Imiquimod-gemcitabine nanoparticles harness immune cells to suppress breast cancer.

Monotherapy with a single chemotherapeutic regimen has met with significant hurdles in terms of clinical efficacy. The complexity of cancer accentuates the need for an alternative approach with a combination of two or more therapeutic regimens to win the battle. However, it is still a challenge to develop a successful combination of drugs with high efficiency and low toxicity to control cancer growth. While gemcitabine monotherapy remains a choice of standard treatment for advanced breast cancer, the approach has not prolonged the median survival time of metastatic breast cancer patients. Here, we report a hyaluronic acid (HA)-based drug combination of gemcitabine (GEM) with imiquimod (IMQ) to stimulate immune cells for anticancer activity. Treatment of the drug combination (IMQ-HA-GEM) showed enhanced anticancer activity against 4T1 breast tumor cells in vitro. Our study with a microfluidics-based 3D, compartmentalized cancer model showed that infiltration of THP-1 monocytes occurred particularly at the site of cancer cells treated with IMQ-HA-GEM. Moreover, IMQ-HA-GEM significantly suppressed the volume of 4T1 breast tumor of mice in vivo. Flow cytometry study displayed a significantly higher activation of CD11b+ immune cells in the blood of mice treated with IMQ-HA-GEM, whereas immunohistochemistry study revealed greater prevalence of CD68+ tumor-associated macrophages in the tumor. Histological examination of isolated tumors of mice treated with IMQ-HA-GEM further confirmed the efficacy of drug combination on cancer cells. This study supports the conclusion that imiquimod potentiates the effect of gemcitabine by activating immune cells to suppress tumors in the form of combination nanoparticles.

Sustained, local delivery of the PARP inhibitor talazoparib prevents the development of mammary gland hyperplasia in Brca1-deficient mice.

Mutations in BRCA genes are the leading cause of hereditary breast cancer. Current options to prevent cancer in these high-risk patients, such as anti-estrogen drugs and radical mastectomy, are limited by lack of efficacy, undesirable toxicities, or physical and emotional challenges. We have previously shown that PARP inhibitors can significantly delay tumor development in BRCA1-deficient mice. Here, we fabricated the PARP inhibitor talazoparib (TLZ) into spacer implants (InCeT-TLZ) for localized and sustained delivery. We hypothesized that this novel formulation will provide an effective chemopreventive strategy with minimal toxicity. TLZ was released gradually over 30 days as implants degraded. InCeT-TLZ significantly decreased proliferation and increased DNA damage in the mammary glands of BRCA1-deficient mice. Notably, the number of mice that developed hyperplasia in the mammary glands was significantly lower with InCeT-TLZ treatment compared to the control group. Meanwhile, InCeT-TLZ was also better tolerated than oral TLZ, without loss of body weight or anemia. This study provides proof of concept for a novel and safe chemopreventive strategy using localized delivery of a PARP inhibitor for high-risk individuals. Future studies will directly evaluate the effects of InCeT-TLZ for preventing tumor development.

Nanoparticle Formulations of Poly (ADP-ribose) Polymerase Inhibitors for Cancer Therapy.

A number of poly(ADP-ribose) polymerase (PARP) inhibitors have been recently approved for clinical use in BRCA mutated and other cancers. However, off-target toxicity of PARP inhibitors and the emergence of drug resistance following prolonged administration of these inhibitors indicate the need for improved methods of drug delivery to the tumors. Nanomedicines based upon nanoparticle formulations of conventional small molecule drugs and inhibitors offer many advantages, such as increased solubility and bioavailability of drugs, reduced toxicity and drug resistance, and improved tissue selectivity and therapeutic efficacy. This review highlights the current trends in formulations of PARP inhibitors developed by nanotechnology approaches and provides an insight into the applications and limitations of these PARP inhibitor nanomedicines for cancer therapies.

Author Correction: Selective Priming of Tumor Blood Vessels by Radiation Therapy Enhances Nanodrug Delivery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Roadmap for metal nanoparticles in radiation therapy: current status, translational challenges, and future directions.

