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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.

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.

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. Keywords: human neural cells, polyplexes, GFAP promoters, intravenous gene delivery, gene therapy, blood-brain barrier

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.

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.

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.

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.

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 (~150 nm vs. the more usual ~320 nm) 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.

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.

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.

Neuronal gene therapy potentially offers an effective therapeutic intervention to cure or slow the progression of neurological diseases. However, neuronal cells are difficult to transfect with nonviral vectors, and in vivo their transport across the blood–brain barrier (BBB) is inefficient. We synthesized a series of arginine-rich oligopeptides, grafted with polyethyleneimine (PEI) and modified with a short-chain polyethylene glycol (PEG). We hypothesized that the arginine would enhance cellular uptake and transport of these polyplexes across the BBB, with PEG imparting biocompatibility and “stealth” properties and PEI facilitating DNA condensation and gene transfection. The optimized composition of the polyplexes demonstrated hemocompatibility with red blood cells, causing no lysis or aggregation, and showed significantly better cytocompatibility than PEI in vitro . Polyplexes formulated with luciferase-expressing plasmid DNA could transfect rat primary astrocytes and neurons in vitro . Confocal imaging data showed efficient cellular uptake of DNA and its sustained intracellular retention and nuclear localization with polyplexes. Intravenous administration of the optimized polyplexes in mice led to gene expression in the brain, which upon further immunohistochemical analysis demonstrated gene expression in neurons. In conclusion, we have successfully designed a nonviral vector for in vitro and in vivo neuronal gene delivery.

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.

In this review, we focus on the biophysics of cell membrane lipids, particularly when cancers develop acquired drug resistance, and how biophysical changes in resistant cell membrane influence drug transport and nanoparticle-mediated drug delivery. Recent advances in membrane lipid research show the varied roles of lipids in regulating membrane P-glycoprotein function, membrane trafficking, apoptotic pathways, drug transport, and endocytic functions, particularly endocytosis, the primary mechanism of cellular uptake of nanoparticle-based drug delivery systems. Since acquired drug resistance alters lipid biosynthesis, understanding the role of lipids in cell membrane biophysics and its effect on drug transport is critical for developing effective therapeutic and drug delivery approaches to overcoming drug resistance. Here we discuss novel strategies for (a) modulating the biophysical properties of membrane lipids of resistant cells to facilitate drug transport and regain endocytic function and (b) developing effective nanoparticles based on their biophysical interactions with membrane lipids to enhance drug delivery and overcome drug resistance.

Nanoparticles have emerged in the medical field as a technology well-suited for the diagnosis and treatment of a variety of disease states. They have been heralded as efficacious owing to both in terms of improved therapeutic efficacy as well as reduction of treatment side effects in some cases. Various nanomaterials have been developed which can be tagged with targeting moieties, and with drug delivery and imaging capability or combination of both as theranostic agent. These nanomaterials have been investigated for treatment and detection of various pathological conditions. The emphasis of this review is to demonstrate current research and clinical applications for nanoparticles in the diagnosis and treatment of renal diseases.

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.

Cancer stem cell (CSC) population in solid human breast tumor was identified by CD44(+)/CD24(-) phenotype, characterized by high tumorigenicity, invasiveness and drug resistance. In this study, we characterized drug resistant breast cancer cell line-MCF-7/Adr and a number of breast cancer cell lines using flow cytometry, immunofluorescence, mammosphere formation assay and migration assay, examining their CSC immunophenotypes, presence of CSC proteins, tumorigenicity in vitro and migratory rates, respectively. Our results show that MCF-7/Adr cells uniformly display CSC characteristics yet retain low migratory rate. They are also able to self-renew and differentiate under floating culture conditions. Furthermore, MCF-7/Adr is selectively sensitive to epigenetic drug, suberoylanilide hydroxamic acid (SAHA), losing drug resistance and changes morphology yet retaining CSC immunophenotypes. In conclusion, we show that resistant breast cancer cell line MCF-7/Adr demonstrates uniform CSC like characteristics and are sensitive to epigenetic drug treatment.

