo was not as evident. Further micellar aggregation studies indicated that the relatively higher hydrophobic property of PSMA-Cy7.5_2 may lower its bioavailability and tissue distribution following systemic injection, limiting its ability of targeting PSMA tumour in vivo. Nevertheless, the excellent binding capability of PSMA-Cy7.5_2 renders this probe a valid lead for further structural optimisation to develop imaging analogues with high affinity and specificity for PSMA, as required for effective NIR fluorescence-guided applications pre-clinically and clinically.
Assuntos
Corantes Fluorescentes , Neoplasias da Próstata , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Masculino , Camundongos , Imagem Óptica/métodos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , UreiaRESUMO
Rational: Tumor immunogenic cell death (ICD), induced by certain chemotherapeutic drugs such as doxorubicin (Dox), is a form of apoptosis potentiating a protective immune response. One of the hallmarks of ICD is the translocation of calreticulin to the cell surface acting as an 'eat me' signal. This manuscript describes the development of a stable nucleic acid-lipid particles (SNALPs) formulation for the simultaneous delivery of ICD inducing drug (Dox) with small interfering RNA (siRNA) knocking down CD47 (siCD47), the dominant 'don't eat me' marker, for synergistic enhancement of ICD. Methods: SNALPs loaded with Dox or siCD47 either mono or combinatory platforms were prepared by ethanol injection method. The proposed systems were characterized for particle size, surface charge, entrapment efficiency and in vitro drug release. The ability of the SNALPs to preserve the siRNA integrity in presence of serum and RNAse were assessed over 48 h. The in vitro cellular uptake and gene silencing of the prepared SNALPs was assessed in CT26 cells. The immunological responses of the SNALPs were defined in vitro in terms of surface calreticulin expression and macrophage-mediated phagocytosis induction. In vivo therapeutic studies were performed in CT26 bearing mice where the therapeutic outcomes were expressed as tumor volume, expression of CD4 and CD8 as well as in vivo silencing. Results: The optimized SNALPs had a particle size 122 ±6 nm and an entrapment efficiency > 65% for both siRNA and Dox with improved serum stability. SNALPs were able to improve siRNA and Dox uptake in CT26 cells with enhanced cytotoxicity. siCD47 SNALPs were able to knockdown CD47 by approximately 70% with no interference from the presence of Dox. The siCD47 and Dox combination SNALPs were able to induce surface calreticulin expression leading to a synergistic effect on macrophage-mediated phagocytosis of treated cells. In a tumor challenge model, 50% of mice receiving siCD47 and Dox containing SNALPs were able to clear the tumor, while the remaining animals showed significantly lower tumor burden as compared to either monotreatment. Conclusion: Therefore, the combination of siCD47 and Dox in a particulate system showed potent anti-tumor activity which merits further investigation in future clinical studies.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Morte Celular Imunogênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Calreticulina/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Imunoterapia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sistemas de Liberação de Fármacos por Nanopartículas/química , Sistemas de Liberação de Fármacos por Nanopartículas/farmacologia , Fagocitose/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologiaRESUMO
Black porous silicon nanoparticles (BPSi NPs) are known as highly efficient infrared light absorbers that are well-suitable for photothermal therapy (PTT) and photoacoustic imaging (PAI). PTT and PAI require a sufficient number of effectively light-absorbing NPs to be accumulated in tumor after intravenous administration. Herein, biodistribution of PEGylated BPSi NPs with different sizes (i.e., 140, 200, and 300 nm in diameter) is investigated after intravenous administration in mice. BPSi NPs were conjugated with fluorescent dyes Cy5.5 and Cy7.5 to track them in vitro and in vivo, respectively. Optical imaging with an in vivo imaging system (IVIS) was found to be an inadequate technique to assess the biodistribution of the dye-labeled BPSi NPs in vivo because the intrinsic strong absorbance of the BPSi NPs interfered fluorescence detection. This challenge was resolved via the use of inductively coupled plasma optical emission spectrometry to analyze ex vivo the silicon content in different tissues and tumors. The results indicated that most of the polyethylene glycol-coated BPSi NPs were found to accumulate in the liver and spleen after intravenous injection. The smallest 140 nm particles accumulated the most in tumors at an amount of 9.5 ± 3.4% of the injected dose (concentration of 0.18 ± 0.08 mg/mL), the amount known to produce sufficient heat for cancer PTT. Furthermore, the findings from the present study also suggest that techniques other than optical imaging should be considered to study the organ biodistribution of NPs with strong light absorbance properties.
