Tinagl1 Gene Therapy Suppresses Growth and Remodels the Microenvironment of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) remains one of the most challenging subtypes of breast cancer to treat and is responsible for approximately 12% of breast cancer cases in the US per year. In 2019, the protein Tinagl1 was identified as a key factor for improved prognoses in certain TNBC patients. While the intracellular mechanism of action has been thoroughly studied, little is known about the role of Tinagl1 in the tumor microenvironment. In this study, we developed a lipid nanoparticle-based gene therapy to directly target the expression of Tinagl1 in tumor cells for localized expression. Additionally, we sought to characterize the changes to the tumor microenvironment induced by Tinagl1 treatment, with the goal of informing future choices for combination therapies including Tinagl1. We found that Tinagl1 gene therapy was able to slow tumor growth from the first dose and that the effects held steady for nearly a week following the final dose. No toxicity was found with this treatment. Additionally, the use of Tinagl1 increases the tumor vasculature by 3-fold but does not increase the tumor permeability or risk of metastasis. However, the increase in vasculature arising from Tinagl1 therapy reduced the expression of Hif1a significantly (p < 0.01), which may decrease the risk of drug resistance.

Biomolecule-assisted green synthesis of nanostructured calcium phosphates and their biomedical applications.

Calcium phosphates (CaPs) are ubiquitous in nature and vertebrate bones and teeth, and have high biocompatibility and promising applications in various biomedical fields. Nanostructured calcium phosphates (NCaPs) are recognized as promising nanocarriers for drug/gene/protein delivery owing to their high specific surface area, pH-responsive degradability, high drug/gene/protein loading capacity and sustained release performance. In order to control the structure and surface properties of NCaPs, various biomolecules with high biocompatibility such as nucleic acids, proteins, peptides, liposomes and phosphorus-containing biomolecules are used in the synthesis of NCaPs. Moreover, biomolecules play important roles in the synthesis processes, resulting in the formation of various NCaPs with different sizes and morphologies. At room temperature, biomolecules can play the following roles: (1) acting as a biocompatible organic phase to form biomolecule/CaP hybrid nanostructured materials; (2) serving as a biotemplate for the biomimetic mineralization of NCaPs; (3) acting as a biocompatible modifier to coat the surface of NCaPs, preventing their aggregation and increasing their colloidal stability. Under heating conditions, biomolecules can (1) control the crystallization process of NCaPs by forming biomolecule/CaP nanocomposites before heating; (2) prevent the rapid and disordered growth of NCaPs by chelating with Ca2+ ions to form precursors; (3) provide the phosphorus source for the controlled synthesis of NCaPs by using phosphorus-containing biomolecules. This review focuses on the important roles of biomolecules in the synthesis of NCaPs, which are expected to guide the design and controlled synthesis of NCaPs. Moreover, we will also summarize the biomedical applications of NCaPs in nanomedicine and tissue engineering, and discuss their current research trends and future prospects.

Liposomal Nanostructures for Drug Delivery in Gastrointestinal Cancers.

Gastrointestinal (GI) cancers like liver, pancreatic, colorectal, and gastric cancer remain some of the most difficult and aggressive cancers. Nanoparticles like liposomes had been approved in the clinic for cancer therapy dating as far back as 1995. Over the years, liposomal formulations have come a long way, facing several roadblocks and failures, and advancing by optimizing formulations and incorporating novel design approaches to navigate therapeutic delivery challenges. The first liposomal formulation for a GI cancer drug was approved recently in 2015, setting the stage for further clinical developments of liposome-based delivery systems for therapies against GI malignancies. This article reviews the design considerations and strategies that can be used to deliver drugs to GI tumors, the wide range of therapeutic agents that have been explored in preclinical as well as clinical studies, and the current therapies that are being investigated in the clinic against GI malignancies.

Dual Functional LipoMET Mediates Envelope-type Nanoparticles to Combinational Oncogene Silencing and Tumor Growth Inhibition.

