CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

VCAM-1-targeted nanoparticles to diagnose, monitor and treat atherosclerosis.

Vascular cell adhesion molecule-1 (VCAM-1) was identified over 2 decades ago as an endothelial adhesion receptor involved in leukocyte recruitment and cell-based immune responses. In atherosclerosis, a chronic inflammatory disease of the blood vessels that is the leading cause of death in the USA, endothelial VCAM-1 is robustly expressed beginning in the early stages of the disease. The interactions of circulating immune cells with VCAM-1 on the activated endothelial cell surface promote the uptake of monocytes and the progression of atherosclerotic lesions in susceptible vessels. Herein, we review the role of VCAM-1 in atherosclerosis and the use of VCAM-1 binding peptides, antibodies and aptamers as targeting agents for nanoplatforms for early detection and treatment of atherosclerotic disease.

Reactive Oxygen Species-Triggered Hydrogen Sulfide Release and Cancer-Selective Antiproliferative Effect of Anethole Dithiolethione-Containing Polymeric Micelles.

Hydrogen sulfide (H2 S) is a gaseous signaling molecule in the human body and has attracted attention in cancer therapy due to its regulatory roles in cancer cell proliferation and migration. Accumulating evidence suggests that continuous delivery of H2 S to cancer cells for extended periods of time suppresses cancer progression. However, one major challenge in therapeutic applications of H2 S is its controlled delivery. To solve this problem, polymeric micelles are developed containing H2 S donating-anethole dithiolethione (ADT) groups, with H2 S release profiles optimal for suppressing cancer cell proliferation. The micelles release H2 S upon oxidation by reactive oxygens species (ROS) that are present inside the cells. The H2 S release profiles can be controlled by changing the polymer design. Furthermore, the micelles that show a moderate H2 S release rate exert the strongest anti-proliferative effect in human colon cancer cells in in vitro assays as well as the chick chorioallantoic membrane cancer model, while the micelles do not affect proliferation of human umbilical vein endothelial cells. This study shows the importance of fine-tuning H2 S release profiles using a micelle approach for realizing the full therapeutic potential of H2 S in cancer treatment.

Aptamer-Targeted Calcium Phosphosilicate Nanoparticles for Effective Imaging of Pancreatic and Prostate Cancer.

PURPOSE: Accurate tumor identification and staging can be difficult. Aptamer-targeted indocyanine green (ICG)-nanoparticles can enhance near-infrared fluorescent imaging of pancreatic and prostate tumors and could improve early cancer detection. This project explored whether calcium-phosphosilicate nanoparticles, also known as NanoJackets (NJs), that were bioconjugated with a tumor-specific targeting DNA aptamer could improve the non-invasive detection of pancreatic and prostate tumors. METHODS: Using in vivo near-infrared optical imaging and ex vivo fluorescence analysis, DNA aptamer-targeted ICG-loaded NJs were compared to untargeted NJs for detection of tumors. RESULTS: Nanoparticles were bioconjugated with the DNA aptamer AP1153, which binds to the CCK-B receptor (CCKBR). Aptamer bioconjugated NJs were not significantly increased in size compared with unconjugated nanoparticles. AP1153-ICG-NJ accumulation in orthotopic pancreatic tumors peaked at 18 h post-injection and the ICG signal was cleared by 36 h with no evidence on uptake by non-tumor tissues. Ex vivo tumor imaging confirmed the aptamer-targeted NJs accumulated to higher levels than untargeted NJs, were not taken up by normal pancreas, exited from the tumor vasculature, and were well-dispersed throughout pancreatic and prostate tumors despite extensive fibrosis. Specificity for AP1153-NJ binding to the CCK-B receptor on pancreatic tumor cells was confirmed by pre-treating tumor-bearing mice with the CCK receptor antagonist proglumide. Proglumide pre-treatment reduced the in vivo tumoral accumulation of AP1153-NJs to levels comparable to that of untargeted NJs. CONCLUSION: Through specific interactions with CCK-B receptors, tumor-targeted nanoparticles containing either ICG or rhodamine WT were well distributed throughout the matrix of both pancreatic and prostate tumors. Tumor-targeted NJs carrying various imaging agents can enhance tumor detection.

