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Supplementary Figure S1: Mesoporous silica-based nanoconstruct for targeted delivery of siRNA; Supplementary Figure S2: Screening of siPLK1 by mRNA knockdown and viability; Supplementary Figure S3: ROS intensity and NOX1-5 mRNA levels of TNBC cell lines; Supplementary Figure S4: HER2 signaling in LM2-4luc+/H2N cells; Supplementary Figure S5: Representative whole body bioluminescent IVIS imaging of a mouse injected with cancer cells; Supplementary Figure S6: Representative images of lung nodes of mice from short-term study; Supplementary Figure S7: Representative images of the whole lung, H&E, and anti-human vimentin staining of mouse lungs from the long-term study.

Immune checkpoint inhibitors (ICIs) targeting PD-L1 and PD-1 have improved survival in a subset of patients with advanced non-small cell lung cancer (NSCLC). However, only a minority of NSCLC patients respond to ICIs, highlighting the need for superior immunotherapy. Herein, we developed a nanoparticle-based immunotherapy termed ARAC (Antigen Release Agent and Checkpoint Inhibitor) to enhance the efficacy of PD-L1 inhibitor. ARAC is a nanoparticle co-delivering PLK1 inhibitor (volasertib) and PD-L1 antibody. PLK1 is a key mitotic kinase that is overexpressed in various cancers including NSCLC and drives cancer growth. Inhibition of PLK1 selectively kills cancer cells and upregulates PD-L1 expression in surviving cancer cells thereby providing opportunity for ARAC targeted delivery in a positive feedback manner. ARAC reduced effective doses of volasertib and PD-L1 antibody by 5-fold in a metastatic lung tumor model (LLC-JSP) and the effect was mainly mediated by CD8 + T cells. Notably, ARAC also showed efficacy in another lung tumor model (KLN-205), which does not respond to CTLA-4 and PD-1 inhibitor combination. Further, the nanoparticle construct was well-tolerated in non-human primates. This study highlights a rationale combination strategy to augment existing therapies by utilizing our nanoparticle platform that can load multiple cargo types at once.

Given the complexity of cancer, it is becoming apparent that combination therapy - in which complementary biological pathways are simultaneously targeted - is required to achieve clinically meaningful responses. To that end, PDX Pharma, in collaboration with Oregon Health & Science University, has developed a proprietary nanoparticle platform (Pdx-NP™) that can effectively co-deliver a plethora of therapeutic modalities while maintaining a small size in saline (100 nm), suitable for infusion and tumor accumulation. This enables the targeting of complementary cancer and immune pathways, leading to synergistic clinical benefits. Herein, we report on the second generation ARAC-02 (Antigen Release Agent and Checkpoint Inhibitor) which is designed to improve the efficacy of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC). While ICIs have improved survival in a subset of patients with advanced NSCLC, only a minority of patients respond to ICIs, highlighting the need for combination immunotherapy. ARAC-02 co-delivers a polo-like kinase 1 (PLK1)-targeted therapy (volasertib), a PD-L1 antibody, and the immune-stimulant CpG. These three agents were carefully selected based on their roles in cancer and immune pathways. Volasertib 1) selectively kills cancer cells, 2) modulates the immune-suppressive tumor microenvironment, and 3) upregulates PD-L1 expression in cancer cells, providing opportunity for targeted delivery with PD-L1 antibody on the nanoparticles. CpG is a TLR9 agonist that enhances antigen presentation to generate tumor-specific T cells. PD-L1 antibodies serve as tumor targeting agent for nanoparticle delivery as well as an ICI (i.e., PD-L1 degradation upon internalization with the nanoparticles), releasing the brakes and allowing T cells to attack the cancer. We found that the optimized ARAC-02 is preferentially internalized in PD-L1 expressing NSCLC cells, demonstrating a 10-fold greater uptake to PD-L1(high) NSCLC cells than PD-L1(low) normal cells. ARAC-02 effectively kills NSCLC cells regardless of their mutational status, while sparing normal cells. In a metastatic lung tumor model, ARAC-02 treatments reduced the tumor burden by 30-fold vs. saline control and 8-fold vs. the first generation ARAC (not containing CpG), whose effect was mainly mediated by CD8+ T cells. ARAC-02 was found to be safe to mice as assessed by body weight, serum biomarkers, and histology. Importantly, intravenous infusions of the platform was also found to be safe in a preliminary toxicology study in non-human primates. Due to its unique ability to catalyze various steps of the adaptive immune response, ARAC-02 is anticipated to provide superior outcomes in NSCLC and a broad range of tumor types regardless of baseline PD-L1 expression. Citation Format: Moataz Reda, Worapol Ngamcherdtrakul, Ruijie Wang, Noah Crumrine, Cole Baker, Alyssa Wallstrum, Jeremy Saito, Natalie White, Wassana Yantasee. Nano-immunotherapy targeting PD-L1, PLK1, and TLR9 for treatment of non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5119.

