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1.
J Control Release ; 342: 337-344, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34973307

RESUMO

Systemic administration of toll-like receptor (TLR) agonists have demonstrated impressive preclinical results as an anti-cancer therapy due to their potent innate immune-stimulatory properties. The clinical advancement has, however, been hindered by severe adverse effects due to systemic activation of the immune system. Liposomal drug delivery systems may modify biodistribution, cellular uptake, and extend blood circulation, and thus, potentially enable systemic administration of TLR agonists at therapeutic doses. In this study, we investigated potential barriers for the administration of TLR agonists formulated in polyethylene glycosylated (PEGylated) liposomes with regards to liposome formulation, TLR agonist, administration route, administration schedule, biodistribution, blood clearance, and anti-PEG antibodies. We found that administration of TLR agonists formulated in PEGylated liposomes led to high anti-PEG antibody titers, which upon multiple intravenous administrations, resulted in accelerated blood clearance and acute hypersensitivity reactions. The latter was found to be associated with anti-PEG IgG antibody and not anti-PEG IgM antibody opsonization. This study highlights the need to carefully design and evaluate nanoparticle delivery systems for immunotherapy as anti-nanoparticle immune responses may challenge the therapeutic application.


Assuntos
Lipossomos , Nanopartículas , Imunoglobulina M , Polietilenoglicóis , Distribuição Tecidual
2.
Sci Rep ; 11(1): 19794, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34611284

RESUMO

Adoptive T-cell transfer (ACT) offers a curative therapeutic option for subsets of melanoma and hematological cancer patients. To increase response rates and broaden the applicability of ACT, it is necessary to improve the post-infusion performance of the transferred T cells. The design of improved treatment strategies includes transfer of cells with a less differentiated phenotype. Such T cell subsets have high proliferative potential but require stimulatory signals in vivo to differentiate into tumor-reactive effector T cells. Thus, combination strategies are needed to support the therapeutic implementation of less differentiated T cells. Here we show that systemic delivery of tumor-associated antigens (TAAs) facilitates in vivo priming and expansion of previously non-activated T cells and enhance the cytotoxicity of activated T cells. To achieve this in vivo priming, we use flexible delivery vehicles of TAAs and a TLR7/8 agonist. Contrasting subcutaneous delivery systems, these vehicles accumulate TAAs in the spleen, thereby achieving close proximity to both cross-presenting dendritic cells and transferred T cells, resulting in robust T-cell expansion and anti-tumor reactivity. This TAA delivery platform offers a strategy to safely potentiate the post-infusion performance of T cells using low doses of antigen and TLR7/8 agonist, and thereby enhance the effect of ACT.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Imunoterapia Adotiva , Neoplasias/imunologia , Neoplasias/terapia , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/administração & dosagem , Biomarcadores , Terapia Combinada , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Epitopos/administração & dosagem , Epitopos/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunomodulação , Imunoterapia Adotiva/métodos , Interferon Tipo I/biossíntese , Lipossomos , Ativação Linfocitária/imunologia , Neoplasias/diagnóstico , Neoplasias/mortalidade , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Resultado do Tratamento , Evasão Tumoral/imunologia
3.
Nanotheranostics ; 5(4): 448-460, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34055574

RESUMO

Increasing numbers of lung tumors are identified at early disease stages by diagnostic imaging in screening programs, but difficulties in locating these during surgical intervention has prevented an improved treatment outcome. Surgical biomarkers that are visible on diagnostic images, and that provide the surgeon with real-time image guidance during the intervention are thus highly warranted to bridge diagnostic precision into enhanced therapeutic outcome. In this paper, a liquid soft tissue marker for near infrared fluorescence and radio-guidance is presented. The biocompatible marker is based on the carbohydrate ester, sucrose acetate isobutyrate, ethanol, and a multifunctional naphthalocyanine dye, which enable near infrared fluorescence image-guided resection at short, medium and long tissue depths. Naphthalocyanine dyes have high quantum yields and may further act as chelators of radionuclides. Upon injection of the liquid marker, a gel-like depot is formed in situ at the site of injection, wherein the fluorescent dye and radionuclide is retained. The radiolabeled markers were optimized for minimal fluorescence quenching and high retention of the positron emission tomography radionuclide 64Cu. The performance of the radiolabeled marker was tested in vivo in mice, where it displayed high photostability over a period of 4 weeks, and high retention of 64Cu for 48 hours. The retention and biodistribution of 64Cu was quantified via PET/CT, and the fluorescence emission by an in vivo imaging system. The presented data demonstrate proof-of-concept for naphthalocyanine markers as multimodal imaging agents that can bridge the precision of diagnostic imaging into surgical interventions.