The success of small interfering RNA (siRNA)-mediated gene silencing for cancer therapy is still limited because of its instability and poor intracellular internalization. Traditional cationic carriers cannot adequately meet the need for clinical application of siRNA. We herein report a dual-functional liposome containing a cholesterol derivative of metformin, i.e., LipoMET, which takes advantage of the fusogenic activity as well as intrinsic tumor apoptosis inducing ability of biguanide moiety to achieve a combinational anti-oncogenic effect. In this study, the vascular endothelial growth factor (VEGF)-specific siRNAs were first electrostatically condensed into a ternary nanocomplex composed of polycation and hyaluronate, which was subsequently enveloped by LipoMET through membrane fusion. In comparison with common cationic control group, the resulting envelope-type nanoparticles (PH@LipoMET nanoparticles [NPs]) showed the ability of rapid cellular internalization and effective endosomal escape of siRNA during intracellular trafficking studies. Systemic administration of the targeted LipoMETs was capable of inducing apoptosis and tumor growth inhibition in the NCI-H460 xenograft model. When carrying VEGF-specific siRNAs, PH@LipoMET NPs remarkably downregulated the expression of VEGF and led to even more tumor suppression in vivo. Thus, LipoMET originated envelope-type nanoparticles may serve as a potential dual-functional siRNA delivery system to improve therapeutic effect of oncogene silencing.

Curcumin Micelles Remodel Tumor Microenvironment and Enhance Vaccine Activity in an Advanced Melanoma Model.

Previously, we have reported a lipid-based Trp2 peptide vaccine for immunotherapy against melanoma. The suppressive immune microenvironment in the tumor is a major hurdle for an effective vaccine therapy. We hypothesized that curcumin (CUR) would remodel the tumor microenvironment to improve the vaccine activity. Curcumin-polyethylene glycol conjugate (CUR-PEG), an amphiphilic CUR-based micelle, was delivered intravenously (i.v.) to the tumor. Indeed, in the B16F10 tumor-bearing mice, the combination of CUR-PEG and vaccine treatment resulted in a synergistic antitumor effect (P < 0.001) compared to individual treatments. In the immune organs, the combination therapy significantly boosted in vivo cytotoxic T-lymphocyte response (41.0 ± 5.0% specific killing) and interferon-γ (IFN-γ) production (sevenfold increase). In the tumor microenvironment, the combination therapy led to significantly downregulated levels of immunosuppressive factors, such as decreased numbers of myeloid-derived suppressor cells and regulatory T cells (Treg) cells and declined levels of interleukin-6 and chemokine ligand 2-in correlation with increased levels of proinflammatory cytokines, including tumor necrosis factor-α and IFN-γ as well as an elevation in the CD8(+) T-cell population. The results indicated a distinct M2 to M1 phenotype switch in the treated tumors. Combining CUR-PEG and vaccine also dramatically downregulated the signal transducer and activator of transcription 3 pathway (76% reduction). Thus, we conclude that CUR-PEG is an effective agent to improve immunotherapy for advanced melanoma.

Smart polymeric nanoparticles for cancer gene delivery.

The massive amount of human genetic information already available has accelerated the identification of target genes, making gene and nucleic acid therapy the next generation of medicine. Nanoparticle (NP)-based anticancer gene therapy treatment has received significant interest in this evolving field. Recent advances in vector technology have improved gene transfection efficiencies of nonviral vectors to a level similar to viruses. This review serves as an introduction to surface modifications of NPs based on polymeric structural improvements and target moieties. A discussion regarding the future perspective of multifunctional NPs in cancer therapy is also included.

Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis.

CD47 is a "self marker" that is usually overexpressed on the surface of cancer cells to enable them to escape immunosurveillance. Recognition of CD47 by its receptor, signal regulatory protein α (SIRPα), which is expressed in the macrophages, inhibits phagocytic destruction of cancer cells by the macrophages. In this study, we have first shown that clinical isolates of human melanoma significantly upregulate CD47, possibly as a mechanism to defend themselves against the macrophages. We then exploited RNA interference (RNAi) technology to test the hypothesis that knocking down CD47 in the tumor cells will render them targets for macrophage destruction; hence, creating a novel anti-cancer therapy. Anti-CD47 siRNA was encapsulated in a liposome-protamine-hyaluronic acid (LPH) nanoparticle (NP) formulation to address the challenge of targeted delivery of siRNA-based therapeutics in vivo. Efficient silencing of CD47 in tumor tissues with systemic administration of LPH(CD47) also significantly inhibited the growth of melanoma tumors. In a lung metastasis model, LPH(CD47) efficiently inhibited lung metastasis to about 27% of the untreated control. Moreover, no hematopoietic toxicity was observed in the animals that received multiple doses of LPH(CD47). Our findings indicate CD47 as a potential prognostic marker for melanoma development as well as a target for therapeutic intervention with RNAi-based nanomedicines.

Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy.

Failure of clinical trials of nonviral vector-mediated gene therapy arises primarily from either an insufficient transgene expression level or immunostimulation concerns caused by the genetic information carrier (e.g., bacteria-generated, double-stranded DNA (dsDNA)). Neither of these issues could be addressed through engineering-sophisticated gene delivery vehicles. Therefore, we propose a systemic delivery of chemically modified messenger RNA (mRNA) as an alternative to plasmid DNA (pDNA) in cancer gene therapy. Modified mRNA evaded recognition by the innate immune system and was less immunostimulating than dsDNA or regular mRNA. Moreover, the cytoplasmic delivery of mRNA circumvented the nuclear envelope, which resulted in a higher gene expression level. When formulated in the nanoparticle formulation liposome-protamine-RNA (LPR), modified mRNA showed increased nuclease tolerance and was more effectively taken up by tumor cells after systemic administration. The use of LPR resulted in a substantial increase of the gene expression level compared with the equivalent pDNA in the human lung cancer NCI-H460 carcinoma. In a therapeutic model, when modified mRNA encoding herpes simplex virus 1-thymidine kinase (HSV1-tk) was systemically delivered to H460 xenograft-bearing nude mice, it was significantly more effective in suppressing tumor growth than pDNA.

Recent advances in nonviral vectors for gene delivery.

Gene therapy has long been regarded a promising treatment for many diseases, whether acquired (such as AIDS or cancer) or inherited through a genetic disorder. A drug based on a nucleic acid, however, must be delivered to the interior of the target cell while surviving an array of biological defenses honed by evolution. Successful gene therapy is thus dependent on the development of an efficient delivery vector. Researchers have pursued two major vehicles for gene delivery: viral and nonviral (synthetic) vectors. Although viral vectors currently offer greater efficiency, nonviral vectors, which are typically based on cationic lipids or polymers, are preferred because of safety concerns with viral vectors. So far, nonviral vectors can readily transfect cells in culture, but efficient nanomedicines remain far removed from the clinic. Overcoming the obstacles associated with nonviral vectors to improve the delivery efficiency and therapeutic effect of nucleic acids is thus an active area of current research. The difficulties are manifold, including the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, and managing the targeting ability of the carriers with respect to the cells of interest. Modifying the surface with poly(ethylene glycol), that is, PEGylation, is the predominant method used to reduce the binding of plasma proteins to nonviral vectors and minimize clearance by the RES after intravenous administration. Nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors because of the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral and anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal models. Other approaches include (i) designing and synthesizing novel cationic lipids and polymers, (ii) chemically coupling the nucleic acid to peptides, targeting ligands, polymers, or environmentally sensitive moieties, and (iii) utilizing inorganic nanoparticles in nucleic acid delivery. Recently, the different classes of nonviral vectors appear to be converging, and the ability to combine features of different classes of nonviral vectors in a single strategy has emerged. With the strengths of several approaches working in concert, more hurdles associated with efficient nucleic acid delivery might therefore be overcome. In this Account, we focus on these novel nonviral vectors, which are classified as multifunctional hybrid nucleic acid vectors, novel membrane/core nanoparticles for nucleic acid delivery, and ultrasound-responsive nucleic acid vectors. We highlight systemic delivery studies and consider the future prospects for nucleic acid delivery. A better understanding of the fate of the nanoparticles inside the cell and of the interactions between the parts of hybrid particles should lead to a delivery system suitable for clinical use. We also underscore the value of sustained release of a nucleic acid in this endeavor; making vectors targeted to cells with sustained release in vivo should provide an interesting research challenge.

