Magnetic Nanoparticle-based Targeted Drug Delivery for Treatment of Neuro-AIDS and Drug Addiction

Brain is one of the safe sanctuaries for HIV and, in turn, continuously supplies active viruses to the periphery. Additionally, HIV infection in brain results in several mild-to-severe neuro-immunological complications termed neuroAIDS. One-tenth of HIV-infected population is addicted to recreational drugs such as opiates, alcohol, nicotine, marijuana, etc. which share common target-areas in the brain with HIV. Interestingly, intensity of neuropathogenesis is remarkably enhanced due to exposure of recreational drugs during HIV infection. Current treatments to alleviate either the individual or synergistic effects of abusive drugs and HIV on neuronal modulations are less effective at CNS level, basically due to impermeability of therapeutic molecules across blood-brain barrier (BBB). Despite exciting advancement of nanotechnology in drug delivery, existing nanovehicles such as dendrimers, polymers, micelles, etc. suffer from the lack of adequate BBB penetrability before the drugs are engulfed by the reticuloendothelial system cells as well as the uncertainty that if and when the nanocarrier reaches the brain. Therefore, in order to develop a fast, target-specific, safe, and effective approach for brain delivery of anti-addiction, anti-viral and neuroprotective drugs, we exploited the potential of magnetic nanoparticles (MNPs) which, in recent years, has attracted significant importance in biomedical applications. We hypothesize that under the influence of external (non-invasive) magnetic force, MNPs can deliver these drugs across BBB in most effective manner. Accordingly, in this dissertation, I delineated the pharmacokinetics and dynamics of MNPs bound anti-opioid, anti-HIV and neuroprotective drugs for delivery in brain. I have developed a liposome-based novel magnetized nanovehicle which, under the influence of external magnetic forces, can transmigrate and effectively deliver drugs across BBB without compromising its integrity. It is expected that the developed nanoformulations may be of high therapeutic significance for neuroAIDS and for drug addiction as well.

Advanced Bioactive Inorganic Materials for Bone Regeneration and Drug Delivery

Bioceramics play an important role in repairing and regenerating bone defects. Annually, more than 500,000 bone graft procedures are performed in the United states and approximately 2.2 million are conducted worldwide. The estimated cost of these procedures approaches $2.5billion per year. Around 60% of the bone graft substitutes available on the market involve bioceramics. It is reported that bioceramics in the world market increase by 9% per year. For this reason, the research of bioceramics has been one of the most active areas during, the past several years. Considering the significant importance of bioceramics, our goal was to compile this book to review the latest research advances in the field of bioceramics. The text also summarizes our work during the past 10 years in an effort to share innovative concepts, design of bioceramisc, and methods for material synthesis and drug delivery. We anticipate that this text will provide some useful information and guidance in the bioceramics field for biomedical engineering researchers and material scientists. Information on novel mesoporous bioactive glasses and silicate-based ceramics for bone regeneration and drug delivery are presented. Mesoporous bioactive glasses have shown multifunctional characteristics of bone regeneration and drug delivery due to their special mesopore structures,whereas silicated-based bioceramics, as typical third-generation biomaterials,possess significant osteostimulation properties. Silica nanospheres with a core-shell structure and specific properties for controllable drug delivery have been carefully reviewed-a variety of advanced synthetic strategies have been developed to construct functional mesoporous silica nanoparticles with a core-shell structure, including hollow, magnetic, or luminescent, and other multifunctional core-shell mesoporous silica nanoparticles. In addition, multifunctional drug delivery systems based on these nanoparticles have been designed and optimized to deliver the drugs into the targeted organs or cells,with a controllable release fashioned by virtue of various internal and external triggers. The novel 3D-printing technique to prepare advanced bioceramic scaffolds for bone tissue engineering applications has been highlighted, including the preparation, mechanical strength, and biological properties of 3D-printed porous scaffolds of calcium phosphate cement and silicate bioceramics. Three-dimensional printing techniques offer improved large-pore structure and mechanical strength. In addition , biomimetic preparation and controllable crystal growth as well as biomineralization of bioceramics are summarized, showing the latest research progress in this area. Finally, inorganic and organic composite materials are reviewed for bone regeneration and gene delivery. Bioactive inorganic and organic composite materials offer unique biological, electrical, and mechanical properties for designing excellent bone regeneration or gene delivery systems. It is our sincere hope that this book will updated the reader as to the research progress of bioceramics and their applications in bone repair and regeneration. It will be the best reward to all the contributors of this book if their efforts herein in some way help reader in any part of their study, research, and career development.