Assuntos
Radioisótopos de Cobre , Corantes Fluorescentes , Neoplasias Pulmonares , Imagem Óptica , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos , Animais , Carboidratos/química , Carboidratos/farmacocinética , Carboidratos/farmacologia , Radioisótopos de Cobre/química , Radioisótopos de Cobre/farmacocinética , Radioisótopos de Cobre/farmacologia , Feminino , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Corantes Fluorescentes/farmacologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/cirurgia , Camundongos , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia
4.
Adv Healthc Mater ; 10(10): e2002009, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33763995

RESUMO

Local application of radioactive sources as brachytherapy is well established in oncology. This treatment is highly invasive however, due to the insertion of millimeter sized metal seeds. The authors report the development of a new concept for brachytherapy, based on gold-palladium (AuPd) alloy nanoparticles, intrinsically radiolabeled with 103 Pd. These are formulated in a carbohydrate-ester based liquid, capable of forming biodegradable gel-like implants upon injection. This allows for less invasive administration through small-gauge needles. [103 Pd]AuPd nanoparticles with sizes around 20 nm are prepared with radiolabeling efficiencies ranging from 79% to >99%. Coating with the hydrophobic polymer poly(N-isopropylacrylamide) leads to nanoparticle diameters below 40 nm. Dispersing the nanoparticles in ethanol with water insoluble carbohydrate esters gives "nanogels", a low viscosity liquid capable of solidifying upon injection into aqueous environments. Both nanoparticles and radioactivity are stably retained in the nanogel over 25 days (>99%) after formation in aqueous buffers. Animals bearing CT26 murine tumors are injected intratumorally with 25 MBq of the 103 Pd-nanogel, and display tumor growth delay and significantly increase median survival times compared with control groups. Excellent retention in the tumor of both the 103 Pd and the nanoparticle matrix itself is observed, demonstrating a potential for replacing currently used brachytherapy seeds.


Assuntos
Braquiterapia , Nanopartículas Metálicas , Nanopartículas , Ligas , Animais , Ouro , Camundongos , Paládio
5.
Nanoscale ; 12(43): 22298-22306, 2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33146209

RESUMO

The main structural element defining the cell is the lipid membrane, which is an integral part of regulating the fluxes of ion and nutrition molecules in and out of the cell. Surprisingly, copper ions were found to have anomalous membrane permeability. This led us to consider a broader spectrum of cations and further a new approach for using liposomes as nanoreactors for synthesis of metal and metal alloy nanoparticles. In the present study, the high membrane permeability of Cu2+ and its neighbouring transition elements in the periodic table was investigated. The permeability of Ni2+, Cu2+, Zn2+, Ag+, Au3+, Mg2+, Ca2+ and Lu3+ was assessed, and we report that Zn2+, Cu2+, Ag+ and Au3+ surprisingly are able to cross lipid bilayers. This knowledge is highly relevant for understanding trafficking of cations in biological systems, as well as for design of novel nanoparticle and nanoreactor systems. An example of its use is presented as a platform for synthesizing single highly uniform gold nanoparticles inside liposomal nanoreactors. We envision that this approach could provide a new nanoreactor methodology for forming highly structurally constrained uniform metal and metal alloy nanoparticles, as well as new methods for in vivo tracking of liposomes.