In vivo gene delivery by nonviral vectors: overcoming hurdles?

The promise of cancer gene therapeutics is hampered by difficulties in the in vivo delivery to the targeted tumor cells, and systemic delivery remains to be the biggest challenge to be overcome. Here, we concentrate on systemic in vivo gene delivery for cancer therapy using nonviral vectors. In this review, we summarize the existing delivery barriers together with the requirements and strategies to overcome these problems. We will also introduce the current progress in the design of nonviral vectors, and briefly discuss their safety issues.

Systemic delivery of siRNA via LCP nanoparticle efficiently inhibits lung metastasis.

Targeted delivery remains the major challenge for the application of small interfering RNA (siRNA). We have developed a lipid/calcium/phosphate (LCP) nanoparticle (NP) to improve siRNA delivery efficiency. The LCP NP was prepared by using microemulsion technology to form calcium/phosphate (CaP) core and further coated with cationic lipids. The final NP was grafted with polyethylene glycol (PEG) and anisamide (AA) ligand on the surface to target sigma receptor-expressing B16F10 melanoma cells. The LCP NP exhibited a 40 nm particle size, a +25 mV zeta-potential, and 91% siRNA encapsulation efficiency. After a single intravenous (i.v.) injection of antiluciferase siRNA (0.12 mg siRNA/kg) formulated in targeted LCP NP, luciferase activity in metastatic B16F10 tumor-loaded lungs decreased by 78% in C57BL/6 mice. In a therapeutic experiment, siRNA against MDM2, c-myc, and VEGF coformulated in the targeted LCP NP resulted in simultaneous silencing of the respective oncogenes in metastatic nodules. Treatment with siRNA in the targeted NP significantly reduced lung metastases (~70-80%) at a relatively low dose (0.36 mg/kg), whereas control group showed little therapeutic effect. Moreover, this targeted LCP NP significantly prolonged the mean survival time of the animals by 27.8% compared to control group without showing any toxicity at the therapeutic dose.

In vivo delivery of RNAi with lipid-based nanoparticles.

RNA interference (RNAi) technology represents a fundamentally new category of treatments for human disease by addressing targets that are traditionally considered undruggable with existing medicines. The major challenge for RNAi-based therapy is the delivery system that meets human therapeutic needs. Therefore, engineering vectors with good delivery efficiency and safety profile is an intense area of research. Lipid-based nanoparticles for RNAi have yielded successful advances in vivo and to an extent in clinical trials. In this review, we discuss the barriers in developing lipid-based nanoparticles for in vivo RNAi and different strategies to overcome them. Rational designs that address safety concerns and ensure effective delivery will aid the translation of engineered lipid-based nanoparticles toward the clinic in the foreseeable future.

Formulation of two lipid-based membrane-core nanoparticles for FOLFOX combination therapy.

FOLFOX is a combination of folinic acid (FnA), 5-fluorouracil (5-Fu) and oxaliplatin (OxP). It has been used as the standard treatment for colorectal cancer (CRC) and hepatocellular carcinoma (HCC). This treatment is effective, but its high toxicity is dose limiting, and the drugs need to be taken for a long time. To lower the toxicity so that higher doses can be administered with minimal side effects, two lipid-based membrane-core (MC) nanoformulations, Nano-Folox and Nano-FdUMP, have recently been developed by using the nanoprecipitation technique. The combination of Nano-Folox (containing platinum drug and FnA) and Nano-FdUMP (containing fluorine drug) significantly improves the antitumor effect against CRC and HCC relative to FOLFOX (the combination of free drugs), resulting in long-term survival of animals without significant toxic signs. Here, we describe two formulation protocols. First, for Nano-Folox, a Pt(DACH)•FnA nanoprecipitate is formed by [Pt(DACH)(H2O)2]2+ (the active form of OxP) and FnA2-, and the resultant nanoprecipitate is encapsulated inside the lipid nanoparticles (NPs) modified with the PEGylated aminoethyl anisamide (AEAA, a targeting ligand for sigma-1 receptor overexpressing on CRC and HCC). Second, for Nano-FdUMP, FdUMP (the active metabolite of 5-Fu) is entrapped inside the amorphous Ca3(PO4)2 nanoprecipitate, and the resultant Ca3(PO4)2•FdUMP nanoprecipitate is encapsulated into the AEAA-targeted PEGylated lipid NPs. The procedures for Nano-Folox and Nano-FdUMP take ~17 h and ~4 h, respectively (~17 h if they are prepared simultaneously). Procedures for the physicochemical (~30 h) and cytotoxic (~54 h) characterization are also described.

