Name: a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines
Uploaded: 2017-03-04T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines at JoVE.com

The authors would like to thank Drs. Helene Salmon and Miriam Merad from Icahn School of Medicine at Mount Sinai for providing the B16-F10-YFP cells and for their expert advice on melanoma mouse models. The authors further thank the Animal Imaging Core Facility, the Radiochemistry and Molecular Imaging Probes Core Facility, and the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center (MSK) for their support. This work was supported by National Institutes of Health grants NIH 1 R01 HL125703 (W.J.M.M.), R01CA155432 (W.J.M.M.), K25 EB016673 (T.R.) and P30 CA008748 (MSK Center Grant). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) at MSK for their financial support (T.R.).

O processo consiste na marcação radioactiva e dupla fluorescente de nanopartículas, in vivo imagiologia PET-CT, ex vivo de biodistribuição medições, e imunocoloração ex vivo e análises de citometria de fluxo. Todos os experimentos com animais foram aprovados pelo Comitê Cuidado e Uso Institucional Animal do Memorial Sloan Kettering Cancer Center. 1. Preparação de lipossomas duplamente marcadas NOTA: Os tumores de melanoma B16 singeneicas pode ser induzida por injecção de 300.000 células B16-F10 nos flancos traseiros de ratinhos C57BL / 6 sob anestesia de inalação 2% contendo -isoflurane oxigénio. Utilizar os reagentes e instrumentos esterilizados em um espaço de trabalho estéril durante a cirurgia. Após a cirurgia, observar cuidadosamente os animais até à sua recuperação da anestesia. Coloque os animais de volta em suas gaiolas apenas quando recuperar a plena consciência e mobilidade. Abrigá-los em salas estéreis para animais com tumores xenoenxerto. Os tumores com um tamanhode 300 mm a 3 são ideais para a caracterização de nanopartículas, devido aos seus efeitos de permeabilidade e retenção aumentadas pronunciadas (EPR), que podem aumentar a acumulação de nanopartículas. Os protocolos detalhados são fornecidos abaixo. <ol><li> Em um balão de fundo redondo, dissolver uma mistura de 20 mg de lípidos em 2 ml de clorofórmio contendo 1,2-dipalmitoil-sn-glicero-3-phophocholine (DPPC), colesterol, 1,2-sn-glicero-disearoyl- 3-fosfoetanolamina-poli (etileno-glicol) 2000 (DSPE-PEG2000), DSPE-deferoxamina (DFO-DSPE) 8, e ácido 1,1-diododecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-dissulfónico ( DiIC12 [5] -ds) em proporções molares de 61,3%, 33,4%, 5, 0,2% e 0,1%, respectivamente. NOTA: Esta composição de lípido é muito semelhante para a formulação lipossómica de doxorrubicina actualmente utilizado na clínica 2. Os lipossomas resultantes do nosso processo vai imitar de perto o desempenho in vivo do nanomedic clínicaine. </li><li> Usar um evaporador rotativo para remover o solvente orgânico e formar uma película fina de lípidos, à temperatura ambiente. Adiciona-se 20 mL de PBS e sonicado durante 30 min para gerar nanopartículas de lipossomas utilizando uma ponta ultra-sons de 3,8 mm e uma amplitude de 30 W, com arrefecimento em gelo suficiente. </li><li> Centrifugar a solução de lipossomas a 4.000 xg durante 10 minutos para remover os agregados e lavam as nanopartículas com PBS utilizando filtração centrífuga (peso molecular de corte (MWCO:. 