Assuntos
Nanopartículas Metálicas , Cobre , Ouro , Nanotecnologia , Permeabilidade , Prata , Zinco
6.
Sci Adv ; 6(34): eabb5353, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32875113

RESUMO

Diagnostic imaging often outperforms the surgeon's ability to identify small structures during therapeutic procedures. Smart soft tissue markers that translate the sensitivity of diagnostic imaging into optimal therapeutic intervention are therefore highly warranted. This paper presents a unique adaptable liquid soft tissue marker system based on functionalized carbohydrates (Carbo-gel). The liquid state of these markers allows for high-precision placement under image guidance using thin needles. Based on step-by-step modifications, the image features and mechanical properties of markers can be optimized to bridge diagnostic imaging and specific therapeutic interventions. The performance of Carbo-gel is demonstrated for markers that (i) have radiographic, magnetic resonance, and ultrasound visibility; (ii) are palpable and visible; and (iii) are localizable by near-infrared fluorescence and radio guidance. The study demonstrates encouraging proof of concept for the liquid marker system as a well-tolerated multimodal imaging marker that can improve image-guided radiotherapy and surgical interventions, including robotic surgery.


Assuntos
Marcadores Fiduciais , Radioterapia Guiada por Imagem , Imageamento por Ressonância Magnética/métodos , Agulhas , Imagens de Fantasmas , Radioterapia Guiada por Imagem/métodos
7.
Nanotheranostics ; 4(3): 142-155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32483520

RESUMO

Liquid brachytherapy is an emerging technology for internal radiation therapy where liquids containing radionuclides are administered directly into solid tumors. These technologies are less invasive than conventional brachytherapy, and can potentially improve the dose coverage and homogeneity of the radioactivity distribution within the tumor. For this purpose, we have developed a novel cationic micelle system for delivery of a range of radionuclides. The system is applicable for emitters of alpha, beta or photon radiation, and enables dose-mapping via theranostic nuclear imaging. Methods: The cationic micelles were developed as linear surfactants comprising the chelator DOTA, a triarginine sequence and a palmitoyl or stearoyl fatty acid chain. The critical micelle concentration of the surfactants was determined, and the micelles were radiolabelled with 64Cu or 177Lu in high radiochemical purity (>95%). The tumor retention and biodistribution of the 64Cu-radiolabeled surfactants, administered as micelles or formulated in liposomes, were investigated in vivo by PET/CT in a tumor bearing mouse model. Results: The interaction of the micelles with anionic lipid membranes was demonstrated to be favourable, using a liposome partition assay. In vivo, the surfactants formulated both as cationic micelles and liposomes displayed the best intratumoral retention, with micelles providing more homogeneous activity distribution. Conclusion: A cationic, surfactant-based drug delivery system was developed and demonstrated promise as a vehicle for liquid brachytherapy when formulated as micelles or in liposomes. The system enables accurate dosimetry due to the flexible radiochemistry of DOTA.


Assuntos
Braquiterapia/métodos , Sistemas de Liberação de Medicamentos/métodos , Compostos Heterocíclicos com 1 Anel/química , Nanoconjugados/química , Nanomedicina Teranóstica/métodos , Animais , Arginina/química , Linhagem Celular Tumoral , Radioisótopos de Cobre , Feminino , Lipídeos/química , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Tomografia por Emissão de Pósitrons , Tensoativos/química , Distribuição Tecidual
8.
Int J Nanomedicine ; 13: 7647-7656, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30538449

RESUMO

BACKGROUND: Active, ligand-mediated, targeting of functionalized liposomes to folate receptors (FRs) overexpressed on cancer cells could potentially improve drug delivery and specificity. Studies on folate-targeting liposomes (FTLs) have, however, yielded varying results and generally fail to display a clear benefit of FR targeting. METHOD: Tumor accumulating potential of FTLs and NTLs were investigated in a FR overex-pressing xenograft model by positron emission tomography/computed tomography imaging. RESULTS: Tumors displayed significantly lower activity of FTLs than NTLs. Furthermore, FTLs displayed worse circulating properties and increased liver-accumulation than NTLs. CONCLUSION: This study underlines that long-circulating properties of liposomes must be achieved to take advantage of EPR-dependent tumor accumulation which may be lost by functionalization. FR-functionalization negatively affected both tumor accumulation and circulation properties.


