ojun Xia

High Capacity Nanoporous Silicon Carrier for Systemic Delivery of Gene Silencing Therapeutics

Gene silencing agents such as small interfering RNA (siRNA) and microRNA offer the promise to modulate expression of almost every gene for the treatment of human diseases including cancer. However, lack of vehicles for effective systemic delivery to the disease organs has greatly limited their in vivo applications. In this study, we developed a high capacity polycation-functionalized nanoporous silicon (PCPS) platform comprised of nanoporous silicon microparticles functionalized with arginine-polyethyleneimine inside the nanopores for effective delivery of gene silencing agents. Incubation of MDA-MB-231 human breast cancer cells with PCPS loaded with STAT3 siRNA (PCPS/STAT3) or GRP78 siRNA (PCPS/GRP78) resulted in 91 and 83% reduction of STAT3 and GRP78 gene expression in vitro. Treatment of cells with a microRNA-18a mimic in PCPS (PCPS/miR-18) knocked down 90% expression of the microRNA-18a target gene ATM. Systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells, causing significant reduction of cancer stem cells in the residual tumor tissue. At the therapeutic dosage, PCPS/STAT3 siRNA did not trigger acute immune response in FVB mice, including changes in serum cytokines, chemokines, and colony-stimulating factors. In addition, weekly dosing of PCPS/STAT3 siRNA for four weeks did not cause signs of subacute toxicity based on changes in body weight, hematology, blood chemistry, and major organ histology. Collectively, the results suggest that we have developed a safe vehicle for effective delivery of gene silencing agents.

Multistage Vectored siRNA Targeting Ataxia-Telangiectasia Mutated for Breast Cancer Therapy

The ataxia-telangiectasia mutated (ATM) protein plays a central role in DNA damage response and cell cycle checkpoints, and may be a promising target for cancer therapy if normal tissue toxicity could be avoided. The strategy presented here to target ATM for breast cancer therapy involves the use of liposomal-encapsulated, gene-specific ATM siRNA delivered with a well-characterized porous silicon-based multistage vector (MSV) delivery system (MSV/ATM). Biweekly treatment of MSV/ATM suppressed ATM expression in tumor tissues, and consequently inhibited growth of MDA-MB-231 orthotopic tumor in nude mice. At the therapeutic dosage, neither free liposomal ATM siRNA nor MSV/ATM triggered an acute immune response in BALB/c mice, including changes in serum cytokines, chemokines or colony-stimulating factors. Weekly treatments of mice with free liposomal ATM siRNA or MSV/ATM for 4 weeks did not cause significant changes in body weight, hematology, blood biochemistry, or major organ histology. These results indicate that MSV/ATM is biocompatible and efficacious in inhibiting tumor growth, and that further preclinical evaluation is warranted for the development of MSV/ATM as a potential therapeutic agent.

Polycation-functionalized nanoporous silicon particles for gene silencing on breast cancer cells.

Nanoporous silicon particles (pSi), with a pore size in the range of 20-60 nm, were modified with polyethyleneimine (PEI) to yield pSi-PEI particles, which were subsequently complexed with siRNA. Thus, pSi-PEI/siRNA particles were fabricated, with the PEI/siRNA nanocomplexes mainly anchored inside the nanopore of the pSi particles. These hybrid particles were used as carriers to deliver siRNA to human breast cancer cells. Due to the gradual degradation of the pSi matrix under physiological conditions, the PEI/siRNA nanocomplexes were released from the pore interior in a sustained manner. Physicochemical characterization revealed that the released PEI/siRNA nanocomplexes exhibited well-defined spherical shape and narrow particle size distribution between 15 and 30 nm. Gene knockdown against the ataxia telangiectasia mutated (ATM) cancer gene showed dramatic gene silencing efficacy. Moreover, comprehensive biocompatibility studies were performed for the pSi-PEI/siRNA particles both in vitro and in vivo and demonstrated that the pSi-PEI particles exhibited significantly enhanced biocompatibility. As a consequence, PEI-modified porous silicon particles may have substantial potential as safe and effective siRNA delivery systems.

Porous Silicon Microparticle Potentiates Anti-Tumor Immunity by Enhancing Cross-Presentation and Inducing Type I Interferon Response

Micro- and nanometer-size particles have become popular candidates for cancer vaccine adjuvants. However, the mechanism by which such particles enhance immune responses remains unclear. Here, we report a porous silicon microparticle (PSM)-based cancer vaccine that greatly enhances cross-presentation and activates type I interferon (IFN-I) response in dendritic cells (DCs). PSM-loaded antigen exhibited prolonged early endosome localization and enhanced cross-presentation through both proteasome- and lysosome-dependent pathways. Phagocytosis of PSM by DCs induced IFN-I responses through a TRIF- and MAVS-dependent pathway. DCs primed with PSM-loaded HER2 antigen produced robust CD8 T cell-dependent anti-tumor immunity in mice bearing HER2+ mammary gland tumors. Importantly, this vaccination activated the tumor immune microenvironment with elevated levels of intra-tumor IFN-I and MHCII expression, abundant CD11c+ DC infiltration, and tumor-specific cytotoxic T cell responses. These findings highlight the potential of PSM as an immune adjuvant to potentiate DC-based cancer immunotherapy.

