Efficacy and Safety of Dual Paclitaxel and Sirolimus Nanoparticle-Coated Balloon.

We evaluated a novel dual active pharmaceutical ingredient (API) drug-coated balloon (DCB), which consists of a coating of nanoparticles encapsulating low-dose paclitaxel (PTX) in combination with sirolimus in a synergistic ratio. Compared to the PTX DCB, the dual API DCB demonstrated similar inhibition of cell proliferation in vitro but at a significantly lower total drug dose (over 13 times lower than sirolimus nanoparticles). Animal experiments demonstrated that the dual API DCB is more effective in inhibiting intimal cell proliferation with insignificant downstream embolic effects and myocardial damage compared to the PTX DCB. These findings indicate that dual API DCBs have a high potential to demonstrate improved clinical outcomes and a greater safety profile than the PTX DCBs.

Nanoparticle-mediated delivery of tetrahydrobiopterin restores endothelial function in diabetic rats.

Endothelial dysfunction, underlying the vascular complications of diabetes and other cardiovascular disorders, may result from uncoupling of endothelial nitric oxide synthase (eNOS) activity due to decreased levels of tetrahydrobiopterin (BH4), a critical co-factor for eNOS. Some clinical trials attempting to deliver exogenous BH4 as a potential therapeutic strategy in vascular disease states have failed due to oxidation of BH4 in the circulation. We sought to develop a means of protecting BH4 from oxidation while delivering it to dysfunctional endothelial cells. Polymeric and solid lipid nanoparticles (NPs) loaded with BH4 were delivered by injection or oral gavage, respectively, to streptozotocin-induced diabetic rats. BH4 was measured in coronary endothelial cells and endothelium-dependent vascular reactivity was assessed in vascular rings. Lymphatic uptake of orally delivered lipid NPs was verified by sampling mesenteric lymph. BH4-loaded polymeric NPs maintained nitric oxide production by cultured endothelial cells under conditions of oxidative stress. BH4-loaded NPs, delivered via injection or ingestion, increased coronary endothelial BH4 concentration and improved endothelium-dependent vasorelaxation in diabetic rats. Pharmacodynamics assessment indicated peak concentration of solid lipid NPs in the systemic bloodstream 6 hours after ingestion, with disappearance noted by 48 hours. These studies support the feasibility of utilizing NPs to deliver BH4 to dysfunctional endothelial cells to increase nitric oxide bioavailability. BH4-loaded NPs could provide an innovative tool to restore redox balance in blood vessels and modulate eNOS-mediated vascular function to reverse or retard vascular disease in diabetes.

Catalytic antioxidant nanoparticles mitigate secondary injury progression and promote functional recovery in spinal cord injury model.

Traumatic spinal cord injury exacerbates disability with time due to secondary injury cascade triggered largely by overproduction of reactive oxygen species (ROS) at the lesion site, causing oxidative stress. This study explored nanoparticles containing antioxidant enzymes (antioxidant NPs) to neutralize excess ROS at the lesion site and its impact. When tested in a rat contusion model of spinal cord injury, a single dose of antioxidant NPs, administered intravenously three hours after injury, effectively restored the redox balance at the lesion site, interrupting the secondary injury progression. This led to reduced spinal cord tissue inflammation, apoptosis, cavitation, and inhibition of syringomyelia. Moreover, the treatment reduced scar tissue forming collagen at the lesion site, protected axons from demyelination, and stimulated lesion healing, with further analysis indicating the formation of immature neurons. The ultimate effect of the treatment was improved motor and sensory functions and rapid post-injury weight loss recovery. Histological analysis revealed activated microglia in the spinal cord displaying rod-shaped anti-inflammatory and regenerative phenotype in treated animals, contrasting with amoeboid inflammatory and degenerative phenotype in untreated control. Overall data suggest that restoring redox balance at the lesion site shifts the dynamics in the injured spinal cord microenvironment from degenerative to regenerative, potentially by promoting endogenous repair mechanisms. Antioxidant NPs show promise to be developed as an early therapeutic intervention in stabilizing injured spinal cord for enhanced recovery.