In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane’s biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug, 5-aza-2′-deoxycytidine (decitabine), significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

Mutation of the p53 tumor suppressor gene, the most common genetic alteration in human cancers, results in more aggressive disease and increased resistance to conventional therapies. Aggressiveness may be related to the increased angiogenic activity of cancer cells containing mutant p53. To restore wild-type p53 function in cancer cells, we developed polymeric nanoparticles (NPs) for p53 gene delivery. Previous in vitro and in vivo studies demonstrated the ability of these NPs to provide sustained intracellular release of DNA, thus sustained gene transfection and decreased tumor cell proliferation. We investigated in vivo mechanisms involved in NP-mediated p53 tumor inhibition, with focus on angiogenesis. We hypothesize that sustained p53 gene delivery will help decrease tumor angiogenic activity and thus reduce tumor growth and improve animal survival. Xenografts of p53 mutant tumors were treated with a single intratumoral injection of p53 gene-loaded NPs (p53NPs). We observed intratumoral p53 gene expression corresponding to tumor growth inhibition, over 5 weeks. Treated tumors showed upregulation of thrombospondin-1, a potent antiangiogenic factor, and a decrease in microvessel density vs controls (saline, p53 DNA alone, and control NPs). Greater levels of apoptosis were also observed in p53NP-treated tumors. Overall, this led to significantly improved survival in p53NP-treated animals. NP-mediated p53 gene delivery slowed cancer progression and improved survival in an in vivo cancer model. One mechanism by which this was accomplished was disruption of tumor angiogenesis. We conclude that the NP-mediated sustained tumor p53 gene therapy can effectively be used for tumor growth inhibition.

Iron-oxide nanoparticles facilitate cancer diagnosis through enhanced contrast, selectively enhance tumor cell death with magnetic hyperthermia, and improve drug delivery with magnetic drug targeting. One application that remains largely unexplored is using the iron-oxide nanoparticles themselves to selectively inhibit tumor growth. In this leading opinion paper, we propose that high doses of iron-oxide nanoparticles can be used as a treatment for cancer by generating an oxidative assault against cancer. This proposal may be met with resistance considering the controversy surrounding iron in the field of cancer. Iron generates reactive oxygen species through the Fenton reaction, which may both cause - or cure cancer. Additionally, high demand for iron by cancer cells leads to contradictory therapeutic approaches: iron deprivation or overdose are both potential cancer therapies.

Stroke is a leading cause of death, long-term disability, and socioeconomic costs, highlighting the urgent need for more effective treatments. Intravenous administration of tissue plasminogen activator (t-PA) is the only FDA-approved therapy to re-establish cerebral blood flow. However, because of increased risk of hemorrhage beyond 3 h post stroke, few stroke patients (1-2%) benefit from t-PA; t-PA, which has neurotoxic effects, can also aggravate the extent of reperfusion injury by increasing blood-brain barrier permeability. An alternative strategy is needed to extend the window of intervention, minimize damage from reperfusion injury, and promote brain repair leading to neurological recovery. Reactive oxygen species (ROS), generated soon after ischemia and during reperfusion and thereafter, are considered the main mediators of ischemic injury. Antioxidant enzymes such as catalase, superoxide dismutase, etc. can neutralize ROS-mediated injury but their effective delivery to the brain remains a challenge. In this article, we review various therapeutic approaches including surgical interventions, and discuss the potential of nanoparticle-mediated delivery of antioxidants for stroke therapy.

Poly(vinyl alcohol) (PVA) is commonly used as an emulsifier in the formulation of nanoparticles (NPs) comprising poly DL-lactide co-glycolide and polylactide-based biodegradable polymers for drug and gene delivery applications. A fraction of PVA remains associated with the NPs at the interface despite their repeated washing (residual PVA). We hypothesize that this residual PVA influences the interfacial properties of NPs and hence their biophysical interactions with membrane lipids. In this study, we formulated NPs using PVA of different molecular weights to determine the effects of the residual PVA on biophysical interactions of the formulated NPs with the endothelial cell model membrane using a Langmuir balance. Despite similar physical properties (particle size and zeta potential), NPs formulated with different PVA demonstrated significant variations in their intrinsic surface properties and biophysical interactions with the model membrane. This was evident from the difference in the surface pressure–area (π–A) isotherms prepared in the presence of different formulations of NPs and the change in surface pressure of the model membrane following interaction with NPs. The variation in the biophysical properties was observed even with the NPs formulated using the same molecular weight PVA but obtained from different lots. Since the interfacial properties of NPs can significantly influence NP interactions with cells and tissue, their biophysical characterization could prove to be an important parameter not only to obtain consistent results with NPs but also to optimize their properties for drug/gene delivery applications.

