Triagem biológica de inibidores de sirtuína 2 (TcSir2rp1) candidatos a antichagásicos / Biological screening of sirtuin 2 inhibitors (TcSir2rp1) antichagasic candidates

A doença de Chagas é causada pelo Trypanosoma cruzi, e atualmente, acomete entre 6 a 7 milhões de pessoas em todo o mundo. A quimioterapia disponível para seu o tratamento se baseia apenas em dois fármacos, nifurtimox e benznidazol, com mais de 50 anos de descoberto. Estes fármacos apresentam eficácia limitada, pois são pouco efetivos na fase crônica e apresentam alta toxicidade, resultando em efeitos adversos graves. Esse panorama mostra a necessidade de novas abordagens terapêuticas contra essa doença. Nesse sentido, a inibição de vias bioquímicas essencias para o parasita se mostram como uma boa sugestão para identificação de compostos promissores candidatos a novos agentes quimioterápicos. A sirtuína 2 (Sir2) são enzimas reguladoras que participam de mecanismos epigenéticos em tripanossomatídeos, e no T. cruzi possuem um papel fundamental em todos os seus estágios evolutivos, devido a este fato, se apresentam como um alvo promissor na busca por novos fármacos contra a doença de Chagas. Neste sentido propomos a busca de inibidores da Sir2 proteína 1 do T. cruzi (TcSir2rp1) que é geneticamente validada como alvo farmacológico, por meio da estratégia de triagem biológica. Realizou-se a expressão da enzima recombinante por biologia molecular em um sistema de transformação utilizando cepa de Escherichia coli Artic Express (DE3). Foi feita a purificação e a confirmação da obtenção da proteína recombinante se deu por gel SDS-PAGE. Após a obtenção da enzima os parâmetros cinéticos foram determinados por experimentos de fluorimetria. A triagem foi realizada para um conjunto de 82 compostos, previamente sintetizados pelo nosso grupo de pesquisa, como inibidores da TcSir2p1 em dose única de 100 µM. Os ensaios foram realizados em triplicata e em experimentos independentes. Dentre os 82 compostos testados, 20 apresentaram inibições maior que 50% contra a enzima TcSir2rp1, na dose de 100 µM. Dentre estes, se destacaram 3 compostos derivados de chalconas, para os quais foi determinada a potência. O composto 1 foi o que mais potente, apresentando valor de IC50 de 11,65 µM, já os compostos 3 e 5 foram menos potentes (IC50= 38,50 µM e 19,85 µM, respectivamente). Diante dos resultados obtidos, pode-se concluir que a estratégia de triagem biológica é promissora na identificação de inibidores da TcSir2p1 candidatos a agentes anti- T. cruzi

Chagas disease is caused by Trypanosoma cruzi, and currently affects 6 to 7 million people worldwide. The chemotherapy available for its treatment is based on only two drugs, nifurtimox and benznidazole, with more than 50 years of discovery. These drugs have limited efficacy, as they are ineffective in the chronic phase and have high toxicity, resulting in serious adverse effects. This panorama shows the need for new therapeutic approaches against this disease. In this sense, the inhibition of essential biochemical pathways for the parasite proves to be a good suggestion for the identification of promising compounds candidates for new chemotherapeutic agents. Sirtuin 2 (Sir2) are regulatory enzymes that participate in epigenetic mechanisms in trypanosomatids, and in T. cruzi they have a fundamental role in all their evolutionary stages, due to this fact, they present themselves as a promising target in the search for new drugs against Chagas disease. In this sense, we propose the search for inhibitors of Sir2 protein 1 of T. cruzi (TcSir2rp1) which is genetically validated as a pharmacological target, through the biological screening strategy. The expression of the recombinant enzyme was performed by molecular biology in a transformation system using strain of Escherichia coli Artic Express (DE3). Purification was performed and confirmation of obtaining the recombinant protein was performed by SDS-PAGE gel. After obtaining the enzyme, the kinetic parameters were determined by fluorimetry experiments. Screening was performed for a set of 82 compounds, previously synthesized by our research group, as TcSir2p1 inhibitors in a single dose of 100 µM. Assays were performed in triplicate and in independent experiments. Among the 82 compounds tested, 20 showed inhibitions greater than 50% against the enzyme TcSir2rp1, at a dose of 100 µM. Among these, 3 compounds derived from chalcones stood out, for which the potency was determined. Compound 1 was the most potent, with an IC50 value of 11.65 µM, while compounds 3 and 5 were less potent (IC50= 38.50 µM and 19.88 µM, respectively). In view of the results obtained, it can be concluded that the biological screening strategy is promising in the identification of TcSir2p1 inhibitors candidates for anti-T. cruzi agents