Nanoparticle-mediated synergistic drug combination for treating bone metastasis.

Bone metastasis at an advanced disease stage is common in most solid tumors and is untreatable. Overexpression of receptor activator of nuclear factor κB ligand (RANKL) in tumor-bone marrow microenvironment drives a vicious cycle of tumor progression and bone resorption. Biodegradable nanoparticles (NPs), designed to localize in the tumor tissue in bone marrow, were evaluated in a prostate cancer model of bone metastasis. The combination treatment, encapsulating docetaxel, an anticancer drug (TXT-NPs), and Denosumab, a monoclonal antibody that binds to RANKL (DNmb-NPs), administered intravenously regressed the tumor completely, preventing bone resorption, without causing any mortality. With TXT-NPs alone treatment, after an initial regression, the tumor relapsed and acquired resistance, whereas DNmb-NPs alone treatment was ineffective. Only in the combination treatment, RANKL was not detected in the tumor tibia, thus negating its role in tumor progression and bone resorption. The combination treatment was determined to be safe as the vital organ tissue showed no increase in inflammatory cytokine or the liver ALT/AST levels, and animals gained weight. Overall, dual drug treatment acted synergistically to modulate the tumor-bone microenvironment with encapsulation enhancing their therapeutic potency to achieve tumor regression.

Nanotechnology in Stroke: New Trails with Smaller Scales.

Stroke is a leading cause of death, long-term disability, and socioeconomic costs, highlighting the urgent need for effective treatment. During acute phase, intravenous administration of recombinant tissue plasminogen activator (tPA), a thrombolytic agent, and endovascular thrombectomy (EVT), a mechanical intervention to retrieve clots, are the only FDA-approved treatments to re-establish cerebral blood flow. Due to a short therapeutic time window and high potential risk of cerebral hemorrhage, a limited number of acute stroke patients benefit from tPA treatment. EVT can be performed within an extended time window, but such intervention is performed only in patients with occlusion in a larger, anatomically more proximal vasculature and is carried out at specialty centers. Regardless of the method, in case of successful recanalization, ischemia-reperfusion injury represents an additional challenge. Further, tPA disrupts the blood-brain barrier integrity and is neurotoxic, aggravating reperfusion injury. Nanoparticle-based approaches have the potential to circumvent some of the above issues and develop a thrombolytic agent that can be administered safely beyond the time window for tPA treatment. Different attributes of nanoparticles are also being explored to develop a multifunctional thrombolytic agent that, in addition to a thrombolytic agent, can contain therapeutics such as an anti-inflammatory, antioxidant, neuro/vasoprotective, or imaging agent, i.e., a theragnostic agent. The focus of this review is to highlight these advances as they relate to cerebrovascular conditions to improve clinical outcomes in stroke patients.

Antioxidant Therapy in Oxidative Stress-Induced Neurodegenerative Diseases: Role of Nanoparticle-Based Drug Delivery Systems in Clinical Translation.