Immune checkpoint inhibitors (ICIs) have provided long-lasting response but only in a small subset of cancer patients. To address this shortcoming, we developed a nanotherapeutic (AIRISE-02) that can manipulate both cancer cells and immunosuppressive tumor microenvironment (TME), leading to successful production of anti-tumor T cells that promote ICIs’ response. AIRISE-02 (Augmenting Immune Response and Inhibiting Suppressive Environment of tumors) is our patented polymer-coated mesoporous silica nanoparticle (Pdx-NPTM) that co-delivers immunostimulant CpG and siRNA against signal transducer and activator of transcription 3 (siSTAT3). CpG is a widely used adjuvant that activates antigen presenting cells (APCs) to process and present tumor antigens. STAT3 contributes to progression, metastasis, and immune escape of cancer cells. STAT3 also has roles in immunosuppressive TME, by inhibiting maturation and activation of APCs, suppressing M1 macrophage polarization, and mediating immunosuppressive function of myeloid-derived suppressor cells. AIRISE-02 can be given both intratumorally and systemically. In bilateral B16F10 melanoma and MC38 colon cancer models, three intratumoral injections of AIRISE-02 to one tumor inhibit both treated and untreated distal tumors. When combined with ICIs against PD-1 and CTLA-4, AIRISE-02 leads to long term cure in 63% of mice with B16F10 tumors and 43% of mice with MC38 colon tumors (while ICIs alone provides no cure in B16F10 and 17% cure in MC38 tumor mice). In the tumor rechallenge study at 6 months post-cure, B16F10 mice reject new B16F10 tumors, while allowing LLC-JSP lung tumors to grow, suggesting tumor-specific immune memory effect. Through antibody depletion studies, AIRISE-02’s activity is shown to be highly dependent on CD8+ T cells and IFN-ɣ, while slightly dependent on B cells and NK cells. Strikingly, depleting CD4+ T cells strongly boosts AIRISE-02’s activity, which is likely due to depleting regulatory T cells. Effectiveness of AIRISE-02 is owing to the ability of Pdx-NPTM to retain and protect oligonucleotide cargos in the tumors; e.g., 50% of injected siRNA dose remained at three days with Pdx-NP vs. 2% when given as free siRNA. In addition to STAT3, our platform technology can be tailored to other important therapeutic targets with the power of siRNA. Ongoing works to attack other hard-to-drug targets, such as KRAS, Myc, and AR will also be presented. Citation Format: Worapol Ngamcherdtrakul, Moataz Reda, Cole Baker, Noah Crumrine, Ruijie Wang, Alyssa Wallstrum, Natalie White, Jeremy Saito, Husam Zaidan, Justin Rehwaldt, Molly Nelson, Joe Gray, Wassana Yantasee. Triggering anti-tumor immune response with a nanotherapeutic that targets TLR9 and STAT3 pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3229.

The first-line treatment of advanced and metastatic human epidermal growth factor receptor type 2 (HER2+) breast cancer requires two HER2-targeting antibodies (trastuzumab and pertuzumab) and a taxane (docetaxel or paclitaxel). The three-drug regimen costs over $320,000 per treatment course, requires a 4-hour infusion time, and has many adverse side effects, while achieving only 18 months of progression-free survival. To replace this regimen, reduce infusion time, and enhance efficacy, a single therapeutic was developed based on trastuzumab-conjugated nanoparticles for co-delivering docetaxel and siRNA against HER2 (siHER2). The optimal nanoconstruct has a hydrodynamic size of 100 nm and specifically treats HER2+ breast cancer cells over organ-derived normal cells. In a drug-resistant orthotopic HER2+ HCC1954 tumor mouse model, the nanoconstruct inhibited tumor growth more effectively than the docetaxel and trastuzumab combination. When coupled with microbubble-assisted focused ultrasound that transiently disrupted the blood brain barrier, the nanoconstruct inhibited the growth of trastuzumab-resistant HER2+ BT474 tumors residing in brains of mice. The nanoconstruct has a favorable safety profile in cells and in mice. Combination therapies have become the cornerstone of cancer treatment and this versatile nanoparticle platform can co-deliver multiple therapeutic types to ensure that they reach the target cells at the same time to realize their synergy.

