Cancer Letters 359 (2015) 178–186
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Therapeutic potential of targeted multifunctional nanocomplex co-delivery of siRNA and low-dose doxorubicin in breast cancer Dawen Dong, Wei Gao, Yujie Liu, Xian-Rong Qi * State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
A R T I C L E
I N F O
Article history: Received 31 October 2014 Received in revised form 31 December 2014 Accepted 8 January 2015 Keywords: Synergistic treatment Co-delivery of siRNA and doxorubicin Multifunctional nanocomplex Antiangiogenic Cytotoxicity
A B S T R A C T
Malignant tumors remain a major health burden throughout the world, and effective therapeutic strategies are urgently needed. Combining gene therapy with chemotherapeutics in a single delivery system is more effective than co-treatment of cancer with individual delivery systems carrying either gene or drug. In this study, a multifunctional folate-decorated and pH-responsive PHD/PPF/siVEGF nanocomplex is developed via a self-assembly process utilizing ternary pre-functionalized polymers with vascular endothelial growth factor targeted siRNA. Antitumor effects of the combination therapy are evaluated in both in vitro and in vivo orthotopic xenograft models of breast cancer with systemic administration. The improved therapeutic response was supported by the observation of over 70% and 55% downregulation of VEGF mRNA expressed in vitro and in vivo, effective antiproliferation and inhibition of tumor spheroids in vitro, significant decrease in tumor microvessel density in vivo, dramatic increase in life span of mice with a tumor xenograft and a decrease in toxicity in vivo. In addition, the current studies demonstrated the potential of combination of antiangiogenic therapy of siVEGF and killing off tumor cells of DOX, with the incorporation of tumor microenvironment sensitivity and target modified into a single nanoparticulate formulation for profound therapeutic effect. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Malignant tumors remain a major health burden throughout the world, and effective therapeutic strategies are urgently needed. Chemotherapy is one of the most common types of therapy employed in oncology. However, many of these chemotherapeutic agents often cause severe side effects because they produce similar cytotoxicity in both cancerous and healthy cells. For example, doxorubicin (DOX) exemplifies a potent chemotherapeutic agent to efficiently kill off tumor cells but with highly problematic systemic toxicity [1]. The use of RNA interference (RNAi) as a tumor-specific gene therapy is considered as one of the most promising platforms for cancer therapy [2–4]. Moreover, the combination of several types of therapeutic approaches with distinct mechanisms is considered to be a potential strategy for the effective treatment of cancers [5]. Besides, co-delivering chemotherapeutic drug and small interfering RNA (siRNA) in the same delivery system is more effective than co-treatment of cancer cells with delivery systems carrying either siRNA or drug [6,7]. Both angiogenesis and proliferation of tumor cells are the main features of tumor tissues [8]. Angiogenesis is a crucial component of tumor growth and metastasis [9]. In most solid tumors, the newly
* Corresponding author. Tel.: +86 10 82801584; fax: +86 10 82801584. E-mail address: [email protected] (X.-R. Qi). http://dx.doi.org/10.1016/j.canlet.2015.01.011 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
formed vessels are plagued by structural and functional abnormalities owing to an imbalance between levels of proangiogenic and antiangiogenic molecules [10]. Vascular endothelial growth factor (VEGF), a ligand produced by tumor cells and associated stroma, binds two highly related receptor tyrosine kinases, VEGF receptor 1 (VEGFR-1) and VEGFR-2, which activate multiple downstream pathways, and ultimately results in endothelial cell proliferation and migration [11–14]. The fact that tumor progression can be arrested by angiogenesis inhibitors has been confirmed. Some angiogenesis inhibitors including bevacizumab, sunitinib, sorafenib, pazopanib, and vandetanib have been approved by the Food and Drug Administration for solid tumor therapy [15,16]. RNAi-mediated silencing VEGF expression has been proven to inhibit the expression of VEGF successfully, resulting in decreased blood vessel density and delayed tumor growth [17,18]. Although inhibitors of the VEGF pathway have shown only modest efficacy as a monotherapy [19,20], they hold tremendous promise when given in combination with cytotoxic agents [21–23]. It was suggested that anti-angiogenic therapy, to some extent, corrects the flaw in structure and function of tumor blood vessels. This normalization of vessels decreases interstitial fluid pressure (IFP) of solid tumors and improves perfusion resulting in enhanced delivery and efficacy of cytotoxic agents [10,15,16,24–28]. Though highly effective, the efficiency of combination therapy is restrained by the limited duration of normalization window [26]. However, literature reports have been somewhat equivocal about the utility of this technique [29,30]. Some of these
