RESUMEN
Nanomedicines have been touted as the future of cancer therapy for decades. However, the field of tumor-targeted nanomedicine has failed to significantly advance toward becoming the primary choice for cancer intervention. One of the largest obstacles that has yet to be overcome is off-target accumulation of the nanoparticles. We propose a novel approach to tumor delivery by focusing on decreasing off-target accumulation of nanomedicines rather than directly increasing tumor delivery. Acknowledging a poorly understood "refractory" response to intravenously injected gene therapy vectors observed in ours and other studies, we hypothesize that virus-like particles (lipoplexes) can be utilized to initiate an anti-viral innate immune response that limits off-target accumulation of subsequently administered nanoparticles. Indeed, our results show a significant reduction in the deposition of both dextran and Doxil® in major organs with a concurrent increase in plasma and tumor accumulation when injection occurred 24 h after a lipoplex injection. Furthermore, our data showing that the direct injection of interferon lambda (IFN-λ) is capable of eliciting this response demonstrates a central role for this type III interferon in limiting accumulation in non-tumor tissues.
Asunto(s)
Liposomas , Neoplasias , Humanos , Interferón lambda , Sistemas de Liberación de Medicamentos , Neoplasias/terapia , Inmunidad Innata , NanomedicinaRESUMEN
It is becoming increasingly clear that the intravenous administration of nanoparticles elicits an immune response that compromises delivery efficiency and can be life threatening. This study investigated both the systemic and tissue-level cytokine response to repeat administration of lipoplexes coated with either lactose or PEG. We report that blood cytokine levels differ significantly from that observed in individual tissues. While we consistently observed a reduced cytokine response to lactosylated particles, this did not result in enhanced delivery or expression as compared to PEGylated formulations. We also document that repeat injection did not increase plasmid levels in the liver, lung, or spleen, but delivery to the tumor was enhanced under these conditions. In addition, we show that changes in neither blood nor tissue cytokines correlated strongly with reporter gene expression, and we observed relatively constant expression efficiencies (RLU/ng plasmid) across all tissues despite a considerably reduced cytokine response in the tumor. Together, these results indicate that both biodistribution and cytokine responses are dramatically altered by a repeat intravenous injection of lipoplexes, and that the mechanisms regulating reporter gene expression are not straightforward.
Asunto(s)
Citocinas , Neoplasias , Animales , Técnicas de Transferencia de Gen , Liposomas , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Plásmidos , Distribución Tisular , TransfecciónRESUMEN
Studies have suggested imatinib mesylate (ImM) as a potential treatment for systemic lupus erythematosus nephritis (SLEN). However, ImM has limited renal excretion. The goal of the current research was to develop an ImM containing nanoformulation, conduct studies to evaluate pharmacokinetics, and determine whether kidney deposition can be enhanced in a mouse model of SLEN. A fish oil-based ImM oil-in-water nanoemulsion was developed and characterized for particle size, zeta potential, pH, and stability. MRL/MpJ-Faslrp (model of SLEN) and MRL/MpJ (control) mice (12-13 weeks) received one dose of ImM as either a nanoemulsion or naked drug. Pharmacokinetics and kidney deposition studies were performed. Statistics were conducted with a student's T-test. The nanoemulsion characteristics included particle size range of 60-80 nm, zeta potential of -6.6 to -7.8 mV, polydispersity index < 0.3, 3-day stability at 4 °C, and limited ImM leakage from the nanoemulsion in serum. Pharmacokinetics of the nanoformulation showed changes to pharmacokinetic parameters suggesting reduced systemic exposures (with reduced potential for toxicities) to ImM. Kidney deposition of ImM was threefold higher after 4 h in the MRL/MpJ-Faslrp mice that received the nanoformulation vs. naked drug. The current study showed encouraging results for development of a stable and well-characterized nanoemulsion for optimizing kidney deposition of ImM. Future strategies will define dose-efficacy and dose-toxicity relationships and evaluate approaches to further enhance kidney delivery and optimize deposition to the mesangial location of the kidney.
