Formulation and Evaluation of Anisamide-Targeted Amphiphilic Cyclodextrin Nanoparticles To Promote Therapeutic Gene Silencing in a 3D Prostate Cancer Bone Metastases Model.

In recent years, RNA interference (RNAi) has emerged as a potential therapeutic offering the opportunity to treat a wide range of diseases, including prostate cancer. Modified cyclodextrins have emerged as effective gene delivery vectors in a range of disease models. The main objective of the current study was to formulate anisamide-targeted cyclodextrin nanoparticles to interact with the sigma receptor (overexpressed on the surface of prostate cancer cells). The inclusion of octaarginine in the nanoparticle optimized uptake and endosomal release of siRNA in two different prostate cancer cell lines (PC3 and DU145 cells). Resulting nanoparticles were less than 200 nm in size with a cationic surface charge (∼+20 mV). In sigma receptor-positive cell lines, the uptake of anisamide-targeted nanoparticles was reduced in the presence of the sigma receptor competitive ligand, haloperidol. When cells were transfected in 2D, the levels of PLK1 mRNA knockdown elicited by targeted versus untargeted nanoparticles tended to be greater but the differences were not statistically different. In contrast, when cells were grown on 3D scaffolds, recapitulating bone metastasis, targeted formulations showed significantly higher levels of PLK1 mRNA knockdown (46% for PC3 and 37% for DU145, p < 0.05). To our knowledge, this is the first time that a targeted cyclodextrin has been used to transfect prostate cancer cells in a 3D model of bone metastasis.

Motivational Interviewing: Improving Confidence With Self-care Management in Postoperative Thoracolumbar Spine Patients.

Patients undergoing thoracic or lumbar spine surgery often lack confidence with self-care management of symptoms contributing to disability, such as pain, lack of sleep, depression, and immobility. The purpose of this pilot study was to examine whether a targeted motivational interview, focused on evidence-based recommendations to manage postoperative symptoms related to thoracolumbar spine surgery, would improve patient confidence with self-care management of their symptom-related disability. A quasiexperimental, 1-group, pretest-posttest design was used on a convenience sample of 15 adult surgical spine patients at a large university spine center. Level of disability was measured using the Oswestry Disability Index (ODI). Confidence with self-care management of symptom-related disability was measured using the Health Confidence Index (HCI). Paired samples t tests were completed on participants' preintervention and postintervention scores on the HCI and ODI and on each of the 10 items that the ODI questionnaire addresses. The results of the paired samples t test on participants' HCI scores showed a statistically significant improvement in participants' confidence with self-care management of symptom-related disability from pretest (mean [SD], 6.73 [2.12]) to posttest (mean [SD], 8.73 [1.43]), conditions: t14 = -3.80, P = .002. Motivational interviewing is a beneficial intervention for health professionals to incorporate into practice to encourage the implementation of various health promoting behaviors that improve confidence with self-care management of symptoms in postoperative thoracolumbar spine patients.

Anisamide-targeted gold nanoparticles for siRNA delivery in prostate cancer - synthesis, physicochemical characterisation and in vitro evaluation.

Metastatic prostate cancer is a leading cause of cancer-related death in men and current chemotherapies are largely inadequate in terms of efficacy and toxicity. Hence improved treatments are required. The application of siRNA as a cancer therapeutic holds great promise. However, translation of siRNA into the clinic is dependent on the availability of an effective delivery system. Gold nanoparticles (AuNPs) are known to be effective and non-toxic siRNA delivery agents. In this study, a stable gold nanosphere coated with poly(ethylenimine) (PEI) was prepared to yield PEI capped AuNPs (Au-PEI). The PEI was further conjugated with the targeting ligand anisamide (AA, is known to bind to the sigma receptor overexpressed on the surface of prostate cancer cells) to produce an anisamide-targeted nanoparticle (Au-PEI-AA). The resulting untargeted and targeted nanoparticles (Au-PEI and Au-PEI-AA respectively) were positively charged and efficiently complexed siRNA. Au-PEI-AA mediated siRNA uptake into PC3 prostate cancer cells via binding to the sigma receptor. In addition, the Au-PEI-AA·siRNA complexes resulted in highly efficient knockdown of the RelA gene (∼70%) when cells were transfected in serum-free medium. In contrast, no knockdown was observed in the presence of serum, suggesting that adsorption of serum proteins inhibits the binding of the anisamide moiety to the sigma receptor. This study provides (for the first time) proof of principle that anisamide-labelled gold nanoparticles can target the sigma receptor. Further optimisation of the formulation to increase serum stability will enhance its potential to treat prostate cancer.

