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Recent developments in the co-delivery of siRNA and small molecule anticancer drugs for cancer treatment
Materials Today Volume 00, Number 00 May 2014
RESEARCH
RESEARCH: Review
Recent developments in the co-delivery of siRNA and small molecule anticancer drugs for cancer treatment Manju Saraswathy and Shaoqin Gong* Department of Biomedical Engineering and Wisconsin Institutes for Discovery, University of Wisconsin – Madison, Madison, WI 53706, USA
Because of the complexity of cancer, combination therapy is becoming increasingly important to overcome multidrug resistance in cancer and to enhance apoptosis. Cancer treatment using nanocarriers to co-deliver small interfering RNA (siRNA) and small molecule anticancer drugs has gained more attention because of its ability to generate synergistic anticancer effects via different mechanisms of action. This article provides a brief review on the recent developments of nanotechnology-based anticancer drug and/or siRNA delivery systems for cancer therapy. Particularly, the synergistic effects of combinatorial anticancer drug and siRNA therapy in various cancer models employing multifunctional drug/siRNA co-delivery nanocarriers have been discussed.
Introduction Cancer is a complex disorder that results from multiple genetic changes and cellular abnormalities. The complexity of cancer progression mechanisms and heterogeneity promote the aggressive growth of cancer cells leading to significant mortality in cancer patients [1,2]. According to global cancer statistics, cancer is the second leading cause of death worldwide [3]. The World Health Organization (WHO) estimates that 27 million new cancer cases and 17.5 million cancer deaths will occur per year by 2050 [4]. There has been tremendous progress over the past few decades in the prevention, detection, and treatment of cancer. However, heterogeneity of tumor microenvironment and complexity of signaling pathways that regulate cancer progression and metastasis remains a major hurdle for effective cancer therapy [5]. In addition, because single drug therapy is typically not very effective in treating cancers, combining two or more therapeutic approaches with different mechanisms of action has been gaining more attention for successful cancer treatment. In fact, various drug combinations have already been utilized in clinical practice, such as the co-administration of two or more anticancer drugs that differ in their anticancer mechanisms, the co-administration of multidrug resistance (MDR) modulators with anticancer drugs, and the co-administration of pro-apoptotic compounds with anticancer drugs [6–9]. For instance, *Corresponding author:. Gong, S. ([email protected])
when multiple therapeutic agents with different molecular targets are applied, the cancer adaptation process can be delayed [10]. In addition, when multiple therapeutic agents target the same cellular pathway, they can function synergistically for higher therapeutic efficacy and higher target selectivity [11]. Gene therapy has emerged as a powerful strategy for cancer treatment over the past several decades because of the genetic link associated with tumor development and progression. With a deeper understanding of the genetic aberrations involved in cancer cells, the delivery of nucleic acid therapeutics (e.g. DNA, siRNA, shRNA, antisense oligonucleotides) to down-regulate or replace mutated genes, and to silence unwanted gene expression, is becoming a highly attractive approach to suppressing tumor cell growth and invasion. There have been intensive efforts to develop safe and efficient gene delivery materials to provide high transfection efficiency at the desired site of action. While initial preclinical and clinical studies are promising, the molecular complexity of cancer suggests that the use of a single therapeutic gene may be insufficient to halt the progression of most cancers. Thus, combination therapies using nucleic acid and anticancer drugs represent a more promising approach [12,7]. This review will mostly cover advances in the co-delivery of siRNA and anticancer drugs using nanocarriers to enhance the efficacy of cancer treatment. Figure 1 summarizes some of the key advantages offered by combined chemotherapy and siRNA therapy.
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Materials Today Volume 00, Number 00 May 2014
RESEARCH: Review FIGURE 1
Major advantages offered by combined chemotherapy and siRNA therapy (adapted with modifications from Ref. [11]).
Nanotechnology-based cancer treatments Nanocarriers are desirable delivery vehicles for anticancer agents because of their passive and active tumor targeting abilities, which can enhance therapeutic efficacy, reduce systemic toxicity, and potentially circumvent drug resistance [13]. The rapid growth of solid tumors results in altered physiology at the tumor site. By contrast to normal vessels, tumor vasculature walls have many ‘openings’ (endothelial fenestrae, vesicles, and transcellular holes), widened interendothelial junctions, discontinuous or absent basement membranes, and abnormal endothelial cells, thereby making the tumor vessels leaky [14,15]. The increased vascular permeability, coupled with impaired lymphatic drainage in the tumor, induces an enhanced permeability and retention (EPR) effect, thereby allowing the nanoparticles to penetrate into and accumulate at the tumor site; this is often referred to as passive targeting [16,17]. Physicochemical factors – including nanoparticle size and size distribution, shape, surface charge, and surface hydrophilicity, as well as nanoparticle chemistry – play important roles in passive targeting [18]. Particles less than 100 nm can pass through the fenestrations in the liver endothelium and the sieve plates of sinusoids to localize in the liver, spleen, and bone marrow [19]. The natural tendency of nanocarriers to be taken up by the reticuloendothelial system (RES) provides an excellent opportunity to deliver therapeutic agents more specifically to the macrophages present in the liver and spleen [18–20]. However, it
becomes a major hurdle for therapeutic agents whose site of action is located in tissues other than the RES. Surface coating using biocompatible and hydrophilic polymers – including polyethylene glycol (PEG), poly (vinyl alcohol), poly(acrylamide), and poly(vinylpyrrolidone) – can improve the solubility and stability of the nanocarriers in an aqueous solution while minimizing opsonization during circulation in the bloodstream. This will significantly increase the circulation time of the nanocarriers, thereby enhancing the in vivo tumor accumulation of the therapeutic agent via the EPR effect [21,22]. However, passive tumor targeting alone is often not sufficient to achieve high-level in vivo tumor drug accumulation. To overcome this deficiency, nanocarriers are frequently conjugated with various tumor-targeting ligands including peptides, antibodies, aptamers, and some other small molecules (e.g. folic acid) that can bind to specific receptors overexpressed on cancer cell surfaces. The cellular uptake of such tumor-targeted drug nanocarriers is generally much higher than their non-targeted counterparts because of receptor-mediated endocytosis [23,24].
siRNA delivery systems for cancer treatment siRNA has the ability to inhibit or silence different cellular pathways by the destruction of specific mRNA molecules. In fact, RNAi therapies using various siRNA molecules have already been shown to be effective in inhibiting different signaling pathways in cell
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proliferation and apoptosis [25–27]. In addition, siRNA has the ability to down-regulate the expression of different multidrug resistant genes to enhance the accumulation of anticancer drugs at the tumor site [28]. However, delivering siRNA efficiently to the target cell/tissue/organ has been a challenge. Naked siRNAs can be rapidly degraded by serum ribonucleases and can hardly cross the cell membrane because of their polyanionic nature along with their relatively large molecular weight [29]. Various viral and nonviral delivery vectors have been developed to improve the efficacy of RNAi therapy in vivo [30–33]. Although viral vectors (e.g. adenoassociated virus, adenovirus, alphavirus, herpes simplex virus, lentivirus, and retrovirus) provide high transfection efficiency, concerns associated with insertional mutagenesis and immunogenicity limit their use in RNAi therapy [34]. Non-viral vectors offer a safer alternative to viral vectors. An ideal non-viral vector should have good biocompatibility, can effectively complex with siRNA, provide better half-life in the blood stream, specifically target to the tissue site of action, and facilitate its intracellular uptake and subsequent gene silencing [35] (Table 1). Nanotechnology offers tremendous promise in addressing most of the challenges associated with siRNA delivery. A large variety of nanocarriers such as liposomes, polymer micelles, polymer vesicles, dendrimers, and functionalized inorganic nanoparticles have been developed for the delivery of siRNA [36–41]. Nanocarriers have to enter the cells and navigate through intracellular trafficking pathways to deliver siRNA to the cytoplasm. Nanocarriers are taken up by cells via different endocytosis pathways such as
clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and other clathrin- and caveolae-independent endocytosis [42,24]. Most often nanocarriers internalized in the endosome end up in the lysosome (pH 4.8), where the acidic environment and degradative enzymes facilitate the degradation of the nanocarrier as well as its payloads (e.g. siRNA). Therefore, it is important for the siRNA nanocarriers to escape from the endosome at an early stage to avoid lysosomal degradation [43,44]. Different strategies have been devised to facilitate the endosomal escape of nanocarriers including proton sponge effect, endosomal membrane fusion, pore formation, flip flop mechanism, and photochemical internalization, among others [43,45]. Xiong et al. reported biodegradable poly(ethyleneoxide)-blockpoly(3-caprolactone) (PEO-b-PCL) based copolymers with different polyamine side chains grafted onto the PCL block (i.e. PEO-bPCL grafted with spermine (PEO-b-P(CL-g-SP)), tetraethylenepentamine (PEO-b-P(CL-g-TP)), or N,N-dimethyldipropylenetriamine (PEO-b-P(CL-g-DP))) for siRNA delivery [46]. MDR-1-targeted siRNA complexed onto nanocarriers formed by PEO-b-P(CL-gSP) and PEO-b-P(CL-g-TP) effectively induced down-regulation of P-gp in a dose-dependent manner. In addition, PEO-b-P(CL-gSP) nanocarriers conjugated with RGD4C and cell-penetrating peptide (TAT) targeting ligands enhanced the cellular uptake and intracellular targeting of siRNA. Layer-by-layer (LbL)-assembled nanocarriers have emerged as a promising class of materials for applications in nanomedicine including siRNA delivery. LbL assembly provides flexibility in terms of the constituent
TABLE 1
Various multifunctional anticancer drug/siRNA co-delivery nanocarriers reported for cancer treatment. Nanosystem
siRNA
Anticancer drug
Target
Ref.