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Nefritis Lúpica , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Mesilato de Imatinib/uso terapéutico , Riñón , Nefritis Lúpica/tratamiento farmacológico , Masculino , Ratones , Tamaño de la PartículaRESUMEN
As mRNA vaccines became the frontrunners in late-stage clinical trials to fight the COVID-19 pandemic, challenges surrounding their formulation and stability became readily apparent. In this commentary, we first describe company proposals, based on available public information, for the (frozen) storage of mRNA vaccine drug products across the vaccine supply chain. We then review the literature on the pharmaceutical stability of mRNA vaccine candidates, including attempts to improve their stability, analytical techniques to monitor their stability, and regulatory guidelines covering product characterization and storage stability. We conclude that systematic approaches to identify the key physicochemical degradation mechanism(s) of formulated mRNA vaccine candidates are currently lacking. Rational design of optimally stabilized mRNA vaccine formulations during storage, transport, and administration at refrigerated or ambient temperatures should thus have top priority in the pharmaceutical development community. In addition to evidence of human immunogenicity against multiple viral pathogens, including compelling efficacy results against COVID-19, another key strength of the mRNA vaccine approach is that it is readily adaptable to rapidly address future outbreaks of new emerging infectious diseases. Consequently, we should not wait for the next pandemic to address and solve the challenges associated with the stability and storage of formulated mRNA vaccines.
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Vacunas contra la COVID-19/química , COVID-19/prevención & control , Potencia de la Vacuna , Vacunas Sintéticas/química , Vacuna nCoV-2019 mRNA-1273 , Vacuna BNT162 , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Frío , Estabilidad de Medicamentos , Almacenaje de Medicamentos/métodos , Humanos , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/inmunología , SARS-CoV-2/inmunología , Vacunas Sintéticas/inmunología , Vacunas de ARNmRESUMEN
Nanoparticle-mediated drug delivery has long utilized PEGylation as a mechanism for reducing uptake by the reticuloendothelial system and extending circulation lifetimes. However, studies over the past 2 decades have established that immune responses to PEG can promote clearance on repeat injection and elicit life-threatening anaphylactic reactions in some patients. As a potential alternative to PEGylation, we explored the ability of utilizing lactose, a naturally occurring sugar that is common on the surface of blood cells, as a coating for lipoplexes. Our data indicate that lactose imparts similar effects as PEG in terms of reducing leukocyte uptake, extending circulation half-life, and enhancing delivery to the tumor and other organs. In addition, measurements of blood cytokine levels after repeat injection indicate that reduced levels of inflammatory cytokines (IL-6, IFN-γ, TNFα) are elicited in response to lipoplexes coated with lactose as compared to PEG. These data indicate that a lactose coating on lipoplexes results in slightly improved tumor accumulation as compared to PEGylated formulations while eliciting a reduced innate immune response.
Asunto(s)
Nanopartículas , Neoplasias , Humanos , Lactosa , PolietilenglicolesRESUMEN
AIM: Conventional conjugation reactions often involve the use of activated PEG as a linker, but concerns about PEG-mediated reduction in intracellular delivery and enhanced immunogenicity have generated interest in developing methods that eliminate the need for a PEG linker. MATERIALS & METHODS: Reaction conditions were identified that specifically couples the terminal amine of a cyclic iRGD peptide (CRGDRGPDC) to the hydroxyl moiety of cholesterol through a short carbamate linker. RESULTS & CONCLUSION: Using this method for synthesizing iRGD-cholesterol, peptide ligands can be incorporated into lipid-based delivery systems, thereby eliminating concerns about adverse reactions to PEG. Toxicity and stability data indicate low toxicity and adequate serum stability at low ligand levels.