The success of immunotherapy with immune checkpoint inhibitors (ICIs) in a subset of individuals has been very exciting. However, in many cancers, responses to current ICIs are modest and are seen only in a small subsets of patients. Herein, a widely applicable approach that increases the benefit of ICIs is reported. Intratumoral administration of augmenting immune response and inhibiting suppressive environment of tumors-AIRISE-02 nanotherapeutic that co-delivers CpG and STAT3 siRNA-results in not only regression of the injected tumor, but also tumors at distant sites in multiple tumor model systems. In particular, three doses of AIRISE-02 in combination with systemic ICIs completely cure both treated and untreated aggressive melanoma tumors in 63% of mice, while ICIs alone do not cure any mice. A long-term memory immune effect is also reported. AIRISE-02 is effective in breast and colon tumor models as well. Lastly, AIRISE-02 is well tolerated in mice and nonhuman primates. This approach combines multiple therapeutic agents into a single nanoconstruct to create whole-body immune responses across multiple cancer types. Being a local therapeutic, AIRISE-02 circumvents regulatory challenges of systemic nanoparticle delivery, facilitating rapid translation to the clinic. AIRISE-02 is under investigational new drug (IND)-enabling studies, and clinical trials will soon follow.

Immune checkpoint inhibitors (ICIs) targeting PD-L1 and PD-1 have improved survival in a subset of patients with advanced non-small cell lung cancer (NSCLC). However, only a minority of NSCLC patients respond to ICIs, highlighting the need for superior immunotherapy. Herein, we developed a nanoparticle-based immunotherapy termed ARAC (Antigen Release Agent and Checkpoint Inhibitor) to enhance the efficacy of PD-L1 inhibitor. ARAC is nanoparticle co-delivering PLK1 inhibitor, volasertib, and PD-L1 antibody. PLK1 is a key mitotic kinase that is overexpressed in various cancers including NSCLC and drives cancer growth. Inhibition of PLK1 selectively kills cancer cells and upregulates PD-L1 expression in surviving cancer cells thereby providing opportunity for ARAC targeted delivery in a positive feedback manner. ARAC reduced effective doses of volasertib and PD-L1 antibody by 5-fold in a metastatic lung tumor model and the effect was mainly mediated by CD8 + T cells. We also observed abscopal effect of ARAC in bilateral NSCLC tumor model and achieved complete cures in some mice when incorporating immune-stimulant CpG onto ARAC. Further, ARAC was well-tolerated in non-human primates. This study highlights a rationale combination strategy to augment existing therapies by utilizing our nanoparticle platform that can load multiple cargo types at once.

Gadolinium based contrast agents (GBCAs) have been linked to toxicity in patients, regardless of having impaired or normal renal function. Currently, no therapy is considered highly effective for removing gadolinium (Gd) from the body. We propose a new strategy to reduce blood Gd content that facilitates whole body removal of Gd using a hemoperfusion system consisting of a cartridge of porous silica beads (Davisil®) functionalized with 1,2-hydroxypyridinone (1,2-HOPO). Herein, we report optimization of the hemoperfusion system using an ex vivo blood and an in vivo rat model of chronic kidney disease (CKD). In our ex vivo system, 1,2-HOPO-Davisil outperformed Gambro activated charcoal (AC), which is commonly used in clinical hemoperfusion of aqueous toxins, in terms of Gd capture capacity and rate. In the CKD rat model, the 1,2-HOPO-Davisil hemoperfusion system removed Gd by 3.4 times over the Gambro AC system. 1,2-HOPO-Davisil did not change complete blood counts and common blood biochemistry. Thus, this strategy has great potential for clinical translation to manage GBCAs after magnetic resonance imaging (MRI), before Gd can deposit in the body and cause long-term toxicity. Although gadodiamide was used as a proof of concept model for GBCAs in this study, 1,2-HOPO functionalized mesoporous silica could also capture dissociated Gd and other GBCAs.

