RESUMO
The starting hypothesis for this work was that microwave synthesis could enable the rapid assembly of polymers into size-specific nanoparticles (NPs). The Zapped Assembly of Polymeric (ZAP) NPs was initially realized using poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) block copolymers and distinct microwave reaction parameters. A library of polymeric NPs was generated with sizes ranging from sub-20 nm to 350 nm and low polydispersity. Select ZAP NPs were synthesized in 30 seconds at different scales and concentrations, up to 200 mg and 100 mg mL-1, without substantial size variation. ZAP NPs with diameters of 25 nm, 50 nm, and 100 nm were loaded with the chemotherapeutic paclitaxel (PXL), demonstrated unique release profiles, and exhibited dose-dependent cytotoxicity similar to Taxol. Incorporation of d-alpha tocopheryl polyethylene glycol succinate (TPGS) and PLGA33k allowed for the production of a sub-40 nm NP with an exceptionally high loading of PXL (12.6 wt%, ca. 7 times the original NP) and a slower release profile. This ZAP NP platform demonstrated scalable, flexible, and tunable synthesis with potential toward clinical scale production of size-specific drug carriers.
Assuntos
Antineoplásicos Fitogênicos/química , Portadores de Fármacos/química , Nanopartículas/química , Poliésteres/química , Polietilenoglicóis/química , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Células HeLa , Humanos , Micro-Ondas , Paclitaxel/química , Paclitaxel/farmacologia , Tamanho da PartículaRESUMO
The safety and efficacy of anticancer antibody-drug conjugates (ADCs) depend on the selection of tumor-targeting monoclonal antibody (mAb), linker, and drug, as well as their specific chemical arrangement and linkage chemistry. In this study, we used a heterobifunctional cross-linker to conjugate docetaxel (DX) to cetuximab (CET) or panitumumab (PAN). The resulting ADCs were investigated for their in vitro EGFR-specific cytotoxicity and in vivo anticancer activity. Reaction conditions, such as reducing agent, time, temperature, and alkylation buffer, were optimized to yield potent and stable ADCs with consistent batch-to-batch drug-to-antibody ratios (DARs). ADCs were synthesized with DARs from 0.4 to 3.0, and all retained their EGFR affinity and specificity after modification. ADCs were sensitive to cell surface wildtype EGFR expression, demonstrating more cytotoxicity in EGFR-expressing A431 and MDA-MB-231 cell lines compared to U87MG cells. A431 tumor-bearing mice treated once weekly for four weeks with 100 mg/kg cetuximab-docetaxel ADC (C-SC-DX, DAR 2.5) showed durable anticancer responses and improved overall survival compared to the same treatment regimen with 1 mg/kg DX, 100 mg/kg CET, or a combination 1 mg/kg DX and 100 mg/kg CET. New treatment options are emerging for patients with both wild-type and mutated EGFR-overexpressing cancers, and these studies highlight the potential role of EGFR-targeted ADC therapies as a promising new treatment option.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Cetuximab/química , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Reagentes de Ligações Cruzadas/química , Docetaxel/química , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Camundongos , Camundongos Nus , Neoplasias/mortalidade , Neoplasias/patologia , Panitumumabe/química , Panitumumabe/farmacologia , Panitumumabe/uso terapêutico , Análise de Sobrevida , Distribuição Tecidual , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ability to non-invasively monitor tumor-infiltrating T cells in vivo could provide a powerful tool to visualize and quantify tumor immune infiltrates. For non-invasive evaluations in vivo, an anti-CD3 mAb was modified with desferrioxamine (DFO) and radiolabeled with zirconium-89 (Zr-89 or 89Zr). Radiolabeled 89Zr-DFO-anti-CD3 was tested for T cell detection using positron emission tomography (PET) in both healthy mice and mice bearing syngeneic bladder cancer BBN975. In vivo PET/CT and ex vivo biodistribution demonstrated preferential accumulation and visualization of tracer in the spleen, thymus, lymph nodes, and bone marrow. In tumor bearing mice, 89Zr-DFO-anti-CD3 demonstrated an 11.5-fold increase in tumor-to-blood signal compared to isotype control. Immunological profiling demonstrated no significant change to total T cell count, but observed CD4+ T cell depletion and CD8+ T cell expansion to the central and effector memory. This was very encouraging since a high CD8+ to CD4+ T cell ratio has already been associated with better patient prognosis. Ultimately, this anti-CD3 mAb allowed for in vivo imaging of homeostatic T cell distribution, and more specifically tumor-infiltrating T cells. Future applications of this radiolabeled mAb against CD3 could include prediction and monitoring of patient response to immunotherapy.