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Técnicas de Química Sintética/métodos , Colesterol/química , Portadores de Fármacos/síntesis química , Oligopéptidos/química , Animales , Línea Celular Tumoral , Colesterol/toxicidad , Portadores de Fármacos/toxicidad , Ligandos , Ratones , Oligopéptidos/toxicidad , Tamaño de la Partícula , Polietilenglicoles/efectos adversos , Polietilenglicoles/química , Pruebas de ToxicidadRESUMEN
Many pharmaceuticals must be administered intravenously due to their poor oral bioavailability. In addition to issues associated with sterility and inconvenience, the cost of repeated infusion over a 6-week course of therapy costs the health care system tens of billions of dollars per year. Attempts to improve oral bioavailability have traditionally focused on enhancing drug solubility and membrane permeability, and the use of synthetic nanoparticles has also been investigated. As an alternative strategy, some recent reports have clearly demonstrated that exosomes from cow milk are absorbed from the gastrointestinal tract in humans and could potentially be used for oral delivery of drugs that are traditionally administered intravenously. Our previous work has shown that antibodies are present in exosome preparations, and the current work with milk exosomes suggests that absorption from the gastrointestinal tract occurs via the "neonatal" Fc receptor, FcRn. Furthermore, our results demonstrate that milk exosomes are absorbed from the gut as intact particles that can be modified with ligands to promote retention in target tissues.
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Portadores de Fármacos/farmacocinética , Exosomas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Mucosa Intestinal/metabolismo , Leche/citología , Receptores Fc/metabolismo , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Disponibilidad Biológica , Carbocianinas , Bovinos , Línea Celular Tumoral/trasplante , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Exosomas/química , Femenino , Colorantes Fluorescentes/química , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Permeabilidad , Solubilidad , Distribución TisularRESUMEN
Decades of research into improving drug delivery to tumors has documented uptake of particulate delivery systems by resident macrophages in the lung, liver, and spleen, and correlated short circulation times with reduced tumor accumulation. An implicit assumption in these studies is that nanoparticles present in the blood are available for distribution to the tumor. This study documents significant levels of lipoplex uptake by circulating leukocytes, and its effect on distribution to the tumor and other organs. In agreement with previous studies, PEGylation dramatically extends circulation times and enhances tumor delivery. However, our studies suggest that this relationship is not straightforward, and that particle sequestration by leukocytes can significantly alter biodistribution, especially with non-PEGylated nanoparticle formulations. We conclude that leukocyte uptake should be considered in biodistribution studies, and that delivery to these circulating cells may present opportunities for treating viral infections and leukemia.
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Leucocitos/metabolismo , Nanopartículas/metabolismo , Polietilenglicoles/metabolismo , Animales , ADN/administración & dosificación , ADN/farmacocinética , Femenino , Técnicas de Transferencia de Gen , Ratones Endogámicos BALB C , Ratones SCID , Neoplasias/metabolismo , Plásmidos/administración & dosificación , Plásmidos/farmacocinética , Distribución TisularRESUMEN
Glioblastoma multiforme (GBM) is a devastating tumour with abysmal prognoses. We desperately need novel approaches to understand GBM biology and therapeutic vulnerabilities. Extracellular vesicles (EVs) are membrane-enclosed nanospheres released locally and systemically by all cells, including tumours, with tremendous potential for intercellular communication. Tumour EVs manipulate their local environments as well as distal targets; EVs may be a mechanism for tumourigenesis in the recurrent GBM setting. We hypothesized that GBM EVs drive molecular changes in normal human astrocytes (NHAs), yielding phenotypically tumour-promoting, or even tumourigenic, entities. We incubated NHAs with GBM EVs and examined the astrocytes for changes in cell migration, cytokine release and tumour cell growth promotion via the conditioned media. We measured alterations in intracellular signalling and transformation capacity (astrocyte growth in soft agar). GBM EV-treated NHAs displayed increased migratory capacity, along with enhanced cytokine production which promoted tumour cell growth. GBM EV-treated NHAs developed tumour-like signalling patterns and exhibited colony formation in soft agar, reminiscent of tumour cells themselves. GBM EVs modify the local environment to benefit the tumour itself, co-opting neighbouring astrocytes to promote tumour growth, and perhaps even driving astrocytes to a tumourigenic phenotype. Such biological activities could have profound impacts in the recurrent GBM setting.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.