Radiation therapy is a cornerstone of modern cancer therapy alongside surgery, chemotherapy, and immunotherapy, with over half of all cancer patients receiving radiation therapy as part of their treatment regimen. Development of novel radiation sensitizers that can improve the therapeutic window of radiation therapy are sought after, particularly for tumors at an elevated risk of local and regional recurrence such as locally-advanced lung, head and neck, and gastrointestinal tumors. This review discusses clinical strategies to enhance radiotherapy efficacy and decrease toxicity, hence, increasing the overall therapeutic window. A focus is given to the molecular targets that have been identified and their associated mechanisms of action in enhancing radiotherapy. Examples include cell survival and proliferation signaling such as the EGFR and PI3K/AKT/mTOR pathways, DNA repair genes including PARP and ATM/ATR, angiogenic growth factors, epigenetic regulators, and immune checkpoint proteins. By manipulating various mechanisms of tumor resistance to ionizing radiation (IR), targeted therapies hold significant value to increase the therapeutic window of radiotherapy. Further, the use of novel nanoparticles to enhance radiotherapy is also reviewed, including nanoparticle delivery of chemotherapies, metallic (high-Z) nanoparticles, and nanoparticle delivery of targeted therapies - all of which may improve the therapeutic window of radiotherapy by enhancing the tumor response to IR or reducing normal tissue toxicity.

Radiation sensitizers that can selectively act on cancer cells hold great promise to patients who receive radiation therapy. We developed a novel targeted therapy and radiation sensitizer for non-small cell lung cancer (NSCLC) based on cetuximab conjugated nanoparticle that targets epidermal growth factor receptor (EGFR) and delivers small interfering RNA (siRNA) against polo-like kinase 1 (PLK1). EGFR is overexpressed in 50% of lung cancer patients and a mediator of DNA repair, while PLK1 is a key mitotic regulator whose inhibition enhances radiation sensitivity. The nanoparticle construct (C-siPLK1-NP) effectively targets EGFR + NSCLC cells and reduces PLK1 expression, leading to G2/M arrest and cell death. Furthermore, we show a synergistic combination between C-siPLK1-NP and radiation, which was confirmed in vivo in A549 flank tumors. We also demonstrate the translational potential of C-siPLK1-NP as a systemic therapeutic in an orthotopic lung tumor model, where administration of C-siPLK1-NP reduced tumor growth and led to prolonged survival. Our findings demonstrate that C-siPLK1-NP is effective as a targeted therapy and as a potent radiation sensitizer for NSCLC. Potential application to other EGFR + cancer types such as colorectal and breast cancer is also demonstrated.

Multiparametric and high-content protein analysis of single cells or tissues cannot be accomplished with the currently available flow cytometry or imaging techniques utilizing fluorophore-labelled antibodies, because the number of spectrally resolvable fluorochromes is limited. In contrast, mass cytometry can resolve more signals by exploiting lanthanide-tagged antibodies; however, only about 100 metal reporters can be attached to an antibody molecule. This makes the sensitivity of lanthanide-tagged antibodies substantially lower than fluorescent reporters. A new probe that can carry more lanthanide molecules per antibody is a desirable way to enhance the sensitivity needed for the detection of protein with low cellular abundance. Herein, we report on the development of new probes utilizing mesoporous silica nanoparticles (MSNPs) with hydroxyl, amine, or phosphonate functional groups. The phosphonated MSNPs proved to be best at loading lanthanides for up to 1.4 × 106 molecules per particle, and could be loaded with various lanthanide elements (Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, and Lu) at relatively similar molar extents. The modified MSNPs can also load a fluorescent dye, allowing bimodal mass and fluorescence-based detection. We achieved specificity of antibody-conjugated nanoparticles (at 1.4 × 103 antibodies per nanoparticle) for targeting proteins on the cell surface. The new materials can potentially be used as mass cytometry probes and provide a method for simultaneous monitoring of a large host of factors comprising the tumor microenvironment (e.g., extracellular matrix, cancer cells, and immune cells). These novel probes may also benefit personalized medicine by allowing for high-throughput analysis of multiple proteins in the same specimen.