This roadmap outlines the potential roles of metallic nanoparticles (MNPs) in the field of radiation therapy. MNPs made up of a wide range of materials (from Titanium, Z = 22, to Bismuth, Z = 83) and a similarly wide spectrum of potential clinical applications, including diagnostic, therapeutic (radiation dose enhancers, hyperthermia inducers, drug delivery vehicles, vaccine adjuvants, photosensitizers, enhancers of immunotherapy) and theranostic (combining both diagnostic and therapeutic), are being fabricated and evaluated. This roadmap covers contributions from experts in these topics summarizing their view of the current status and challenges, as well as expected advancements in technology to address these challenges.

Harnessing cells to deliver nanoparticle drugs to treat cancer.

Clinical translation of nanoparticle drug (nanodrug) delivery systems for cancer therapy is primarily hindered by short half-life of nanodrugs in blood circulation and their poor ability of tumor targeting and penetration in vivo. Circulatory cells have garnered much attention in cancer therapy as drug delivery vehicles due to their biocompatibility, high mobility, biodegradability, tissue targeting capability, high drug loading capacity, ability to cross biological barriers and inherent ability to remain in blood circulation long enough to accumulate within the tumors. Here, we review the progress and potential of circulatory cells as nanodrug delivery vehicles, especially for cancer therapy.

Selective Priming of Tumor Blood Vessels by Radiation Therapy Enhances Nanodrug Delivery.

Effective drug delivery is restricted by pathophysiological barriers in solid tumors. In human pancreatic adenocarcinoma, poorly-permeable blood vessels limit the intratumoral permeation and penetration of chemo or nanotherapeutic drugs. New and clinically viable strategies are urgently sought to breach the neoplastic barriers that prevent effective drug delivery. Here, we present an original idea to boost drug delivery by selectively knocking down the tumor vascular barrier in a human pancreatic cancer model. Clinical radiation activates the tumor endothelial-targeted gold nanoparticles to induce a physical vascular damage due to the high photoelectric interactions. Active modulation of these tumor neovessels lead to distinct changes in tumor vascular permeability. Noninvasive MRI and fluorescence studies, using a short-circulating nanocarrier with MR-sensitive gadolinium and a long-circulating nanocarrier with fluorescence-sensitive nearinfrared dye, demonstrate more than two-fold increase in nanodrug delivery, post tumor vascular modulation. Functional changes in altered tumor blood vessels and its downstream parameters, particularly, changes in Ktrans (permeability), Kep (flux rate), and Ve (extracellular interstitial volume), reflect changes that relate to augmented drug delivery. The proposed dual-targeted therapy effectively invades the tumor vascular barrier and improve nanodrug delivery in a human pancreatic tumor model and it may also be applied to other nonresectable, intransigent tumors that barely respond to standard drug therapies.

Self-Assembled, Adjuvant/Antigen-Based Nanovaccine Mediates Anti-Tumor Immune Response against Melanoma Tumor.

Malignant melanoma is a highly aggressive type of cancer that requires radical treatment strategies to inhibit the cancer cell progression and metastasis. In recent years, preclinical research and clinical trials on melanoma treatment have been considerably focused on the adjuvant-based immunotherapy for enhancing the immune response of innate immune cells against cancer cells. However, the clinical outcome of these adjuvant-based treatments is inadequate due to an improper delivery system for these immune activators to reach the target site. Hence, we developed a vaccine formulation containing tumor lysate protein (TL) and poly I:C (PIC) complexed with positively charged poly (sorbitol-co-polyethylenimine (PEI) (PSPEI). The resulting ionic PSPEI-polyplexed antigen/adjuvant (PAA) (PSPEI-PAA) nanocomplexes were stable at the physiological condition, are non-toxic, and have enhanced intracellular uptake of antigen and adjuvant in immature dendritic cells leading to dendritic cell maturation. In the murine B16F10 tumor xenograft model, PSPEI-PAA nanocomplexes significantly suppressed tumor growth and did not exhibit any noticeable sign of toxicity. The level of matured dendritic cells (CD80+/CD86+ cells) in the tumor draining lymph node of PSPEI-PAA treated tumor mice were enhanced and therefore CD8+ T cells infiltration in the tumor were enriched. Additionally, the cytotoxic T lymphocytes (CTLs) assay involving co-culturing of splenocytes isolated from the PSPEI-PAA-treated mice with that of B16F10 cells significantly revealed enhanced cancer killing by the TL-reactivated CTLs compared to untreated control mice bearing tumor. Therefore, we strongly believe that PSPEI-PAA nanocomplexes could be an efficient antigen/adjuvant delivery system and enhance the antitumor immune response against melanoma tumor in the future clinical trials.