Assuntos
Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Bexiga Urinária/diagnóstico por imagem , Neoplasias da Bexiga Urinária/imunologia , Animais , Anticorpos , Butilidroxibutilnitrosamina , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Desferroxamina , Citometria de Fluxo , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Radioisótopos , Compostos Radiofarmacêuticos , Distribuição Tecidual , Neoplasias da Bexiga Urinária/patologia , ZircônioRESUMO
HYPOTHESIS: Accessing the phase inversion temperature by microwave heating may enable the rapid synthesis of small lipid nanoparticles. EXPERIMENTS: Nanoparticle formulations consisted of surfactants Brij 78 and Vitamin E TPGS, and trilaurin, trimyristin, or miglyol 812 as nanoparticle lipid cores. Each formulation was placed in water and heated by microwave irradiation at temperatures ranging from 65°C to 245°C. We observed a phase inversion temperature (PIT) for these formulations based on a dramatic decrease in particle Z-average diameters. Subsequently, nanoparticles were manufactured above and below the PIT and studied for (a) stability toward dilution, (b) stability over time, (c) fabrication as a function of reaction time, and (d) transmittance of lipid nanoparticle dispersions. FINDINGS: Lipid-based nanoparticles with distinct sizes down to 20-30nm and low polydispersity could be attained by a simple, one-pot microwave synthesis. This was carried out by accessing the phase inversion temperature using microwave heating. Nanoparticles could be synthesized in just one minute and select compositions demonstrated high stability. The notable stability of these particles may be explained by the combination of van der Waals interactions and steric repulsion. 20-30nm nanoparticles were found to be optically transparent.
RESUMO
Monoclonal antibodies (mAbs) offer promise as effective tumor targeting and drug delivery agents for cancer therapy. However, comparative biological and clinical characteristics of mAbs targeting the same tumor-associated antigen (TAA) often differ widely. This study examined the characteristics of mAbs that impact tumor targeting using a panel of mAb clones specific to the cancer-associated cell-surface receptor and cancer stem cell marker CD44. CD44 mAbs were screened for cell-surface binding, antigen affinity, internalization, and CD44-mediated tumor uptake by CD44-positive A549 cells. It was hypothesized that high-affinity, rapidly internalizing CD44 mAbs would result in high tumor uptake and prolonged tumor retention. Although high-affinity clones rapidly bound and were internalized by A549 cells in vitro, an intermediate-affinity clone demonstrated significantly greater tumor uptake and retention than high-affinity clones in vivo. Systemic exposure, rather than high antigen affinity or rapid internalization, best associated with tumor targeting of CD44 mAbs in A549 tumor-bearing mice.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Receptores de Hialuronatos/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos NusRESUMO
The biological activity of nanoparticle-directed therapies critically depends on cellular targeting. We examined the subtumoral fate of Particle Replication in Non-Wetting Templates (PRINT) nanoparticles in a xenografted melanoma tumor model by multi-color flow cytometry and in vivo confocal tumor imaging. These approaches were compared with the typical method of whole-organ quantification by radiolabeling. In contrast to radioactivity based detection which demonstrated a linear dose-dependent accumulation in the organ, flow cytometry revealed that particle association with cancer cells became dose-independent with increased particle doses and that the majority of the nanoparticles in the tumor were associated with cancer cells despite a low fractional association. In vivo imaging demonstrated an inverse relationship between tumor cell association and other immune cells, likely macrophages. Finally, variation in particle size nonuniformly affected subtumoral association. This study demonstrates the importance of subtumoral targeting when assessing nanoparticle activity within tumors. FROM THE CLINICAL EDITOR: Particle Replication in Non-Wetting Templates (PRINT) technology allows the production of nanoparticles with uniform size. The authors in the study utilized PRINT-produced nanoparticles to investigate specific tumor uptake by multi-color flow cytometry and in vivo confocal tumor imaging. This approach allowed further in-depth correlation between nanoparticle properties and tumor cells and should improve future design.
Assuntos
Citometria de Fluxo , Melanoma/diagnóstico por imagem , Nanomedicina , Nanopartículas/efeitos adversos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Melanoma/patologia , Nanopartículas/administração & dosagem , Tamanho da Partícula , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gold nanostars (AuNSs) are unique systems that can provide a novel multifunctional nanoplatform for molecular sensing and diagnostics. The plasmonic absorption band of AuNSs can be tuned to the near infrared spectral range, often referred to as the "tissue optical window", where light exhibits minimal absorption and deep penetration in tissue. AuNSs have been applied for detecting disease biomarkers and for biomedical imaging using multi-modality methods including surface-enhanced Raman scattering (SERS), two-photon photoluminescence (TPL), magnetic resonance imaging (MRI), positron emission tomography (PET), and X-ray computer tomography (CT) imaging. In this paper, we provide an overview of the recent development of plasmonic AuNSs in our laboratory for biomedical applications and highlight their potential for future translational medicine as a multifunctional nanoplatform.