Human immunodeficiency virus (HIV)-1 targets mononuclear phagocytes (MP), which disseminate infection to organs such as brain, spleen and lymph. Thus MP, which include microglia, tissue macrophages and infiltrating monocyte-derived macrophages (MDM), are important target of anti-HIV-1 drug therapy. Most of the currently used antiretroviral drugs are effective in reducing viral loadin the periphery but cannot effectively eradicate infection from tissue reservoirs such as brain MP. HIV-1 infection of the central nervous system can lead to a wide variety of HIV-1-associated neurocognitive disorders. In this study, we demonstrate that ritonavir-loaded nanoparticles (RNPs) are effective in inhibiting HIV-1 infection of MDM. Reduced infection is observed in multiple read-out systems including reduction of cytopathic effects, HIV-1 p24 protein secretion and production of progeny virions. Furthermore, the RNPs retained antiretroviral efficacy after being removed from MDM cultures. As HIV-1-infected cells in the brain are likely to survive for a long period of time, both acute and chronic infection paradigms were evaluated. Tat-peptide-conjugated RNPs (Tat-RNP) were effective in both short-term and long-term HIV-1-infected MDM. Importantly, we confirm that delivery of RNPs, both with and without Tat-peptide conjugation, is toxic neither to MDM nor to neural cells, which may be bystander targets of the nanoformulations. In conclusion, Tat-NPs could be a safe and effective way of delivering anti-HIV-1 drugs for controlling viral replication occurring within brain MP.

Understanding the role of lipids in drug transport is critical in cancer chemotherapy to overcome drug resistance. In this study, we isolated lipids from doxorubicin-sensitive (MCF-7) and -resistant (MCF-7/ADR) breast cancer cells to characterize the biophysical properties of membrane lipids (particularly lipid packing and membrane fluidity) and to understand the role of the interaction of cell membrane lipids with drug/nanocarrier on drug uptake and efficacy. Resistant cell membrane lipids showed significantly different composition and formed more condensed, less fluid monolayers than did lipids from sensitive cells. Doxorubicin, used as a model anticancer agent, showed a strong hydrophobic interaction with resistant cell membrane lipids but significantly less interaction, as well as a different pattern of interaction (i.e., ionic), with sensitive ones. The threshold intracellular doxorubicin concentration required to produce an antiproliferative effect was similar for both sensitive and resistant cell lines, suggesting that drug transport is a major barrier in determining drug efficacy in resistant cells. In addition to the biophysical characteristics of resistant cell membrane lipids, lipid-doxorubicin interactions appear to decrease intracellular drug transport via diffusion as the drug is trapped in the lipid bilayer. The rigid nature of resistant cell membranes also seems to influence endosomal functions that inhibit drug uptake when a liposomal formulation of doxorubicin is used. In conclusion, biophysical properties of resistant cell membrane lipids significantly influence drug transport, and hence drug efficacy. A better understanding of the mechanisms of cancer drug resistance is vital to developing more effective therapeutic interventions. In this regard, biophysical interaction studies with cell membrane lipids might be helpful to improve drug transport and efficacy through drug discovery and/or drug delivery approaches by overcoming the lipid barrier in resistant cells.

Next to surgical resection, tumor ablation is a commonly used intervention in the treatment of solid tumors. Tumor ablation methods include thermal therapies, photodynamic therapy, and reactive oxygen species (ROS) producing agents. Thermal therapies induce tumor cell death via thermal energy and include radiofrequency, microwave, high intensity focused ultrasound, and cryoablation. Photodynamic therapy and ROS producing agents cause increased oxidative stress in tumor cells leading to apoptosis. While these therapies are safe and viable alternatives when resection of malignancies is not feasible, they do have associated limitations that prevent their widespread use in clinical applications. To improve the efficacy of these treatments, nanoparticles are being studied in combination with nonsurgical ablation regimens. In addition to better thermal effect on tumor ablation, nanoparticles can deliver anticancer therapeutics that show a synergistic antitumor effect in the presence of heat and can also be imaged to achieve precision in therapy. Understanding the molecular mechanism of nanoparticle-mediated tumor ablation could further help engineer nanoparticles of appropriate composition and properties to synergize the ablation effect. This review aims to explore the various types of nonsurgical tumor ablation methods currently used in cancer treatment and potential improvements by nanotechnology applications.