N-((2-hydroxy-3-trimethyl ammonium)propyl) chitosan chloride
mTERT siRNA (telomerase reverse transcriptase specific siRNA)
Paclitaxel
Caco-2 cells (in vitro and in vivo)
[89]
Triblock co-polymeric system based on poly(amidoamine) dendrimer-poly(ethylene glycol)-1,2-dioleoyl-sn-glycero3-phosphoethanolamine (G(4)-D-PEG2K-DOPE).
anti-GFP siRNA
Doxorubicin
C166-GFP cells, A549 (in vitro)
[95]
Cationic solid lipid nanoparticles based on 1,2-diphytanoylsn-glycero-3-phosphatidylethanolamine (DPhPE), 3b[N-(N0 ,N0 -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), and methoxypolyethylene glycol 2000-disteraaroyl phosphatidylethanolamine (mPEG-DSPE)
siMcl1 (myeloid cell leukemia sequence-1 specific siRNA)
Paclitaxel
Human epithelial carcinoma (KB) cells (in vitro and in vivo)
[96]
Polyamidoamine-hyaluronic acid complex
MVP siRNA (major vault protein specific siRNA)
Doxorubicin
NCI-ADR-RES (in vitro and in vivo)
[97]
Octreotide conjugated polyethylene glycol and polyarginine coated gold nanorod
ASCL1 siRNA (achaete-scute complex-like 1 gene specific siRNA)
Doxorubicin
Human carcinoid cell line (in vitro)
[98]
Self-assembly of copolymer polyethyleneimine-3maleimidopropionic acid-hydrazide-doxorubicin and polyethyleneimine-polyethylene glycol-folate
anti-GFP siRNA
Doxorubicin
HeLa cells (in vitro and in vivo)
[99]
Poly(N-methyl diethaneamine sebacate)-co-[(cholesteryl oxocarbonyl amidoethyl)methyl bis(ethylene)ammonium bromide] sebacate
Bcl-2 siRNA
Paclitaxel
HeLa, HepG2, MDA-MB-231 cell lines (in vitro)
[57]
PEI grafted graphene oxide
Bcl-2 siRNA
Doxorubicin
HeLa cells (in vitro)
[69]
Folic acid conjugated poly(ethylene glycol)-blockpoly(glutamic acid) coated PEI-PCL
Bcl-2 siRNA
Doxorubicin
Human hepatic cancer cell line Bel-7402 (in vitro)
[100]
PEI coated mesoporous silica nanoparticle
MDR-1 siRNA
Doxorubicin
KB-V1 cells (in vitro)
[101] 3
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components, drug loading level, surface characteristics, and morphology of the nanocarriers [47,48]. Elbakry et al. modified the surface of gold nanoparticle (AuNPs) (15.5 nm in diameter) using oppositely charged polyethyleneimine (PEI: 25 kDa) and siRNA via electrostatic interactions [49]. The PEI/siRNA/PEI-AuNPs nanosystem showed no serious toxicity in cell culture. It was reasoned that effective removal of free PEI during the LBL construction of the nanosystem significantly reduced the toxicity. Reducible disulfide (S-S) linkages have also been explored for siRNA delivery. Polymer micelles with disulfide crosslinked cores can maintain a stable micellar structure at physiological ionic strength, but collapse in the presence of intracellular glutathione (GSH), thereby facilitating the delivery of siRNA [50,51]. For instance, Matsumoto et al. developed iminothiolane-modified poly(ethylene glycol)-blockpoly(L-lysine) [PEG-b-(PLL-IM)] and siRNA complex [52]. The reducible PEG-b-(PLL-IM) and siRNA complex showed 100-fold higher siRNA transfection efficiency than the non-crosslinked PEG-b–PLL and siRNA complex. Direct conjugation of siRNA onto PEG molecules via disulfide linkage was also reported [53]. In this case, the PEG-siRNA conjugate could be further complexed with cationic polymers or peptides as core condensing agents to form colloidal nanoparticles, also referred to as polyelectrolyte complex micelles (PEC micelles). Kim et al. employed PEC micelles as a siRNA delivery system to silence the VEGF gene in human prostate carcinoma cells [54]. VEGF targeted siRNA was conjugated onto PEG via a disulfide linkage that was intended to cleave in a reductive cytosolic environment. Branched polyethylenimine (PEI) was used as a cationic core condensing agent to form the self-assembled PEC micelles. The resulting siRNA–PEG/PEI PEC micelles demonstrated excellent resistance against nuclease-mediated siRNA degradation and effectively silenced the expression of VEGF in human prostate carcinoma (PC-3) cells in vitro. Kim et al. also studied the cancer targeting effect of a luteinizing hormone-releasing hormone (LHRH) peptide analog coupled onto the siRNA–PEG/ PEI PEC micelles [55]. PEC micelles with LHRH induced a higher inhibition of VEGF expression (139.4 15.2 pg/mL) than those without LHRH (214.1 25.4 pg/mL) in LHRH receptor-overexpressing cell line (A2780) because of receptor-mediated endocytosis.
Co-delivery of siRNA and small molecule anticancer drugs siRNA and small molecule drugs have very different physical, chemical, and biological characteristics (e.g. different molecular weight, hydrophobicity/hydrophilicity, and systemic stability, among others), thus designing an effective siRNA/drug co-delivery system can be challenging. In general, siRNA and drug nanocarriers should be nontoxic and non-immunogenic for systemic administration. siRNA nanocarriers should condense siRNA effectively via electrostatic interaction or chemical conjugation, while drug nanocarriers should encapsulate anticancer drugs effectively via physical encapsulation, formation of an inclusion complex, or direct chemical conjugation. In addition to these characteristics, siRNA/drug co-delivery nanocarriers should be able to simultaneously deliver siRNA and small molecule drug to the target cancer cells/tissues with no adverse effect on their individual release kinetics and pharmacological action. To date, a number of promising nanocarriers have been developed for the co-delivery of siRNA and anticancer drugs [56].
Materials Today Volume 00, Number 00 May 2014
Co-delivery of siRNA and anticancer drugs allows a specific amount of siRNA and anticancer drugs at a designated ratio to be delivered into the same population of cancer cells at the same time, thereby creating synergistic effects [57,58]. Judiciously designed co-delivery systems may also allow simultaneous delivery of multiple drugs to the target site with specific time-programmed release profiles [59].
Co-delivery of siRNA and small molecule anticancer drugs to overcome drug efflux mechanisms As discussed earlier, MDR is a major obstacle to the successful treatment of cancer. Several factors are involved in the development of MDR in cancer, including decreased drug influx in the cell, increased drug efflux, increased DNA repair activation, detoxification/drug metabolism, and the blockage of apoptosis. In particular, MDR is highly associated with drug efflux mechanisms involving the ATP binding cassette (ABC) family of transporters on cancer cell membrane that can detect and expel a large variety of hydrophobic compounds including anthracyclines, epipodophyllotoxins, vinca alkaloids, and taxanes, thus resulting in lower intracellular drug concentrations [60,61]. P-glycoprotein (P-gp; also known as ABCB1 or MDR1 protein), which is the most characterized drug efflux protein, is encoded by the MDR1 gene. P-gp is overexpressed in many types of human cancers, including liver, pancreas, ovary, breast, and brain cancers [62,63]. Other important ABC membrane transporter proteins that are involved in cancer MDR include multidrug resistance associated protein-1 (MRP1 or ABCC1), multidrug resistance associated protein-2 (MRP2, ABCC2), and breast cancer resistant proteins (e.g. ABCG2) [64]. Developments of many high-throughput ‘omic’ techniques, such as genomics, transcriptomics, proteomics, and metabolomics, along with advances in system biology, have enabled the identification of most of the genes/proteins associated with drug resistance in cancer [65]. Several studies have been carried out during the past few years to enhance the efficacy of chemotherapy by suppressing or evading the drug efflux mechanism via the co-delivery of siRNA and anticancer drugs. siRNA can down-regulate the proteins associated with drug efflux and subsequently enhance the accumulation of anticancer drugs at the cancer cell for effective anticancer action [31]. Xiong et al. reported the co-delivery of siRNA against P-gp (MDR-1 siRNA) and doxorubicin (Dox) using polymer micelles formed by poly (ethylene oxide)-block-poly(e-caprolactone) (PEOb-PCL) amphiphilic block copolymers to improve the anticancer effect of Dox in a multidrug resistant human breast cancer cell line (MDA-MB-435/LCC6MDR1) that overexpressed P-gp [66]. Polyamines (i.e. spermine (SP), or N,N-dimethyldipropylenetriamine (DP)) were introduced onto the PCL block for siRNA complexation or chemical conjugation of Dox via pH-sensitive hydrazine linkages. The PEO shell was conjugated with integrin avb3-specific RGD4C peptide for active cancer targeting and/or cell penetrating peptide (TAT) for enhanced cellular uptake. To avoid siRNA binding to the positively charged TAT or RGD4C, complexation with siRNA was performed before RGD4C and/or TAT peptides were conjugated onto the PEO shell of the polymer micelles. As shown in Fig. 2, the intracellular accumulation of Dox in the MDA-MB-435/LCC6 cells treated with RGD4C/TAT-conjugated micelles co-loaded with Dox (5 mg/mL) and MDR-1 siRNA was
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FIGURE 2
Dox distribution in MDA-MB-435/LCC6 MDR-1 resistant cancer cells after 72 hours of treatment using nanoparticle complexed with MDR-1 siRNA and loaded with Dox (adapted from Ref. [64]).