Assuntos
Técnicas Biossensoriais , Diagnóstico por Imagem , Ouro/química , Nanopartículas/química , Humanos , Análise Espectral RamanRESUMO
The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.
Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Ativação Plaquetária/fisiologia , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidores , Animais , Senescência Celular , Clopidogrel , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/deficiência , Hemostasia , Linfopenia/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores Purinérgicos P2Y12/fisiologia , Veia Safena/lesões , Esplenectomia , Trombocitopenia/genética , Trombocitopenia/cirurgia , Trombopoese , Ticlopidina/análogos & derivados , Ticlopidina/farmacologia , Proteínas rap1 de Ligação ao GTP/fisiologiaRESUMO
Asymmetric bifunctional silyl ether (ABS) prodrugs of chemotherapeutics were synthesized and incorporated within 200 nm × 200 nm particles. ABS prodrugs of gemcitabine were selected as model compounds because of the difficulty to encapsulate a water-soluble drug within a hydrogel. The resulting drug delivery systems were degraded under acidic conditions and were found to release only the parent or active drug. Furthermore, changing the steric bulk of the alkyl substituents on the silicon atom could regulate the rate of drug release and, therefore, the intracellular toxicity of the gemcitabine-loaded particles. This yielded a family of novel nanoparticles that could be tuned to release drug over the course of hours, days, or months.
Assuntos
Antineoplásicos/administração & dosagem , Camptotecina/administração & dosagem , Preparações de Ação Retardada/química , Desoxicitidina/análogos & derivados , Nanopartículas/química , Pró-Fármacos/administração & dosagem , Pirimidinas/administração & dosagem , Tiazóis/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Linhagem Celular Tumoral , Dasatinibe , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Éteres/administração & dosagem , Éteres/farmacologia , Humanos , Nanopartículas/ultraestrutura , Neoplasias/tratamento farmacológico , Pró-Fármacos/farmacologia , Pirimidinas/farmacologia , Silanos/administração & dosagem , Silanos/farmacologia , Tiazóis/farmacologia , GencitabinaRESUMO
Lipid-based oil-filled nanoparticles (NPs) with a high concentration of surface-chelated nickel (Ni-NPs) were successfully prepared using a Brij 78-NTA-Ni conjugate synthesized with Brij 78 (Polyoxyethylene (20) stearyl ether) and nitrilotriacetic acid (NTA). The facile incorporation of the Brij 78-NTA-Ni conjugate into the NP formulation allowed up to 90% Ni incorporation, which was a significant improvement over the previously used standard agent DOGS-NTA-Ni which led to ~6% Ni incorporation. The Ni-NPs were targeted to the highly epidermal growth factor receptor (EGFR)-overexpressing epidermoid carcinoma cells A431. This was accomplished using a novel high affinity histidine×6-tagged EGFR-binding Z domain (heptameric Z(EGFR) domain). In vitro cell uptake studies showed enhanced internalization (up to 90%) of the targeted Ni-NPs in A431 cells with only ≤10% internalization of the untargeted Ni-NPs. ICP-MS analysis used to quantify the amount of Ni in the cells were in close agreement with flow cytometry studies, which showed a dose dependent increase in the amount of Ni with the targeted Ni-NPs. Cell uptake competition studies showed that internalization of the targeted Ni-NPs within the cells was competed off with free heptameric Z(EGFR) domain at concentrations of 8.75ng/mL or higher. In vivo studies were carried out in nude mice bearing A431 tumors to determine the biodistribution and intracellular delivery. Near Infrared (NIR) optical imaging studies using Alexa750-labeled heptameric Z(EGFR) domain showed localization of 19% of the total detected fluorescence intensity in the tumor tissue, 28% in the liver and 42% in the kidneys 16h post i.v. injection. ICP-MS analysis showed almost a two-fold increase in the amount of intracellular Ni with the targeted Ni-NPs. These new Ni-NPs could be a very useful tool for targeting and drug delivery to a wide range of EGFR positive cancers.