Combination therapy with doxorubicin-loaded galactosylated poly(ethyleneglycol)-lithocholic acid to suppress the tumor growth in an orthotopic mouse model of liver cancer.

Despite advances in technology, neither conventional anti-cancer drugs nor current nanoparticle (NP) drugs have gained substantial success in cancer treatment. While conventional chemotherapy drugs have several limitations such as low potency, poor in vivo stability and limited bioavailability, non-specific targeting of NP drugs diminishes their potency at actual target sites. In addition, the development of drug resistance to anti-cancer drugs is another challenging problem. To overcome these limitations, we aimed to develop a polymer-drug conjugate, which functions as an active NP drug and drug carrier both, to deliver a chemotherapeutic drug for combination therapy. Accordingly, we made targeting NP carrier of lithocholic acid-poly(ethylene glycol)-lactobionic acid (LPL) loading doxorubicin (Dox) to produce Dox/LPL NPs. The cellular uptake of Dox/LPL NPs was relatively higher in human liver cancer cell line (SK-HEP-1) due to galactose ligand-asialoglycoprotein receptor interaction. Consequently, the cellular uptake of Dox/LPL NPs led to massive cell death of SK-HEP-1 cells by two different mechanisms, particularly apoptotic activity by LPL and mitotic catastrophe by Dox. Most importantly, Dox/LPL NPs, when administered to orthotopic xenograft model of liver cancer, greatly reduced proliferation, invasion, migration, and angiogenesis of liver tumor in vivo. Thus, this study exemplifies the superiority of combination therapy over individual NP drug or conventional small molecule drug for cancer therapy. Overall, we present a promising approach of combinatorial therapy to inhibit the hepatic tumor growth and metastasis in the orthotopic xenograft model mice, thus representing an effective weapon for cancer treatment.

Suppression of Tobacco Carcinogen-Induced Lung Tumorigenesis by Aerosol-Delivered Glycerol Propoxylate Triacrylate-Spermine Copolymer/Short Hairpin Rab25 RNA Complexes in Female A/J Mice.

BACKGROUND: Rab25, a member of Rab family of small guanosine triphosphatase, is associated with progression of various types of human cancers, including lung cancer, the leading cause of cancer-associated deaths around the globe. METHODS: In this study, we report the gene therapeutic effect of short hairpin Rab25 RNA (shRab25) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in female A/J mice. Initially, mice (6 weeks old) were injected with single dose of NNK (2 mg/0.1 mL saline/mouse) by intraperitoneal injection to induce the tumor. Eight weeks later, shRab25 was complexed with glycerol propoxylate triacrylate-spermine (GPT-SPE) copolymer and delivered into tobacco-induced lung cancer models through a nose-only inhalation system twice a week for 2 months. RESULTS: GPT-SPE/shRab25 largely decreased the tobacco-induced tumor numbers and tumor volume in the lungs compared to GPT-SPE- or GPT-SPE/shScr-delivered groups. Remarkably, aerosol-delivered GPT-SPE/shRab25 significantly decreased the expression level of Rab25 and other prominent apoptosis-related proteins in female A/J mice. The apoptosis in these mice was determined by detecting the expression level of Bcl-2, proliferating cell nuclear antigen, Bax, and further confirmed by TUNEL assay. CONCLUSIONS: Our results strongly confirm the tumorigenic role of Rab25 in tobacco carcinogen-induced lung cancer and hence demonstrate aerosol delivery of shRab25 as a therapeutic target for lung cancer treatment.