higher than that treated with RGD4C/TAT conjugated micelles loaded with Dox (5 mg/mL) and scrambled siRNA. Similarly, the cytotoxicity of RGD4C/TAT-conjugated micelles co-loaded with Dox and MDR-1 siRNA was also higher than that of RGD4C/TAT conjugated micelles loaded with Dox and scrambled siRNA in MDA-MB-435/LCC6 cells (approximately 70% versus 40% cell growth inhibition). Navarro et al. demonstrated the co-delivery of MDR-1 siRNA and Dox using dioleoylphosphatidylethanolamine modified polyethylenimine (DOPE-modified PEI) nanoparticles in NCI/ADR-RES cell lines (referred to as Dox-resistant MDR-1 overexpressing MCF-7 human breast adenocarcinoma cell lines in this paper) [67]. Multidrug resistant MCF-7/AdrR cell lines were believed to be derived from MCF-7 breast adenocarcinoma cell lines, but several studies found that MCF-7/AdrR cell lines were actually derived from OVCAR-8 ovarian adenocarcinoma cells and were subsequently re-designated as NCI-ADR-RES [68]. Down-regulation of P-gp by MDR-1 siRNA led to the inhibition of Dox efflux activity and subsequent enhancement of intracellular Dox accumulation, and thus a significant increase of drug toxicity in NCI-ADR-RES cell lines. Meng et al. developed surface engineered mesoporous silica nanoparticles (MSN) to co-deliver MDR-1 siRNA and Dox in a drug resistant human cervical carcinoma (KB-V1) cell line [69]. The MSN were modified with 3-trihydroxysilylpropyl methyl phosphonate to introduce negative charge and facilitate Dox loading into the porous interior via electrostatic interaction, as well as polyethyleneimine (PEI) for MDR-1 siRNA complexation. A significant increase in cytotoxicity was observed in KB-V1 cells treated with MSN-siRNA-Dox in comparison to free Dox or MSN-Dox in vitro. Down-regulation of P-gp allowed the intracellular Dox accumulation level to increase above the threshold required for inducing apoptosis and cell death. Li et al. reported multifunctional quantum dots (CdSe/ZnSe) modified with b-cyclodextrin
and L-arginine/L-histidine to co-deliver Dox and MDR1-siRNA [70]. Co-localization of MDR-1 siRNA and Dox in drug resistant HeLa cells (HeLa/Dox) reduced the levels of MDR1 gene expression, and thus enhanced the intracellular accumulation of Dox to induce apoptosis in HeLa/Dox cells. The schematic representation of the co-delivery of MDR-1 siRNA and Dox using multifunctional QDs is shown in Fig. 3.
Co-delivery of siRNA and anticancer drugs to activate apoptosis In addition to the enhanced drug efflux observed in cancer cells caused by different drug transporters, alterations in apoptotic mechanisms can play a major role in reversing the efficacy of cancer treatment [71]. Apoptosis is a process of programmed cell death that is regulated by several cellular pathways. It involves mainly tumor-suppressor genes (p53), proapoptotic genes, and antiapoptotic genes [72]. p53 is the most frequently mutated tumor-suppressor gene in human cancers. Several reports suggest that the loss of p53 function in cells with mutated p53 genes induces several oncogenic properties such as increased genomic instability and cell proliferation, MDR, and inhibition of apoptosis [73]. Silencing the mutated p53 gene by siRNA could reduce MDR and induce apoptosis in certain cancer cell lines. For example, Zhu et al. reported the co-delivery of p53 targeting siRNA (sip53) and cisplatin in human bladder cancer (T24 and T5637) cell lines in vitro [74]. Effective knockdown (>70%) of p53 mRNA expression, and subsequent G2 phase cell cycle arrest, were observed at a sip53 concentration of 50 nm/L after 48 hours incubation. The average reduction in cell viability in cells treated with both sip53 and cisplatin (72.3%) was much higher than the reduction in cells treated with only sip53 (38.7%) or cisplatin (44.9%). The Bcl-2 family of proteins, including both anti-apoptotic (e.g. Bcl-2, and Bcl-xL) and pro-apoptotic (e.g. Bax) proteins, play a central role in the regulation of apoptosis. The pro-apoptotic 5
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Materials Today Volume 00, Number 00 May 2014
RESEARCH: Review FIGURE 3
Co-administration of MRD-1 siRNA and Dox using b-cyclodextrin-modified CdSe/ZnSe quantum dots (adapted from Ref. [70]).
proteins act as a mitochondrial gateway to induce the release of cytochrome c to trigger programmed cell death. However, overexpression of anti-apoptotic proteins inhibits apoptosis [75,76]. It is also evident that overexpression of anti-apoptotic proteins (e.g. Bcl-2) in cancer cells results in resistance to certain drugs including Dox, paclitaxel, etoposide, camptothecin, mitoxantrone, and cisplatin [77,78]. Therefore, knockdown of anti-apoptotic proteins in cancer cells may effectively enhance the efficacy of cancer treatment. It has been shown that siRNA-mediated Bcl-2 inhibition caused apoptosis and reversed the drug resistance associated with various cancer cell lines [79]. Zheng et al. reported the co-delivery of siRNA targeting Bcl-2 (siRNA-Bcl-2) and docetaxel (DTX) using polymer micelles formed by poly (ethylene glycol)-bpoly(L-lysine)-b-poly(L-leucine) triblock copolymers into NCIADR-RES cell lines (referred to as MCF-7 human breast cancer cells that overexpress Bcl-2 protein in this paper) [80]. The hydrophobic poly(L-leucine) core was used to load docetaxel via a hydrophobic interaction. siRNA Bcl-2 was subsequently absorbed into the drug loaded polymer micelles via electrostatic interactions with the cationic poly(L-lysine) moiety. Using Bcl-2 mRNA, a knockdown of approximately 32% and 78% was obtained at a siRNA-Bcl-2 concentration of 50 and 100 nM, respectively. A higher siRNABcl-2 concentration led to more effective Bcl-2 mRNA knockdown. Significantly reduced cell proliferation was also demonstrated via synergistic inhibitory effect through the co-delivery of siRNA-Bcl-2 with DTX both in vitro and in vivo. Cheng et al. studied the co-delivery of Bcl-2 siRNA and Dox in rat C6 glioma cells using polymer micelles formed by poly(e-caprolactone)-poly(ethyleneimine) (PCL-PEI) block copolymers [81]. After siRNA and Dox were loaded into the micelles, the resulting micelleplexes were further modified with folic acid-conjugated poly(ethylene glycol)poly(glutamic acid) block polymer. Folate-targeted nanoparticle
co-delivery of Bcl-2 siRNA (25 nM) and Dox (0.5 mg/mL) showed the highest apoptosis level (85.96%) because of the effective suppression of the Bcl-2/Bax mRNA ratio and subsequent increase in the Dox accumulation in comparison with folate-targeted nanoparticle delivery of Dox (66.04%) and free Dox (20%) in vitro. Folate-targeted nanoparticle co-delivery of BCL-2 siRNA and Dox was also much more effective in suppressing tumor growth in vivo than folatetargeted nanoparticle delivery of Dox alone (tumor volume 8.93 versus 166.48 mm3). Kim et al. reported the co-delivery of Bcl-xL siRNA and Dox using methoxy poly(ethylene glycol)-poly(D,L-lactic acid) (mPEG-PLA) block copolymers [82]. Bcl-xL siRNA and Dox coencapsulated polymersome (CPSomes) effectively silenced the targeted Bcl-xL mRNA and activated Bax mRNA expression levels in human gastric cancer cell lines (MKN-45 and MKN-28) in vitro. Co-delivery of siRNA and Dox using CPSomes into MKN-45 and MKN-28 human gastric cancer cell lines also significantly improved the in vitro anti-proliferation efficacy compared to individual treatment groups. CPSome containing a very small amount of Dox (200 nM) and Bcl-xL siRNA (500 nM) induced effective in vitro cytotoxicity of 73.3% and 75.2% for the MKN-45 and MKN-28 cells, respectively. In comparison, Dox-only loaded polymersomes showed an in vitro cytotoxicity of 42.3% and 45.4% for the MKN45 and MKN-28 cells, respectively. Furthermore, the IC50 value of CPSome was 106 nM, which was 80-fold lower than that of Bcl-xL siRNA loaded lipofectamine and free Dox against the MKN-45 and MKN-28 cells. Taratula et al. reported the co-delivery of siRNA targeting Bcl-2/ MRP-1 and Dox/paclitaxel (TAX) using LHRH (luteinizing hormone-releasing hormone) conjugated nanostructured lipid carriers (NLCs) for lung cancer in vivo [83]. Here the lipid phase consisted of precitrol ATO 5, squalene, and soybean phosphatidylcholine, whereas the aqueous phase was composed of Tween-80,
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FIGURE 4
Co-delivery system based on mPEG-PLGA and EPL co-polymers to deliver Dox, paclitaxel (TAX), and survivin siRNA to B16F10 tumors for enhanced chemotherapeutic efficacy (adapted from Ref. [88]).