Planejamento, síntese e avaliação biológica de candidatos a fármacos: busca de inibidores epigenéticos da Sirtuína 2 Sir2 / Design, synthesis and biological evaluation of drug candidates: search for Sirtuin 2 (Sir 2) epigenetic inhibitors

As doenças negligenciadas são causadas por agentes infecciosos e parasitários, como vírus, bactérias, protozoários e helmintos. Essas doenças são prevalentes em populações de baixa renda que vivem em países em desenvolvimento e são responsáveis por incapacitar e levar milhares de pessoas à morte. Este nome se dá pois, apesar de sua grande relevância médica, recebem pouca atenção dos governos e indústrias farmacêuticas. Dentre essas doenças podemos destacar a Doença de Chagas, doença infecciosa causada pelo parasita hemoflagelado Trypanosoma cruzi. Endêmica em 21 países, com 6 a 7 milhões de pessoas infectadas resultando em 7500 mortes por ano. A quimioterapia disponível contra essa parasitose é baseada em apenas dois medicamentos, o benznidazol e o nifurtimox, ativos principalmente na fase aguda da doença e com efeitos adversos graves que comprometem a adesão ao tratamento e, além disso, apesar dos enormes esforços na pesquisa de novos agentes antichagásicos em nível nacional e internacional, na maioria realizada academicamente, ainda não foram encontradas alternativas terapêuticas para a doença, persistindo, assim, a necessidade de descoberta e desenvolvimento de novos fármacos. O início de um planejamento de um novo fármaco se dá pela definição de um alvo bioquímico a ser utilizado na busca de moléculas que possam exercer a função de inibidores ou moduladores, conforme a atividade biológica desejada. Neste sentido, as sirtuínas 2 (Sir2) são enzimas que se mostraram essenciais para o crescimento in vitro do T. cruzi em suas formas amastigota e epimastigota. No caso de tripanossomatídeos, em geral, a superexpressão de Sir2 está relacionada à sobrevivência de formas amastigotas. Assim, essas evidências indicam que a Sir2 de tripanosomatídeos tem grande potencial como alvo biológico na busca e desenvolvimento de novos fármacos antichagásicos. O objetivo principal deste projeto foi identificar moléculas que apresentaram atividade inibitória para a sirtuína 2 de T. cruzi por meio da utilização da estratégia de Planejamento de Fármacos Baseada no Ligante - Ligand Based Drug Design (LBDD) e o desenvolvimento de análogos dos inibidores da Sir2. A modificação molecular está entre algumas das técnicas tradicionais usadas no desenvolvimento racional de um fármaco, e é usada principalmente no desenvolvimento de análogos, e busca melhorar as propriedades farmacocinéticas e/ou farmacodinâmicas de um protótipo, obter propriedades de interação semelhantes ao alvo e, em alguns casos, revelar uma atividade biológica. Com este intuito, análogos do sirtinol e da salermida foram sintetizados e uma nova rota sintética utilizando o microrreator em fluxo contínuo foi desenvolvida e apresentou rendimento superior quando comparado à síntese em bancada. A partir desta metodologia foram obtidos 20 compostos. Os ensaios in vitro contra formas amastigotas do T. cruzi indicaram que 8 compostos inibiram a atividade parasitária em mais de 50%, na dose de 10 µM, sendo que alguns destes apresentaram maior inibição parasitária quando comparados ao benznidazol, o fármaco de referência e único disponível no Brasil. Com estes resultados preliminares, novos ensaios estão sendo realizados para identificar potência e mecanismo de ação destes candidatos a agentes tripanomicidas