Assuntos
Carcinoma/patologia , Receptores de Folato com Âncoras de GPI/metabolismo , Compostos Radiofarmacêuticos/química , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Feminino , Ácido Fólico/administração & dosagem , Humanos , Cinética , Ligantes , Lipossomos , Camundongos , Distribuição Tecidual
9.
ACS Nano ; 12(11): 11386-11398, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30372038

RESUMO

The enhanced permeability and retention (EPR) effect increases tumor accumulation of liposomal chemotherapy and should, in theory, increase anticancer effects and lower toxicity. Unfortunately, liposomal chemotherapy has generally not met the expected potential, perhaps because the EPR effect is not ubiquitous. PET imaging using radiolabeled liposomes can identify cancers positive for the EPR effect. In the current study, we show in clinical canine cancer patients that repeated imaging with radiolabeled liposomes (64Cu-liposome) induces the accelerated blood clearance (ABC) phenomenon. This was observed even with very long intervals between PEGylated liposome injections, which contradict previous reporting in experimental animal models. The induction of ABC may be devastating for the theranostic use of liposomal imaging, as this could vaccinate patients against therapeutic efficacy. To investigate and solve this important problem, an additional study part was designed in which rats were subjected to repeated liposomal administrations, including stealth 64Cu-liposome PET imaging and Caelyx chemotherapy. Most importantly, it was found that, by increasing the lipid dose at the first injection or by supplying a small predose before the second 64Cu-liposome injection, ABC could be prevented. Importantly, signs of liposome tracer breakdown with subsequent renal excretion were observed. These findings highlight the importance of the ABC phenomenon for liposomal predictive imaging in a clinically relevant setting and show that carefully planned application is central to avoid potential detrimental effects on patient benefit.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , Neoplasias/tratamento farmacológico , Polietilenoglicóis/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Nanomedicina Teranóstica , Vacinação , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/química , Modelos Animais de Doenças , Cães , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/farmacocinética , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Ratos , Ratos Wistar , Distribuição Tecidual
10.
Theranostics ; 8(21): 5828-5841, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30613265

RESUMO

Long circulating liposomes entrapping iodinated and radioiodinated compounds offer a highly versatile theranostic platform. Here we report a new methodology for efficient and high-yield loading of such compounds into liposomes, enabling CT/SPECT/PET imaging and 131I-radiotherapy. Methods: The CT contrast agent diatrizoate was synthetically functionalized with a primary amine, which enabled its remote loading into PEGylated liposomes by either an ammonium sulfate- or a citrate-based pH transmembrane gradient. Further, the amino-diatrizoate was radiolabeled with either 124I (t1/2 = 4.18 days) for PET or 125I (t1/2 = 59.5 days) for SPECT, through an aromatic Finkelstein reaction. Results: Quantitative loading efficiencies (>99%) were achieved at optimized conditions. The 124I-labeled compound was remote-loaded into liposomes, with an overall radiolabeling efficiency of 77 ± 1%, and imaged in vivo in a CT26 murine colon cancer tumor model by PET/CT. A prolonged blood circulation half-life of 19.5 h was observed for the radiolabeled liposomes, whereas injections of the free compound were rapidly cleared. Lower accumulation was observed in the spleen, liver, kidney and tumor than what is usually seen for long-circulating liposomes. Conclusion: The lower accumulation was interpreted as release of the tracer from the liposomes within these organs after accumulation. These results may guide the design of systems for controlled release of remote loadable drugs from liposomes.


Assuntos
Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/terapia , Meios de Contraste/administração & dosagem , Lipossomos/administração & dosagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Radioterapia/métodos , Animais , Neoplasias do Colo/patologia , Diatrizoato/administração & dosagem , Modelos Animais de Doenças , Radioisótopos do Iodo/administração & dosagem , Camundongos , Nanomedicina Teranóstica/métodos
11.
J Control Release ; 269: 100-109, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29122662