1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-distearoylsn-glycero-3-phosphoehtanolamine-N-[carboxy(polyethylene glycol)-2000]. Preferential accumulation of siRNA/Dox in the lung cancer cells was observed in vivo using a mouse orthotopic model of human lung cancer via inhalation administration. The tumor size in animals treated with LHRH-conjugated NLCs loaded with both Bcl-2 siRNA and TAX shrank down to 2.6 3.0 mm3 after 24 days of treatment. By contrast, the tumor volume of the mice treated with free TAX was 82 9.8 mm3 after 24 days of treatment. Oh et al. reported the co-delivery of Bcl-2 siRNA and paclitaxel using a radioopaque nanoemulsion system. Nanoemulsions were prepared using a mixture of lipiodol, cholesterol, linear polyethyleneimine grafted cholesterol (LPEI-g-Chol), and 1,2-distearoyl-sn-glycero-3phosphoethanolamine polyethylene glycol (DSPE-PEG) [84]. Paclitaxel, which was dissolved in lipiodol and LPEI-g-Chol in an aqueous solution, served as a positively charged shell stabilizer to enable siRNA complexation. Co-delivery of paclitaxel (140 nM) and Bcl-2
siRNA (38 nM) in MCF-7 cells induced enhanced apoptosis (60.1%) compared to individual treatments using 38 nM siRNA (12.8%) and 140 nM paclitaxel (33.8%) delivered with the nanoemulsions. Survivin is one of the most highly expressed anti-apoptotic proteins in human cancer tissues (e.g. breast, colon, pancreas, and lung). It inhibits caspase activation [85]. It is reported that the mitotic regulatory activity of survivin leads to enhanced cell proliferation [86,87]. Wang et al. studied the co-delivery of Dox, paclitaxel, and survivin siRNA (siRNA targeting survivin: GAA UUA ACC CUU GGU GAA UTT) using a nanoemulsion system made of an amphiphilic block copolymer of methoxy poly(ethylene glycol)–poly(lactide-co-glycolide) (mPEG–PLGA) and e-polylysine (EPL) [88]. As shown in Fig. 4, Dox was packaged into the hydrophilic core of the nanoemulsion, while paclitaxel was encapsulated into the hydrophobic layer. Survivin siRNA was complexed onto the surface of the nanoemulsion through electrostatic interactions with EPL. Strong synergistic antitumor effects on the co-delivery of Dox 7
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FIGURE 5
Schematic representation of the co-delivery of EGFRvIII siRNA and an anticancer drug (suberoylanilide hydroxamic acid (SAHA) or erlotinib) using DexAMs (adapted from Ref. [82]).
(8.62 mmol/kg), paclitaxel (17.24 mmol/kg), and survivin-siRNA (1.5 mg/kg) were demonstrated in B16-F10 melanoma bearing mice. Kim et al. demonstrated synergistic induction of apoptosis in brain cancer cells by the targeted co-delivery of siRNA against oncogenic receptor EGFRvIII and an anticancer drug (suberoylanilide hydroxamic acid (SAHA) or erlotinib) using a dendritic polyamine core conjugated with b-cyclodextrin (DexAMs) (Fig. 5) [82]. The presence of the b-cyclodextrin moiety facilitated the loading of SAHA/erlotinib via inclusion complex. A synergistic anti-cancer effect on the co-administration of SAHA/erlotinib and EGFRvIII siRNA was demonstrated using U87-EGFP glioblastoma cells. Co-delivery of SAHA (5 mM)/EGFRvIII siRNA (200 nM) or erlotinib (30 mM)/siRNA (200 nM) enhanced the cytotoxicity (% cell death: 85% and 70%, respectively) as compared to SAHA (68%) or erlotinib (51%) alone. Polo-like kinase 1 (Plk1), which is overexpressed in a broad range of tumors, has a pivotal role in cell mitosis. The inhibition of Plk1 causes mitotic arrest and leads to apoptosis [89]. Zhao et al. investigated the co-delivery of docetaxel and siPlk1 (siRNA that down-regulates Plk1 proteins by post-transcriptional degradation of Plk1 mRNA) using a herceptin-conjugated vitamin E TPGS (D-a-tocopheryl polyethylene glycol 1000 succinate)-based immunomicelle [90]. Compared to the docetaxel-loaded TPGS micelle, the IC50 of docetaxel-loaded TPGS-siPlk1/TPGS micelle after 72 hours incubation was reduced by 99.8%, 83.0%, and 90.4% for NIH3T3 fibroblast cells, MCF-7 cells, and SK-BR-3 cells in vitro, respectively. Nuclear factor-kb (NF-kb) is another important transcription factor that regulates the expression of various genes to inhibit apoptosis in cancer models [91]. It is also evident that NF-kb has a major role in developing MDR in cancer cells [92]. Zhao et al. studied the co-delivery of Dox and NF-kb p65 siRNA using spermine-grafted poly-g-benzyl-L-glutamate (PGS) polyelectrolyte brushes in vitro [93]. PGS polyelectrolyte brushes complexed with NF-kb p65 siRNA subsequently enhanced the bioavailability of Dox in the cancer cells because of NF-kB p65 inhibition. The total apoptosis of Dox with pre-transfection of PGS polyelectrolyte brushes/NF-kB p65 siRNA polyplex was 170% of that treated with the same dose of doxorubicin alone. Y-box binding protein-1 (YB-1) is another protein that is overexpressed in cancer. It was reported that YB-1 translocation from the cytoplasm to the nucleus stimulated MDR proteins [94]. Zeng et al. investigated the co-delivery of rapamycin and siRNA
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targeting YB-1 (siYB-1) using poly(ethylene glycol)-block-poly(bbenzyl-L-aspartate)-block-poly(e-caprolactone) triblock copolymers [80]. Compared to individual administration (rapamycin (1 nM)/NPs: 40% cell death; siYB-1 (50 nM)/NPs: <9% cell death), co-delivery of rapamycin (1 nM) and siYB-1 (50 nM) induced around 62% cell death after 48 hours incubation in human PC3 prostate cancer cells in vitro. In addition, a 75% tumor volume reduction in a PC3 xenograft nude mice model of human prostate cancer was observed on the 10th day after co-delivery of rapamycin (30 mg/kg) and siYB-1 (2 mg/kg).
Conclusions Various types of nanocarriers have been investigated for the co-administration of siRNA and anticancer drugs in an effort to enhance anticancer effects by overcoming MDR or inducing different apoptosis pathways. Judiciously engineered multifunctional drug/siRNA co-delivery nanocarriers can significantly increase their in vivo tumor accumulation via both passive (i.e. the EPR effect) and active (i.e. via proper conjugation of active tumortargeting ligands such as peptides, antibodies, aptamers, and certain small molecules) tumor-targeting abilities. Thus, multifunctional nanomedicines offer great promise in overcoming the drawbacks of current treatment modalities, including chemotherapy. Nevertheless, a deeper understanding of several factors, including the optimal ratio of each therapeutic agent (e.g. siRNA versus anticancer drug) as well as their pharmacological fate at the tumor site, and the nature of cancer heterogeneity, is needed to achieve the maximum synergistic effect of co-administering siRNA with anticancer drugs. Additionally, further studies are needed to avoid unexpected immune stimulation with the simultaneous administration of siRNA and anticancer drugs.