Research roadmap report : Integrated Design and Delivery Solutions (IDDS)

Changes in the construction sector are creating opportunities in research to maximise the benefits of those changes and to continue the exciting developments in improved people skills, new processes and developing technologies. There are many research centres around the world investigating aspects of the current changes to drive their particular expertise forward. However, the CIB Integrated Design and Delivery Solutions (IDDS) priority research theme takes a higher-level view of the changes and then focuses down on a prioritised set of research targets. These targets have been investigated, re-focussed and validated over a period of four years through many workshops, conferences and meetings by a wide ranging group of representatives from approximately 90 industry and research organisations. This roadmap prioritises and details the research to be performed, why and by whom. In particular, some 25 CIB Working Commissions and Task Groups are explained as having potential roles in the delivery of this research theme. We are extremely privileged to have been urged on by such distinguished construction professionals in their forewords and the case for research. The outcomes of such research, once put into practice should be significantly shortened timespans from conception of need to occupation of new or revised structures. As time is money, the owners will get their investments into productive use sooner, which means a shorter payback time. In addition, there will inevitably be a reduction in construction costs as productivity increases. The improvements in reliable delivery and improved quality currently being seen in relatively simplistic use of Building information Modelling (BIM) (compared to full IDDS) will inevitably continue its on-going trajectory of improvement. We should also consider the wider economic contribution to society that will stem from such improvements and, finally, and by no means unimportantly, the reliable modelling and delivery of sustainability at both the building and estate/ area scale will significantly improve carbon footprints and other sustainable outcomes. Whilst there are huge opportunities for early adopters, the primary risk will be the expansion of the gap between those working in this way and those who are not so advanced or who even refuse to progress1. However, a similar issue arises between industry, clients, educators and trainers; the latter have particular challenges, having existed for many years in a sector that has had relatively few technological changes. However, the opportunities to address the significant and widely varying wastes within the structure of the construction sector and within and across projects are huge and timely. Whilst this Roadmap is specifically targeted at the Standing Commissions and Task Groups of the CIB, it is hoped that there are elements for research and applied research across academia and industry.

Porous silica-based nanocapsules for targeted and controlled release of biocides

Develops a new technology for the delivery of biocides against agricultural pests, with biocides contained within silica nanocapsules which are themselves protected by an outer envelope, capable of being selectively broken down by the target pest. Will reduce the amount of biocide escaping into the environment, prolong the life of the biocide, reduce biocide usage rates, and reduce undesirable effects on non-target organisms.

A multisite managed environment facility for targeted trait and germplasm phenotyping

Field evaluation of germplasm for performance under water and heat stress is challenging. Field environments are variable and unpredictable, and genotype x environment interactions are difficult to interpret if environments are not well characterised. Numerous traits, genes and quantitative trait loci have been proposed for improving performance but few have been used in variety development. This reflects the limited capacity of commercial breeding companies to screen for these traits and the absence of validation in field environments relevant to breeding companies, and because little is known about the economic benefit of selecting one particular trait over another. The value of the proposed traits or genes is commonly not demonstrated in genetic backgrounds of value to breeding companies. To overcome this disconnection between physiological trait breeding and uptake by breeding companies, three field sites representing the main environment types encountered across the Australian wheatbelt were selected to form a set of managed environment facilities (MEFs). Each MEF manages soil moisture stress through irrigation, and the effects of heat stress through variable sowing dates. Field trials are monitored continuously for weather variables and changes in soil water and canopy temperature in selected probe genotypes, which aids in decisions guiding irrigation scheduling and sampling times. Protocols have been standardised for an essential core set of measurements so that phenotyping yield and other traits are consistent across sites and seasons. MEFs enable assessment of a large number of traits across multiple genetic backgrounds in relevant environments, determine relative trait value, and facilitate delivery of promising germplasm and high value traits into commercial breeding programs.