Assuntos
Nanopartículas/química , Silício/farmacocinética , Animais , Carbocianinas/química , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Imagem Óptica , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Porosidade , Células RAW 264.7 , Silício/química , Baço/metabolismo , Distribuição TecidualRESUMO
This paper reports the use of a self-assembling hydrogel as a delivery vehicle for the Parkinson's disease drug l-DOPA. Based on a two-component combination of an l-glutamine amide derivative and benzaldehyde, this gel has very soft rheological properties and self-healing characteristics. It is demonstrated that the gel can be formulated to encapsulate l-DOPA. These drug-loaded gels are characterized, and rapid release of the drug is obtained from the gel network. This drug-loaded hydrogel has appropriate rheological characteristics to be amenable for injection. This system is therefore tested as a vehicle for nasal delivery of neurologically-active drugs-a drug delivery strategy that can potentially avoid first pass liver metabolism and bypass the blood-brain barrier, hence enhancing brain uptake. In vitro tests indicate that the gel has biocompatibility with respect to nasal epithelial cells. Furthermore, animal studies demonstrate that the nasal delivery of a gel loaded with 3 H-labeled l-DOPA out-performed a simple intranasal l-DOPA solution. This is attributed to longer residence times of the gel in the nasal cavity resulting in increased blood and brain concentrations. It is demonstrated that the likely routes of brain penetration of intranasally-delivered l-DOPA gel involve the trigeminal and olfactory nerves connecting to other brain regions.
Assuntos
Antiparkinsonianos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Hidrogéis/administração & dosagem , Levodopa/administração & dosagem , Administração Intranasal , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/metabolismoRESUMO
This study investigated the feasibility of lipid polymer hybrid nanoparticles (LPH) as a platform for the combinatorial delivery of small interfering RNA (siRNA) and etoposide (Eto). Different Eto loaded LPH formulations (LPH Eto ) are prepared. The optimized cationic LPH Eto with a particle size of 109.66 ± 5.17 nm and Eto entrapment efficiency (EE %) of 80.33 ± 2.55 is used to incorporate siRNA targeting CD47 (siCD47), a do not eat me marker on the surface of cancer cells. The siRNA-encapsulating LPH (LPH siNEG-Eto ) has a particle size of 115.9 ± 4.11 nm and siRNA EE % of 63.54 ± 4.36 %. LPHs improved the cellular uptake of siRNA in a dose- and concentration-dependent manner. Enhanced cytotoxicity (3.8-fold higher than Eto solution) and high siRNA transfection efficiency (≈50 %) are obtained. An in vivo biodistribution study showed a preferential uptake of the nanosystem into lung, liver, and spleen. In an experimental pseudo-metastatic B16F10 lung tumor model, a superior therapeutic outcome can be observed in mice treated with combinatory therapy. Immunological studies revealed elevated CD4+, CD8+ cells, and macrophages in the lung following combinatory treatment. The study suggests the potential of the current system for combinatory chemotherapy and immunotherapy for the treatment of lung cancer or lung metastasis.
Assuntos
Neoplasias Pulmonares , Melanoma Experimental , Nanopartículas , Animais , Linhagem Celular Tumoral , Etoposídeo/farmacologia , Lipídeos , Pulmão , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Polímeros , RNA Interferente Pequeno , Distribuição TecidualRESUMO
Poly(lactic-co-glycolic acid) (PLGA) is the most commonly described biocompatible copolymer used in biomedical applications. In this work, a green synthetic approach based on the biocompatible zinc proline complex, as an initiator for PLGA synthesis, is reported for the first time for the synthesis of methoxy-poly(ethylene glycol)-block-poly(l-lactic-co-glycolic acid) (mPEG-PLGA). mPEG-PLGA with controlled molecular weight and narrow polydispersity was synthesised. Its potential for delivery of irinotecan (Ir), a poorly water-soluble chemotherapeutic drug used for the treatment of colon and pancreatic cancer, was studied. Nanoparticles of controlled size (140-160 nm), surface charge (â¼-10 mV), release properties and cytotoxicity against CT-26 (colon) and BxPC-3 (pancreatic) cancer cells, were prepared. Tumor accumulation was confirmed by optical imaging of fluorescently labelled nanoparticles. Unlike Tween® 80 coated NP-Ir, the Pluronic® F-127 coated NP-Ir exhibits significant tumor growth delay compared to untreated and blank formulation treated groups in the CT-26 subcutaneous tumor model, after 4 treatments of 30 mg irinotecan per kg dose. Overall, this proof-of-concept study demonstrates that the newly synthesized copolymer, via a green route, is proven to be nontoxic, requires fewer purification steps and has potential applications in drug delivery.