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Recent developments in the co-delivery of siRNA and small molecule anticancer drugs for cancer treatment Manju Saraswathy and Shaoqin Gong* Department of Biomedical Engineering and Wisconsin Institutes for Discovery, University of Wisconsin – Madison, Madison, WI 53706, USA
Because of the complexity of cancer, combination therapy is becoming increasingly important to overcome multidrug resistance in cancer and to enhance apoptosis. Cancer treatment using nanocarriers to co-deliver small interfering RNA (siRNA) and small molecule anticancer drugs has gained more attention because of its ability to generate synergistic anticancer effects via different mechanisms of action. This article provides a brief review on the recent developments of nanotechnology-based anticancer drug and/or siRNA delivery systems for cancer therapy. Particularly, the synergistic effects of combinatorial anticancer drug and siRNA therapy in various cancer models employing multifunctional drug/siRNA co-delivery nanocarriers have been discussed.
Introduction Cancer is a complex disorder that results from multiple genetic changes and cellular abnormalities. The complexity of cancer progression mechanisms and heterogeneity promote the aggressive growth of cancer cells leading to significant mortality in cancer patients [1,2]. According to global cancer statistics, cancer is the second leading cause of death worldwide [3]. The World Health Organization (WHO) estimates that 27 million new cancer cases and 17.5 million cancer deaths will occur per year by 2050 [4]. There has been tremendous progress over the past few decades in the prevention, detection, and treatment of cancer. However, heterogeneity of tumor microenvironment and complexity of signaling pathways that regulate cancer progression and metastasis remains a major hurdle for effective cancer therapy [5]. In addition, because single drug therapy is typically not very effective in treating cancers, combining two or more therapeutic approaches with different mechanisms of action has been gaining more attention for successful cancer treatment. In fact, various drug combinations have already been utilized in clinical practice, such as the co-administration of two or more anticancer drugs that differ in their anticancer mechanisms, the co-administration of multidrug resistance (MDR) modulators with anticancer drugs, and the co-administration of pro-apoptotic compounds with anticancer drugs [6–9]. For instance, *Corresponding author:. Gong, S. ([email protected])
when multiple therapeutic agents with different molecular targets are applied, the cancer adaptation process can be delayed [10]. In addition, when multiple therapeutic agents target the same cellular pathway, they can function synergistically for higher therapeutic efficacy and higher target selectivity [11]. Gene therapy has emerged as a powerful strategy for cancer treatment over the past several decades because of the genetic link associated with tumor development and progression. With a deeper understanding of the genetic aberrations involved in cancer cells, the delivery of nucleic acid therapeutics (e.g. DNA, siRNA, shRNA, antisense oligonucleotides) to down-regulate or replace mutated genes, and to silence unwanted gene expression, is becoming a highly attractive approach to suppressing tumor cell growth and invasion. There have been intensive efforts to develop safe and efficient gene delivery materials to provide high transfection efficiency at the desired site of action. While initial preclinical and clinical studies are promising, the molecular complexity of cancer suggests that the use of a single therapeutic gene may be insufficient to halt the progression of most cancers. Thus, combination therapies using nucleic acid and anticancer drugs represent a more promising approach [12,7]. This review will mostly cover advances in the co-delivery of siRNA and anticancer drugs using nanocarriers to enhance the efficacy of cancer treatment. Figure 1 summarizes some of the key advantages offered by combined chemotherapy and siRNA therapy.
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Major advantages offered by combined chemotherapy and siRNA therapy (adapted with modifications from Ref. [11]).
Nanotechnology-based cancer treatments Nanocarriers are desirable delivery vehicles for anticancer agents because of their passive and active tumor targeting abilities, which can enhance therapeutic efficacy, reduce systemic toxicity, and potentially circumvent drug resistance [13]. The rapid growth of solid tumors results in altered physiology at the tumor site. By contrast to normal vessels, tumor vasculature walls have many ‘openings’ (endothelial fenestrae, vesicles, and transcellular holes), widened interendothelial junctions, discontinuous or absent basement membranes, and abnormal endothelial cells, thereby making the tumor vessels leaky [14,15]. The increased vascular permeability, coupled with impaired lymphatic drainage in the tumor, induces an enhanced permeability and retention (EPR) effect, thereby allowing the nanoparticles to penetrate into and accumulate at the tumor site; this is often referred to as passive targeting [16,17]. Physicochemical factors – including nanoparticle size and size distribution, shape, surface charge, and surface hydrophilicity, as well as nanoparticle chemistry – play important roles in passive targeting [18]. Particles less than 100 nm can pass through the fenestrations in the liver endothelium and the sieve plates of sinusoids to localize in the liver, spleen, and bone marrow [19]. The natural tendency of nanocarriers to be taken up by the reticuloendothelial system (RES) provides an excellent opportunity to deliver therapeutic agents more specifically to the macrophages present in the liver and spleen [18–20]. However, it
becomes a major hurdle for therapeutic agents whose site of action is located in tissues other than the RES. Surface coating using biocompatible and hydrophilic polymers – including polyethylene glycol (PEG), poly (vinyl alcohol), poly(acrylamide), and poly(vinylpyrrolidone) – can improve the solubility and stability of the nanocarriers in an aqueous solution while minimizing opsonization during circulation in the bloodstream. This will significantly increase the circulation time of the nanocarriers, thereby enhancing the in vivo tumor accumulation of the therapeutic agent via the EPR effect [21,22]. However, passive tumor targeting alone is often not sufficient to achieve high-level in vivo tumor drug accumulation. To overcome this deficiency, nanocarriers are frequently conjugated with various tumor-targeting ligands including peptides, antibodies, aptamers, and some other small molecules (e.g. folic acid) that can bind to specific receptors overexpressed on cancer cell surfaces. The cellular uptake of such tumor-targeted drug nanocarriers is generally much higher than their non-targeted counterparts because of receptor-mediated endocytosis [23,24].
siRNA delivery systems for cancer treatment siRNA has the ability to inhibit or silence different cellular pathways by the destruction of specific mRNA molecules. In fact, RNAi therapies using various siRNA molecules have already been shown to be effective in inhibiting different signaling pathways in cell
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proliferation and apoptosis [25–27]. In addition, siRNA has the ability to down-regulate the expression of different multidrug resistant genes to enhance the accumulation of anticancer drugs at the tumor site [28]. However, delivering siRNA efficiently to the target cell/tissue/organ has been a challenge. Naked siRNAs can be rapidly degraded by serum ribonucleases and can hardly cross the cell membrane because of their polyanionic nature along with their relatively large molecular weight [29]. Various viral and nonviral delivery vectors have been developed to improve the efficacy of RNAi therapy in vivo [30–33]. Although viral vectors (e.g. adenoassociated virus, adenovirus, alphavirus, herpes simplex virus, lentivirus, and retrovirus) provide high transfection efficiency, concerns associated with insertional mutagenesis and immunogenicity limit their use in RNAi therapy [34]. Non-viral vectors offer a safer alternative to viral vectors. An ideal non-viral vector should have good biocompatibility, can effectively complex with siRNA, provide better half-life in the blood stream, specifically target to the tissue site of action, and facilitate its intracellular uptake and subsequent gene silencing [35] (Table 1). Nanotechnology offers tremendous promise in addressing most of the challenges associated with siRNA delivery. A large variety of nanocarriers such as liposomes, polymer micelles, polymer vesicles, dendrimers, and functionalized inorganic nanoparticles have been developed for the delivery of siRNA [36–41]. Nanocarriers have to enter the cells and navigate through intracellular trafficking pathways to deliver siRNA to the cytoplasm. Nanocarriers are taken up by cells via different endocytosis pathways such as
clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and other clathrin- and caveolae-independent endocytosis [42,24]. Most often nanocarriers internalized in the endosome end up in the lysosome (pH 4.8), where the acidic environment and degradative enzymes facilitate the degradation of the nanocarrier as well as its payloads (e.g. siRNA). Therefore, it is important for the siRNA nanocarriers to escape from the endosome at an early stage to avoid lysosomal degradation [43,44]. Different strategies have been devised to facilitate the endosomal escape of nanocarriers including proton sponge effect, endosomal membrane fusion, pore formation, flip flop mechanism, and photochemical internalization, among others [43,45]. Xiong et al. reported biodegradable poly(ethyleneoxide)-blockpoly(3-caprolactone) (PEO-b-PCL) based copolymers with different polyamine side chains grafted onto the PCL block (i.e. PEO-bPCL grafted with spermine (PEO-b-P(CL-g-SP)), tetraethylenepentamine (PEO-b-P(CL-g-TP)), or N,N-dimethyldipropylenetriamine (PEO-b-P(CL-g-DP))) for siRNA delivery [46]. MDR-1-targeted siRNA complexed onto nanocarriers formed by PEO-b-P(CL-gSP) and PEO-b-P(CL-g-TP) effectively induced down-regulation of P-gp in a dose-dependent manner. In addition, PEO-b-P(CL-gSP) nanocarriers conjugated with RGD4C and cell-penetrating peptide (TAT) targeting ligands enhanced the cellular uptake and intracellular targeting of siRNA. Layer-by-layer (LbL)-assembled nanocarriers have emerged as a promising class of materials for applications in nanomedicine including siRNA delivery. LbL assembly provides flexibility in terms of the constituent
TABLE 1
Various multifunctional anticancer drug/siRNA co-delivery nanocarriers reported for cancer treatment. Nanosystem
siRNA
Anticancer drug
Target
Ref.