Systemic administration of RANKL overcomes the bottleneck of oral vaccine delivery through microfold cells in ileum.

A successful delivery of antigen through oral route requires to overcome several barriers, such as enzymatic barrier of gastrointestinal tract and epithelial barrier that constitutes of microfold cells (M cells) for antigen uptake. Although each barrier represents a critical step in determining the final efficiency of antigen delivery, the transcytosis of antigen by M cells in the follicle-associated epithelium (FAE) to Peyer's patches appears to be a major bottleneck. Considering the systemic administration of receptor activator of nuclear factor (NF)-ĸB ligand (RANKL) induces differentiation of receptor activator of nuclear factor (NF)-ĸB (RANK)-expressing enterocytes into M cells, here, we illustrated a promising approach of antigen delivery using full length transmembrane RANKL (mRANKL). The results showed that the intraperitoneal injection of mRANKL increased the population of dendritic cells and macrophages in mesenteric lymph nodes and spleen. Subsequently, systemic administration of mRANKL resulted in significantly higher number of functional GP2(+) M cells leading higher transcytosis of fluorescent beads through them. To corroborate the effect of mRANKL in antigen delivery through M cells, we orally delivered microparticulate antigen to mice treated with mRANKL. Oral immunization induced strong protective IgA and systemic IgG antibody responses against orally delivered antigen in mRANKL-treated mice. The higher antibody responses are attributed to the higher transcytosis of antigens through M cells. Ultimately, the higher memory B cells and effector memory CD4 T cells after oral immunization in RANKL-treated mice confirmed potency of RANKL-mediated antigen delivery. To the best of our knowledge, this is the first study to demonstrate significant induction of mucosal and humoral immune responses to M cell targeted oral vaccines after the systemic administration of RANKL.

Non Surgical Correction of Mandibular Deviation and Neuromuscular Coordination after Two years of Mandibular Guidance Therapy: A Case Report.

Carcinomas of the mandible may require resection of a segment of bone (continuity defect), partial removal of bone (discontinuity defect), tongue, and floor of the mouth and muscle attachments. Patients undergoing such treatment suffer from facial disfigurement, loss of muscle function, loss of neuromuscular coordination resulting in inability to masticate and swallow acceptably. Surgical reconstruction may not always be possible because of high reoccurrence rate, inability of the patient to cope with another surgery etc. The treatment of choice in non surgical cases is prosthetic rehabilitation using guiding flange prosthesis. This article describes the management of a patient who had undergone hemimandibulectomy and was not willing for a surgical reconstruction. Interim maxillary ramp prosthesis was given to the patient 15 days postoperatively followed by definitive guiding flange prosthesis for two years after which the patient was able to occlude in centric occlusal position without any aid.

Image-Guided Nanoparticle-Based siRNA Delivery for Cancer Therapy.

With the discovery of RNA interference technology, small-interfering RNA (siRNA) has emerged as new powerful tool for gene therapy because of its high targeting specificity and selectivity. However, one of the limitations to successful gene therapy is the inability to monitor delivery of genes and therapeutic responses at the targeted site. Hence, a combinatorial approach of gene therapy with molecular imaging has been crucial in optimizing gene therapy. Recent advances in nanotechnology have made tremendous efforts to develop multifunctional nanoparticles that contain imaging and therapeutic agents together for image-guided therapy. The nanoparticles serve as contrast agents in imaging for disease detection with simultaneous delivery of therapeutics to cure the diseases. The therapy also helps to monitor the drug accumulation and assimilation in the body, thereby facilitating the evaluation of treatment effects. Here, we present an overview of polymer and lipid-based carriers for siRNA delivery, along with imaging agents as image guided therapy, in the treatment of breast, lung, liver, ovarian, cervical, and prostate cancers.