Assuntos
Neoplasias do Colo , Nanopartículas , Preparações Farmacêuticas , Neoplasias do Colo/tratamento farmacológico , Dioxanos , Portadores de Fármacos , Humanos , Irinotecano , Tamanho da Partícula , Polietilenoglicóis , Prolina , ZincoRESUMO
Ion-pairing a lifesaving drug such as theophylline with a targeting moiety could have a significant impact on medical emergencies such as status asthmaticus or COVID-19 induced pneumomediastinum. However, to achieve rapid drug targeting in vivo the ion-pair must be protected against breakdown before the entry into the target tissue. This study aims to investigate if inserting theophylline, when ion-paired to the polyamine transporter substrate spermine, into a cyclodextrin (CD), to form a triplex, could direct the bronchodilator to the lungs selectively after intravenous administration. NMR demonstrates that upon the formation of the triplex spermine protruded from the CD cavity and this results in energy-dependent uptake in A549 cells (1.8-fold enhancement), which persists for more than 20 min. In vivo, the triplex produces a 2.4-fold and 2.2-fold increase in theophylline in the lungs 20 min after injection in rats and mice, respectively (p < 0.05). The lung targeting is selective with no increase in uptake into the brain or the heart where the side-effects of theophylline are treatment-limiting. Selectively doubling the concentration of theophylline in the lungs could improve the benefit-risk ratio of this narrow therapeutic index medicine, which continues to be important in critical care.
RESUMO
Exosomes (Exo)-based therapy holds promise for treatment of lethal pancreatic cancer (PC). Limited understanding of key factors affecting Exo uptake in PC cells restricts better design of Exo-based therapy. This work aims to study the uptake properties of different Exo by PC cells. Exo from pancreatic carcinoma, melanoma and non-cancer cell lines were isolated and characterised for yield, size, morphology and exosomal marker expression. Isolated Exo were fluorescently labelled using a novel in-house developed method based on copper-free click chemistry to enable intracellular tracking and uptake quantification in cells. Important factors influencing Exo uptake were initially predicted by Design of Experiments (DoE) approach to facilitate subsequent actual experimental investigations. Uptake of all Exo types by PC cells (PANC-1) showed time- and dose-dependence as predicted by the DoE model. PANC-1 cell-derived exosomes (PANC-1 Exo) showed significantly higher uptake in PANC-1 cells than that of other Exo types at the longest incubation time and highest Exo dose. In vivo biodistribution studies in subcutaneous tumour-bearing mice similarly showed favoured accumulation of PANC-1 Exo in self-tissue (i.e. PANC-1 tumour mass) over the more vascularised melanoma (B16-F10) tumours, suggesting intrinsic tropism of PC-derived Exo for their parent cells. This study provides a simple, universal and reliable surface modification approach via click chemistry for in vitro and in vivo exosome uptake studies and can serve as a basis for a rationalised design approach for pre-clinical Exo cancer therapies.
RESUMO
In this study, enteric coatings based exclusively on naturally occurring ingredients were reported. Alginate (Alg) and pectin (Pec) blends with or without naturally occurring glyceride, glycerol monostearate (GMS), were initially used to produce solvent-casted films. Incorporating GMS in the natural polymeric films significantly enhanced the acid-resistance properties in gastric medium. Theophylline tablets coated with Alg-Pec blends without GMS disintegrated shortly after incubation in gastric medium (pH 1.2), leading to a premature and complete release of theophylline. Interestingly, tablets coated with Alg-Pec blends that contain the natural glyceride (GMS) resisted the gastric environment for 2 h with minimal drug release (<5%) and disintegrated rapidly following introduction to the intestinal medium, allowing a fast and complete drug release. Furthermore, the coating system proved to be stable for six months under accelerated conditions. These findings are particularly appealing to nutraceutical industry as they provide the foundation to produce naturally-occurring GRAS based enteric coatings.
Assuntos
Alginatos/química , Química Farmacêutica/métodos , Suplementos Nutricionais , Pectinas/química , Comprimidos com Revestimento Entérico/química , Teofilina/administração & dosagem , Varredura Diferencial de Calorimetria , Liberação Controlada de Fármacos , Ácido Gástrico , Glicerídeos/química , Glicerol/química , Concentração de Íons de Hidrogênio , Ácidos Polimetacrílicos , Solubilidade , Teofilina/químicaRESUMO
In this work we describe the formulation and characterisation of red-emitting polymeric nanocapsules (NCs) incorporating superparamagnetic iron oxide nanoparticles (SPIONs) for magnetic tumour targeting. The self-fluorescent oligomers were synthesised and chemically conjugated to PLGA which was confirmed by NMR spectroscopy, FT-IR spectroscopy and mass spectrometry. Hydrophobic SPIONs were synthesised through thermal decomposition and their magnetic and heating properties were assessed by SQUID magnetometry and calorimetric measurements, respectively. Magnetic nanocapsules (m-NCs) were prepared by a single emulsification/solvent evaporation method. Their in vitro cytotoxicity was examined in CT26 colon cancer cells. The formulated fluorescent m-NCs showed good stability and biocompatibility both in vitro and in vivo in CT 26 colon cancer models. Following intravenous injection, accumulation of m-NCs in tumours was observed by optical imaging. A higher iron content in the tumours exposed to a magnetic field, compared to the contralateral tumours without magnetic exposure in the same animal, further confirmed the magnetic tumour targeting in vivo. The overall results show that the engineered red-emitting m-NCs have great potential as multifunctional nanocarriers for multi-model bioimaging and magnetic-targeted drug delivery.