RESUMO

Liposomes are nanoparticles used in drug delivery that distribute over several days in humans and larger animals. Radiolabeling with long-lived positron emission tomography (PET) radionuclides, such as manganese-52 (52Mn, T½=5.6days), allow the imaging of this biodistribution. We report optimized protocols for radiolabeling liposomes with 52Mn, through both remote-loading and surface labeling. For comparison, liposomes were also remote-loaded and surface labeled with copper-64 (64Cu, T½=12.7h) through conventional means. The chelator DOTA was used in all cases. The in vivo stability of radiometal chelates is widely debated but studies that mimic a realistic in vivo setting are lacking. Therefore, we employed these four radiolabeled liposome types as platforms to demonstrate a new concept for such in vivo evaluation, here of the chelates 52Mn-DOTA and 64Cu-DOTA. This was done by comparing "shielded" remote-loaded with "exposed" surface labeled variants in a CT26 tumor-bearing mouse model. Remote loading (90min at 55°C) and surface labeling (55°C for 2h) of 52Mn gave excellent radiolabeling efficiencies of 97-100% and 98-100% respectively, and the liposome biodistribution was imaged by PET for up to 8days. Liposomes with surface-conjugated 52Mn-DOTA exhibited a significantly shorter plasma half-life (T½=14.4h) when compared to the remote-loaded counterpart (T½=21.3h), whereas surface-conjugated 64Cu-DOTA cleared only slightly faster and non-significantly, when compared to remote-loaded (17.2±2.9h versus 20.3±1.2h). From our data, we conclude the successful remote-loading of liposomes with 52Mn, and furthermore that 52Mn-DOTA may be unstable in vivo whereas 64Cu-DOTA appears suitable for quantitative imaging.


Assuntos
Quelantes/administração & dosagem , Radioisótopos de Cobre/administração & dosagem , Compostos Heterocíclicos com 1 Anel/administração & dosagem , Manganês/administração & dosagem , Radioisótopos/administração & dosagem , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Linhagem Celular Tumoral , Quelantes/farmacocinética , Radioisótopos de Cobre/farmacocinética , Compostos Heterocíclicos com 1 Anel/farmacocinética , Lipossomos , Manganês/farmacocinética , Camundongos Endogâmicos BALB C , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual
12.
Methods Mol Biol ; 1548: 159-180, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28013503

RESUMO

There is considerable interest in understanding the interactions of antimicrobial peptides with phospholipid membranes. Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that can be used to gain insight into these interactions. Specifically, FCS can be used to quantify leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles, thereby providing a tool for estimating the size of peptide-induced membrane disruptions. If fluorescently labeled lipids are incorporated into the membranes of the vesicles, FCS can also be used to obtain information about whether leakage occurs due to localized membrane perturbations or global membrane destabilization. Here, we outline a detailed step-by-step protocol on how to optimally implement an FCS-based leakage assay. To make the protocol easily accessible to other researchers, it has been supplemented with a number of practical tips and tricks.


Assuntos
Membrana Celular/química , Peptídeos/química , Espectrometria de Fluorescência , Algoritmos , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Modelos Químicos , Peptídeos/metabolismo , Fosfolipídeos/química , Espectrometria de Fluorescência/métodos
13.
ACS Appl Mater Interfaces ; 7(41): 22796-806, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26426093

RESUMO

Due to low ion permeability of lipid bilayers, it has been and still is common practice to use transporter molecules such as ionophores or lipophilic chelators to increase transmembrane diffusion rates and loading efficiencies of radionuclides into liposomes. Here, we report a novel and very simple method for loading the positron emitter (64)Cu(2+) into liposomes, which is important for in vivo positron emission tomography (PET) imaging. By this approach, copper is added to liposomes entrapping a chelator, which causes spontaneous diffusion of copper across the lipid bilayer where it is trapped. Using this method, we achieve highly efficient (64)Cu(2+) loading (>95%), high radionuclide retention (>95%), and favorable loading kinetics, excluding the use of transporter molecule additives. Therefore, clinically relevant activities of 200-400 MBq/patient can be loaded fast (60-75 min) and efficiently into preformed stealth liposomes avoiding subsequent purification steps. We investigate the molecular coordination of entrapped copper using X-ray absorption spectroscopy and demonstrate high adaptability of the loading method to pegylated, nonpegylated, gel- or fluid-like, cholesterol rich or cholesterol depleted, cationic, anionic, and zwitterionic lipid compositions. We demonstrate high in vivo stability of (64)Cu-liposomes in a large canine model observing a blood circulation half-life of 24 h and show a tumor accumulation of 6% ID/g in FaDu xenograft mice using PET imaging. With this work, it is demonstrated that copper ions are capable of crossing a lipid membrane unassisted. This method is highly valuable for characterizing the in vivo performance of liposome-based nanomedicine with great potential in diagnostic imaging applications.