100 kDa), para remover os lípidos livres e solvente orgânico residual Concentra-se a lipossomas, o que vai permanecem na câmara de topo, e transferi-las para um novo tubo. os lipossomas podem ser armazenados num frigorífico em PBS durante até 1 semana antes dos passos subsequentes. </li><li> Para controle de qualidade, caracterizam os lipossomas por espalhamento dinâmico de luz (DLS). Especificamente, misturar 50 mL de lipossomas com 950 uL de PBS, e em seguida, transferir a mistura para a cuvete de dimensionamento. Colocar a cuba no analisador e medir a partículatamanhos e sua distribuição de tamanho. Os tamanhos das partículas e a sua distribuição de tamanho (índice de polidispersidade (PDI)) deverão ser 90-120 nm e 0,1-0,2, respectivamente. </li><li> Para a marcação radioactiva, misturar os lipossomas purificados, o equivalente a 2 mg de lípidos, em PBS, a um pH entre 6,9 e 7,1 com 1 mCi de 89 Zr-oxalato a 37 ° C durante 2 h, num tubo de 1,5 ml, (volume total: ~ 100 mL). Remover o livre, que não reagiu, 89 Zr-se por filtração centrífuga (MWCO = 100 kDa), semelhante ao passo 1.3. Lavar o retentado com PBS estéril e diluir até ao volume desejado. O rendimento de marcação deve ser de 80 - 95%. CUIDADO! Trabalhando com materiais radioativos é altamente regulado. As instituições devem ser certificadas e proporcionar as facilidades necessárias, treinamento de pessoal, e um controlo rigoroso do uso de radioatividade. Os pesquisadores precisam receber treinamento adequado e usar equipamento de protecção pessoal e anéis de dosimetria / badges ao manusear radioatividade. </li><li&gt; Determinar a pureza radioquímica dos lipossomas marcados radioactivamente por exclusão de tamanho de rádio-HPLC, que normalmente deve ser superior a 98% 10. As purezas radioquímicas inferiores a 95% são indicativos de lavagem insuficiente no passo 1.5. </li><li> Após a marcação radioactiva, determinar o tamanho das nanopartículas por dispersão dinâmica da luz. O tamanho e PDI Espera-se que esteja dentro da mesma gama que as medições no passo 1.4 10. NOTA: Se amostras radioactivas não são permitidos no citômetro de fluxo ou microscópio de fluorescência, nat não-radioativo Zr-oxalato pode ser usado para rotular lipossomas na etapa 1.5. Estes lipossomas não radioactivos tem características idênticas aos seus análogos radioactivos. Uma descrição deste procedimento é apresentada na Figura 2. </li></ol> 2. In vivo de imagens PET-CT e biodistribuição de lipossomas duplamente marcada <ol><li> Injectar cerca de 100 ul de lipossomas duplamente marcadas wom cerca de 300 uCi de radioactividade em ratos melanoma impedido (C57BL / 6, fêmeas, de 10 - 16 semanas de idade), através das suas veias da cauda, a cerca de 10 mCi de 89 Zr / kg de peso corporal. </li><li> Para determinar a meia-vida, a obtenção de sangue da veia da cauda de animais anestesiados com menos de 2% de isoflurano contendo oxigénio utilizando 28-G seringas de insulina. Recolhe-se o sangue (10-20 uL) em tubos pré-pesados ​​aos 2 min, 15 min, 1 h, 4 h, 8 h, 24 h e 48 h após a injecção. Pesa-se o sangue por diferença, determinar o teor de radioactividade utilizando um contador gama, e calcular a concentração de radioactividade como percentagem de dose injectada por grama (% ID / g). NOTA: Execute o procedimento em uma sala equipada com um sistema de anestesia, gaiolas de recuperação, aquecimento, e um capuz de risco biológico. Certifique-se de que os animais se recuperar totalmente e ganhar mobilidade normal após o procedimento antes de transferi-los de gaiolas de recuperação para segurando gaiolas. Casa os animais portadores de tumor em salas designadas para os animais administrados commateriais radioativos. </li><li> 24 ou 48 h após a injecção do lipossomas, a imagem dos camundongos em um pequeno animal MicroPET-CT scanner de 10. Coloque o animal anestesiado horizontalmente em sua barriga e mantê-lo anestesiado. Administrar 2% de isoflurano em oxigênio através de um cone do nariz. Aplicar pomada oftálmica aos seus olhos antes de a digitalização para impedi-los de secagem. </li><li> Executar uma de corpo inteiro PET gravação verificação estática, pelo menos, 50 milhões eventos coincidentes, com uma duração aproximada de 15 minutos. Definir