Preferential uptake of antibody targeted calcium phosphosilicate nanoparticles by metastatic triple negative breast cancer cells in co-cultures of human metastatic breast cancer cells plus bone osteoblasts.

Calcium phosphosilicate nanoparticles (CPSNPs) are bioresorbable nanoparticles that can be bioconjugated with targeting molecules and encapsulate active agents and deliver them to tumor cells without causing damage to adjacent healthy tissue. Data obtained in this study demonstrated that an anti-CD71 antibody on CPSNPs targets these nanoparticles and enhances their internalization by triple negative breast cancer cells in-vitro. Caspase 3,7 activation, DNA damage, and fluorescent microscopy confirmed the apoptotic breast cancer response caused by targeted anti-CD71-CPSNPs encapsulated with gemcitabine monophosphate, the active metabolite of the chemotherapeutic gemcitabine used to treat cancers including breast and ovarian. Targeted anti-CD71-CPSNPs encapsulated with the fluorophore, Rhodamine WT, were preferentially internalized by breast cancer cells in co-cultures with osteoblasts. While osteoblasts partially internalized anti-CD71-GemMP-CPSNPs, their cell growth was not affected. These results suggest that CPSNPs may be used as imaging tools and selective drug delivery systems for breast cancer that has metastasized to bone.

The use of nanoparticulates to treat breast cancer.

Breast cancer is a major ongoing public health issue among women in both developing and developed countries. Significant progress has been made to improve the breast cancer treatment in the past decades. However, the current clinical approaches are invasive, of low specificity and can generate severe side effects. As a rapidly developing field, nanotechnology brings promising opportunities to human cancer diagnosis and treatment. The use of nanoparticulate-based platforms overcomes biological barriers and allows prolonged blood circulation time, simultaneous tumor targeting and enhanced accumulation of drugs in tumors. Currently available and clinically applicable innovative nanoparticulate-based systems for breast cancer nanotherapies are discussed in this review.

Effective encapsulation and biological activity of phosphorylated chemotherapeutics in calcium phosphosilicate nanoparticles for the treatment of pancreatic cancer.

Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive, phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained biological activity. Analysis of orthotopic tumors from mice treated systemically with tumor-targeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells and release of active drug cargos intracellularly. These findings demonstrate a novel methodology to efficiently encapsulate chemotherapeutic agents into the CPSNPs and to effectively deliver them to pancreatic tumor cells.

A Cholecystokinin B Receptor-Specific DNA Aptamer for Targeting Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against "exposed" CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bioconjugation of AP1153 to the surface of fluorescent NPs greatly facilitated delivery of NPs to PDAC tumors in vivo. The selectivity of this AP-targeted NP delivery system holds promise for enhanced early detection of PDAC lesions as well as improved chemotherapeutic treatments for PDAC patients.

3D Printing of Personalized Artificial Bone Scaffolds.

Additive manufacturing technologies, including three-dimensional printing (3DP), have unlocked new possibilities for bone tissue engineering. Long-term regeneration of normal anatomic structure, shape, and function is clinically important subsequent to bone trauma, tumor, infection, nonunion after fracture, or congenital abnormality. Due to the great complexity in structure and properties of bone across the population, along with variation in the type of injury or defect, currently available treatments for larger bone defects that support load often fail in replicating the anatomic shape and structure of the lost bone tissue. 3DP could provide the ability to print bone substitute materials with a controlled chemistry, shape, porosity, and topography, thus allowing printing of personalized bone grafts customized to the patient and the specific clinical condition. 3DP and related fabrication approaches of bone grafts may one day revolutionize the way clinicians currently treat bone defects. This article gives a brief overview of the current advances in 3DP and existing materials with an emphasis on ceramics used for 3DP of bone scaffolds. Furthermore, it addresses some of the current limitations of this technique and discusses potential future directions and strategies for improving fabrication of personalized artificial bone constructs.

Novel strategies for managing pancreatic cancer.