Free radicals are formed as a part of normal metabolic activities but are neutralized by the endogenous antioxidants present in cells/tissue, thus maintaining the redox balance. This redox balance is disrupted in certain neuropathophysiological conditions, causing oxidative stress, which is implicated in several progressive neurodegenerative diseases. Following neuronal injury, secondary injury progression is also caused by excessive production of free radicals. Highly reactive free radicals, mainly the reactive oxygen species (ROS) and reactive nitrogen species (RNS), damage the cell membrane, proteins, and DNA, which triggers a self-propagating inflammatory cascade of degenerative events. Dysfunctional mitochondria under oxidative stress conditions are considered a key mediator in progressive neurodegeneration. Exogenous delivery of antioxidants holds promise to alleviate oxidative stress to regain the redox balance. In this regard, natural and synthetic antioxidants have been evaluated. Despite promising results in preclinical studies, clinical translation of antioxidants as a therapy to treat neurodegenerative diseases remains elusive. The issues could be their low bioavailability, instability, limited transport to the target tissue, and/or poor antioxidant capacity, requiring repeated and high dosing, which cannot be administered to humans because of dose-limiting toxicity. Our laboratory is investigating nanoparticle-mediated delivery of antioxidant enzymes to address some of the above issues. Apart from being endogenous, the main advantage of antioxidant enzymes is their catalytic mechanism of action; hence, they are significantly more effective at lower doses in detoxifying the deleterious effects of free radicals than nonenzymatic antioxidants. This review provides a comprehensive analysis of the potential of antioxidant therapy, challenges in their clinical translation, and the role nanoparticles/drug delivery systems could play in addressing these challenges.

Arginine-Modified Polymers Facilitate Poly (Lactide-Co-Glycolide)-Based Nanoparticle Gene Delivery to Primary Human Astrocytes.

PURPOSE: Astrocyte dysfunction is a hallmark of central nervous system injury or infection. As a primary contributor to neurodegeneration, astrocytes are an ideal therapeutic target to combat neurodegenerative conditions. Gene therapy has arisen as an innovative technique that provides excellent prospect for disease intervention. Poly (lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) are polymeric nanoparticles commonly used in gene delivery, each manifesting their own set of advantages and disadvantages. As a clinically approved polymer by the Federal Drug Administration, well characterized for its biodegradability and biocompatibility, PLGA-based nanoparticles (PLGA-NPs) are appealing for translational gene delivery systems. However, our investigations revealed PLGA-NPs were ineffective at facilitating exogenous gene expression in primary human astrocytes, despite their success in other cell lines. Furthermore, PEI polymers illustrate high delivery efficiency but induce cytotoxicity. The purpose of this study is to develop viable and biocompatible NPsystem for astrocyte-targeted gene therapy. MATERIALS AND METHODS: Successful gene expression by PLGA-NPs alone or in combination with arginine-modified PEI polymers (AnPn) was assessed by a luciferase reporter gene encapsulated in PLGA-NPs. Cytoplasmic release and nuclear localization of DNA were investigated using fluorescent confocal imaging with YOYO-labeled plasmid DNA (pDNA). NP-mediated cytotoxicity was assessed via lactate dehydrogenase in primary human astrocytes and neurons. RESULTS: Confocal imaging of YOYO-labeled pDNA confirmed PLGA-NPs delivered pDNA to the cytoplasm in a dose and time-dependent manner. However, co-staining revealed pDNA delivered by PLGA-NPs did not localize to the nucleus. The addition of AnPn significantly improved nuclear localization of pDNA and successfully achieved gene expression in primary human astrocytes. Moreover, these formulations were biocompatible with both astrocytes and neurons. CONCLUSION: By co-transfecting two polymeric NPs, we developed an improved system for gene delivery and expression in primary human astrocytes. These findings provide a basis for a biocompatible and clinically translatable method to regulate astrocyte function during neurodegenerative diseases and disorders.

Synergistic combination treatment to break cross talk between cancer cells and bone cells to inhibit progression of bone metastasis.