N-((2-hydroxy-3-trimethyl ammonium)propyl) chitosan chloride
mTERT siRNA (telomerase reverse transcriptase specific siRNA)
Paclitaxel
Caco-2 cells (in vitro and in vivo)
[89]
Triblock co-polymeric system based on poly(amidoamine) dendrimer-poly(ethylene glycol)-1,2-dioleoyl-sn-glycero3-phosphoethanolamine (G(4)-D-PEG2K-DOPE).
anti-GFP siRNA
Doxorubicin
C166-GFP cells, A549 (in vitro)
[95]
Cationic solid lipid nanoparticles based on 1,2-diphytanoylsn-glycero-3-phosphatidylethanolamine (DPhPE), 3b[N-(N0 ,N0 -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), and methoxypolyethylene glycol 2000-disteraaroyl phosphatidylethanolamine (mPEG-DSPE)
siMcl1 (myeloid cell leukemia sequence-1 specific siRNA)
Paclitaxel
Human epithelial carcinoma (KB) cells (in vitro and in vivo)
[96]
Polyamidoamine-hyaluronic acid complex
MVP siRNA (major vault protein specific siRNA)
Doxorubicin
NCI-ADR-RES (in vitro and in vivo)
[97]
Octreotide conjugated polyethylene glycol and polyarginine coated gold nanorod
ASCL1 siRNA (achaete-scute complex-like 1 gene specific siRNA)
Doxorubicin
Human carcinoid cell line (in vitro)
[98]
Self-assembly of copolymer polyethyleneimine-3maleimidopropionic acid-hydrazide-doxorubicin and polyethyleneimine-polyethylene glycol-folate
anti-GFP siRNA
Doxorubicin
HeLa cells (in vitro and in vivo)
[99]
Poly(N-methyl diethaneamine sebacate)-co-[(cholesteryl oxocarbonyl amidoethyl)methyl bis(ethylene)ammonium bromide] sebacate
Bcl-2 siRNA
Paclitaxel
HeLa, HepG2, MDA-MB-231 cell lines (in vitro)
[57]
PEI grafted graphene oxide
Bcl-2 siRNA
Doxorubicin
HeLa cells (in vitro)
[69]
Folic acid conjugated poly(ethylene glycol)-blockpoly(glutamic acid) coated PEI-PCL
Bcl-2 siRNA
Doxorubicin
Human hepatic cancer cell line Bel-7402 (in vitro)
[100]
PEI coated mesoporous silica nanoparticle
MDR-1 siRNA
Doxorubicin
KB-V1 cells (in vitro)
[101] 3
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components, drug loading level, surface characteristics, and morphology of the nanocarriers [47,48]. Elbakry et al. modified the surface of gold nanoparticle (AuNPs) (15.5 nm in diameter) using oppositely charged polyethyleneimine (PEI: 25 kDa) and siRNA via electrostatic interactions [49]. The PEI/siRNA/PEI-AuNPs nanosystem showed no serious toxicity in cell culture. It was reasoned that effective removal of free PEI during the LBL construction of the nanosystem significantly reduced the toxicity. Reducible disulfide (S-S) linkages have also been explored for siRNA delivery. Polymer micelles with disulfide crosslinked cores can maintain a stable micellar structure at physiological ionic strength, but collapse in the presence of intracellular glutathione (GSH), thereby facilitating the delivery of siRNA [50,51]. For instance, Matsumoto et al. developed iminothiolane-modified poly(ethylene glycol)-blockpoly(L-lysine) [PEG-b-(PLL-IM)] and siRNA complex [52]. The reducible PEG-b-(PLL-IM) and siRNA complex showed 100-fold higher siRNA transfection efficiency than the non-crosslinked PEG-b–PLL and siRNA complex. Direct conjugation of siRNA onto PEG molecules via disulfide linkage was also reported [53]. In this case, the PEG-siRNA conjugate could be further complexed with cationic polymers or peptides as core condensing agents to form colloidal nanoparticles, also referred to as polyelectrolyte complex micelles (PEC micelles). Kim et al. employed PEC micelles as a siRNA delivery system to silence the VEGF gene in human prostate carcinoma cells [54]. VEGF targeted siRNA was conjugated onto PEG via a disulfide linkage that was intended to cleave in a reductive cytosolic environment. Branched polyethylenimine (PEI) was used as a cationic core condensing agent to form the self-assembled PEC micelles. The resulting siRNA–PEG/PEI PEC micelles demonstrated excellent resistance against nuclease-mediated siRNA degradation and effectively silenced the expression of VEGF in human prostate carcinoma (PC-3) cells in vitro. Kim et al. also studied the cancer targeting effect of a luteinizing hormone-releasing hormone (LHRH) peptide analog coupled onto the siRNA–PEG/ PEI PEC micelles [55]. PEC micelles with LHRH induced a higher inhibition of VEGF expression (139.4 15.2 pg/mL) than those without LHRH (214.1 25.4 pg/mL) in LHRH receptor-overexpressing cell line (A2780) because of receptor-mediated endocytosis.
Co-delivery of siRNA and small molecule anticancer drugs siRNA and small molecule drugs have very different physical, chemical, and biological characteristics (e.g. different molecular weight, hydrophobicity/hydrophilicity, and systemic stability, among others), thus designing an effective siRNA/drug co-delivery system can be challenging. In general, siRNA and drug nanocarriers should be nontoxic and non-immunogenic for systemic administration. siRNA nanocarriers should condense siRNA effectively via electrostatic interaction or chemical conjugation, while drug nanocarriers should encapsulate anticancer drugs effectively via physical encapsulation, formation of an inclusion complex, or direct chemical conjugation. In addition to these characteristics, siRNA/drug co-delivery nanocarriers should be able to simultaneously deliver siRNA and small molecule drug to the target cancer cells/tissues with no adverse effect on their individual release kinetics and pharmacological action. To date, a number of promising nanocarriers have been developed for the co-delivery of siRNA and anticancer drugs [56].
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Co-delivery of siRNA and anticancer drugs allows a specific amount of siRNA and anticancer drugs at a designated ratio to be delivered into the same population of cancer cells at the same time, thereby creating synergistic effects [57,58]. Judiciously designed co-delivery systems may also allow simultaneous delivery of multiple drugs to the target site with specific time-programmed release profiles [59].
Co-delivery of siRNA and small molecule anticancer drugs to overcome drug efflux mechanisms As discussed earlier, MDR is a major obstacle to the successful treatment of cancer. Several factors are involved in the development of MDR in cancer, including decreased drug influx in the cell, increased drug efflux, increased DNA repair activation, detoxification/drug metabolism, and the blockage of apoptosis. In particular, MDR is highly associated with drug efflux mechanisms involving the ATP binding cassette (ABC) family of transporters on cancer cell membrane that can detect and expel a large variety of hydrophobic compounds including anthracyclines, epipodophyllotoxins, vinca alkaloids, and taxanes, thus resulting in lower intracellular drug concentrations [60,61]. P-glycoprotein (P-gp; also known as ABCB1 or MDR1 protein), which is the most characterized drug efflux protein, is encoded by the MDR1 gene. P-gp is overexpressed in many types of human cancers, including liver, pancreas, ovary, breast, and brain cancers [62,63]. Other important ABC membrane transporter proteins that are involved in cancer MDR include multidrug resistance associated protein-1 (MRP1 or ABCC1), multidrug resistance associated protein-2 (MRP2, ABCC2), and breast cancer resistant proteins (e.g. ABCG2) [64]. Developments of many high-throughput ‘omic’ techniques, such as genomics, transcriptomics, proteomics, and metabolomics, along with advances in system biology, have enabled the identification of most of the genes/proteins associated with drug resistance in cancer [65]. Several studies have been carried out during the past few years to enhance the efficacy of chemotherapy by suppressing or evading the drug efflux mechanism via the co-delivery of siRNA and anticancer drugs. siRNA can down-regulate the proteins associated with drug efflux and subsequently enhance the accumulation of anticancer drugs at the cancer cell for effective anticancer action [31]. Xiong et al. reported the co-delivery of siRNA against P-gp (MDR-1 siRNA) and doxorubicin (Dox) using polymer micelles formed by poly (ethylene oxide)-block-poly(e-caprolactone) (PEOb-PCL) amphiphilic block copolymers to improve the anticancer effect of Dox in a multidrug resistant human breast cancer cell line (MDA-MB-435/LCC6MDR1) that overexpressed P-gp [66]. Polyamines (i.e. spermine (SP), or N,N-dimethyldipropylenetriamine (DP)) were introduced onto the PCL block for siRNA complexation or chemical conjugation of Dox via pH-sensitive hydrazine linkages. The PEO shell was conjugated with integrin avb3-specific RGD4C peptide for active cancer targeting and/or cell penetrating peptide (TAT) for enhanced cellular uptake. To avoid siRNA binding to the positively charged TAT or RGD4C, complexation with siRNA was performed before RGD4C and/or TAT peptides were conjugated onto the PEO shell of the polymer micelles. As shown in Fig. 2, the intracellular accumulation of Dox in the MDA-MB-435/LCC6 cells treated with RGD4C/TAT-conjugated micelles co-loaded with Dox (5 mg/mL) and MDR-1 siRNA was
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FIGURE 2
Dox distribution in MDA-MB-435/LCC6 MDR-1 resistant cancer cells after 72 hours of treatment using nanoparticle complexed with MDR-1 siRNA and loaded with Dox (adapted from Ref. [64]).