Evaluation of polymeric nanomedicines targeted to PSMA: Effect of ligand on targeting efficiency

Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA−). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle.

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/ 32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/ 32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/ 32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.

Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System

Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective.

Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP-PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection.

Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration.

In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo.

Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment.

Targeted RNA interference of cyclin A(2) mediated by functionalized single-walled carbon nanotubes induces proliferation arrest and apoptosis in chronic myelogenous leukemia K562 cells

Cyclin A(2) plays critical role in DNA replication, transcription, and cell cycle regulation. Its overexpression has been detected and related to many types of cancers including leukemia, suggesting that suppression of cyclin A(2) would be an attractive strategy to prevent tumor progression. Herein, we apply functionalized single wall carbon nanotubes (f-SWNTs) to carry small interfering RNA (siRNA) into K562 cells and determine whether inhibition of cyclin A(2) would be a potential therapeutic target for chronic myelogenous leukemia.

Glycyrrhetinic acid-modified nanoparticles for drug delivery: Preparation and characterization

In this work, glycyrrhetinic acid-modified chitosan (mGA-suc-CTS) used as liver targeted carrier for drug delivery, was prepared via hemisuccinate as a bridged group. The structure of the product was confirmed by IR and NMR methods and the degree of substitution (DS) of glycyrrhetinic acid groups was estimated via elemental analysis. Nanoparticles were formed by ionic gelation methold. The drug-loading and release behavior of the nanoparticles were investigated using BSA as the model drug. The results indicated that the carrier with a highest DS of 5.19% could be got and the DS was controlled by changing reaction temperature or feed ratio. BSA could be entrapped into the nanoparticles with the drug-loading ratio of 26.3% and the encapsulation efficiency of 81.5%. A sustained release over an 11-day period was observed in pH 7.4 in vitro.

Design and evaluation of novel microencapsulation technologies to enhance delivery of Ciclosporin

Drug delivery systems influence the various processes of release, absorption, distribution and elimination of drug. Conventional delivery methods administer drug through the mouth, the skin, transmucosal areas, inhalation or injection. However, one of the current challenges is the lack of effective and targeted oral drug administration. Development of sophisticated strategies, such as micro- and nanotechnology that can integrate the design and synthesis of drug delivery systems in a one-step, scalable process is fundamental in advancing the limitations of conventional processing techniques. Thus, the objective of this thesis is to evaluate novel microencapsulation technologies in the production of size-specific and target-specific drug-loaded particles. The first part of this thesis describes the utility of PDMS and silicon microfluidic flow focusing devices (MFFDs) to produce PLGA-based microparticles. The formation of uniform droplets was dependent on the surface of PDMS remaining hydrophilic. However, the durability of PDMS was limited to no more than 1 hour before wetting of the microchannel walls with dichloromethane and subsequent swelling occurred. Critically, silicon MFFDs revealed very good solvent compatibility and was sufficiently robust to withstand elevated fluid flow rates. Silicon MFFDs facilitated experiments to run over days with continuous use and re-use of the device with a narrower microparticle size distribution, relative to conventional production techniques. The second part of this thesis demonstrates an alternative microencapsulation technology, SmPill® minispheres, to target CsA delivery to the colon. Characterisation of CsA release in vitro and in vivo was performed. By modulating the ethylcellulose:pectin coating thickness, release of CsA in-vivo was more effectively controlled compared to current commercial CsA formulations and demonstrated a linear in-vitro in-vivo relationship. Coated minispheres were shown to limit CsA release in the upper small intestine and enhance localised CsA delivery to the colon.

Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K(D) for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC(50)s that decrease from 15000 nM (1500 microg mL(-1)) for the 25 mol % functionalized polymers to 11 nM (1 microg mL(-1)) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.