Neglected diseases are caused by infectious and parasitic agents such as viruses, bacteria, protozoa and helminths. These diseases are prevalent in low-income populations living in developing countries and are responsible for disabling and killing thousands of people. They get this name because, despite their great medical relevance, they end up receiving little attention from governments and pharmaceutical industries. Among these diseases, we can highlight Chagas disease, an infectious endemic disease caused by the hemoflagellate parasite Trypanosoma cruzi. This disease is endemic in 21 countries, with 6 to 7 million people infected resulting in 7,500 deaths per year. Chemotherapy is based on just two drugs, benznidazole and nifurtimox, which are mainly active in the acute phase of the disease. These drugs have adverse effects that compromise adherence, even more, considering that they are not effective from the point of view of the chronic phase of the disease. Despite the enormous efforts in researching new anti-chagasic agents at the national and international level, and mostly carried out academically, therapeutic alternatives for the disease have not yet been found, thus, the need for the discovery and development of new drugs persists. Sirtuins 2 (Sir2) are enzymes that have been shown to be essential for the in vitro growth of T. cruzi in its amastigote and epimastigote forms. In the case of trypanosomatids in general, Sir2 overexpression is related to the survival of amastigote forms. Sir2 inhibitors, such as sirtinol, have shown efficacy in leishmanicides. Thus, these evidences indicate that Sir2 from trypanosomatids can be considered as a biological target in the search and development of new anti-chagasic drugs. The beginning of a new drug planning study is the definition of a biochemical target to be used in the search for molecules that can play the role of inhibitors or modulators, according to the desired biological activity. The main objective of this project was to identify molecules that presented inhibitory activity to sirtuin 2 of T. cruzi using the Ligand Based Drug Design (LBDD) strategy of planning and the development of analogues of Sir2 inhibitors. Molecular modification is a traditional technique used in the rational development of a drug, as well as the use of natural products, combinatorial chemistry, high-throughput screening (HTS), among others. Mainly used in the development of analogues, molecular modification is applied for different purposes, among them, it seeks to improve the pharmacokinetic and/or pharmacodynamic properties of a prototype, obtain target-like interaction properties and, in some cases, reveal an activity biological. For this purpose, analogues of sirtinol and salermide were synthesized and a new synthetic route using the microreactor in continuous flow was developed and presented superior yield when compared to benchtop synthesis. From this methodology, 20 compounds were obtained. in vitro assays against amastigote forms of T. cruzi indicated that 8 compounds inhibited parasitic activity by more than 50% at a dose of 10 µM, and some of these showed greater parasitic inhibition when compared to benznidazole, the reference drug, and only available in Brazil. With these preliminary results, new assays are being carried out to identify the potency and mechanism of action of these candidate trypanocidal agents

Inhibiting silence information regulator 2 and glutaminase in the amygdala can improve social behavior in autistic rats / 浙江大学学报·医学版

OBJECTIVE@#To investigate the underlying molecular mechanisms by which silence information regulator (SIRT) 2 and glutaminase (GLS) in the amygdala regulate social behaviors in autistic rats.@*METHODS@#Rat models of autism were established by maternal sodium valproic acid (VPA) exposure in wild-type rats and SIRT2-knockout ( SIRT2 -/-) rats. Glutamate (Glu) content, brain weight, and expression levels of SIRT2, GLS proteins and apoptosis-associated proteins in rat amygdala at different developmental stages were examined, and the social behaviors of VPA rats were assessed by a three-chamber test. Then, lentiviral overexpression or interference vectors of GLS were injected into the amygdala of VPA rats. Brain weight, Glu content and expression level of GLS protein were measured, and the social behaviors assessed.@*RESULTS@#Brain weight, amygdala Glu content and the levels of SIRT2, GLS protein and pro-apoptotic protein caspase-3 in the amygdala were increased in VPA rats, while the level of anti-apoptotic protein Bcl-2 was decreased (all P<0.01). Compared with the wild-type rats, SIRT2 -/- rats displayed decreased expression of SIRT2 and GLS proteins in the amygdala, reduced Glu content, and improved social dysfunction (all P<0.01). Overexpression of GLS increased brain weight and Glu content, and aggravated social dysfunction in VPA rats (all P<0.01). Knockdown of GLS decreased brain weight and Glu content, and improved social dysfunction in VPA rats (all P<0.01).@*CONCLUSIONS@#The glutamate circulatory system in the amygdala of VPA induced autistic rats is abnormal. This is associated with the upregulation of SIRT2 expression and its induced increase of GLS production; knocking out SIRT2 gene or inhibiting the expression of GLS is helpful in maintaining the balanced glutamate cycle and in improving the social behavior disorder of rats.