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Astrocitos/patología , Proliferación Celular , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/fisiopatología , Microambiente Tumoral , Línea Celular Tumoral , Movimiento Celular , Humanos , FenotipoAsunto(s)
Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Polietilenglicoles/química , Polímeros/química , Composición de Medicamentos , Liberación de Fármacos , Estabilidad de Medicamentos , Humanos , Tamaño de la Partícula , Proteínas/efectos de los fármacos , Proteínas/farmacocinética , Propiedades de SuperficieRESUMEN
Remarkable progress has recently been made in the synthesis and characterization of engineered nanoparticles for imaging and treatment of cancers, resulting in several promising candidates in clinical trials. Despite these advances, clinical applications of nanoparticle-based therapeutic/imaging agents remain limited by biological, immunological, and translational barriers. In order to overcome the existing status quo in drug delivery, there is a need for open and frank discussion in the nanomedicine community on what is needed to make qualitative leaps toward translation. In this Nano Focus, we present the main discussion topics and conclusions from a recent workshop: "Mechanisms and Barriers in Nanomedicine". The focus of this informal meeting was on biological, toxicological, immunological, and translational aspects of nanomedicine and approaches to move the field forward productively. We believe that these topics reflect the most important issues in cancer nanomedicine.
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Nanopartículas/química , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Medios de Contraste/administración & dosificación , Medios de Contraste/química , Sistemas de Liberación de Medicamentos , Humanos , Terapia Molecular Dirigida/métodos , NanomedicinaRESUMEN
The relevance of using nude mouse models for evaluating drug delivery to human tumors has recently been questioned by numerous researchers. While the immune response is known to play a critical role in cancer, this study assesses the effect of using immunocompromised "nude" mice on drug delivery. By inoculating both nude and immunocompetent mice with a mouse mammary carcinoma cell line (4T1), differences in the "first pass effect", distribution, and reporter gene expression due to the use of the nude mouse model could be elucidated. Our results indicate that initial tumor deposition (5 min) was slightly lower in nude mice but comparable after 24 h. In addition, some small differences in tissue deposition/accumulation and reporter gene expression were observed between the two mouse models. The results with this one tumor model suggest that delivery studies conducted in nude mice can provide comparable results to those in immunocompetent mouse models.
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Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Neoplasias/terapia , Animales , Línea Celular Tumoral , ADN/administración & dosificación , Femenino , Genes Reporteros , Luciferasas/genética , Luciferasas/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/administración & dosificación , Trasplante de Neoplasias , Neoplasias/metabolismo , Neoplasias/patología , Plásmidos , Carga TumoralRESUMEN
Repetitive administration is routinely used to maintain therapeutic drug levels, but previous studies have documented an accelerated blood clearance of some lipid-based delivery systems under these conditions. To assess the effect of repetitive administration, non-PEGylated lipoplexes (+/-0.5) were administered 4 times via tail vein injection at 3-day intervals to immunocompetent BALB/c mice bearing 4T1 tumors. This study measured the effect of repeat administration of nontargeted lipoplexes on clearance, cytokine/chemokine response, plasmid distribution, reporter gene expression, and liver toxicity. We do not observe a refractory period or a statistically significant difference in blood clearance between the first administration and subsequent injections of this lipoplex formulation, consistent with the absence of a cytokine/chemokine response. However, we do see a significant effect on both plasmid accumulation and expression, an enhancement of 26-fold and 10-fold in tumor plasmid levels and expression, respectively, after 4 injections as compared to that after a single injection. In addition, in vivo imaging suggests that expression in other organs had diminished rapidly 72 h after each administration, in contrast to relatively constant expression in the tumor. Taken together, the findings indicate that gene delivery to tumors can be dramatically enhanced by employing repetitive administration.