This Review discusses the various types of non-coding oligonucleotides, which have garnered extensive interest as new alternatives for targeted cancer therapies over small molecule inhibitors and monoclonal antibodies. These oligonucleotides can target any hallmark of cancer, no longer limited to so-called “druggable” targets. Thus, any identified gene that plays a key role in cancer progression or drug resistance can be exploited with oligonucleotides. Among them, small-interfering RNAs (siRNAs) are frequently utilized for gene silencing due to the robust and well established mechanism of RNA interference. Despite promising advantages, clinical translation of siRNAs is hindered by the lack of effective delivery platforms. This Review provides general criteria and consideration of nanoparticle development for systemic siRNA delivery. Different classes of nanoparticle candidates for siRNA delivery are discussed, and the progress in clinical trials for systemic cancer treatment is reviewed. Lastly, this Review presents HER2 (human epidermal growth factor receptor type 2)-positive breast cancer as one example that could benefit significantly from siRNA technology. How siRNA-based therapeutics can overcome cancer resistance to such therapies is discussed.

Worapol Ngamcherdtrakul,1,2,* Thanapon Sangvanich,1,* Moataz Reda,1 Shenda Gu,1 Daniel Bejan,2 Wassana Yantasee1,2 1Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR, USA; 2Nanomedicine Research Unit, PDX Pharmaceuticals, LLC, Portland, OR, USA *These authors contributed equally to this work Introduction: Long-term stability of therapeutic candidates is necessary toward their clinical applications. For most nanoparticle systems formulated in aqueous solutions, lyophilization or freeze-drying is a common method to ensure long-term stability. While lyophilization of lipid, polymeric, or inorganic nanoparticles have been studied, little has been reported on lyophilization and stability of hybrid nanoparticle systems, consisting of polymers, inorganic particles, and antibody. Lyophilization of complex nanoparticle systems can be challenging with respect to preserving physicochemical properties and the biological activities of the materials. We recently reported an effective small-interfering RNA (siRNA) nanoparticle carrier consisting of 50-nm mesoporous silica nanoparticles decorated with a copolymer of polyethylenimine and polyethyleneglycol, and antibody. Materials and methods: Toward future personalized medicine, the nanoparticle carriers were lyophilized alone and loaded with siRNA upon reconstitution by a few minutes of simple mixing in phosphate-buffered saline. Herein, we optimize the lyophilization of the nanoparticles in terms of buffers, lyoprotectants, reconstitution, and time and temperature of freezing and drying steps, and monitor the physical and chemical properties (reconstitution, hydrodynamic size, charge, and siRNA loading) and biological activities (gene silencing, cancer cell killing) of the materials after storing at various temperatures and times. Results: The material was best formulated in Tris-HCl buffer with 5% w/w trehalose. Freezing step was performed at -55°C for 3 h, followed by a primary drying step at -40°C (100 μBar) for 24 h and a secondary drying step at 20°C (20 μBar) for 12 h. The lyophilized material can be stored stably for 2 months at 4°C and at least 6 months at -20°C. Conclusion: We successfully developed the lyophilization process that should be applicable to other similar nanoparticle systems consisting of inorganic nanoparticle cores modified with cationic polymers, PEG, and antibodies. Keywords: nanoparticles, lyophilization, cancer, mesoporous silica, antibody, siRNA

HER2 is overexpressed in about 20% of breast cancers and contributes to poor prognosis. Unfortunately, a large fraction of patients have primary or acquired resistance to the HER2-targeted therapy trastuzumab, thus a multi-drug combination is utilized in the clinic, putting significant burden on patients. We systematically identified an optimal HER2 siRNA from 76 potential sequences and demonstrated its utility in overcoming intrinsic and acquired resistance to trastuzumab and lapatinib in 18 HER2-positive cancer cell lines. We provided evidence that the drug-resistant cancer maintains dependence on HER2 for survival. Importantly, cell lines did not readily develop resistance following extended treatment with HER2 siRNA. Using our recently developed nanoparticle platform, systemic delivery of HER2 siRNA to trastuzumab-resistant tumors resulted in significant growth inhibition. Moreover, the optimal HER2 siRNA could also silence an exon 16 skipped HER2 splice variant reported to be highly oncogenic and linked to trastuzumab resistance.