janela de tempo a energia ea coincidência 350-700 keV e 6 ns, respectivamente. Em seguida, execute uma varredura 5 min tomografia computadorizada (CT). Ajuste o tubo de raios-X para uma voltagem de 80 kV e uma corrente de 500 mA; a tomografia computadorizada usa 120 passos de rotação para um total de 220 graus, com cerca de 145 ms por fotograma 10. </li><li> Após o exame PET-CT, sacrificar imediatamente os ratos por asfixia com dióxido de carbono e confirmar as mortes por pinching as patas dos animais. Uma vez confirmada, realizar luxações cervicais. </li><li> Remover o sangue em tecidos de ratinho por primeiro corte aberto do átrio direito e, em seguida, injectando 20 mL de PBS para o ventrículo esquerdo, e posteriormente recolher os tecidos relevantes, tais como tumor, fígado, baço, pulmão, rim, músculo, e outros 16. </li><li> Pesar os tecidos, medir o seu teor de radioactividade por contagem gama 8, 9, e calcular a acumulação de radioactividade do tecido como uma percentagem da dose injectada por grama (% ID / g). </li></ol> 3. Ex Vivo citometria de fluxo e imunocoloração NOTA: Injectar cerca de 100? L de lipossomas fluorescentes de melanoma em ratinhos através da sua veia da cauda a uma dose de 0,5 mg de corante por kg de peso corporal. Se as amostras radioactivas não são permitidos no citômetro de fluxo e microscópio de fluorescência, deve ser utilizado lipossomas não-radioativos. <ol><li> Sacrificar o h pós-injecção ratos 24, como no passo 2.5, e recolher os tumores, que devem ser armazenados em PBS frio. </li><li> Por citometria de fluxo, siga estas etapas 17: <ol><li> Prepara-se uma mistura de enzimas consiste de uma mistura de colagenase I purificada e II (4 U / mL), hialuronidase (60 U / mL), e a ADNase I (60 U / mL) em citometria de fluxo tampão (PBS com 0,5% de BSA e 1 mM de EDTA). </li><li> Misturar 100 mg de tecido tumoral com 200 uL de tampão de cocktail de enzima num tubo de 1,5 mL e cortar o tecido em pequenos pedaços com uma tesoura fina. Adicione outro 1,3 ml de mistura de enzimas para dentro do tubo e transferir o seu conteúdo para um prato de 6 poços. </li><li> Agitar a placa de 6 poços, num agitador horizontal colocado dentro de um forno de 37 ° C durante 60 min a 60 rpm. </li><li> Lavar o tecido homogeneizado com tampão de citometria de fluxo de 3 vezes para se obter uma suspensão de célula única. </li><li> Corar as células com um cocktail de anticorpos específicos para biomarcadores incluindo CD45, CD11b, Ly6C, CD11c,CD64, CD3, MHCII, e CD31 (diluição 1: 200 para todos os anticorpos; clone informação é fornecida na folha de cálculo materiais). Identificar tipos de células relevantes e determinar a sua intensidade de fluorescência média (MFI) de DiIC12 (5) -DS em cada população de células com um fluxo adequado citometria de software de análise. </li></ol></li><li> Para imunocoloração, siga estes passos: <ol><li> Coloque um tumor em uma cassete cheia com o meio de corte óptima (OCT) e congelá-lo em gelo seco. </li><li> Faça 6 mícrons seções congeladas de tecido tumoral e colocá-los em lâminas de histologia de vidro; as secções deverão contemplar as maiores secções transversais do tumor, que fornecem as informações mais representativo no tecido. </li><li> Manchar os biomarcadores (por exemplo, CD31 para células endoteliais) de interesse com os anticorpos correspondentes (por exemplo, anti-CD31, clone MEC13.3). Fixar as seções com paraformaldeído; bloqueá-las com soro correspondentes a origem das espécies de anticorpos secundários; incubcomeu com anticorpos primários durante 12 h a 4 ° C e, em seguida, com anticorpos secundários durante 2 h; e, finalmente, lavou-se com PBS e cobrir com lamelas. Imagem as lâminas coradas com uma Leica confocal na vertical SP5 microscópio 13, 16. </li></ol></li></ol>