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Inmunocompetencia/efectos de los fármacos , Neoplasias/metabolismo , Polietilenglicoles/administración & dosificación , Polietilenglicoles/metabolismo , Animales , Esquema de Medicación , Inmunocompetencia/fisiología , Lípidos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológicoRESUMEN
BACKGROUND: Agents targeting HSP90 and GRP94 are seldom tested in stressed contexts such as heat shock (HS) or the unfolded protein response (UPR). Tumor stress often activates HSPs and the UPR as pro-survival mechanisms. This begs the question of stress effects on chemotherapeutic efficacy, particularly with drugs targeting chaperones such as HSP90 or GRP94. We tested the utility of several HSP90 inhibitors, including PU-H71 (targeting GRP94), on a primary canine lung cancer line under HS/UPR stress compared to control conditions. METHODS: We cultured canine bronchoalveolar adenocarcinoma cells that showed high endogenous HSP90 and GRP94 expression; these levels substantially increased upon HS or UPR induction. We treated cells with HSP90 inhibitors 17-DMAG, 17-AAG or PU-H71 under standard conditions, HS or UPR. Cell viability/survival was assayed. Antibody arrays measured intracellular signalling and apoptosis profiles. RESULTS: HS and UPR had varying effects on cells treated with different HSP90 inhibitors; in particular, HS and UPR promoted resistance to inhibitors in short-term assays, but combinations of UPR stress and PU-H571 showed potent cytotoxic activity in longer-term assays. Array data indicated altered signalling pathways, with apoptotic and pro-survival implications. UPR induction + dual targeting of HSP90 and GRP94 swayed the balance toward apoptosis. CONCLUSION: Cellular stresses, endemic to tumors, or interventionally inducible, can deflect or enhance chemo-efficacy, particularly with chaperone-targeting drugs. Stress is likely not held accountable when testing new pharmacologics or assessing currently-used drugs. A better understanding of stress impacts on drug activities should be critical in improving therapeutic targeting and in discerning mechanisms of drug resistance.
RESUMEN
Brain tumors such as high grade gliomas are among the deadliest forms of human cancers. The tumor environment is subject to a number of cellular stressors such as hypoxia and glucose deprivation. The persistence of the stressors activates the unfolded proteins response (UPR) and results in global alterations in transcriptional and translational activity of the cell. Although the UPR is known to effect tumorigenesis in some epithelial cancers, relatively little is known about the role of the UPR in brain tumors. Here, we evaluated the changes at the molecular level under homeostatic and stress conditions in two glioma cell lines of differing tumor grade. Using mass spectrometry analysis, we identified proteins unique to each condition (unstressed/stressed) and within each cell line (U87MG and UPN933). Comparing the two, we find differences between both the conditions and cell lines indicating a unique profile for each. Finally, we used our proteomic data to identify the predominant pathways within these cells under unstressed and stressed conditions. Numerous predominant pathways are the same in both cell lines, but there are differences in biological and molecular classifications of the identified proteins, including signaling mechanisms, following UPR induction; we see that relatively minimal proteomic alterations can lead to signaling changes that ultimately promote cell survival.