Suppression of tumor growth in lung cancer xenograft model mice by poly(sorbitol-co-PEI)-mediated delivery of osteopontin siRNA.

Small interfering RNA (siRNA)-mediated gene silencing represents a promising strategy for treating diseases such as cancer; however, specific gene silencing requires an effective delivery system to overcome the instability and low transfection efficiency of siRNAs. To address this issue, a polysorbitol-based transporter (PSOT) was prepared by low molecular weight branched polyethylenimine (bPEI) crosslinked with sorbitol diacrylate (SDA). Osteopontin (OPN) gene, which is highly associated with non-small cell lung cancer (NSCLC) was targeted by siRNA therapy using siRNA targeting OPN (siOPN). Characterization study confirmed that PSOT formed compact complexes with siOPN and protected siOPN against degradation by RNase. PSOT/siOPN complexes demonstrated low cytotoxicity and enhanced transfection efficiency in vitro, suggesting that this carrier may be suitable for gene silencing. In the A549 and H460 lung cancer cell lines, PSOT/siOPN complexes demonstrated significant silencing efficiency at both RNA and protein levels. To study in vivo tumor growth suppression, two lung cancer cell-xenograft mouse models were prepared and PSOT/siOPN complexes were delivered into the mice through intravenous injection. The siOPN-treated groups demonstrated significantly reduced OPN expression at both the RNA and protein levels as well as suppression of tumor volume and weight. Taken together, siOPN delivery using PSOT may present an effective and novel therapeutic system for lung cancer treatment.

Nanoparticle-mediated delivery of siRNA for effective lung cancer therapy.

Lung cancer is one of the most lethal diseases worldwide, and the survival rate is less than 15% even after the treatment. Unfortunately, chemotherapeutic treatments for lung cancer are accompanied by severe side effects, lack of selectivity and multidrug resistance. In order to overcome the limitations of conventional chemotherapy, nanoparticle-mediated RNA interference drugs represent a potential new approach due to selective silencing effect of oncogenes and multidrug resistance related genes. In this review, we provide recent advancements on nanoparticle-mediated siRNA delivery strategies including lipid system, polymeric system and rigid nanoparticles for lung cancer therapies. Importantly, codelivery of siRNA with conventional anticancer drugs and recent theranostic agents that offer great potential for lung cancer therapy is covered.

Galactosylated poly(ethyleneglycol)-lithocholic Acid selectively kills hepatoma cells, while sparing normal liver cells.

Delivering drugs selectively to cancer cells but not to nearby normal cells is a major obstacle in drug therapy. In this study, lithocholic acid (LCA), a potent anti-cancer drug, is converted to two forms of poly(ethyleneglycol) (PEG) conjugates, viz., PEG-LCA (PL) and lactobionic acid (LBA) conjugated PEG-LCA (LPL). The latter form contains a galactose ligand in LBA to target the hepatocytes. Both forms are self-assembled to form nanoparticle formulation, and they have high potency than LCA to kill HepG2 cancer cells, sparing normal LO2 cells. Besides, LPL has high specificity to mouse liver cells in vivo. Western blot results confirm that the cell death is occurred through apoptosis induced by LPL nanoparticles. In conclusion, the induction of apoptosis and cell death is much more efficient with LPL nanoparticles than LCA molecules.

Tuning the buffering capacity of polyethylenimine with glycerol molecules for efficient gene delivery: staying in or out of the endosomes.