Assuntos
Radioisótopos de Cobre/química , Lipossomos/química , Animais , Linhagem Celular Tumoral , Cromatografia em Gel , Cães , Humanos , Transporte de Íons , Cinética , Lipídeos/química , Masculino , Camundongos Nus , Compostos Organometálicos/química , Tomografia por Emissão de Pósitrons , Distribuição Tecidual , Tomografia Computadorizada por Raios X , Espectroscopia por Absorção de Raios X , Ensaios Antitumorais Modelo de Xenoenxerto
15.
PLoS One ; 10(5): e0122419, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25932639

RESUMO

Cationic membrane-active peptides have been studied for years in the hope of developing them into novel types of therapeutics. In this article, we investigate an effect that might have significant experimental implications for investigators who wish to study these peptides, namely, that the peptides adsorb to solid surfaces of glass and plastic. Specifically, we use analytical HPLC to systematically quantify the adsorption of the three cationic membrane-active peptides mastoparan X, melittin, and magainin 2 to the walls of commonly used glass and plastic sample containers. Our results show that, at typical experimental peptide concentrations, 90% or more of the peptides might be lost from solution due to rapid adsorption to the walls of the sample containers. Thus, our results emphasize that investigators should always keep these adsorption effects in mind when designing and interpreting experiments on cationic membrane-active peptides. We conclude the article by discussing different strategies for reducing the experimental impact of these adsorption effects.


Assuntos
Vidro/química , Peptídeos/isolamento & purificação , Plásticos/química , Adsorção , Sequência de Aminoácidos , Cátions , Cromatografia Líquida de Alta Pressão , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Dados de Sequência Molecular , Peptídeos/química , Padrões de Referência , Cloreto de Sódio/química , Soluções , Propriedades de Superfície
16.
ACS Nano ; 9(7): 6985-95, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26022907

RESUMO

Since the first report of the enhanced permeability and retention (EPR) effect, the research in nanocarrier based antitumor drugs has been intense. The field has been devoted to treatment of cancer by exploiting EPR-based accumulation of nanocarriers in solid tumors, which for many years was considered to be a ubiquitous phenomenon. However, the understanding of differences in the EPR-effect between tumor types, heterogeneities within each patient group, and dependency on tumor development stage in humans is sparse. It is therefore important to enhance our understanding of the EPR-effect in large animals and humans with spontaneously developed cancer. In the present paper, we describe a novel loading method of copper-64 into PEGylated liposomes and use these liposomes to evaluate the EPR-effect in 11 canine cancer patients with spontaneous solid tumors by PET/CT imaging. We thereby provide the first high-resolution analysis of EPR-based tumor accumulation in large animals. We find that the EPR-effect is strong in some tumor types but cannot be considered a general feature of solid malignant tumors since we observed a high degree of accumulation heterogeneity between tumors. Six of seven included carcinomas displayed high uptake levels of liposomes, whereas one of four sarcomas displayed signs of liposome retention. We conclude that nanocarrier-radiotracers could be important in identifying cancer patients that will benefit from nanocarrier-based therapeutics in clinical practice.