Bone marrow endothelium-targeted therapeutics for metastatic breast cancer

Effective treatment of cancer metastasis to the bone relies on bone marrow drug accumulation. The surface proteins in the bone marrow vascular endothelium provide docking sites for targeted drug delivery. We have developed a thioaptamer that specifically binds to E-selectin that is overexpressed in the vasculature of tumor and inflammatory tissues. In this study, we tested targeted delivery of therapeutic siRNA loaded in the E-selectin thioaptamer-conjugated multistage vector (ESTA-MSV) drug carrier to bone marrow for the treatment of breast cancer bone metastasis. We evaluated tumor type- and tumor growth stage-dependent targeting in mice bearing metastatic breast cancer in the bone, and carried out studies to identify factors that determine targeting efficiency. In a subsequent study, we delivered siRNA to knock down expression of the human STAT3 gene in murine xenograft models of human MDA-MB-231 breast tumor, and assessed therapeutic efficacy. Our studies revealed that the CD31(+)E-selectin(+) population accounted for 20.8%, 26.4% and 29.9% of total endothelial cells respectively inside the femur of mice bearing early, middle and late stage metastatic MDA-MB-231 tumors. In comparison, the double positive cells remained at a basal level in mice with early stage MCF-7 tumors, and jumped to 23.9% and 28.2% when tumor growth progressed to middle and late stages. Accumulation of ESTA-MSV inside the bone marrow correlated with the E-selectin expression pattern. There was up to 5-fold enrichment of the targeted MSV in the bone marrow of mice bearing early or late stage MDA-MB-231 tumors and of mice with late stage, but not early stage, MCF-7 tumors. Targeted delivery of STAT3 siRNA in ESTA-MSV resulted in knockdown of STAT3 expression in 48.7% of cancer cells inside the bone marrow. Weekly systemic administration of ESTA-MSV/STAT3 siRNA significantly extended survival of mice with MDA-MB-231 bone metastasis. In conclusion, targeting the overexpressed E-selectin provides an effective approach for tissue-specific drug delivery to the bone marrow. Tumor growth in the bone can be effectively inhibited by blockage of the STAT3 signaling.

Serum peptidomic biomarkers for pulmonary metastatic melanoma identified by means of a nanopore-based assay.

The significant mortality rate associated with metastatic melanoma, which exceeds the number of deaths attributed to the primary tumor, is primarily due to poor diagnosis and increased resistance to systemic therapy. Early detection and treatment of invasive melanoma are therefore crucial to increase survival rates. Low molecular weight proteins and peptides have garnered significant interest as biomarker candidates as they potentially represent a snap shot of pathological condition within the body and, by extension, the organism as a whole. We have developed a nanoporous silica-based platform to segregate the low molecular weight from the high molecular weight protein fraction to aid in the detection of peptides from serum samples using mass spectrometry. The combination of sample treatment with our platform, MALDI-TOF MS and following biostatistical analysis led to the discovery and identification of 27 peptides that are potential biomarkers associated with the development of pulmonary metastatic melanoma. We strongly believe our findings can assist to discover stage-specific peptide signatures and lead to more specific and personalized treatments for patients suffering from pulmonary metastatic melanoma.

Effective Concentration of a Multikinase Inhibitor within Bone Marrow Correlates with In Vitro Cell Killing in Therapy-Resistant Chronic Myeloid Leukemia

Leukemia cells escape BCR-ABL–targeted therapy by developing mutations, such as T315I, in the p210BCR-ABL fusion protein in Philadelphia chromosome–positive chronic myeloid leukemia (CML). Although most effort has been focused on development of new tyrosine kinase inhibitors, enrichment of these small-molecule inhibitors in the tumor tissue can also have a profound impact on treatment outcomes. Here, we report that a 2-hour exposure of the T315I-mutant CML cells to 10 μmol/L of the multikinase inhibitor TG101209 suppressed BCR-ABL–independent signaling and caused cell-cycle arrest at G2–M. Further increase in drug concentration to 17.5 μmol/L blocked phosphorylation of the mutant BCR-ABL kinase and its downstream JAK2 and STAT5. The effective dosage to overcome therapy resistance identified in an in vitro setting serves as a guidance to develop the proper drug formulation for in vivo efficacy. A targeted formulation was developed to achieve sustained bone marrow TG101209 concentration at or above 17.5 μmol/L for effective killing of CML cells in vivo. Potent inhibition of leukemia cell growth and extended survival were observed in two murine models of CML treated with 40 mg/kg intravenously administered targeted TG101209, but not with the untargeted drug at the same dosage. Our finding provides a unique approach to develop treatments for therapy-resistant CML. Mol Cancer Ther; 15(5); 899–910. ©2016 AACR.