Photosensitiser delivery for photodynamic therapy. Part 2: systemic carrier platforms

Background: The treatment of solid tumours and angiogenic ocular diseases by photodynamic therapy (PDT) requires the injection of a photosensitiser (PS) to destroy target cells through a combination of visible light irradiation and molecular oxygen. There is currently great interest in the development of efficient and specific carrier delivery platforms for systemic PDT. Objective: This article aims to review recent developments in systemic carrier delivery platforms for PDT, with an emphasis on target specificity. Methods: Recent publications, spanning the last five years, concerning delivery carrier platforms for systemic PDT were reviewed, including PS conjugates, dendrimers, micelles, liposomes and nanoparticles. Results/conclusion: PS conjugates and supramolecular delivery platforms can improve PDT selectivity by exploiting cellular and physiological specificities of the targeted tissue. Overexpression of receptors in cancer and angiogenic endothelial cells allows their targeting by affinity-based moieties for the selective uptake of PS conjugates and encapsulating delivery carriers, while the abnormal tumour neovascularisation induces a specific accumulation of heavy weighted PS carriers by enhanced permeability and retention (EPR) effect. in addition, polymeric prodrug delivery platforms triggered by the acidic nature of the tumour environment or the expression of proteases can be designed. Promising results obtained with recent systemic carrier platforms will, in due course, be translated into the clinic for highly efficient and selective PDT protocols.

Gene delivery using dimethyldidodecylammonium bromide-coated PLGA nanoparticles

In this present work we describe a poly(lactic-co-glycolic acid) (PLGA) nanoparticle formulation for intracellular delivery of plasmid DNA. This formulation was developed to encapsulate DNA within PLGA nanoparticles that combined salting out and emulsion evaporation processes. This process reduced the requirement for sonication which can induce degradation of the DNA. A monodispersed nanoparticle population with a mean diameter of approximately 240 nm was produced, entrapping a model plasmid DNA in both supercoiled and open circular structures. To induce endosomal escape of the nanoparticles, a superficial cationic charge was introduced using positively charged surfactants cetyl trimethylammonium bromide (CTAB) and dimethyldidodecylammonium bromide (DMAB), which resulted in elevated zeta potentials. As expected, both cationic coatings reduced cell viability, but at equivalent positive zeta potentials, the DMAB coated nanoparticles induced significantly less cytotoxicity than those coated with CTAB. Fluorescence and transmission electron microscopy demonstrated that the DMAB coated cationic nanoparticles were able to evade the endosomal lumen and localise in the cytosol of treated cells. Consequently, DMAB coated PLGA nanoparticles loaded with a GFP reporter plasmid exhibited significant improvements in transfection efficiencies with comparison to non-modified particles, highlighting their functional usefulness. These nanoparticles may be useful in delivery of gene therapies to targeted cells. (C) 2010 Elsevier Ltd. All rights reserved.

Evaluation of a multi-functional nanocarrier for targeted breast cancer iNOS gene therapy.

The present study determines whether the novel designer biomimetic vector (DBV) can condense anddeliver the cytotoxic iNOS gene to breast cancer cells to achieve a therapeutic effect. We have previouslyshown the benefits of iNOS for cancer gene therapy but the stumbling block to future development hasbeen the delivery system.The DBV was expressed, purified and complexed with the iNOS gene. The particle size and chargewere determined via dynamic light scattering techniques. The toxicity of the DBV/iNOS nanoparticleswas quantified using the cell toxicity and clonogenic assays. Over expression of iNOS was confirmed viaWestern blotting and Griess test.The DBV delivery system fully condensed the iNOS gene with nanoparticles less than 100 nm. Transfectionwith the DBV/iNOS nanoparticles resulted in a maximum of 62% cell killing and less than 20%clonogenicity. INOS overexpression was confirmed and total nitrite levels were in the range of 18M.We report for the first time that the DBV can successfully deliver iNOS and achieve a therapeuticeffect. There is significant cytotoxicity coupled with evidence of a bystander effect. We concludethat the success of the DBV fusion protein in the delivery of iNOS in vitro is worthy of future in vivo experiments.