With the incidence reports of pancreatic cancer increasing every year, research over the last several decades has been focused on the means to achieve early diagnosis in patients that are at a high risk of developing the malignancy. This review covers current strategies for managing pancreatic cancer and further discusses efforts in understanding the role of early onset symptoms leading to tumor progression. Recent investigations in this discussion include type 3c diabetes, selected biomarkers and pathways related to pancreatic intraepithelial neoplasia lesions, drug resistance, and advances in nanomedicine which may provide significant solutions for improving early detection and treatments in future medicine.

PhotoImmunoNanoTherapy reveals an anticancer role for sphingosine kinase 2 and dihydrosphingosine-1-phosphate.

Tumor-associated inflammation mediates the development of a systemic immunosuppressive milieu that is a major obstacle to effective treatment of cancer. Inflammation has been shown to promote the systemic expansion of immature myeloid cells which have been shown to exert immunosuppressive activity in laboratory models of cancer as well as cancer patients. Consequentially, significant effort is underway toward the development of therapies that decrease tumor-associated inflammation and immunosuppressive cells. The current study demonstrated that a previously described deep tissue imaging modality, which utilized indocyanine green-loaded calcium phosphosilicate nanoparticles (ICG-CPSNPs), could be utilized as an immunoregulatory agent. The theranostic application of ICG-CPSNPs as photosensitizers for photodynamic therapy was shown to block tumor growth in murine models of breast cancer, pancreatic cancer, and metastatic osteosarcoma by decreasing inflammation-expanded immature myeloid cells. Therefore, this therapeutic modality was termed PhotoImmunoNanoTherapy. As phosphorylated sphingolipid metabolites have been shown to have immunomodulatory roles, it was hypothesized that the reduction of immature myeloid cells by PhotoImmunoNanoTherapy was dependent upon bioactive sphingolipids. Mechanistically, PhotoImmunoNanoTherapy induced a sphingosine kinase 2-dependent increase in sphingosine-1-phosphate and dihydrosphingosine-1-phosphate. Furthermore, dihydrosphingosine-1-phosphate was shown to selectively abrogate myeloid lineage cells while concomitantly allowing the expansion of lymphocytes that exerted an antitumor effect. Collectively, these findings revealed that PhotoImmunoNanoTherapy, utilizing the novel nontoxic theranostic agent ICG-CPSNP, can decrease tumor-associated inflammation and immature myeloid cells in a sphingosine kinase 2-dependent manner. These findings further defined a novel myeloid regulatory role for dihydrosphingosine-1-phosphate. PhotoImmunoNanoTherapy holds the potential to be a revolutionary treatment for cancers with inflammatory and immunosuppressive phenotypes.

Calcium phosphosilicate nanoparticles for imaging and photodynamic therapy of cancer.

Photodynamic therapy (PDT) has emerged as an alternative modality for cancer treatment. PDT works by initiating damaging oxidation or redox-sensitive pathways to trigger cell death. PDT can also regulate tumor angiogenesis and modulate systemic antitumor immunity. The drawbacks to PDT--photosensitizer toxicity, a lack of selectivity and efficacy of photosensitizers, and a limited penetrance of light through deep tissues--are the same pitfalls associated with diagnostic imaging. Developments in the field of nanotechnology have generated novel platforms for optimizing the advantages while minimizing the disadvantages of PDT. Calcium phosphosilicate nanoparticles (CPSNPs) represent an optimal nano-system for both diagnostic imaging and PDT. In this review, we will discuss how CPSNPs can enhance optical agents and serve as selective, non-toxic, and functionally stable photosensitizers for PDT. We will also examine novel applications of CPSNPs and PDT for the treatment of leukemia to illustrate their potential utility in cancer therapeutics.

Calcium phosphate-based composite nanoparticles in bioimaging and therapeutic delivery applications.