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Passos críticos dentro do Protocolo: A elevada qualidade dos lipossomas duplamente marcadas é a chave para a produção de resultados consistentes durante um longo período de tempo. Corantes fluorescentes livres ou 89 iões Zr pode gerar totalmente diferentes padrões de segmentação e tem de ser completamente removido durante o passo de purificação. Além disso, se o sistema imunológico afecta significativamente o desempenho experimental nanomedicina cancro, a utilização de...

Nanomedicine está mudando o paradigma do desenvolvimento tratamento do câncer 1. Inspirado pelo impacto clínico enorme de nanomedicamentos câncer anteriores, como liposome- e nanotherapies à base de albumina 2, 3, muitas formulações inovadoras foram produzidas na última década. No entanto, estudos recentes do sucesso clínico tradução destes nanomedicamentos cancerosas indicam que apenas alguns deles têm sido aprovadas para o uso clínico de 4, 5. Um grande obstáculo para a tradução clínica de novas nanomedicamentos câncer é a sua melhoria limitada do índice terapêutico em comparação com a administração direta dos compostos terapêuticos livre 6. Como tal avaliação, exacta do comportamento in vivo de nanomedicamentos em sistémica, tecido, e níveis celulares em modelos animais pré-clínicos é essencial para identify aqueles com índices terapêuticos ideais para futura tradução clínica. Nanomateriais podem ser radiomarcados para a caracterização quantitativa em animais vivos com a tomografia por emissão de pósitrons de imagem (PET), que tem excelente sensibilidade e reprodutibilidade entre todas as modalidades de imagem clínicos 7. Por exemplo, 89 Zr-marcado de longa circulação nanomedicamentos foram caracterizadas em modelos de ratinho para o cancro 8, 9, 10, bem como em outros modelos de doenças 11. Além disso, a semi-vida no sangue e a biodistribuição dos nanomedicamentos pode ser extensivamente avaliada pela utilização ex vivo medições de radioactividade em tecidos individuais 8. Portanto, a marcação radioactiva permite a avaliação quantitativa de nanomedicamentos em níveis sistémicos e tecidos. Importante, radiolabeled nanomedicamentos geralmente não podem ser analisados ​​no unicelular ou níveis subcelulares devido à limitada resolução espacial do sinal radioactivo. Assim, a rotulagem fluorescente revela-se uma modalidade complementar para a avaliação de nanopartículas com técnicas de imagiologia óptica, tais como citometria de fluxo e microscopia de fluorescência 12. Para este fim, as nanopartículas marcadas com radioisótopos e marcadores fluorescentes podem ser quantitativamente avaliado in vivo por imagiologia nuclear e ex vivo por contagem de radioactividade, e que também pode ser amplamente caracterizada a nível celular por imagiologia óptica. Anteriormente, temos desenvolvido procedimentos modulares para incorporar marcadores radioactivos e fluorescentes em várias nanopartículas, incluindo a lipoproteína de alta densidade (HDL), 11, 9, 10 lipossomas, nanopartículas poliméricas, fragmentos de anticorpo, e nanoemulsions 10, 13. Estas nanopartículas marcadas têm permitido a caracterização quantitativa em modelos animais relevantes em diferentes níveis, que orientaram a otimização desses nanomateriais para suas aplicações específicas. No estudo atual, o objetivo é usar nanopartículas-lipossomal plataforma nanomedicina mais estabelecido 14 -como um exemplo para demonstrar os procedimentos abrangentes para gerar uma nanopartícula duplamente marcada e caracterizá-la completamente em um melanoma singênica rato modelo B16-F10 clássico 15 . A partir dos resultados, estamos confiantes de que essa abordagem caracterização de nanopartículas pode ser adaptado para avaliar outros nanomedicamentos cancerosas em modelos relevantes do mouse.

<table><tbody><tr><td>DPPC</td><td>Avantilipids</td><td>850355</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C8667</td><td></td></tr><tr><td>DSPE-PEG2000</td><td>Avantilipids</td><td>880120P</td><td></td></tr><tr><td>DSPE-DFO</td><td>Home made</td><td>110634</td><td>Perez-Medina &lt;em&gt;et al.&lt;/em&gt;, JNM, 2014</td></tr><tr><td>DiIC12[5]-DS</td><td>AAT Bioquest</td><td>22051</td><td></td></tr><tr><td>Centrifugal filter</td><td>Vivaproducts</td><td>VS2061</td><td></td></tr><tr><td>Rotary evaporator</td><td>Buchi</td><td>R-100</td><td></td></tr><tr><td>Radio-HPLC</td><td>Shimadzu HPLC with 2 LC-10AT pumps</td><td>N/A</td><td></td></tr><tr><td>&lt;sup&gt;89&lt;/sup&gt;Zr-oxalate</td><td>MSKCC</td><td>Synthesized in house</td><td>TR19/9 variable beam cyclotron (Ebco Industries Inc.)</td></tr><tr><td>Micro PET-CT</td><td>Siemens</td><td>Inveon Micro-PET/CT</td><td></td></tr><tr><td>Gamma counter</td><td>PerkinElmer</td><td>2470-0150</td><td></td></tr><tr><td>Flow cytometry</td><td>BD Biosciences</td><td>Fortessa</td><td>Any multi-parametric flow cytometry analyzers would suffice</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Laboratories</td><td></td><td></td></tr><tr><td>B16-YFP melanoma cells</td><td>Home made</td><td>N/A</td><td>Salmon &lt;em&gt;et al.&lt;/em&gt;, Immunity, 2016</td></tr><tr><td>Ly6C (clone HK1.4)--APC-Cy7</td><td>128025</td><td>Biolegend</td><td></td></tr><tr><td>MHCII (M5/114/152)--APC</td><td>107613</td><td>Biolegend</td><td></td></tr><tr><td>CD45 (30-F11)--BV510</td><td>103137</td><td>Biolegend</td><td></td></tr><tr><td>CD64 (X54-5/7.1)--PE-Cy7</td><td>139313</td><td>Biolegend</td><td></td></tr><tr><td>CD11b (M1/70)--BV605</td><td>101237</td><td>Biolegend</td><td></td></tr><tr><td>CD3 (17A2)--BV711</td><td>100241</td><td>Biolegend</td><td></td></tr><tr><td>CD31 (13.3)--PE</td><td>561073</td><td>Biolegend</td><td></td></tr><tr><td>CD11c (M418)--PerCP-Cy5.5</td><td>117327</td><td>BD Biosciences</td><td></td></tr><tr><td>CD31 (13.3) no fluorophore</td><td>550274</td><td>BD Biosciences</td><td></td></tr></tbody></table>