Particulate matter 2.5 triggers airway inflammation and bronchial hyperresponsiveness in mice by activating the SIRT2-p65 pathway / 医学前沿

Exposure to particulate matter 2.5 (PM2.5) potentially triggers airway inflammation by activating nuclear factor-κB (NF-κB). Sirtuin 2 (SIRT2) is a key modulator in inflammation. However, the function and specific mechanisms of SIRT2 in PM2.5-induced airway inflammation are largely understudied. Therefore, this work investigated the mechanisms of SIRT2 in regulating the phosphorylation and acetylation of p65 influenced by PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Results revealed that PM2.5 exposure lowered the expression and activity of SIRT2 in bronchial tissues. Subsequently, SIRT2 impairment promoted the phosphorylation and acetylation of p65 and activated the NF-κB signaling pathway. The activation of p65 triggered airway inflammation, increment of mucus secretion by goblet cells, and acceleration of tracheal stenosis. Meanwhile, p65 phosphorylation and acetylation, airway inflammation, and bronchial hyperresponsiveness were deteriorated in SIRT2 knockout mice exposed to PM2.5. Triptolide (a specific p65 inhibitor) reversed p65 activation and ameliorated PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Our findings provide novel insights into the molecular mechanisms underlying the toxicity of PM2.5 exposure. Triptolide inhibition of p65 phosphorylation and acetylation could be an effective therapeutic approach in averting PM2.5-induced airway inflammation and bronchial hyperresponsiveness.

SIRT2-Reverses Drug-Resistance of HL-60/A through Autophagy Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of targeting the silent information regulator 2 hemolog 2 (SIRT2) expression on the apoptosis of drug-resistant AML cell line HL-60/A and its mechanism.@*METHODS@#The expression of SIRT2 and antophagy-related protein LCT, P62 in HL-60/A and HL-60 was detected by Western blot, the effect of cytorabine on the apoptosis of HL-60/A cells was detected by using Annexin V/PI double staining after targeting inhibition of SIRT2 expression resulting from transfecting HL-60/A cells with SiRNA. The Western blot and transmission electray microscopy were used to detect the cell autophagy. To further clarify the role of autophragy in the regulatory effect of SIRT2 on the drug-resistance of HL-60/A cells, the autophagy-specific agonist, repamycin, was added into the cell culture medium after SIRT2-siRNA transfection. Then, the autophagy and apoptosis of HL-60/A were detected, respectively.@*RESULTS@#The SIRT2 protein expression obviously increased in HL-60/A cells than that in HL-60 cells. Moreover, the expression rate of LC3II/I was higher, but P62 expression was lower in HL-60/A cells. After siRNA successfully transfecting into HL-60/A cells, quantitative PCR and Western blot should that the expression of SIRT2 significantly decreased. Meawhile, Western blot showed that the expression of LC3 II/I decreased, but P62 increased. Meanwhile, By TEM found that the number of autophagosome also decreased, suggesting the autophagy was inhibited after down-regulation of SIRT2. In addition, the drug senstivity of HL-60/A cells to cytarabine in siRNA-transfection group increased, and the apoptotic rate detected by Annexin V/PI double staining significantly increased. However, after co-culture with rapamycin, the suppressed autophagy in siRNA-trasfect HL-60/A cells was activated, leading to the reappearance of drug resistance of cells to cytarabine, and more significantly decrease of apoptotic rate.@*CONCLUSION@#The high expression of SIRT2 in HL-60/A cells activates the protective autophagy mechanism, which closely related with drug resistance.

Busca por inibidores seletivos de Sirtuína 2 de T. cruzi empregando técnicas de planejamento de fármacos baseadona estrutura do receptor / Search for selective inhibitors of T. cruzi Sirtuin 2 employing drug design techniques based on receptor structure