Assuntos
Compostos Férricos/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Nanocápsulas/administração & dosagem , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Feminino , Compostos Férricos/farmacocinética , Corantes Fluorescentes/farmacocinética , Hipertermia Induzida , Ferro/metabolismo , Fenômenos Magnéticos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacocinética , Poliglactina 910/administração & dosagem , Poliglactina 910/farmacocinética , Distribuição TecidualRESUMO
Asenapine maleate (ASPM) is an antipsychotic drug prescribed for the treatment of schizophrenia and bipolar disorder. ASPM possesses low oral bioavailability due to extensive hepatic metabolism. Therefore, RGD peptide conjugated liposomes loaded with ASPM were prepared to target Peyer's patches in the intestine which in-turn get access into intestinal lymphatic system thereby increasing the oral bioavailability of the drug. Liposomes were evaluated for size, zeta potential, differential scanning calorimetry (DSC), FTIR spectroscopy, X-ray diffraction (XRD), shape and morphology, in vitro drug release, cell line studies, everted intestinal uptake, pharmacodynamics, pharmacokinetics, tissue distribution, targetability and stability studies. In vitro drug release study showed the sustained release of drug from the formulations. Optimized liposomes (size <110 nm) showed greater permeability across the Caco2 + Raji B co-culture model in vitro and everted rat ileum ex vivo. Liposomes showed increase in bioavailability and high efficacy in reducing the L-DOPA-carbidopa induced locomotor count compared to plain drug. Liposomes also showed high concentration of drug in the brain after their oral administration. Imaging studies showed that RGD peptide conjugated liposomes were successful in targeting the Peyer's patches, both in vivo and ex vivo. The study successfully demonstrated the improved pharmacokinetics and efficacy profile of ASPM by using a ligand conjugated targeted liposomal system.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis , Nanoestruturas , Nódulos Linfáticos Agregados/metabolismo , Animais , Células CACO-2 , Dibenzocicloeptenos , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Lipossomos , Masculino , Nanoestruturas/química , Nanoestruturas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Propriedades de SuperfícieRESUMO
Apoptotic cells and cell fragments, especially those produced as a result of immunogenic cell death (ICD), are known to be a potential source of cancer vaccine immunogen. However, due to variation between tumours and between individuals, methods to generate such preparations may require extensive ex vivo personalisation. To address this, we have utilised the concept of in situ vaccination whereby an ICD inducing drug is injected locally to generate immunogenic apoptotic fragments/cells. These fragments are then adjuvanted by a co-administered cell reactive CpG adjuvant. We first evaluate means of labelling tumour cells with CpG adjuvant, we then go on to demonstrate in vitro that labelling is preserved following apoptosis and, furthermore, that the apoptotic body-adjuvant complexes are readily transferred to macrophages. In in vivo studies we observe synergistic tumour growth delays and elevated levels of CD4+ and CD8+ cells in tumours receiving adjuvant drug combination. CD4+/CD8+ cells are likewise elevated in the tumour draining lymph node and activated to a greater extent than individual treatments. This study represents the first steps toward the evaluation of rationally formulated drug-adjuvant combinations for in situ chemo-immunotherapy.
Assuntos
Vacinas Anticâncer , Preparações Farmacêuticas , Linfócitos T CD8-Positivos , Células Dendríticas , Humanos , Morte Celular Imunogênica , ImunoterapiaRESUMO
Three-dimensional (3D) cell cultures and organs-on-a-chip have been developed to construct microenvironments that resemble the environment within the human body and to provide a platform that enables clear observation and accurate assessments of cell behavior. However, direct observation of transendothelial electrical resistance (TEER) has been challenging. To improve the efficiency in monitoring the cell development in organs-on-a-chip, in this study, we designed and integrated commercially available TEER measurement electrodes into an in vitro blood-brain barrier (BBB)-on-chip system to quantify TEER variation. Moreover, a flowing culture medium was added to the monolayered cells to simulate the promotion of continuous shear stress on cerebrovascular cells. Compared with static 3D cell culture, the proposed BBB-on-chip integrated with electrodes could measure TEER in a real-time manner over a long period. It also allowed cell growth angle measurement, providing instant reports of cell growth information online. Overall, the results demonstrated that the developed system can aid in the quantification of the continuous cell-pattern variations for future studies in drug testing.