a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines

Inspirado pelo sucesso de nanomedicamentos câncer anteriores na clínica, os pesquisadores têm gerado um grande número de novas formulações na última década. No entanto, apenas um pequeno número de nanomedicamentos foram aprovados para uso clínico, ao passo que a maioria dos nanomedicamentos sob desenvolvimento clínico têm produzido resultados decepcionantes. Um grande obstáculo para a tradução clínica bem sucedida de novas nanomedicamentos câncer é a falta de uma compreensão exata do seu desempenho in vivo. Este artigo apresenta um procedimento rigoroso para caracterizar o comportamento in vivo de nanomedicamentos em ratinhos portadores de tumor em sistémica, de tecido, de uma única célula, e níveis sub-celulares através da integração da emissão de positrões tomografia computadorizada de tomografia (PET-CT), métodos de radioactividade quantificação , citometria de fluxo e microscopia de fluorescência. Usando essa abordagem, os pesquisadores podem avaliar com precisão novas formulações em nano-escala em modelos de ratos relevantes do cancer. Estes protocolos podem ter a capacidade de identificar os nanomedicamentos câncer mais promissores com alto potencial translacional ou para ajudar na otimização de nanomedicamentos cancerosas para tradução futuro.

A Figura 1 mostra uma visão geral do procedimento. A Figura 2 apresenta o procedimento de síntese esquemática dos lipossomas duplamente marcadas descritas no passo 1 10. A Figura 3 exibe um representante de PET-CT imagem (Figura 3a), a quantificação de radioactividade a partir de imagens de PET (Figura 3b), uma meia-vida no sangue (Figura 3C), e biodistribui?...

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines at JoVE.com

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Um método completo para avaliar o<em&gt; In Vivo</em&gt; Performance of Cancer Nanomedicinas

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

a-comprehensive-procedure-to-evaluate-vivo-performance-cancer

Research

JoVE Journal

Cancer Research

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

8.8K visualizações.  Memorial Sloan Kettering Cancer Center. The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Vídeo: A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Anticancer eficácia da terapia fotodinâmica com Nanopartículas Lung Cancer segmentadas

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Pesquisa do câncer

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Extração de amostra e quantificação cromatográfica simultânea de doxorrubicina e mitomicina C após entrega de combinação de drogas em nanopartículas para ratos de Tumor-rolamento

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Avaliação de Captação de nanopartículas em tumores em tempo real usando imagens intravital

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Medicina

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Nanopartículas virais para<em&gt; Em vivo</em&gt; Imaging Tumor

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengenharia

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Avaliação da Atividade Antitumoral In vivo de Nanopartículas de Polianidrido IL-1α

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Avaliar a eficácia do Cancer Drogas Sensibilização<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validação de nanocorpo e Antibody Based<em&gt; In Vivo</em&gt; Experimentos Tumor Xenoenxerto NIRF-imagem em ratos usando<em&gt; Ex Vivo</em&gt; Citometria de fluxo e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

<meta charset="utf8"></head><body>Nanofármaco direcionado ao miRNA-10b oncogênico para o tratamento do câncer de mama metastático

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

<meta charset="utf8"></head><body>Monitoramento do acúmulo de nanodrogas em metástases de câncer de mama murino

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Monitoramento do acúmulo de nanodrogas em metástases de câncer de mama murino

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

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