Abstract 2370: Therapeutic nano-DC vaccine For HER2 positive breast cancer

Harnessing a patient9s own immune capacity to eradicate malignant cells is a promising approach for cancer treatment, and therapeutic vaccine represents an important arm of cancer immunotherapy. Our research aims to develop a potent therapeutic cancer vaccine for breast cancer which is traditionally considered as poorly immunogenic. We have recently shown that porous silicon microparticles (PSMs) can stimulate type I interferon (IFN) expression in dendritic cells (DCs), and thus serve as a potent adjuvant for a therapeutic cancer vaccine (Xia et al: Cell Reports 2015, 11:957-966). This action is mediated by the TRIF/MAVS pathways, and is independent of the cell surface or endosomal Toll-like receptors. In addition, the nanometer-size pores in PSMs can serve as a reservoir for sustained antigen release. We have assembled a Nano-DC vaccine comprising of bone marrow-derived DCs internalized with HER2 antigen peptide-loaded PSMs. Treatment of murine models of HER2 positive breast cancer with this Nano-DC vaccine not only activates and expands antigen-specific CD8+ T cells, but also promotes a Th2-to-Th1 transition in the tumor microenvironment to favor anti-tumor activity. Thus, our result supports the development of a therapeutic Nano-DC vaccine for breast cancer. Citation Format: Haifa Shen, Xiaojun Xia. Therapeutic nano-DC vaccine For HER2 positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2370.

Chemotherapy sensitizes therapy-resistant cells to mild hyperthermia by suppressing heat shock protein 27 expression in triple negative breast cancer

Purpose: Triple-negative breast cancer (TNBC) is a clinically aggressive disease with poor prognosis. Conventional chemotherapeutics are generally able to shrink the tumor mass, but often fail to completely eradicate cancer stem–like cells (CSCs) that are responsible for high risk of relapse and frequent metastases. In this study, we examined thermal sensibility of CSCs, developed an approach that enabled concurrent elimination of both the bulk of cancer cells and CSCs, and investigated the underlying mechanism. Experimental Design: We designed a platform consisting of gold nanoparticle-coated porous silicon microparticle (AuPSM) that was also loaded with docetaxel micelles (mDTXs) to enable concurrent killing of the bulk of cancer cells by released mDTX and CSCs by mild hyperthermia upon stimulation of AuPSM with near infrared. In addition, we examined the role of heat shock proteins in sensitizing CSC killing. Finally, we applied mDTX-loaded AuPSM to treat mice with SUM159 and 4T1 orthotopic tumors and evaluated tumor growth and tumor metastasis. Results: MDA-MB-231 and SUM159 TNBC cells treated with mDTX-loaded AuPSM and mild hyperthermia displayed significantly reduced efficiencies in mammosphere formation than those treated with mDTX alone or mild hyperthermia alone. Combination treatment also completely inhibited SUM159 orthotopic tumor growth and 4T1 tumor metastasis. Mechanistically, DTX treatment suppressed expression of heat shock protein 27 in cancer cells including the CSCs, rendering cells sensitive to mild hyperthermia. Conclusions: Our results indicate that chemotherapy sensitizes CSC to mild hyperthermia. We have developed an effective therapeutic approach to eliminate therapy-resistant cells in TNBC. Clin Cancer Res; 24(19); 4900–12. ©2018 AACR.

Abstract 1967: Multi-stage silicon vector-delivered tumor antigen and TLR ligands for dendritic cell-based cancer immunotherapy

Immunotherapy has shown promise in cancer treatment but achieved limited success in clinic trials. With this strategy, eliciting strong antitumor immune response in vivo remains a challenge, largely due to the immunosuppressive tumor microenvironment and inefficient delivery of cancer vaccine. Nanoparticle-delivered antigen together with adjuvants provides a promising strategy to increase the vaccine delivery efficiency. Recently, our group developed a multi-stage silicon vector (MSV) system for protected and prolonged nano-scale drug delivery. MSV delivered siRNA in vivo induced sustained knockdown of target gene expression and subsequent anti-tumor efficacy in mice. Here we tested the effect of MSV-formulated antigen peptides and Toll-like receptor (TLR) ligands on dendritic cells. By formulating HER-2 antigen peptide together with TLR4 ligand (MPLA) and TLR9 ligand (CpG), we found that MSV-delivered antigen and TLR ligands were uptaken by mouse dendritic cells after 1h co-incubation, and started to be released inside the cells at 3 h after co-incubation. Moreover, MSV-delivered TLR ligands strongly activated mouse dendritic cell expression of co-stimulatory molecules such as CD80 and CD86. It also induced dendritic cells to produce large amount of pro-inflammatory cytokines, such as TNF-alpha, IL-6, and IL-12. In summary, MSV-delivered vaccine efficiently delivered antigen and immuno-activating TLR ligands together into dendritic cells, and promoted dendritic cell maturation and activation. Therefore, MSV formulation of tumor antigen and TLR ligands may serve as a nano-scale cancer vaccine with enhanced in vivo efficacy. Ongoing experiments include testing in vivo T cell responses to MSV-delivered HER-2 antigen peptide and TLR ligands, and determining the antitumor efficacy on mouse mammary gland tumor model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1967. doi:1538-7445.AM2012-1967