Advanced-stage cancers often metastasize to bone, and is the major cause of cancer-related morbidity and mortality. Due to poor biodistribution of intravenously administered anticancer drugs within the bone, chemotherapy is not optimally effective in treating bone metastasis. Additionally, overexpression of receptor activator of nuclear factor κB ligand (RANKL) in the bone microenvironment drives the vicious, destructive cycle of progression of bone metastasis and bone resorption. We hypothesized that the combination treatment - with docetaxel (TXT), an anticancer drug encapsulated in sustained release biodegradable nanoparticles (TXT-NPs) that are designed to localize in bone marrow, and denosumab monoclonal antibody (DNmb), which binds to RANKL - could be more effective than either treatment alone. We tested our hypothesis in intraosseous prostate cancer (PC-3) cell-induced osteolytic mouse model of bone metastasis with treatments given intravenously. The results demonstrated better efficacy with TXT-NPs than with TXT-CrEL or saline control in inhibiting progression of metastasis and improving survival. TXT-NPs showed ~3-fold higher drug levels in metastasized bone tissue at 1 wk post-administration than TXT-CrEL, thus explaining their efficacy. However, the combination treatment (TXT-NPs + DNmb) given simultaneously was significantly more effective in inhibiting metastatic progression; it caused early tumor regression and improved survival, and caused no body weight loss or tumor relapse, even when the treatment was discontinued, whereas TXT-NPs or DNmb alone treatments showed tumor relapse after an initial regression. Micro-CT analysis of the bone from the combination treatment showed no bone loss and normal bone mineral content, bone density, and bone volume fraction, whereas TXT-NPs or DNmb alone treatments showed bone loss. Confirming the above results, histochemical analysis of the bone from the combination treatment demonstrated normal bone morphology, and osteoblast and osteoclast cell activities. In conclusion, TXT-NPs and DNmb in combination, because of their complementary roles in breaking the cross talk between cancer cells and bone cells, was significantly effective in treating bone metastasis.

Nanoparticles with antioxidant enzymes protect injured spinal cord from neuronal cell apoptosis by attenuating mitochondrial dysfunction.

In spinal cord injury (SCI), the initial damage leads to a rapidly escalating cascade of degenerative events, known as secondary injury. Loss of mitochondrial homeostasis after SCI, mediated primarily by oxidative stress, is considered to play a crucial role in the proliferation of secondary injury cascade. We hypothesized that effective exogenous delivery of antioxidant enzymes - superoxide dismutase (SOD) and catalase (CAT), encapsulated in biodegradable nanoparticles (nano-SOD/CAT) - at the lesion site would protect mitochondria from oxidative stress, and hence the spinal cord from secondary injury. Previously, in a rat contusion model of severe SCI, we demonstrated extravasation and retention of intravenously administered nanoparticles specifically at the lesion site. To test our hypothesis, a single dose of nano-SOD/CAT in saline was administered intravenously 6 h post-injury, and the spinal cords were analyzed one week post-treatment. Mitochondria isolated from the affected region of the spinal cord of nano-SOD/CAT-treated animals demonstrated significantly reduced mitochondrial reactive oxygen species (ROS) activities, increased mitochondrial membrane potential, reduced calcium levels, and also higher adenosine triphosphate (ATP) production capacity than those isolated from the spinal cords of untreated control or SOD/CAT solution treated animals. Although the treatment did not achieve the same mitochondrial function as in the spinal cords of sham control animals, it significantly attenuated mitochondrial dysfunction following SCI. Further, immunohistochemical analyses of the spinal cords of treated animals showed significantly lower ROS, cleaved caspase-3, and cytochrome c activities, leading to reduced spinal cord neuronal cell apoptosis and smaller lesion area than in untreated animals. These results imply that the treatment significantly attenuated progression of secondary injury that was also reflected from less weight loss and improved locomotive recovery of treated vs. untreated animals. In conclusion, nano-SOD/CAT mitigated activation of cascade of degenerating factors by protecting mitochondria and hence the spinal cord from secondary injury. An effective treatment during the acute phase following SCI could potentially have a positive long-term impact on neurological and functional recovery.

Evaluating accessibility of intravenously administered nanoparticles at the lesion site in rat and pig contusion models of spinal cord injury.