Fibrotic diseases such as scleroderma have been linked to increased oxidative stress and upregulation of pro-fibrotic genes. Recent work suggests a role of NADPH oxidase 4 (NOX4) and heat shock protein 47 (HSP47) in inducing excessive collagen synthesis, leading to fibrotic diseases. Herein, we elucidate the relationship between NOX4 and HSP47 in fibrogenesis and propose to modulate them altogether as a new strategy to treat fibrosis. We developed a nanoparticle platform consisting of polyethylenimine (PEI) and polyethylene glycol (PEG) coating on a 50-nm mesoporous silica nanoparticle (MSNP) core. The nanoparticles effectively delivered small interfering RNA (siRNA) targeting HSP47 (siHSP47) in an in vitro model of fibrosis based on TGF-β stimulated fibroblasts. The MSNP core also imparted an antioxidant property by scavenging reactive oxygen species (ROS) and subsequently reducing NOX4 levels in the in vitro fibrogenesis model. The nanoparticle was far superior to n-acetyl cysteine (NAC) at modulating pro-fibrotic markers. In vivo evaluation was performed in a bleomycin-induced scleroderma mouse model, which shares many similarities to human scleroderma disease. Intradermal administration of siHSP47-nanoparticles effectively reduced HSP47 protein expression in skin to normal level. In addition, the antioxidant MSNP also played a prominent role in reducing the pro-fibrotic markers, NOX4, alpha smooth muscle actin (α-SMA), and collagen type I (COL I), as well as skin thickness of the mice.

Metastatic breast cancer is developed in about 20% to 30% of newly diagnosed patients with early-stage breast cancer despite treatments. Herein, we report a novel nanoparticle platform with intrinsic antimetastatic properties for the targeted delivery of Polo-like kinase 1 siRNA (siPLK1). We first evaluated it in a triple-negative breast cancer (TNBC) model, which shows high metastatic potential. PLK1 was identified as the top therapeutic target for TNBC cells and tumor-initiating cells in a kinome-wide screen. The platform consists of a 50-nm mesoporous silica nanoparticle (MSNP) core coated layer-by-layer with bioreducible cross-linked PEI and PEG polymers, conjugated with an antibody for selective uptake into cancer cells. siRNA is loaded last and fully protected under the PEG layer from blood enzymatic degradation. The material has net neutral charge and low nonspecific cytotoxicity. We have also shown for the first time that the MSNP itself inhibited cancer migration and invasion in TNBC cells owing to its ROS- and NOX4-modulating properties. In vivo, siPLK1 nanoconstructs (six doses of 0.5 mg/kg) knocked down about 80% of human PLK1 mRNA expression in metastatic breast cancer cells residing in mouse lungs and reduced tumor incidence and burden in lungs and other organs of an experimental metastasis mouse model. Long-term treatment significantly delayed the onset of death in mice and improved the overall survival. The platform capable of simultaneously inhibiting the proliferative and metastatic hallmarks of cancer progression is unique and has great therapeutic potential to also target other metastatic cancers beyond TNBC. Mol Cancer Ther; 16(4); 763–72. ©2017 AACR.

In vivo delivery of siRNAs designed to inhibit genes important in cancer and other diseases continues to be an important biomedical goal. We now describe a new nanoparticle construct that has been engineered for efficient delivery of siRNA to tumors. The construct is comprised of a 47-nm mesoporous silica nanoparticle (MSNP) core coated with a cross-linked PEI-PEG copolymer, carrying siRNA against the HER2 oncogene, and coupled to the anti-HER2 monoclonal antibody (trastuzumab). The construct has been engineered to increase siRNA blood half-life, enhance tumor-specific cellular uptake, and maximize siRNA knockdown efficacy. The optimized anti-HER2-nanoparticles produced apoptotic death in HER2 positive (HER2+) breast cancer cells grown in vitro, but not in HER2 negative (HER2-) cells. One dose of the siHER2-nanoparticles reduced HER2 protein levels by 60% in trastuzumab-resistant HCC1954 xenografts. Multiple doses administered intravenously over 3 weeks significantly inhibited tumor growth (p < 0.004). The siHER2-nanoparticles have an excellent safety profile in terms of blood compatibility and low cytokine induction, when exposed to human peripheral blood mononuclear cells. The construct can be produced with high batch-to-batch reproducibility and the production methods are suitable for large-scale production. These results suggest that this siHER2-nanoparticle is ready for clinical evaluation.