higher than that treated with RGD4C/TAT conjugated micelles loaded with Dox (5 mg/mL) and scrambled siRNA. Similarly, the cytotoxicity of RGD4C/TAT-conjugated micelles co-loaded with Dox and MDR-1 siRNA was also higher than that of RGD4C/TAT conjugated micelles loaded with Dox and scrambled siRNA in MDA-MB-435/LCC6 cells (approximately 70% versus 40% cell growth inhibition). Navarro et al. demonstrated the co-delivery of MDR-1 siRNA and Dox using dioleoylphosphatidylethanolamine modified polyethylenimine (DOPE-modified PEI) nanoparticles in NCI/ADR-RES cell lines (referred to as Dox-resistant MDR-1 overexpressing MCF-7 human breast adenocarcinoma cell lines in this paper) [67]. Multidrug resistant MCF-7/AdrR cell lines were believed to be derived from MCF-7 breast adenocarcinoma cell lines, but several studies found that MCF-7/AdrR cell lines were actually derived from OVCAR-8 ovarian adenocarcinoma cells and were subsequently re-designated as NCI-ADR-RES [68]. Down-regulation of P-gp by MDR-1 siRNA led to the inhibition of Dox efflux activity and subsequent enhancement of intracellular Dox accumulation, and thus a significant increase of drug toxicity in NCI-ADR-RES cell lines. Meng et al. developed surface engineered mesoporous silica nanoparticles (MSN) to co-deliver MDR-1 siRNA and Dox in a drug resistant human cervical carcinoma (KB-V1) cell line [69]. The MSN were modified with 3-trihydroxysilylpropyl methyl phosphonate to introduce negative charge and facilitate Dox loading into the porous interior via electrostatic interaction, as well as polyethyleneimine (PEI) for MDR-1 siRNA complexation. A significant increase in cytotoxicity was observed in KB-V1 cells treated with MSN-siRNA-Dox in comparison to free Dox or MSN-Dox in vitro. Down-regulation of P-gp allowed the intracellular Dox accumulation level to increase above the threshold required for inducing apoptosis and cell death. Li et al. reported multifunctional quantum dots (CdSe/ZnSe) modified with b-cyclodextrin
and L-arginine/L-histidine to co-deliver Dox and MDR1-siRNA [70]. Co-localization of MDR-1 siRNA and Dox in drug resistant HeLa cells (HeLa/Dox) reduced the levels of MDR1 gene expression, and thus enhanced the intracellular accumulation of Dox to induce apoptosis in HeLa/Dox cells. The schematic representation of the co-delivery of MDR-1 siRNA and Dox using multifunctional QDs is shown in Fig. 3.
Co-delivery of siRNA and anticancer drugs to activate apoptosis In addition to the enhanced drug efflux observed in cancer cells caused by different drug transporters, alterations in apoptotic mechanisms can play a major role in reversing the efficacy of cancer treatment [71]. Apoptosis is a process of programmed cell death that is regulated by several cellular pathways. It involves mainly tumor-suppressor genes (p53), proapoptotic genes, and antiapoptotic genes [72]. p53 is the most frequently mutated tumor-suppressor gene in human cancers. Several reports suggest that the loss of p53 function in cells with mutated p53 genes induces several oncogenic properties such as increased genomic instability and cell proliferation, MDR, and inhibition of apoptosis [73]. Silencing the mutated p53 gene by siRNA could reduce MDR and induce apoptosis in certain cancer cell lines. For example, Zhu et al. reported the co-delivery of p53 targeting siRNA (sip53) and cisplatin in human bladder cancer (T24 and T5637) cell lines in vitro [74]. Effective knockdown (>70%) of p53 mRNA expression, and subsequent G2 phase cell cycle arrest, were observed at a sip53 concentration of 50 nm/L after 48 hours incubation. The average reduction in cell viability in cells treated with both sip53 and cisplatin (72.3%) was much higher than the reduction in cells treated with only sip53 (38.7%) or cisplatin (44.9%). The Bcl-2 family of proteins, including both anti-apoptotic (e.g. Bcl-2, and Bcl-xL) and pro-apoptotic (e.g. Bax) proteins, play a central role in the regulation of apoptosis. The pro-apoptotic 5
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Co-administration of MRD-1 siRNA and Dox using b-cyclodextrin-modified CdSe/ZnSe quantum dots (adapted from Ref. [70]).
proteins act as a mitochondrial gateway to induce the release of cytochrome c to trigger programmed cell death. However, overexpression of anti-apoptotic proteins inhibits apoptosis [75,76]. It is also evident that overexpression of anti-apoptotic proteins (e.g. Bcl-2) in cancer cells results in resistance to certain drugs including Dox, paclitaxel, etoposide, camptothecin, mitoxantrone, and cisplatin [77,78]. Therefore, knockdown of anti-apoptotic proteins in cancer cells may effectively enhance the efficacy of cancer treatment. It has been shown that siRNA-mediated Bcl-2 inhibition caused apoptosis and reversed the drug resistance associated with various cancer cell lines [79]. Zheng et al. reported the co-delivery of siRNA targeting Bcl-2 (siRNA-Bcl-2) and docetaxel (DTX) using polymer micelles formed by poly (ethylene glycol)-bpoly(L-lysine)-b-poly(L-leucine) triblock copolymers into NCIADR-RES cell lines (referred to as MCF-7 human breast cancer cells that overexpress Bcl-2 protein in this paper) [80]. The hydrophobic poly(L-leucine) core was used to load docetaxel via a hydrophobic interaction. siRNA Bcl-2 was subsequently absorbed into the drug loaded polymer micelles via electrostatic interactions with the cationic poly(L-lysine) moiety. Using Bcl-2 mRNA, a knockdown of approximately 32% and 78% was obtained at a siRNA-Bcl-2 concentration of 50 and 100 nM, respectively. A higher siRNABcl-2 concentration led to more effective Bcl-2 mRNA knockdown. Significantly reduced cell proliferation was also demonstrated via synergistic inhibitory effect through the co-delivery of siRNA-Bcl-2 with DTX both in vitro and in vivo. Cheng et al. studied the co-delivery of Bcl-2 siRNA and Dox in rat C6 glioma cells using polymer micelles formed by poly(e-caprolactone)-poly(ethyleneimine) (PCL-PEI) block copolymers [81]. After siRNA and Dox were loaded into the micelles, the resulting micelleplexes were further modified with folic acid-conjugated poly(ethylene glycol)poly(glutamic acid) block polymer. Folate-targeted nanoparticle
co-delivery of Bcl-2 siRNA (25 nM) and Dox (0.5 mg/mL) showed the highest apoptosis level (85.96%) because of the effective suppression of the Bcl-2/Bax mRNA ratio and subsequent increase in the Dox accumulation in comparison with folate-targeted nanoparticle delivery of Dox (66.04%) and free Dox (20%) in vitro. Folate-targeted nanoparticle co-delivery of BCL-2 siRNA and Dox was also much more effective in suppressing tumor growth in vivo than folatetargeted nanoparticle delivery of Dox alone (tumor volume 8.93 versus 166.48 mm3). Kim et al. reported the co-delivery of Bcl-xL siRNA and Dox using methoxy poly(ethylene glycol)-poly(D,L-lactic acid) (mPEG-PLA) block copolymers [82]. Bcl-xL siRNA and Dox coencapsulated polymersome (CPSomes) effectively silenced the targeted Bcl-xL mRNA and activated Bax mRNA expression levels in human gastric cancer cell lines (MKN-45 and MKN-28) in vitro. Co-delivery of siRNA and Dox using CPSomes into MKN-45 and MKN-28 human gastric cancer cell lines also significantly improved the in vitro anti-proliferation efficacy compared to individual treatment groups. CPSome containing a very small amount of Dox (200 nM) and Bcl-xL siRNA (500 nM) induced effective in vitro cytotoxicity of 73.3% and 75.2% for the MKN-45 and MKN-28 cells, respectively. In comparison, Dox-only loaded polymersomes showed an in vitro cytotoxicity of 42.3% and 45.4% for the MKN45 and MKN-28 cells, respectively. Furthermore, the IC50 value of CPSome was 106 nM, which was 80-fold lower than that of Bcl-xL siRNA loaded lipofectamine and free Dox against the MKN-45 and MKN-28 cells. Taratula et al. reported the co-delivery of siRNA targeting Bcl-2/ MRP-1 and Dox/paclitaxel (TAX) using LHRH (luteinizing hormone-releasing hormone) conjugated nanostructured lipid carriers (NLCs) for lung cancer in vivo [83]. Here the lipid phase consisted of precitrol ATO 5, squalene, and soybean phosphatidylcholine, whereas the aqueous phase was composed of Tween-80,
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FIGURE 4
Co-delivery system based on mPEG-PLGA and EPL co-polymers to deliver Dox, paclitaxel (TAX), and survivin siRNA to B16F10 tumors for enhanced chemotherapeutic efficacy (adapted from Ref. [88]).