Bioimaging and therapeutic delivery applications are areas of biomedicine where nanoparticles have had significant impact, but the use of a nanomaterial in these applications can be limited by its physicochemical properties. Calcium phosphate-based composite nanoparticles are nontoxic and biodegradable, and are therefore considered attractive candidates for bioimaging and therapeutic drug delivery applications. Also, the pH-dependent solubility profiles of calcium phosphate materials make this class of nanoparticles especially useful for in vitro and in vivo delivery of dyes, oligonucleotides, and drugs. In this article, we discuss how calcium phosphate-based composite nanoparticles fulfill some of the requirements typically made for nanoparticles in biomedical applications. We also highlight recent studies in bioimaging and therapeutic delivery applications focusing on how these studies have addressed some of the challenges associated with using these nanoparticles in bioimaging and delivery of therapeutics.

Targeted indocyanine-green-loaded calcium phosphosilicate nanoparticles for in vivo photodynamic therapy of leukemia.

Leukemia is one of the most common and aggressive adult cancers, as well as the most prevalent childhood cancer. Leukemia is a cancer of the hematological system and can be divided into a diversity of unique malignancies based on the onset of the disease as well as the specific cell lineages involved. Cancer stem cells, including recently identified leukemia stem cells (LSCs), are hypothesized to be responsible for cancer development, relapse, and resistance to treatment, and new therapeutics targeting these cellular populations are urgently needed. Nontoxic and nonaggregating calcium phosphosilicate nanoparticles (CPSNPs) encapsulating the near-infrared fluoroprobe indocyanine green (ICG) were recently developed for diagnostic imaging and drug delivery as well as for photodynamic therapy (PDT) of solid tumors. Prior studies revealed that specific targeting of CPSNPs allowed for enhanced accumulation within breast cancer tumors, via CD71 targeting, or pancreatic cancer tumors, via gastrin receptor targeting. In the present study, ICG-loaded CPSNPs were evaluated as photosensitizers for PDT of leukemia. Using a novel bioconjugation approach to specifically target CD117 or CD96, surface features enhanced on leukemia stem cells, in vitro ICG-CPSNP PDT of a murine leukemia cell line and human leukemia samples were dramatically improved. Furthermore, the in vivo efficacy of PDT was dramatically enhanced in a murine leukemia model by utilizing CD117-targeted ICG-CPSNPs, resulting in 29% disease-free survival. Altogether, this study demonstrates that leukemia-targeted ICG-loaded CPSNPs offer the promise to effectively treat relapsing and multidrug-resistant leukemia and to improve the life of leukemia patients.

The colloidal stability of fluorescent calcium phosphosilicate nanoparticles: the effects of evaporation and redispersion on particle size distribution.

Understanding the colloidal stability of nanoparticles is important for biological applications, such as bio-imaging and drug delivery. This work combines theoretical calculations with experimental data to elucidate the mechanism of stabilization for calcium phosphosilicate nanoparticles containing Cy3 with both citrate and poly(ethylene glycol) (PEG) surface conjugation. The citrate surface is shown to provide electrosteric dispersion in water-ethanol mixtures as well as the ability to redisperse after evaporating the solvent. Improved colloidal stability is afforded with the addition of PEG with respect to redispersion after drying. Changes in average agglomeration number (AAN) are tracked and explained by DLVO and the Napper electrosteric and steric theories for dispersion, respectively.

Nanoparticulate alternatives for drug delivery.

The ability to apply nanomaterials as targeted delivery agents for drugs and other therapeutics holds promise for a wide variety of diseases, including many types of cancer. A nanodelivery vehicle must demonstrate in vivo efficacy, diminished or no toxicity, stability, improved pharmacokinetics, and controlled-release kinetics. In this issue, Lee et al. construct polymer nanobins that fulfill these requirements and demonstrate effective delivery of doxorubicin in vivo to breast cancer cells. This Perspective explores the outlook for these nanobins as well as other technologies in this field and the challenges that lie ahead.

Near infrared imaging with nanoparticles.