The transport of drugs or drug delivery systems across the cell membrane is a complex biological process, often difficult to understand because of its dynamic nature. In this regard, model lipid membranes, which mimic many aspects of cell-membrane lipids, have been very useful in helping investigators to discern the roles of lipids in cellular interactions. One can use drug-lipid interactions to predict pharmacokinetic properties of drugs, such as their transport, biodistribution, accumulation, and hence efficacy. These interactions can also be used to study the mechanisms of transport, based on the structure and hydrophilicity/hydrophobicity of drug molecules. In recent years, model lipid membranes have also been explored to understand their mechanisms of interactions with peptides, polymers, and nanocarriers. These interaction studies can be used to design and develop efficient drug delivery systems. Changes in the lipid composition of cells and tissue in certain disease conditions may alter biophysical interactions, which could be explored to develop target-specific drugs and drug delivery systems. In this review, we discuss different model membranes, drug-lipid interactions and their significance, studies of model membrane interactions with nanocarriers, and how biophysical interaction studies with lipid model membranes could play an important role in drug discovery and drug delivery.

Understanding the interaction of nanoparticles (NPs) with the cell membrane and their trafficking through cells is imperative to fully explore the use of NPs for efficient intracellular delivery of therapeutics. Here, we report a novel method of measuring the force of NP-cell membrane interactions using atomic force microscopy (AFM). Poly(D,L-lactide-co-glycolide) (PLGA) NPs functionalized with poly-L-lysine were used as a model system to demonstrate that this force determines the adhesive interaction of NPs with the cell membrane and in turn the extent of cellular uptake of NPs, and hence that of the encapsulated therapeutic. Cellular uptake of NPs was monitored using AFM imaging and the dynamics of their intracellular distribution was quantified using confocal microscopy. Results demonstrated that the functionalized NPs have a five-fold greater force of adhesion with the cell membrane and the time-lapse AFM images show their rapid internalization than unmodified NPs. The intracellular trafficking study showed that the functionalized NPs escape more rapidly and efficiently from late endosomes than unmodified NPs and result in 10-fold higher intracellular delivery of the encapsulated model protein. The findings described herein enhance our basic understanding of the NP-cell membrane interaction on the basis of physical phenomena that could have wider applications in developing efficient nanocarrier systems for intracellular delivery of therapeutics.

There is significant interest in recent years in developing magnetic nanoparticles (MNPs) having multifunctional characteristics with complimentary roles. In this study, we investigated the drug delivery and magnetic resonance imaging (MRI) properties of our novel oleic acid-coated iron-oxide and pluronic-stabilized MNPs. The drug incorporation efficiency of doxorubicin and paclitaxel (alone or in combination) in MNPs was 74-95%; the drug release was sustained and the incorporated drugs had marginal effects on physical (size and zeta potential) and magnetization properties of the MNPs. The drugs in combination incorporated in MNPs demonstrated highly synergistic antiproliferative activity in MCF-7 breast cancer cells. The T2 relaxivity (r(2)) was higher for our MNPs than Feridex IV, whereas the T1 relaxivity (r(1)) was better for Feridex IV than for our MNPs, suggesting greater sensitivity of our MNPs than Feridex IV in T2 weighted imaging. The circulation half-life (t(1/2)), determined from the changes in the MRI signal intensity in carotid arteries in mice, was longer for our MNPs than Feridex IV (t(1/2)=31.2 vs. 6.4 min). MNPs with combined characteristics of MRI and drug delivery could be of high clinical significance in the treatment of various disease conditions.