A doença de Chagas, causada pelo parasita Trypanosoma cruzi, acomete entre 6 a 8 milhões de pessoas em todo o mundo. Conhecida como tripanossomíase americana, por ter sido considerada endêmica apenas na América Latina, esta doença, se espalhou para outros continentes devido aos movimentos migratórios se tornando um problema de sáude mundial. Estima-se que 56.000 novos casos e cerca de 12.000 mortes por complicações relacionadas à doença de Chagas anualmente. A quimioterapia disponível para o tratamento é composta apenas por dois fármacos, nifurtimox e benznidazol, no entanto são pouco eficazes na fase crônica da doença. Estes fármacos apresentarem, ainda, efeitos adversos graves e resistência por parte de algumas cepas do parasita. Diante deste panorama, é iminente a necessidade da busca de novos fármacos contra T. cruzi. Para a busca racional de novos quimiterapicos antiparasitários é fundamental a identificação e caracterização de vias metabólicas essenciais à sobrevivência dos parasitas. Assim, a enzima sirtuína 2 - Silent Information Regulator 2 (Sir2), tem importante papel para a infecção por T. cruzi, pois está totalmente envolvida no seu ciclo celular do parasita. Esta é uma enzima NAD+ dependente da classe III histona desacetilases, e se mostra como um interessante alvo bioquímico para o desenvolvimento de antichagásicos. A disponibilidade do sequenciamento genômico da Sir2 nos permite utilizar estratégias de planejamento de fármaco baseado no receptor (SBDD - Structure Based Drug Design) na identificação de candidatos a fármacos para essa doença. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar e selecionar inibidores enzimáticos potentes e seletivos para o alvo escolhido. Assim, neste trabalho, foi construído por meio da técnica de modelagem comparativa o modelo da enzima Sir2 de T. cruzi. Uma simulação por dinâmica molecular de 200ns, foi realizada para averiguar a estabilidade do modelo obtido. Diante da estabilização do modelo a partir de 100ns, o mesmo foi validado utilizando análise de clusters, RMSD (Root-mean-square Deviation) e análises de frequência de ligações de hidrogênio com o Cofator (NAD+) e os aminoácidos do sítio de catálise foram observadas, estes passos de simulação e validação foram realizados no programa DESMOND. Com o modelo robusto, os campos de interações moleculares (MIFs) foram gerados no programa GRID (Molecular Discovery v2.1) com o intuito de elucidar as regiões favoráveis a interação com a enzima em relação a propriedades físico-químicas da Sir2. A partir dos MIFs favoráveis a Sir2 de T. cruzi foi possível a construção de dois modelos farmacofóricos, o qual se baseou nas interações do Cofator (NAD+) e o sítio de catálise (Nicotinamida). O mesmo foi apliacdo como filtro para Triagem Virtual no programa UNITY da plataforma SYBYL X 2.0, utilizando os bancos de dados ZINC15 e GSK. A triagem resultou na seleção de 8 compostos candidatos a inibidores. Destes foram adquiridos 6 compostos por serem considerados mais promissores devido a complementariedade molecular. Estes foram testados contra a enzima de T. cruzi Sri2. Após o ensaio foi possível avaliar a potência de 4 compostos, sendo o composto CDMS-01 (IC50 = 39,9uM) o mais promissor que será submetido à processos de otimização molecular

Chagas disease, caused by the parasite Trypanosoma cruzi, affects between 6 and 8 million people worldwide. Also known as American trypanosomiasis, because it is considered endemic only in Latin America, but has spread to other continents due to migratory movements. It is estimated that 56,000 new cases and about 12,000 deaths from complications related to Chagas disease annually. The chemotherapy available for treatment consists of only two drugs, nifurtimox and benznidazole, however these are poorly effective in the chronic phase. These drugs also have serious adverse effects and resistance from strains of the parasite. Faced with this scenario, the need to search for new drugs against T. cruzi is imminent. For the drug planning for new antiparasitic chemotherapics, the identification and characterization of metabolic pathways essential to the survival of parasites is fundamental. Therewith, the sirtuin 2 - Silent Information Regulator 2 (Sir2) enzyme has an important role for T. cruzi infection, since Sir2 in the parasite is totally involved in its cell cycle. This is an NAD+-dependent enzyme of class III histone deacetylases, and it shows an interesting biochemical target for the development of antichagasic. The availability of Sir2 genomic sequencing allows us to use SBDD (Structure Based Drug Design) strategies in identifying drug candidates for this disease. Among the modern techniques of SBDD used, virtual screening makes it possible to identify and select potent and selective enzyme inhibitors for the chosen target. The model of the T. cruzi Sir2 enzyme was constructed using the comparative modeling technique. A molecular dynamics simulation of 200ns was performed to ascertain the stability of the obtained model. Considering the stabilization of the model from 100ns, it was validated using cluster analysis, Root-mean-square Deviation (RMSD) and hydrogen bond frequency analyzes with Cofator (NAD+) and the amino acids of the catalysis site were observed, these simulation and validation steps were performed in the DESMOND program. With the robust model, the molecular interaction fields (MIFs) were generated in the GRID program (Molecular Discovery v2.1) in order to elucidate the regions favorable to the interaction with the enzyme in relation to the physicalchemical properties of Sir2. From the MIFs favorable to Sir2 of T. cruzi it was possible to construct two pharmacophoric models, which was based on the interactions of Cofator (NAD+) and the catalysis site (Nicotinamide). It was also applied as a Virtual screening filter in the UNITY program of the SYBYL X 2.0 platform, using the ZINC15 and GSK databases. Screening resulted in the selection of 8 inhibitor candidate compounds. Six compounds were obtained from the screening, because they were considered more promising, and were tested against T. cruzi Sri2 enzyme. After the assay it was possible to evaluate the potency of 4 compounds, the most promising compound being CDMS-01 (IC50 = 39.9 µM) that will be submitted to molecular optimization processes