In spinal cord injury (SCI), timely therapeutic intervention is critical to inhibit the post-injury rapidly progressing degeneration of spinal cord. Towards that objective, we determined the accessibility of intravenously administered biodegradable nanoparticles (NPs) as a drug delivery system to the lesion site in rat and pig contusion models of SCI. Poly (d,l-lactide co-glycolide, PLGA)-based NPs loaded with a near-infrared dye as a marker for NPs were used. To analyze and quantify localization of NPs to the lesion site, we mapped the entire spinal cord, segment-by-segment, for the signal count. Our objectives were to determine the NP dose effect and duration of retention of NPs at the lesion site, and the time window post-SCI within which NPs localize at the lesion site. We hypothesized that breakdown of the blood-spinal cord barrier following contusion injury could lead to more specific localization of NPs at the lesion site. The mapping data showed a dose-dependent increase and significantly greater localization of NPs at the lesion site than in the remaining uninjured segment of the spinal cord. Further, NPs were seen to be retained at the lesion site for more than a week. With delayed post-SCI administration, localization of NPs at the lesion site was reduced but still localize even at four weeks post-injury administration. Interestingly, in uninjured animals (sham control), greater accumulation of NPs was seen in the thoracic and lumbar enlargement regions of the spinal cord, which in animals with SCI changed to the lesion site, indicating drastic post-injury hemodynamic changes in the spinal cord. Similar to the rat results, pig contusion model of SCI showed greater NP localization at the lesion site. In conclusion, NPs could potentially be explored as a carrier for delivery of therapeutics to the lesion site to minimize the impact of post-SCI response.

Effectiveness of Small Interfering RNA Delivery via Arginine-Rich Polyethylenimine-Based Polyplex in Metastatic and Doxorubicin-Resistant Breast Cancer Cells.

Poor cellular uptake, rapid degradation in the presence of serum, and inefficient transfection are some of the major barriers in achieving therapeutic efficacy of naked small interfering RNAs (siRNAs). We investigated the efficacy of the polyplex formulated using our synthesized polymer, polyethylene glycol (PEG)-modified l-arginine oligo(-alkylaminosiloxane) that is grafted with poly(ethyleneimine) (PEI) for siRNA delivery. We hypothesized that the polyplex formulated using the polymer with a balanced composition of PEI for siRNA condensation and its protection, PEG for polyplex stability and to minimize the PEI-associated toxicity, and with arginine facilitating cellular uptake would overcome the aforementioned issues with siRNA delivery. We tested our hypothesis using antiluciferase siRNA in luciferase-expressing metastatic breast cancer cells (MDA-MB-231-Luc-D3H2LN) and anti-ABCB1 siRNA against an efflux membrane protein, ABCB1, in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/Adr). The results demonstrated that the polyplex at an optimal nucleotide/polymer ratio is stable in the presence of excess polyanions, has no cellular toxicity, and protects siRNA from RNase degradation. Transfection of MDA-MB-231-Luc-D3H2LN cells with antiluciferase siRNA polyplex showed almost complete knockdown of luciferase expression. In MCF-7/Adr cells, transfection with anti-ABCB1 siRNA effectively downregulated its target efflux protein, ABCB1; increased cellular uptake of DOX; and enhanced its cytotoxic effect. However, the cotreatment did not completely overcome drug resistance, suggesting that further optimization is needed and/or a mechanism(s) other than the efflux protein ABCB1 may be involved in drug resistance. In conclusion, our polyplex is effective for siRNA delivery and can be explored for different therapeutic applications.

Electrical stimulation for neuroregeneration in urology: a new therapeutic paradigm.