The objective of our study was to investigate the neuroprotective efficacy of superoxide dismutase (SOD), loaded in poly(D,L-lactide co-glycolide; PLGA) nanoparticles (NPs), in cultured human neurons challenged with hydrogen peroxide (H(2)O(2))-induced oxidative stress. We hypothesized that the protected and sustained intracellular delivery of SOD encapsulated in NPs would demonstrate better neuroprotection from oxidative stress than either SOD or pegylated SOD (PEG-SOD) in solution. SOD-NPs (approximately 81 +/- 4 nm in diameter, 0.9% w/w SOD loading) released the encapsulated SOD in an active form with 8.2% cumulative release during the first 24 h, followed by a slower release thereafter. The results demonstrated that PLGA-NPs are compatible with human neurons, and the neuroprotective effect of SOD-NPs is dose-dependent, with efficacy seen at100 U SOD, and less significant effects at lower doses. Neither SOD (25-200 U) nor PEG-SOD (100 U) in solution demonstrated the neuroprotective effect under similar conditions. The neuroprotective effect of SOD-NPs was seen up to 6 h after H(2)O(2)-induced oxidative stress, but the effect diminished thereafter. Confocal microscopic studies demonstrated better intracellular neuronal uptake of the encapsulated model protein (fluorescein isothiocyanate-labeled BSA) than the protein in solution. Thus, the mechanism of efficacy of SOD-NPs appears to be due to the stability of the encapsulated enzyme and its better neuronal uptake after encapsulation.

Understanding the biophysical interactions of nanoparticles (NPs) with cell membranes is critical for developing effective nanocarrier systems for drug delivery applications. We developed an endothelial model cell membrane (EMM) using a mixture of lipids and Langmuir balance to study its interaction with NPs. Polystyrene NPs of different surface chemistry and sizes were used as a model nanomaterial, and changes in the membrane's surface pressure (SP) were used as a parameter to monitor its interactions with NPs. Aminated NPs (60 nm) increased SP, plain NPs reduced it, and carboxylated NPs of the same size had no effect. However, smaller NPs (20 nm) increased SP irrespective of surface chemistry, and serum did not influence their SP effect, whereas it masked the effect of larger (60 nm) plain and carboxylated but not that of aminated NPs. Membranes formed with a single phospholipid showed a different pattern of interactions with NPs than that with EMM, signifying the need of using a mixture of lipids representing the respective cells/tissue of interest for a model membrane. The particular effect of NP characteristics on SP, determined using atomic force microscopy and pi- A (surface pressure-area) isotherm, can be explained on the basis of whether the interaction results in condensation of phospholipids (increase in SP) or their displacement from the interface into the subphase (decrease in SP), causing destabilization of the membrane. We conclude that NP characteristics significantly influence biophysical interactions with the membrane. Further, the molecular mechanism(s) of nanoparticle interactions with model membranes can be effectively used for optimizing the characteristics of nanomaterials for particular biological applications.

Many therapeutics require efficient cytosolic delivery either because the receptors for those drugs are located in the cytosol or their site of action is an intracellular organelle that requires transport through the cytosolic compartment. To achieve efficient cytosolic delivery of therapeutics, different nanomaterials have been developed that consider the diverse physicochemical nature of therapeutics (macromolecule to small molecule; water soluble to water insoluble) and various membrane associated and intracellular barriers that these systems need to overcome to efficiently deliver and retain therapeutics in the cytoplasmic compartment. Our interest is in investigating PLGA and PLA-based nanoparticles for intracellular delivery of drugs and genes. The present review discusses the various aspects of our studies and emphasizes the need for understanding of the molecular mechanisms of intracellular trafficking of nanoparticles in order to develop an efficient cytosolic delivery system.

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 [Lβ] 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.

Our study presents a new method for tracking nanoparticle penetration through different layers of the skin using near-infrared dye-loaded nanoparticles (hydrodynamic diameter = 156 nm) and optical imaging. The dye-loaded nanoparticles were mixed in a topical skin cream, applied to human cadaver skin and incubated either for three or 24 h post-application, skin tissue was clipped between glass slides prior to imaging for signal intensity across the skin thickness using an optical imaging system. The data show that nanoparticles penetrate through all the layers of the skin but there is almost an exponential decay in the signal intensity from epidermis to dermis. Depending upon the incubation time, about 55%–59% of the total signal was seen in the epidermis and the remaining through dermis and hypodermis. The advantage of the method is that it allows quantitative analysis of the extent of penetration of nanoparticles through different layers of the skin without interference of any background signal from skin tissue, and without requiring extensive tissue processing. Our method could potentially be used to study the effect of nanoparticle properties and/or the use of different formulation additives on penetration of nanoparticles through different skin layers.