Magnolol-loaded cholesteryl biguanide conjugate hydrochloride nanoparticles for triple-negative breast cancer therapy.

The potential of combination therapy using nanoparticle delivery systems in improving triple-negative breast cancer treatment efficacy remains to be explored. Here, we report a novel nanoparticle system using a cholesterol biguanide conjugate hydrochloride (CBH) as both a drug and carrier to load magnolol (MAG). Poly(ethylene glycol)-poly(lactic-co-glycolic acid) (mPEG-PLGA) and aminoethyl anisamide-poly(ethylene glycol)-poly(lactic-co-glycolic acid) (AEAA-PEG-PLGA) were added to form nanoparticles. Nanoparticles accumulated most in tumor tissues when the weight ratio of AEAA-PEG-PLGA to mPEG-PLGA was 4:1. MAG and CBH exerted a synergistic inhibitory effect on 4 T1 cells. An in vitro study showed that nanoparticles displayed the highest tumor cell uptake rate, highest apoptosis rate, and strongest inhibitory effect on tumor cell migration and monoclonal formation. CBH might promote nanoparticle uptake by cells and lysosomal escape. After intravenous administration to mice with 4 T1 breast tumors in situ, the nanoparticles inhibited tumor growth without obvious toxicity. Western blot results showed that nanoparticles altered the levels of p53, p-AKT, and p-AMPK in the tumor tissue. Moreover, cell apoptosis was found in the same area of H&E-stained and TUNEL-stained tumors treated with the nanoparticles. Collectively, this nanoparticle system provides a novel combination drug delivery strategy for treating triple-negative breast cancer.

Multifunctional nanoparticles delivering small interfering RNA and doxorubicin overcome drug resistance in cancer.

Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N'-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared.

Nanoparticles targeted with NGR motif deliver c-myc siRNA and doxorubicin for anticancer therapy.

We have designed a PEGylated LPD (liposome-polycation-DNA) nanoparticle for systemic, specific, and efficient delivery of small interfering RNA (siRNA) into solid tumors in mice by modification with NGR (aspargine-glycine-arginine) peptide, targeting aminopeptidase N (CD13) expressed in the tumor cells or tumor vascular endothelium. LPD-PEG-NGR efficiently delivered siRNA to the cytoplasm and downregulated the target gene in the HT-1080 cells but not CD13(-) HT-29 cells, whereas nanoparticles containing a control peptide, LPD-PEG-ARA, showed only little siRNA uptake and gene silencing activity. LPD-PEG-NGR efficiently delivered siRNA into the cytoplasm of HT-1080 xenograft tumor 4 hours after intravenous injection. Three daily injections (1.2 mg/kg) of c-myc siRNA formulated in the LPD-PEG-NGR effectively suppressed c-myc expression and triggered cellular apoptosis in the tumor, resulting in a partial tumor growth inhibition. When doxorubicin (DOX) and siRNA were co-formulated in LPD-PEG-NGR, an enhanced therapeutic effect was observed.

Preparation and Characterization of siRNA-Loaded Liposomes.

The major challenge for RNAi-based therapy is the fabrication of the delivery system that meet the requirement of clinical applicability. Liposome-derived nanoparticles (NPs) are one of the best investigated systems for in vivo siRNA delivery. In the recent years, we have successfully redesigned the conventional cationic liposomes into Liposome/Protamine/hyaluronic acid (LPH) NPs and Lipid-Calcium-Phosphate (LCP) NPs in order to increase the in vivo gene silencing effect and reduce the toxicity. Here we describe the preparation of LPH and LCP NPs loaded with siRNA, and characterization analysis including size distribution, trapping efficiency, and in vivo activity. This protocol could be used for in vivo delivery of siRNA to target genes in cancer cells.