Assuntos
Carcinoma/diagnóstico por imagem , Radioisótopos de Cobre/administração & dosagem , Lipossomos/farmacocinética , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Carcinoma/veterinária , Radioisótopos de Cobre/farmacocinética , Cães , Feminino , Lipossomos/química , Masculino , Imagem Multimodal , Polietilenoglicóis/química , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/farmacocinética , Tomografia Computadorizada por Raios X
17.
Langmuir ; 31(8): 2472-83, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25664684

RESUMO

The capability of membrane-active peptides to disrupt phospholipid membranes is often studied by investigating peptide-induced leakage of quenched fluorescent molecules from large unilamellar lipid vesicles. In this article, we explore two fluorescence microscopy-based single-vesicle detection methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles. Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles dispersed in aqueous solution, and we use confocal imaging of surface-immobilized large unilamellar lipid vesicles to investigate whether there are heterogeneities in leakage between individual vesicles. Of importance, we design an experimental protocol that allows us to quantitatively correlate the results of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and magainin 2. The FCS results show that leakage induced by magainin 2 is less dependent on the size of the encapsulated fluorescent molecules than leakage induced by mastoparan X and melittin. The confocal imaging results show that all three peptides induce leakage by a heterogeneous process in which one portion of the vesicles are completely emptied of their contents but another portion of the vesicles are only partially emptied. These pieces of information regarding leakage induced by mastoparan X, melittin, and magainin 2 could not readily have been obtained by the established assays for studying peptide-induced leakage from lipid vesicles.


Assuntos
Bicamadas Lipídicas/química , Peptídeos/química , Fosfolipídeos/química , Microscopia de Fluorescência
18.
Expert Opin Drug Deliv ; 12(7): 1045-58, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25682882

RESUMO

OBJECTIVES: Monocytes are one of the major phagocytic cells that patrol for invading pathogens, and upon activation, differentiate into macrophages or antigen-presenting dendritic cells (DCs) capable of migrating to lymph nodes eliciting an adaptive immune response. The key role in regulating adaptive immune responses has drawn attention to modulate monocyte responses therapeutically within cancer, inflammation and infectious diseases. We present a technology for targeting of monocytes and delivery of a toll-like receptor (TLR) agonist in fresh blood using liposomes with a positively charged surface chemistry. METHODS: Liposomes were extruded at 100 nm, incubated with fresh blood, followed by leukocyte analyses by FACS. Liposomes with and without the TLR7 agonist TMX-202 were incubated with fresh blood, and monocyte activation measured by cytokine secretion by ELISA and CD14 and DC-SIGN expression. RESULTS: The liposomes target monocytes specifically over lymphocytes and granulocytes in human whole blood, and show association with 75 - 95% of the monocytes after 1 h incubation. Formulations of TMX-202 in cationic liposomes were potent in targeting and activation of monocytes, with strong induction of IL-6 and IL-12p40, and differentiation into CD14(+) and DC-SIGN+ DCs. CONCLUSION: Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory cytokines. We envision this technology as a promising tool in future cancer immunotherapy.


Assuntos
Adenina/análogos & derivados , Células Dendríticas/imunologia , Glicerofosfolipídeos/farmacologia , Monócitos/efeitos dos fármacos , Receptor 7 Toll-Like/agonistas , Adenina/administração & dosagem , Adenina/farmacologia , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Glicerofosfolipídeos/administração & dosagem , Humanos , Subunidade p40 da Interleucina-12/metabolismo , Lectinas Tipo C/metabolismo , Lipossomos , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Receptores de Superfície Celular/metabolismo
19.
Nat Protoc ; 9(12): 2841-58, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25411952

RESUMO

Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Nanotecnologia/métodos , Resinas Acrílicas/química , Calibragem , Ácidos Carboxílicos , Endossomos/química , Desenho de Equipamento , Fluoresceína , Corantes Fluorescentes , Células HeLa/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Lisossomos/química , Macrolídeos/farmacologia , Nanopartículas/química , Rodaminas
20.
Biochim Biophys Acta ; 1838(12): 2994-3002, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25135662

RESUMO

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecules from large unilamellar lipid vesicles. Specifically, we derive the mathematical framework required for using FCS to quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, and we describe the appropriate methodology for successful completion of FCS experiments. By use of this methodology, we show that FCS can be used to accurately quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, including leakage of fluorescent molecules of different sizes. To demonstrate the applicability of FCS, we have investigated the antimicrobial peptide mastoparan X. We show that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar lipid vesicles.

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