D. Dong et al./Cancer Letters 359 (2015) 178–186
conflicting results can be attributed to differences in tumor models used, and the inherent heterogeneity between tumors. The rationale used for selecting appropriate drug–nucleic acid combinations as well as nanocarriers suitable for simultaneous delivery of drug– nucleic acid combinations has been found in some literatures [7,31–34]. Furthermore, synergistic combinations of several types of therapeutic approaches with distinct mechanisms can overcome the toxicity and other side effects associated with high doses of single drugs by countering biological compensation mechanisms, which allows the dosage of each compound to be reduced and interferes with context-specific multi-targeting mechanisms [5,35]. Nanotechnology has the potential to dramatically improve cancer therapy while reducing the toxicity associated with current approaches [36]. These nanocarrier systems accumulated in the tumor tissue via a passive targeting mechanism termed the enhanced permeability and retention (EPR) effect [37]. However, several intrinsic limitations in these delivery systems have become apparent due to their lack of specificity. To overcome these limitations such as the delivery of genes or drugs to the desired sites at the appropriate time, a variety of approaches have been adopted. Tumor microenvironmentsensitive nanocarriers that respond to changes in environmental conditions such as pH, temperature, chemicals and biomolecules have received extensive attention for their unique advantages in applications for target drug delivery. The high metabolic rate of tumor tissues often leads to acidosis (pH 20 nm) but small enough to penetrate through the leaky vasculatures in a tumor region (
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Therapeutic potential of targeted multifunctional nanocomplex co-delivery of siRNA and low-dose doxorubicin in breast cancer Dawen Dong, Wei Gao, Yujie Liu, Xian-Rong Qi * State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
A R T I C L E
I N F O
Article history: Received 31 October 2014 Received in revised form 31 December 2014 Accepted 8 January 2015 Keywords: Synergistic treatment Co-delivery of siRNA and doxorubicin Multifunctional nanocomplex Antiangiogenic Cytotoxicity
A B S T R A C T
Malignant tumors remain a major health burden throughout the world, and effective therapeutic strategies are urgently needed. Combining gene therapy with chemotherapeutics in a single delivery system is more effective than co-treatment of cancer with individual delivery systems carrying either gene or drug. In this study, a multifunctional folate-decorated and pH-responsive PHD/PPF/siVEGF nanocomplex is developed via a self-assembly process utilizing ternary pre-functionalized polymers with vascular endothelial growth factor targeted siRNA. Antitumor effects of the combination therapy are evaluated in both in vitro and in vivo orthotopic xenograft models of breast cancer with systemic administration. The improved therapeutic response was supported by the observation of over 70% and 55% downregulation of VEGF mRNA expressed in vitro and in vivo, effective antiproliferation and inhibition of tumor spheroids in vitro, significant decrease in tumor microvessel density in vivo, dramatic increase in life span of mice with a tumor xenograft and a decrease in toxicity in vivo. In addition, the current studies demonstrated the potential of combination of antiangiogenic therapy of siVEGF and killing off tumor cells of DOX, with the incorporation of tumor microenvironment sensitivity and target modified into a single nanoparticulate formulation for profound therapeutic effect. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Malignant tumors remain a major health burden throughout the world, and effective therapeutic strategies are urgently needed. Chemotherapy is one of the most common types of therapy employed in oncology. However, many of these chemotherapeutic agents often cause severe side effects because they produce similar cytotoxicity in both cancerous and healthy cells. For example, doxorubicin (DOX) exemplifies a potent chemotherapeutic agent to efficiently kill off tumor cells but with highly problematic systemic toxicity [1]. The use of RNA interference (RNAi) as a tumor-specific gene therapy is considered as one of the most promising platforms for cancer therapy [2–4]. Moreover, the combination of several types of therapeutic approaches with distinct mechanisms is considered to be a potential strategy for the effective treatment of cancers [5]. Besides, co-delivering chemotherapeutic drug and small interfering RNA (siRNA) in the same delivery system is more effective than co-treatment of cancer cells with delivery systems carrying either siRNA or drug [6,7]. Both angiogenesis and proliferation of tumor cells are the main features of tumor tissues [8]. Angiogenesis is a crucial component of tumor growth and metastasis [9]. In most solid tumors, the newly
* Corresponding author. Tel.: +86 10 82801584; fax: +86 10 82801584. E-mail address: [email protected] (X.-R. Qi). http://dx.doi.org/10.1016/j.canlet.2015.01.011 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
formed vessels are plagued by structural and functional abnormalities owing to an imbalance between levels of proangiogenic and antiangiogenic molecules [10]. Vascular endothelial growth factor (VEGF), a ligand produced by tumor cells and associated stroma, binds two highly related receptor tyrosine kinases, VEGF receptor 1 (VEGFR-1) and VEGFR-2, which activate multiple downstream pathways, and ultimately results in endothelial cell proliferation and migration [11–14]. The fact that tumor progression can be arrested by angiogenesis inhibitors has been confirmed. Some angiogenesis inhibitors including bevacizumab, sunitinib, sorafenib, pazopanib, and vandetanib have been approved by the Food and Drug Administration for solid tumor therapy [15,16]. RNAi-mediated silencing VEGF expression has been proven to inhibit the expression of VEGF successfully, resulting in decreased blood vessel density and delayed tumor growth [17,18]. Although inhibitors of the VEGF pathway have shown only modest efficacy as a monotherapy [19,20], they hold tremendous promise when given in combination with cytotoxic agents [21–23]. It was suggested that anti-angiogenic therapy, to some extent, corrects the flaw in structure and function of tumor blood vessels. This normalization of vessels decreases interstitial fluid pressure (IFP) of solid tumors and improves perfusion resulting in enhanced delivery and efficacy of cytotoxic agents [10,15,16,24–28]. Though highly effective, the efficiency of combination therapy is restrained by the limited duration of normalization window [26]. However, literature reports have been somewhat equivocal about the utility of this technique [29,30]. Some of these
D. Dong et al./Cancer Letters 359 (2015) 178–186
conflicting results can be attributed to differences in tumor models used, and the inherent heterogeneity between tumors. The rationale used for selecting appropriate drug–nucleic acid combinations as well as nanocarriers suitable for simultaneous delivery of drug– nucleic acid combinations has been found in some literatures [7,31–34]. Furthermore, synergistic combinations of several types of therapeutic approaches with distinct mechanisms can overcome the toxicity and other side effects associated with high doses of single drugs by countering biological compensation mechanisms, which allows the dosage of each compound to be reduced and interferes with context-specific multi-targeting mechanisms [5,35]. Nanotechnology has the potential to dramatically improve cancer therapy while reducing the toxicity associated with current approaches [36]. These nanocarrier systems accumulated in the tumor tissue via a passive targeting mechanism termed the enhanced permeability and retention (EPR) effect [37]. However, several intrinsic limitations in these delivery systems have become apparent due to their lack of specificity. To overcome these limitations such as the delivery of genes or drugs to the desired sites at the appropriate time, a variety of approaches have been adopted. Tumor microenvironmentsensitive nanocarriers that respond to changes in environmental conditions such as pH, temperature, chemicals and biomolecules have received extensive attention for their unique advantages in applications for target drug delivery. The high metabolic rate of tumor tissues often leads to acidosis (pH 20 nm) but small enough to penetrate through the leaky vasculatures in a tumor region (