Near infrared imaging has presented itself as a powerful diagnostic technique with potential to serve as a minimally invasive, nonionizing method for sensitive, deep tissue diagnostic imaging. This potential is further realized with the use of nanoparticle (NP)-based near infrared (NIR) contrast agents that are not prone to the rapid photobleaching and instability of their organic counterparts. This review discusses applications that have successfully demonstrated the utility of nanoparticles for NIR imaging, including NIR-emitting semiconductor quantum dots (QDs), resonant gold nanoshells, and dye-encapsulating nanoparticles. NIR QDs demonstrate superior optical performance with exceptional fluorescence brightness stability. However, the heavy metal composition and high propensity for toxicity hinder future application in clinical environments. NIR resonant gold nanoshells also exhibit brilliant signal intensities and likewise have none of the photo- or chemical-instabilities characteristic of organic contrast agents. However, concerns regarding ineffectual clearance and long-term accumulation in nontarget organs are a major issue for this technology. Finally, NIR dye-encapsulating nanoparticles synthesized from calcium phosphate (CP) also demonstrate improved optical performances by shielding the component dye from undesirable environmental influences, thereby enhancing quantum yields, emission brightness, and fluorescent lifetime. Calcium phosphate nanoparticle (CPNP) contrast agents are neither toxic, nor have issues with long-term sequestering, as they are readily dissolved in low pH environments and ultimately absorbed into the system. Though perhaps not as optically superior as QDs or nanoshells, these are a completely nontoxic, bioresorbable option for NP-based NIR imaging that still effectively improves the optical performance of conventional organic agents.

Bioconjugation of calcium phosphosilicate composite nanoparticles for selective targeting of human breast and pancreatic cancers in vivo.

The early diagnosis of cancer is the critical element in successful treatment and long-term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat, and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a approximately 20 nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near-infrared imaging agent, indocyanine green (ICG), is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin or a novel PEG-maleimide coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin-conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are overexpressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting, and imaging of breast and pancreatic cancer tumors.

Yield improvement for lost mould rapid infiltration forming process by a multistage fractional factorial split plot design.

Statistical design of experiments is widely used among scientists and engineers to understand influential factors in a laboratory or manufacturing process. One of the underlying principles of using the statistical design of experiments method is randomisation, each run of experimental settings will be determined completely unsystematically. In practice, especially in a complicated process that consists of multiple stages, randomisation may pose too high a burden on time and cost.In this study, the multistage fraction factorial split plot design is proposed for green yield improvement in a lost mould rapid infiltration process that has been developed to fabricate zirconia ceramic parts. This design allows a relaxation of the randomisation principle so that certain experimental runs can be carried out in convenient groups. The results indicate that the type of immersion chemical and mould coating play a role in improving process yield. Additionally, the results suggest that a mould infiltration machine should be used to improve the reproducibility of the process.

Lost Mold-Rapid Infiltration Forming of Mesoscale Ceramics: Part 2, Geometry and Strength Improvements.

Iterative process improvements have been used to eliminate strength-limiting geometric flaws in mesoscale bend bars composed of yttria-tetragonal zirconia polycrystals (Y-TZP). These improvements led to large quantities of high bend strength material. The metrology of Y-TZP mesoscale bend bars produced using a novel lost mold-rapid infiltration-forming process (LM-RIF) is characterized over several process improvements. These improvements eliminate trapezoidal cross sections in the parts, reduce concave upper surfaces in cross section, and minimize warping along the long axis of 332 x 26 x 17 mum mesoscale bend bars. The trapezoidal cross sections of earlier, first-generation parts were due to the absorption of high-energy ultraviolet (UV) light during the photolithographic mold-forming process, which produced nonvertical mold walls that the parts mirrored. The concave upper surfaces in cross section were eliminated by implementing a RIF-buffing process. Warping during sintering was attributed to impurities in the substrate, which creates localized grain growth and warping as the tetragonal phase becomes destabilized. Precision in the part dimensions is demonstrated using optical profilometry on bend bars and a triangular test component. The bend bar dimensions have a 95% confidence interval of < +/-1 mum, and the tip radius of the triangular test component is 3 mum, consistent with the UV-photolithographic process used to form the mold cavities. The average bend strength of the mesoscale Y-TZP bend exceeds 2 GPa with a Weibull modulus equal to 6.3.