A novel, anisamide-targeted cyclodextrin nanoformulation for siRNA delivery to prostate cancer cells expressing the sigma-1 receptor.

Prostate cancer is a leading cause of cancer-related death in men and RNA interference (RNAi) has emerged as a potential therapeutic option. However, the absence of a safe and specific delivery vector remains a major obstacle to the clinical application of RNAi. Cyclodextrin derivatives are known to be efficient delivery systems with low toxicity in a variety of cell types. In this study, a cationic cyclodextrin derivative was synthesized to complex siRNA. The nanoparticle was then further modified by exploiting the ability of the ß-cyclodextrin cavity to form an inclusion complex with the hydrophobic molecule adamantane. PEGylated adamantane derivatives were synthesized with and without the anisamide-targeting ligand on the terminal end of the PEG chain. Anisamide is known to bind specifically to the sigma receptor which is overexpressed on the surface of prostate cancer cells. The resulting nanocomplexes were slightly cationic and less than 300 nm in size. They successfully protected siRNA from serum-induced nuclease degradation and were non-toxic to prostate cancer cells. In addition, the targeted nanoparticles mediated high levels of siRNA cellular uptake and corresponding PLK1 gene knockdown in prostate cancer cells in vitro. To our knowledge, this is the first time that the ability of cyclodextrins to form inclusion complexes with adamantane derivatives has been exploited for the targeted delivery of siRNA to prostate cancer cells via the sigma receptor.

Nanoparticle-mediated siRNA delivery assessed in a 3D co-culture model simulating prostate cancer bone metastasis.

siRNA has emerged as a potential therapeutic for the treatment of prostate cancer but effective delivery remains a major barrier to its clinical application. This study aimed to develop and characterise a 3D in vitro co-culture model to simulate prostate cancer bone metastasis and to assess the ability of the model to investigate nanoparticle-mediated siRNA delivery and gene knockdown. PC3 or LNCaP prostate cancer cells were co-cultured with hFOB 1.19 osteoblast cells in 2D on plastic tissue culture plates and in 3D on collagen scaffolds mimicking the bone microenvironment. To characterise the co-culture model, cell proliferation, enzyme secretion and the utility of two different gene delivery vectors to mediate siRNA uptake and gene knockdown were assessed. Cell proliferation was reduced by∼50% by day 7 in the co-culture system relative to monoculture (PC3 and LNCaP co-cultures, in 2D and 3D) and an enhanced level of MMP9 (a marker of bone metastasis) was secreted into the media (1.2-4-fold increase depending on the co-culture system). A cationic cyclodextrin gene delivery vector proved significantly less toxic in the co-culture system relative to the commercially available vector Lipofectamine 2000(®). In addition, knockdown of both the GAPDH gene (minimum 15%) and RelA subunit of the NF-κB transcription factor (minimum 20%) was achieved in 2D and 3D cell co-cultures. Results indicate that the prostate cancer-osteoblast in vitro co-culture model was more physiologically relevant vs the monoculture. This model has the potential to help improve the design and efficacy of gene delivery formulations, to more accurately predict in vivo performance and, therefore, to reduce the risk of product failure in late-stage clinical development.

Life in 3D is never flat: 3D models to optimise drug delivery.