Sirtuin-2 inhibition affects hippocampal functions and sodium butyrate ameliorates the reduction in novel object memory, cell proliferation, and neuroblast differentiation / 한국실험동물학회지

We investigated the effects of the sirtuin-2 (SIRT2) inhibitor AK-7 on novel object memory, cell proliferation, and neuroblast differentiation in the dentate gyrus. In addition, we also observed the relationships with sodium butyrate, a histone deacetylase inhibitor, on the hippocampal functions. To investigate the effects of AK-7 on hippocampal functions, 10-week-old C57BL/6 mice were daily injected intraperitoneally with 20 mg/kg AK-7 alone or in combination with subcutaneous administration of 300 mg/kg sodium butyrate, a histone deacetylase inhibitor, for 21 days. A novel object recognition test was conducted on days 20 (training) and 21 (testing) of treatment. Thereafter, the animals were sacrificed for immunohistochemistry for Ki67 (cell proliferation) and doublecortin (DCX, neuroblast differentiation). AK-7 administration significantly reduced the time spent exploring new objects, while treatment in combination with sodium butyrate significantly alleviated this reduction. Additionally, AK-7 administration significantly reduced the number of Ki67-positive cells and DCX-immunoreactive neuroblasts in the dentate gyrus, while the treatment in combination with sodium butyrate ameliorated these changes. This result suggests that the reduction of SIRT2 may be closely related to age-related phenotypes including novel object memory, as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, sodium butyrate reverses SIRT2-related age phenotypes.

Feasibility of integrating tumor therapy with therapeutic effect evaluation using siRNA-loaded microbubbles / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the feasibility of integrating cancer gene therapy with therapeutic effect evaluation using siRNA-loaded nano-scale microbubbles (siRNA-NBs).</p><p><b>METHODS</b>siRNA-NBs were prepared by hetero-assembly of polymeric siRNA micelles and liposomal microbubbles, and the particle sizes and surface potentials were examined with dynamic light scattering. The distributions of cy3-labled siRNA in the tumor tissues were evaluated using confocal laser scanning microscopy. A siRNA targeting the anti-apoptosis gene SIRT2 was designed and its gene silencing effects was tested in vivo using siRNA-NBs with ultrasound exposure. The therapeutic effect of the loaded siRNA-NBs was evaluated by contrast-enhanced ultrasonography.</p><p><b>RESULTS</b>The siRNA-NBs had a mean diameter of 400.7 ± 30.5 nm with a weak positive charge of +8.8 ± 0.8 mV. With ultrasound exposure, siRNA-NBs effectively delivered cy3-siRNA into the cytoplasm of cancer cells and caused SIRT2 suppression and cell apoptosis in tumor tissues, resulting in significantly suppressed tumor growth. In addition, contrast-enhanced ultrasonography of siRNA-NBs provided good imaging quality to allow real-time observation of blood supply during gene therapy.</p><p><b>CONCLUSIONS</b>As a novel ultrasound contrast agent, siRNA-NBs make possible the integration of tumor gene therapy and therapeutic effect evaluation for cancer.</p>