Endosomal escape is a major bottleneck for efficient non-viral gene delivery. This paper presents the development of two novel non-viral vectors by cross-linking glycerol molecules with low molecular weight polyethylenimine (PEI). The vectors, namely, HG-PEI (45 mol% glycerol content) and LG-PEI (9 mol% glycerol content) have apparently similar DNA binding, DNA unpacking and cellular uptake abilities but differ in buffering capacity. The cellular uptake and subsequent transfection efficiency of LG-PEI is superior to commercially available PEI 25 k. Interestingly, although the cellular uptake of HG-PEI is higher than that of PEI 25 k, the transgene expression by HG-PEI-mediated transfection is very low. Inhibitor and co-localization studies demonstrate the mechanism of endocytosis and formation of endosomes prone to lysosomal lysis of HG-PEI polyplexes as a consequence of its weak buffering capacity. Importantly, when the lysosomal lysis is inhibited, the transgene expression of HG-PEI-mediated transfection increases by 9-fold of its initial capacity which is comparable to the transfection efficiency of PEI 25 k. These results indicated that the buffering capacity of the polymers primarily impacts endosomal escape and subsequent transfection efficiency. Furthermore, this study highlights the significance of cross-linkers in optimizing the buffering capacity when designing polymers for gene delivery.

Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli.

Receptor activator of nuclear factor (NF)-κB ligand (RANKL), a master cytokine that drives osteoclast differentiation, activation and survival, exists in both transmembrane and extracellular forms. To date, studies on physiological role of RANKL have been mainly carried out with extracellular RANKL probably due to difficulties in achieving high level expression of functional transmembrane RANKL (mRANKL). In the present study, we took advantage of codon optimization and response surface methodology to optimize the soluble expression of mRANKL in E. coli. We optimized the codon usage of mRANKL sequence to a preferred set of codons for E. coli changing its codon adaptation index from 0.64 to 0.76, tending to increase its expression level in E. coli. Further, we utilized central composite design to predict the optimum combination of variables (cell density before induction, lactose concentration, post-induction temperature and post-induction time) for the expression of mRANKL. Finally, we investigated the effects of various experimental parameters using response surface methodology. The best combination of response variables was 0.6 OD600, 7.5 mM lactose, 26°C post-induction temperature and 5 h post-induction time that produced 52.4 mg/L of fusion mRANKL. Prior to functional analysis of the protein, we purified mRANKL to homogeneity and confirmed the existence of trimeric form of mRANKL by native gel electrophoresis and gel filtration chromatography. Further, the biological activity of mRANKL to induce osteoclast formation on RAW264.7 cells was confirmed by tartrate resistant acid phosphatase assay and quantitative real-time polymerase chain reaction assays. Importantly, a new finding from this study was that the biological activity of mRANKL is higher than its extracellular counterpart. To the best of our knowledge, this is the first time to report heterologous expression of mRANKL in soluble form and to perform a comparative study of functional properties of both forms of RANKL.

Targeted oral delivery of BmpB vaccine using porous PLGA microparticles coated with M cell homing peptide-coupled chitosan.

M cells, the key players of the mucosal immunity induction, are one of the intestinal barriers for the efficient delivery of vaccines to mucosal immune tissues. To overcome the barrier, we have developed an efficient oral vaccine carrier that constitutes poly (lactic-co-glycolic acid) (PLGA) microparticle coated with M cell targeting peptide. In this study, a membrane protein B of Brachyspira hyodysenteriae (BmpB) as a model vaccine against swine dysentery was loaded into porous PLGA microparticles (MPs). The PLGA MPs were further coated with the water-soluble chitosan (WSC) conjugated with M cell homing peptide (CKS9) to prepare BmpB-CKS9-WSC-PLGA MPs. Oral immunization of BmpB vaccine with CKS9-WSC-PLGA MPs in mice showed elevated secretory IgA responses in the mucosal tissues and systemic IgG antibody responses, providing a complete immune response. Specifically, the immunization with these MPs demonstrated to induce both Th1- and Th2-type responses based on elevated IgG1 and IgG2a titers. The elevated immune responses were attributed to the enhanced M cell targeting and transcytosis ability of CKS9-WSC-PLGA MPs to Peyer's patch regions. The high binding affinity of CKS9-WSC-PLGA MPs with the M cells to enter into the Peyer's patch regions of mouse small intestine was investigated by closed ileal loop assay and it was further confirmed by confocal laser scanning microscopy. These results suggest that the M cell targeting approach used in this study is a promising tool for targeted oral vaccine delivery.