Assuntos
Portadores de Fármacos/administração & dosagem , Receptores ErbB/metabolismo , Nanopartículas Metálicas/administração & dosagem , Neoplasias/metabolismo , Níquel/administração & dosagem , Animais , Linhagem Celular Tumoral , Portadores de Fármacos/química , Receptores ErbB/química , Humanos , Nanopartículas Metálicas/química , Camundongos , Camundongos Nus , Níquel/química , Ácido Nitrilotriacético/administração & dosagem , Ácido Nitrilotriacético/química , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Estrutura Terciária de Proteína , Succinimidas/administração & dosagem , Succinimidas/química , Tensoativos/administração & dosagem , Tensoativos/química , Distribuição Tecidual , Triglicerídeos/administração & dosagem , Triglicerídeos/químicaRESUMO
Responsive polymeric biomaterials can be triggered to degrade using localized environments found in vivo. A limited number of biomaterials provide precise control over the rate of degradation and the release rate of entrapped cargo and yield a material that is intrinsically nontoxic. In this work, we designed nontoxic acid-sensitive biomaterials based on silyl ether chemistry. A host of silyl ether cross-linkers were synthesized and molded into relevant medical devices, including Trojan horse particles, sutures, and stents. The resulting devices were engineered to degrade under acidic conditions known to exist in tumor tissue, inflammatory tissue, and diseased cells. The implementation of silyl ether chemistry gave precise control over the rate of degradation and afforded devices that could degrade over the course of hours, days, weeks, or months, depending upon the steric bulk around the silicon atom. These novel materials could be useful for numerous biomedical applications, including drug delivery, tissue repair, and general surgery.
Assuntos
Materiais Biocompatíveis/química , Éteres/química , Compostos de Trimetilsilil/química , Ácidos/química , Materiais Biocompatíveis/síntese química , Reagentes de Ligações Cruzadas , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Concentração de Íons de Hidrogênio , Estrutura MolecularRESUMO
A series of aliphatic polyester dendrons, generations 1 through 8, were prepared with a core p-toluenesulfonyl ethyl (TSe) ester as an easily removable protecting group that can be efficiently replaced with a variety of nucleophiles. Using amidation chemistry, a tridentate bis(pyridyl)amine ligand which is known to form stable complexes with both Tc(I) and Re(I) was introduced at the dendrimer core. Metalation of the core ligand with (99m)Tc was accomplished for generations 5 through 7, and resulted in regioselective radiolabeling of the dendrimers. The distribution of the radiolabeled dendrimers was evaluated in healthy adult Copenhagen rats using dynamic small-animal single photon emission computed tomography (SPECT). The labeled dendrimers were cleanly and rapidly eliminated from the bloodstream via the kidneys with negligible nonspecific binding to organs or tissues being observed. These data were corroborated by a quantitative biodistribution study on the generation 7 dendrimer following necropsy. The quantitative biodistribution results were in excellent agreement with the data obtained from the dynamic SPECT images.
Assuntos
Dendrímeros/química , Compostos de Organotecnécio/química , Poliésteres/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/química , Animais , Dendrímeros/síntese química , Dendrímeros/farmacocinética , Masculino , Compostos de Organotecnécio/síntese química , Compostos de Organotecnécio/farmacocinética , Poliésteres/síntese química , Poliésteres/farmacocinética , Propionatos/química , Ratos , Tecnécio/química , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
Three recently reported aliphatic polyester dendrimers of generations 3, 4, and 5, having 4, 8, and 16 carborane cages within their interior, respectively, were found to exhibit thermally induced, reversible precipitation in aqueous solution. The cloud-point temperatures for these molecules were observed to be between 40 and 80 degrees C, depending on the dendrimer generation. The three dendrimers investigated have a hydroxyl-to-carborane ratio of 8:1, which provides the ideal balance between the hydrophobic interior and the hydrophilic exterior to enable the thermally induced phase transition to occur. It was found that repeated heating/cooling cycles resulted in a decreasing cloud-point temperature and increased dendrimer solubility. Additionally, the effect of pH on the cloud point was investigated, indicating no significant changes as long as the dendrimers remained stable. Size-exclusion chromatography indicated that dendrimer degradation was occurring at pH above 7.0.
RESUMO
The incorporation of multiple p-carborane cages within an aliphatic polyester dendrimer was accomplished through the preparation of a bifunctional carborane synthon. A p-carborane derivative having an acid and a protected alcohol functionality was found to efficiently couple to peripheral hydroxyl groups of low-generation dendrimers under standard esterification conditions. Deprotection of carborane hydroxyl groups allowed for further dendronization through a divergent approach using the highly reactive anhydride of benzylidene-protected 2,2-bis(hydroxymethyl)propanoic acid. This approach was used to prepare fourth- and fifth-generation dendrimers that contain 4, 8, and 16 carborane cages within their interior. Upon peripheral deprotection to liberate a polyhydroxylated dendrimer exterior, these structures exhibited aqueous solubility as long as a minimum of eight hydroxyl groups per carborane were present. Several of the water-soluble structures were found to exhibit a lower critical solution temperature. Additionally, irradiation of these materials with thermal neutrons resulted in emission of gamma radiation that is indicative of boron neutron capture events occurring within the carborane-containing dendrimers.