Nanoparticles evading the reticuloendothelial system: role of the supported bilayer.

We have previously shown that the PEGylated LPD (liposome-polycation-DNA) nanoparticles were highly efficient in delivering siRNA to the tumor with low liver uptake. Its mechanism of evading the reticuloendothelial system (RES) is reported here. In LPD, nucleic acids were condensed with protamine into a compact core, which was then coated by two cationic lipid bilayers with the inner bilayer stabilized by charge-charge interaction (also called the supported bilayer). Finally, a detergent-like molecule, polyethylene glycol (PEG)-phospholipid is post-inserted into the lipid bilayer to modify the surface of LPD. The dynamic light scattering (DLS) data showed that LPD had improved stability compared to cationic liposomes after incubation with a high concentration of DSPE-PEG(2000), which is known to disrupt the bilayer. LPD prepared with a multivalent cationic lipid, DSGLA, had enhanced stability compared to those containing DOTAP, a monovalent cationic lipid, suggesting that stronger charge-charge interaction in the supported bilayer contributed to a higher stability. Distinct nanoparticle structure was found in the PEGylated LPD by transmission electron microscopy, while the cationic liposomes were transformed into tubular micelles. Size exclusion chromatography data showed that approximately 60% of the total cationic lipids, which were located in the outer bilayer of LPD, were stripped off during the PEGylation; and about 20% of the input DSPE-PEG(2000) was incorporated into the inner bilayer with about 10.6 mol% of DSPE-PEG(2000) presented on the particle surface. This led to complete charge shielding, low liver sinusoidal uptake, and 32.5% injected dose delivered to the NCI-H460 tumor in a xenograft model.

Anticancer activities of phytoconstituents and their liposomal targeting strategies against tumor cells and the microenvironment.

Various bioactive ingredients have been extracted from Chinese herbal medicines (CHMs) that affect tumor progression and metastasis. To further understand the mechanisms of CHMs in cancer therapy, this article summarizes the effects of five categories of CHMs and their active ingredients on tumor cells and the tumor microenvironment. Despite their treatment potential, the undesirable physicochemical properties (poor permeability, instability, high hydrophilicity or hydrophobicity, toxicity) and unwanted pharmacokinetic profiles (short half-life in blood and low bioavailability) restrict clinical studies of CHMs. Therefore, development of liposomes through relevant surface modifying techniques to achieve targeted CHM delivery for cancer cells, i.e., extracellular and intracellular targets and targets in tumor microenvironment or vasculature, have been reviewed. Current challenges of liposomal targeting of these phytoconstituents and future perspective of CHM applications are discussed to provide an informative reference for interested readers.

Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA.

The selective delivery of small interfering RNA (siRNA) to metastatic tumors remains a challenging task. We have developed a nanoparticle (NP) formulation composed of siRNA, a carrier DNA, a polycationic peptide, and cationic liposomes. The NP was obtained by a self-assembling process, followed by surface modification with a polyethylene glycol (PEG)-conjugated ligand, anisamide. The NP was PEGylated and a ligand was presented to target sigma receptor-expressing murine melanoma cells, B16F10. The lung metastasis model was established by intravenous (i.v.) injection of the B16F10 cells into C57BL/6 mice. A mixture of siRNA against MDM2, c-myc, and vascular endothelial growth factor (VEGF) co-formulated in the targeted NP caused simultaneous silencing of each of the oncogenes in the metastatic nodules. Two consecutive i.v. injections of siRNA in the targeted NP significantly reduced the lung metastasis (approximately 70-80%) at a relatively low dose (0.45 mg/kg), whereas free siRNA and the nontargeted NP showed little effect. This targeted NP formulation significantly prolonged the mean survival time of the animals by 30% as compared to the untreated controls. At the therapeutic dose, the targeted NP showed little local and systemic immunotoxicity and did not decrease the body weight or damage the major organs.