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Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioma/genética , Glioma/patología , Humanos , Espectrometría de Masas , Proteómica/métodos , Respuesta de Proteína DesplegadaRESUMEN
BACKGROUND: Glioma-related immunosuppression is well documented; however, the mechanisms of suppression are not fully understood. Here we explore a role for glioma extracellular vesicles (EVs) as a means of immune modulation. METHODS: Healthy donor peripheral blood mononuclear cells (PBMCs) were incubated with mitogenic stimuli and various concentrations of glioma-derived EVs. Intracellular signaling and cytokine output were determined by protein microarrays, and phenotypic changes were assessed by flow cytometry. Recall antigen testing, mixed lymphocyte reactions, and migration assays analyzed PBMC functional capacity. RESULTS: Protein microarray data revealed induction of an immunosuppressive phenotype and cytokine output at high tumor-vesicle concentrations but an activated phenotype at low concentrations. T cell activation antigen expression confirmed differential activation profiles. Functional analyses revealed decreased migratory capacity of PBMCs after incubation with EVs; however, recall antigen and mixed lymphocyte tests indicated that activation capacity is still retained in EV-treated cells. CONCLUSION: The differential effects of high and low EV concentrations dictate modulatory effects on PBMCs. These data provide a role for EVs at high concentrations for inducing selective tolerance of an immune response in a tumor setting. This suggests that lymphocytes in patients' circulation are not irreparably impaired, as previously thought, but can be rescued to augment antitumor responses.
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Citocinas/metabolismo , Exosomas/inmunología , Vesículas Extracelulares/inmunología , Glioma/inmunología , Tolerancia Inmunológica/inmunología , Leucocitos Mononucleares/inmunología , Linfocitos T/inmunología , Células Cultivadas , Exosomas/metabolismo , Exosomas/patología , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patología , Glioma/metabolismo , Glioma/patología , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Análisis por Matrices de Proteínas , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
BACKGROUND: The vast majority of studies investigating gene delivery have utilized cationic delivery vehicles, but anionic nanoparticles can also possess high transfection activity, and offer significant benefits in terms of ease of preparation and reduced toxicity. RESULTS: Our study on lipoplexes possessing cholesterol nanodomains demonstrates that in vitro transfection after exposure to serum can be high at anionic charge ratios, and that this effect is also evident in studies assessing delivery to tumors in vivo, despite reduced circulation times. In addition, accumulation in the liver and lungs is reduced as compared with lipoplexes formulated at cationic charge ratios. CONCLUSION: Lipoplexes prepared at anionic charge ratios offer comparable tumor delivery and reduced liver toxicity despite shorter circulation times.
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Hígado/efectos de los fármacos , Nanopartículas/administración & dosificación , Nanopartículas/efectos adversos , Ácidos Nucleicos/administración & dosificación , Ácidos Nucleicos/farmacocinética , Transfección/métodos , Alanina Transaminasa/sangre , Animales , Disponibilidad Biológica , Línea Celular Tumoral , Femenino , Humanos , Iones/administración & dosificación , Iones/efectos adversos , Iones/sangre , Hígado/metabolismo , Pulmón/metabolismo , Ratones , Neoplasias/metabolismo , Ácidos Nucleicos/sangre , Tamaño de la PartículaRESUMEN
In cancer patients, granulocytic myeloid derived suppressor cells (G-MDSCs) expand in number, infiltrating tumor and lymphatic tissues where they suppress an anti-tumor immune response. We report here the development of a liposomal drug delivery system that selectively targets G-MDSCs. The liposomes form a disulfide bond with activated complement C3 after intravenous injection and are taken up by G-MDSCs, which express the receptor for activated C3. In vitro experiments utilizing serum from a C3 knockout mouse demonstrate that G-MDSCs take up these liposomes in a C3-dependent manner. After systemic administration to tumor bearing mice, liposomes were incorporated by 22% of G-MDSCs in the blood and were also present in a percentage of G-MDSCs in the tumor (11%), spleen (22%), liver (35%) and lungs (26%). This liposomal system offers a versatile means of targeted drug delivery to G-MDSCs and could be an important tool for restoring anti-tumor immunity in cancer patients. FROM THE CLINICAL EDITOR: It has been shown that the presence of granulocytic myeloid derived suppressor cells (G-MDSCs) in cancer patients suppress the tumor immune response of T cells. Many drugs can be used to reverse this process. In this article, the authors describe the development of a liposomal drug delivery system for targeted drug delivery to G- MDSCs. This system may prove to be useful adjunct in immunotherapy in the fight against cancers.