PURPOSE OF REVIEW: The present review highlights regenerative electrical stimulation (RES) as potential future treatment options for patients with nerve injuries leading to urological dysfunction, such as urinary incontinence, voiding dysfunction or erectile dysfunction. Additionally, it will highlight the mechanism of nerve injury and regeneration as well as similarities and differences between RES and current electrical stimulation treatments in urology, functional electrical stimulation (FES) and neuromodulation. RECENT FINDINGS: It has been demonstrated that RES upregulates brain-derived neurotrophic factor (BDNF) and its receptor to facilitate neuroregeneration, facilitating accurate reinnervation of muscles by motoneurons. Further, RES upregulates growth factors in glial cells. Within the past 2 years, RES of the pudendal nerve upregulated BDNF in Onuf's nucleus, the cell bodies of motoneurons that course through the pudendal nerve and accelerated functional recovery in an animal model of stress urinary incontinence. Additionally, electrical stimulation of the vaginal tissue in an animal model of stress urinary incontinence accelerated functional recovery. SUMMARY: RES has great potential but future research is needed to expand the potential beneficial effects of RES in the field of urology.

Reaching for the Stars in the Brain: Polymer-Mediated Gene Delivery to Human Astrocytes.

Astrocytes, the "star-shaped" glial cells, are appealing gene-delivery targets to treat neurological diseases due to their diverse roles in brain homeostasis and disease. Cationic polymers have successfully delivered genes to mammalian cells and hence present a viable, non-immunogenic alternative to widely used viral vectors. In this study, we investigated the gene delivery potential of a series of arginine- and polyethylene glycol-modified, siloxane-based polyethylenimine analogs in primary cultured human neural cells (neurons and astrocytes) and in mice. Plasmid DNAs encoding luciferase reporter were used to measure gene expression. We hypothesized that polyplexes with arginine would help in cellular transport of the DNA, including across the blood-brain barrier; polyethylene glycol will stabilize polyethylenimine and reduce its toxicity while maintaining its DNA-condensing ability. Polyplexes were non-toxic to human neural cells and red blood cells. Cellular uptake of polyplexes and sustained gene expression were seen in human astrocytes as well as in mouse brains post-intravenous-injections. The polyplexes also delivered and expressed genes driven by astrocyte-restricted glial fibrillary acidic protein promoters, which are weaker than viral promoters. To our knowledge, the presented work validates a biocompatible and effective polymer-facilitated gene-delivery system for both human brain cells and mice for the first time.

Nanogel-mediated delivery of a cocktail of epigenetic drugs plus doxorubicin overcomes drug resistance in breast cancer cells.

Epigenetic modifications (e.g., DNA methylation or histone deacetylation) are commonly implicated in cancer chemoresistance. We previously showed that pretreating resistant MCF-7/ADR breast cancer cells with a demethylating agent (5-aza-2'-deoxycytidine (DAC)) or with an inhibitor of histone deacetylase (suberoylanilide hydroxamic acid (SAHA)) sensitized resistant cells to doxorubicin (DOX) treatment. However, even with increasing doses of DOX, a fraction of resistant cells remained nonresponsive to this pretreatment (~ 25% pretreated with DAC, ~ 45% with SAHA). We hypothesized that pretreating resistant cells with a combination of epigenetic drugs (DAC + SAHA) could more effectively overcome drug resistance. We postulated that delivery of epigenetic drugs encapsulated in biodegradable nanogels (NGs) would further enhance their efficacy. MCF-7/ADR cells were first treated with a single drug vs. a combination of epigenetic drugs, either as solutions or encapsulated in NGs, then subjected to DOX, either in solution or in NGs. Antiproliferative data showed that pretreatment with epigenetic drugs in NGs, then with DOX in NGs, was most effective in overcoming resistance; this treatment inhibited cell growth by > 90%, even at low doses of DOX. Cell cycle analysis showed that a major fraction of cells treated with a cocktail of epigenetic drugs + DOX, all in NG formulations, remained in the G2/M cell cycle arrest phase for a prolonged period. The mechanism of better efficacy of epigenetic drugs in NGs could be attributed to their sustained effect. A similar strategy could be developed for other cancer cells in which drug resistance is due to epigenetic modifications.

Pro-NP™ protect against TiO2 nanoparticle-induced phototoxicity in zebrafish model: exploring potential application for skin care.