The development of safe, effective and patient-acceptable drug products is an expensive and lengthy process and the risk of failure at different stages of the development life-cycle is high. Improved biopharmaceutical tools which are robust, easy to use and accurately predict the in vivo response are urgently required to help address these issues. In this review the advantages and challenges of in vitro 3D versus 2D cell culture models will be discussed in terms of evaluating new drug products at the pre-clinical development stage. Examples of models with a 3D architecture including scaffolds, cell-derived matrices, multicellular spheroids and biochips will be described. The ability to simulate the microenvironment of tumours and vital organs including the liver, kidney, heart and intestine which have major impact on drug absorption, distribution, metabolism and toxicity will be evaluated. Examples of the application of 3D models including a role in formulation development, pharmacokinetic profiling and toxicity testing will be critically assessed. Although utilisation of 3D cell culture models in the field of drug delivery is still in its infancy, the area is attracting high levels of interest and is likely to become a significant in vitro tool to assist in drug product development thus reducing the requirement for unnecessary animal studies.

The use of collagen-based scaffolds to simulate prostate cancer bone metastases with potential for evaluating delivery of nanoparticulate gene therapeutics.

Prostate cancer bone metastases are a leading cause of cancer-related death in men with current treatments offering only marginally improved rates of survival. Advances in the understanding of the genetic basis of prostate cancer provide the opportunity to develop gene-based medicines capable of treating metastatic disease. The aim of this work was to establish a 3D cell culture model of prostate cancer bone metastasis using collagen-based scaffolds, to characterise this model, and to assess the potential of the model to evaluate delivery of gene therapeutics designed to target bone metastases. Two prostate cancer cell lines (PC3 and LNCaP) were cultured in 2D standard culture and compared to 3D cell growth on three different collagen-based scaffolds (collagen and composites of collagen containing either glycosaminoglycan or nanohydroxyapatite). The 3D model was characterised for cell proliferation, viability and for matrix metalloproteinase (MMP) enzyme and Prostate Specific Antigen (PSA) secretion. Chemosensitivity to docetaxel treatment was assessed in 2D in comparison to 3D. Nanoparticles (NPs) containing siRNA formulated using a modified cyclodextrin were delivered to the cells on the scaffolds and gene silencing was quantified. Both prostate cancer cell lines actively infiltrated and proliferated on the scaffolds. Cell culture in 3D resulted in reduced levels of MMP1 and MMP9 secretion in PC3 cells. In contrast, LNCaP cells grown in 3D secreted elevated levels of PSA, particularly on the scaffold composed of collagen and glycosaminoglycans. Both cell lines grown in 3D displayed increased resistance to docetaxel treatment. The cyclodextrin.siRNA nanoparticles achieved cellular uptake and knocked down the endogenous GAPDH gene in the 3D model. In conclusion, development of a novel 3D cell culture model of prostate cancer bone metastasis has been initiated resulting, for the first time, in the successful delivery of gene therapeutics in a 3D in vitro model. Further enhancement of this model will help elucidate the pathogenesis of prostate cancer and also accelerate the design of effective therapies which can penetrate into the bone microenvironment for prostate cancer therapy.

The role of transcription factors in prostate cancer and potential for future RNA interference therapy.

INTRODUCTION: Prostate cancer is a leading cause of cancer-related death in men and current treatments offer only a modest survival benefit in advanced stages of the disease. RNA interference (RNAi) is a therapeutic option that has received great attention in recent years with the potential to treat a variety of disorders, including prostate cancer. Transcription factors are cellular proteins that can up-regulate or down-regulate the transcription of genes and offer promising therapeutic targets. AREAS COVERED: This review will focus on transcription factors that have been identified as key molecules in drug resistance, disease progression and metastases in prostate cancer, which may offer potential as therapeutic targets for RNAi in the future. EXPERT OPINION: By identifying therapeutically viable transcription factor targets in prostate cancer, it is hoped that treatment strategies using RNAi will augment the effect of current chemotherapy regimens, slow disease progression and reduce metastases in prostate cancer, resulting in disease regression.