1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-distearoylsn-glycero-3-phosphoehtanolamine-N-[carboxy(polyethylene glycol)-2000]. Preferential accumulation of siRNA/Dox in the lung cancer cells was observed in vivo using a mouse orthotopic model of human lung cancer via inhalation administration. The tumor size in animals treated with LHRH-conjugated NLCs loaded with both Bcl-2 siRNA and TAX shrank down to 2.6 3.0 mm3 after 24 days of treatment. By contrast, the tumor volume of the mice treated with free TAX was 82 9.8 mm3 after 24 days of treatment. Oh et al. reported the co-delivery of Bcl-2 siRNA and paclitaxel using a radioopaque nanoemulsion system. Nanoemulsions were prepared using a mixture of lipiodol, cholesterol, linear polyethyleneimine grafted cholesterol (LPEI-g-Chol), and 1,2-distearoyl-sn-glycero-3phosphoethanolamine polyethylene glycol (DSPE-PEG) [84]. Paclitaxel, which was dissolved in lipiodol and LPEI-g-Chol in an aqueous solution, served as a positively charged shell stabilizer to enable siRNA complexation. Co-delivery of paclitaxel (140 nM) and Bcl-2
siRNA (38 nM) in MCF-7 cells induced enhanced apoptosis (60.1%) compared to individual treatments using 38 nM siRNA (12.8%) and 140 nM paclitaxel (33.8%) delivered with the nanoemulsions. Survivin is one of the most highly expressed anti-apoptotic proteins in human cancer tissues (e.g. breast, colon, pancreas, and lung). It inhibits caspase activation [85]. It is reported that the mitotic regulatory activity of survivin leads to enhanced cell proliferation [86,87]. Wang et al. studied the co-delivery of Dox, paclitaxel, and survivin siRNA (siRNA targeting survivin: GAA UUA ACC CUU GGU GAA UTT) using a nanoemulsion system made of an amphiphilic block copolymer of methoxy poly(ethylene glycol)–poly(lactide-co-glycolide) (mPEG–PLGA) and e-polylysine (EPL) [88]. As shown in Fig. 4, Dox was packaged into the hydrophilic core of the nanoemulsion, while paclitaxel was encapsulated into the hydrophobic layer. Survivin siRNA was complexed onto the surface of the nanoemulsion through electrostatic interactions with EPL. Strong synergistic antitumor effects on the co-delivery of Dox 7
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FIGURE 5
Schematic representation of the co-delivery of EGFRvIII siRNA and an anticancer drug (suberoylanilide hydroxamic acid (SAHA) or erlotinib) using DexAMs (adapted from Ref. [82]).
(8.62 mmol/kg), paclitaxel (17.24 mmol/kg), and survivin-siRNA (1.5 mg/kg) were demonstrated in B16-F10 melanoma bearing mice. Kim et al. demonstrated synergistic induction of apoptosis in brain cancer cells by the targeted co-delivery of siRNA against oncogenic receptor EGFRvIII and an anticancer drug (suberoylanilide hydroxamic acid (SAHA) or erlotinib) using a dendritic polyamine core conjugated with b-cyclodextrin (DexAMs) (Fig. 5) [82]. The presence of the b-cyclodextrin moiety facilitated the loading of SAHA/erlotinib via inclusion complex. A synergistic anti-cancer effect on the co-administration of SAHA/erlotinib and EGFRvIII siRNA was demonstrated using U87-EGFP glioblastoma cells. Co-delivery of SAHA (5 mM)/EGFRvIII siRNA (200 nM) or erlotinib (30 mM)/siRNA (200 nM) enhanced the cytotoxicity (% cell death: 85% and 70%, respectively) as compared to SAHA (68%) or erlotinib (51%) alone. Polo-like kinase 1 (Plk1), which is overexpressed in a broad range of tumors, has a pivotal role in cell mitosis. The inhibition of Plk1 causes mitotic arrest and leads to apoptosis [89]. Zhao et al. investigated the co-delivery of docetaxel and siPlk1 (siRNA that down-regulates Plk1 proteins by post-transcriptional degradation of Plk1 mRNA) using a herceptin-conjugated vitamin E TPGS (D-a-tocopheryl polyethylene glycol 1000 succinate)-based immunomicelle [90]. Compared to the docetaxel-loaded TPGS micelle, the IC50 of docetaxel-loaded TPGS-siPlk1/TPGS micelle after 72 hours incubation was reduced by 99.8%, 83.0%, and 90.4% for NIH3T3 fibroblast cells, MCF-7 cells, and SK-BR-3 cells in vitro, respectively. Nuclear factor-kb (NF-kb) is another important transcription factor that regulates the expression of various genes to inhibit apoptosis in cancer models [91]. It is also evident that NF-kb has a major role in developing MDR in cancer cells [92]. Zhao et al. studied the co-delivery of Dox and NF-kb p65 siRNA using spermine-grafted poly-g-benzyl-L-glutamate (PGS) polyelectrolyte brushes in vitro [93]. PGS polyelectrolyte brushes complexed with NF-kb p65 siRNA subsequently enhanced the bioavailability of Dox in the cancer cells because of NF-kB p65 inhibition. The total apoptosis of Dox with pre-transfection of PGS polyelectrolyte brushes/NF-kB p65 siRNA polyplex was 170% of that treated with the same dose of doxorubicin alone. Y-box binding protein-1 (YB-1) is another protein that is overexpressed in cancer. It was reported that YB-1 translocation from the cytoplasm to the nucleus stimulated MDR proteins [94]. Zeng et al. investigated the co-delivery of rapamycin and siRNA
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targeting YB-1 (siYB-1) using poly(ethylene glycol)-block-poly(bbenzyl-L-aspartate)-block-poly(e-caprolactone) triblock copolymers [80]. Compared to individual administration (rapamycin (1 nM)/NPs: 40% cell death; siYB-1 (50 nM)/NPs: <9% cell death), co-delivery of rapamycin (1 nM) and siYB-1 (50 nM) induced around 62% cell death after 48 hours incubation in human PC3 prostate cancer cells in vitro. In addition, a 75% tumor volume reduction in a PC3 xenograft nude mice model of human prostate cancer was observed on the 10th day after co-delivery of rapamycin (30 mg/kg) and siYB-1 (2 mg/kg).
Conclusions Various types of nanocarriers have been investigated for the co-administration of siRNA and anticancer drugs in an effort to enhance anticancer effects by overcoming MDR or inducing different apoptosis pathways. Judiciously engineered multifunctional drug/siRNA co-delivery nanocarriers can significantly increase their in vivo tumor accumulation via both passive (i.e. the EPR effect) and active (i.e. via proper conjugation of active tumortargeting ligands such as peptides, antibodies, aptamers, and certain small molecules) tumor-targeting abilities. Thus, multifunctional nanomedicines offer great promise in overcoming the drawbacks of current treatment modalities, including chemotherapy. Nevertheless, a deeper understanding of several factors, including the optimal ratio of each therapeutic agent (e.g. siRNA versus anticancer drug) as well as their pharmacological fate at the tumor site, and the nature of cancer heterogeneity, is needed to achieve the maximum synergistic effect of co-administering siRNA with anticancer drugs. Additionally, further studies are needed to avoid unexpected immune stimulation with the simultaneous administration of siRNA and anticancer drugs.
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