Titanium dioxide nanoparticles (TiO2NPs) are used in sunscreen products to protect the skin from the sun's ultraviolet rays. However, following exposure to sunlight, the photocatalytic activity of TiO2NPs can produce an excess of reactive oxygen species (ROS), causing skin cell damage, triggering an inflammatory response. In zebrafish model, we evaluated how well Pro-NP™ (biodegradable NPs containing superoxide dismutase and catalase) could protect them from TiO2NP-induced photo-oxidative stress. We hypothesized that the antioxidant properties of Pro-NP™ would protect zebrafish embryos from the phototoxic effects of TiO2NPs, improving overall survival and growth. Dechorionated embryos were treated with TiO2NPs alone or co-treated with Pro-NP™, and then exposed to simulated sunlight. Pro-NP™ by itself caused no toxicity; however, for embryos exposed to 100 µg/ml TiO2NPs, zebrafish survival was reduced to ∼40% and at 500 µg/ml to ∼10%. In contrast, at 100 µg/ml TiO2NP, co-treatment with Pro-NP™ increased zebrafish survival in a dose-dependent manner. Co-treatment also improved percent of embryos hatching and resulted in normal growth of zebrafish. On the other hand, embryos treated with TiO2NPs alone developed deformities, had reduced pigmentation, and showed severely truncated growth. Pro-NP™ afforded a greater level of protection against TiO2NP-induced phototoxicity than other antioxidants (vitamin E or N-acetylcysteine) commonly used in topical skin care formulations. We conclude that Pro-NP™ exert significant protective effects against TiO2NP-induced phototoxicity and could be developed as a safe, effective skin care product, used alone or in combination with sunscreen products to protect the skin from sun's UV radiation.

Destination Brain: the Past, Present, and Future of Therapeutic Gene Delivery.

Neurological diseases and disorders (NDDs) present a significant societal burden and currently available drug- and biological-based therapeutic strategies have proven inadequate to alleviate it. Gene therapy is a suitable alternative to treat NDDs compared to conventional systems since it can be tailored to specifically alter select gene expression, reverse disease phenotype and restore normal function. The scope of gene therapy has broadened over the years with the advent of RNA interference and genome editing technologies. Consequently, encouraging results from central nervous system (CNS)-targeted gene delivery studies have led to their transition from preclinical to clinical trials. As we shift to an exciting gene therapy era, a retrospective of available literature on CNS-associated gene delivery is in order. This review is timely in this regard, since it analyzes key challenges and major findings from the last two decades and evaluates future prospects of brain gene delivery. We emphasize major areas consisting of physiological and pharmacological challenges in gene therapy, function-based selection of a ideal cellular target(s), available therapy modalities, and diversity of viral vectors and nanoparticles as vehicle systems. Further, we present plausible answers to key questions such as strategies to circumvent low blood-brain barrier permeability and most suitable CNS cell types for targeting. We compare and contrast pros and cons of the tested viral vectors in the context of delivery systems used in past and current clinical trials. Gene vector design challenges are also evaluated in the context of cell-specific promoters. Key challenges and findings reported for recent gene therapy clinical trials, assessing viral vectors and nanoparticles are discussed from the perspective of bench to bedside gene therapy translation. We conclude this review by tying together gene delivery challenges, available vehicle systems and comprehensive analyses of neuropathogenesis to outline future prospects of CNS-targeted gene therapies.

Inhibition of bone loss with surface-modulated, drug-loaded nanoparticles in an intraosseous model of prostate cancer.

Advanced-stage prostate cancer usually metastasizes to bone and is untreatable due to poor biodistribution of intravenously administered anticancer drugs to bone. In this study, we modulated the surface charge/composition of biodegradable nanoparticles (NPs) to sustain their blood circulation time and made them small enough to extravasate through the openings of the bone's sinusoidal capillaries and thus localize into marrow. NPs with a neutral surface charge, achieved by modulating the NP surface-associated emulsifier composition, were more effective at localizing to bone marrow than NPs with a cationic or anionic surface charge. These small neutral NPs (~150nm vs. the more usual ~320nm) were also ~7-fold more effective in localizing in bone marrow than large NPs. We hypothesized that NPs that effectively localize to marrow could improve NP-mediated anticancer drug delivery to sites of bone metastasis, thereby inhibiting cancer progression and preventing bone loss. In a PC-3M-luc cell-induced osteolytic intraosseous model of prostate cancer, these small neutral NPs demonstrated greater accumulation in bone within metastatic sites than in normal contralateral bone as well as co-localization with the tumor mass in marrow. Significantly, a single-dose intravenous administration of these small neutral NPs loaded with paclitaxel (PTX-NPs), but not anionic PTX-NPs, slowed the progression of bone metastasis. In addition, neutral PTX-NPs prevented bone loss, whereas animals treated with the rapid-release drug formulation Cremophor EL (PTX-CrEL) or saline (control) showed >50% bone loss. Neutral PTX-NPs did not cause acute toxicity, whereas animals treated with PTX-CrEL experienced weight loss. These results indicate that NPs with appropriate physical and sustained drug-release characteristics could be explored to treat bone metastasis, a significant clinical issue in prostate and other cancers.

Tissue plasminogen activator followed by antioxidant-loaded nanoparticle delivery promotes activation/mobilization of progenitor cells in infarcted rat brain.

Inherent neuronal and circulating progenitor cells play important roles in facilitating neuronal and functional recovery post stroke. However, this endogenous repair process is rather limited, primarily due to unfavorable conditions in the infarcted brain involving reactive oxygen species (ROS)-mediated oxidative stress and inflammation following ischemia/reperfusion injury. We hypothesized that during reperfusion, effective delivery of antioxidants to ischemic brain would create an environment without such oxidative stress and inflammation, thus promoting activation and mobilization of progenitor cells in the infarcted brain. We administered recombinant human tissue-type plasminogen activator (tPA) via carotid artery at 3 h post stroke in a thromboembolic rat model, followed by sequential administration of the antioxidants catalase (CAT) and superoxide dismutase (SOD), encapsulated in biodegradable nanoparticles (nano-CAT/SOD). Brains were harvested at 48 h post stroke for immunohistochemical analysis. Ipsilateral brain slices from animals that had received tPA + nano-CAT/SOD showed a widespread distribution of glial fibrillary acidic protein-positive cells (with morphology resembling radial glia-like neural precursor cells) and nestin-positive cells (indicating the presence of immature neurons); such cells were considerably fewer in untreated animals or those treated with tPA alone. Brain sections from animals receiving tPA + nano-CAT/SOD also showed much greater numbers of SOX2- and nestin-positive progenitor cells migrating from subventricular zone of the lateral ventricle and entering the rostral migratory stream than in t-PA alone treated group or untreated control. Further, animals treated with tPA + nano-CAT/SOD showed far fewer caspase-positive cells and fewer neutrophils than did other groups, as well as an inhibition of hippocampal swelling. These results suggest that the antioxidants mitigated the inflammatory response, protected neuronal cells from undergoing apoptosis, and inhibited edema formation by protecting the blood-brain barrier from ROS-mediated reperfusion injury. A longer-term study would enable us to determine if our approach would assist progenitor cells to undergo neurogenesis and to facilitate neurological and functional recovery following stroke and reperfusion injury.

Sustained Epigenetic Drug Delivery Depletes Cholesterol-Sphingomyelin Rafts from Resistant Breast Cancer Cells, Influencing Biophysical Characteristics of Membrane Lipids.

Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study's objective was to determine the effects of the epigenetic drug, 5-aza-2'-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of cholesterol-sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity, while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lß] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha-structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport and endocytic function, treatment of resistant cells with epigenetic drugs with altered lipid profile could facilitate anticancer drug transport to overcome acquired drug resistance in a combination therapy.