Spinal cord injury (SCI) results in devastating neurological and pathological consequences, causing major dysfunction to the motor, sensory and autonomic systems. The primary traumatic injury to the spinal cord triggers a cascade of acute and chronic degenerative events, leading to further secondary injury. Many therapeutic strategies have been developed to potentially intervene in these progressive neurodegenerative events and minimize secondary damage to the spinal cord. Additionally, significant efforts have been directed towards regenerative therapies that may facilitate neuronal repair and establish connectivity across the injury site. Despite the promise that these approaches have shown in preclinical animal models of SCI, challenges with respect to successful clinical translation still remain. The factors that could have contributed to failure include important biologic and physiologic differences between the preclinical models and the human condition, study designs that do not mirror clinical reality, discrepancies in dosing and the timing of therapeutic interventions, and dose-limiting toxicity. With a better understanding of the pathobiology of events following acute SCI, developing integrated approaches aimed at preventing secondary damage and also facilitating neurodegenerative recovery is possible, and hopefully will lead to effective treatments for this devastating injury. The focus of this review is to highlight the progress that has been made in drug therapies and delivery systems, and also cell-based and tissue engineering approaches for SCI.

Diseases of the aging brain—including Alzheimer’s, Parkinson’s, and Huntington’s—affect the lives of millions and pose a growing threat to public health. These diseases occur as a result of neurodegenerative processes involving the progressive loss of structure or function of neurons, including death of neurons. The population of the USA is aging, and an ever-increasing number of Americans are afflicted with neurodegenerative diseases. It is estimated that approximately one in four Americans will suffer from a neurodegenerative disease, and virtually all Americans will have a family member with one of these conditions. According to the World Health Organization’s estimate, there are currently about 18 million people worldwide with Alzheimer’s disease alone, and this figure is projected to nearly double by 2025.

Nanoparticles, which are submicroscopic particles typically ranging from 100 to 300 nm, are interesting as potential treatment of testicular disorders because they can be engineered to allow delivery to privileged tissues, such as across the blood-brain barrier or theoretically the blood-testis barrier. We compared the effects of anatomical and/or ligand targeting on testicular nanoparticle uptake in a rat model.A total of 48 rats were divided into 6 groups, including a control group and groups that received intra-arterial injection of unconjugated nanoparticles with and without saline flush, intravenous injection of unconjugated nanoparticles, intra-arterial injection of follicle stimulating hormone conjugated nanoparticles, intravenous injection of follicle stimulating hormone conjugated nanoparticles and intra-arterial injection of transactivating transcriptor conjugated nanoparticles. A dose response curve was assessed for intra-arterially injected unconjugated nanoparticles. Using high performance liquid chromatography and histological analysis we determined nanoparticle uptake by the testicle at 4 hours.Intra-arterial injection resulted in a 5.8-fold increase in uptake compared to intravenous injection at 35 mg/kg of unconjugated nanoparticles (3.7 vs 0.6 μg nanoparticles per gm testicle, p = 0.04). Anatomical targeting failed to improve testicular uptake in FSH conjugated nanoparticles (intra-arterial vs intravenous injection 0.33 vs 0.38 μg FSH nanoparticles per gm testicular tissue, p = 0.73). On fluorescence microscopy nanoparticles were noted in the testicular interstitium and seminiferous tubules, and absent from the testicular vasculature.Arterial injection for anatomical targeting of nanoparticles to the testis is feasible, improves unconjugated nanoparticle delivery to testicular tissue and enables nanoparticles to cross the gonadal vascular endothelium and the blood-testis barrier.

Advancements in epigenetic treatments are not only coming from new drugs, but also from modifications or encapsulation of the existing drugs into different formulations leading to greater stability and enhanced delivery to the target site. The epigenome is highly regulated and complex; therefore, it is important that off-target effects of epigenetic drugs be minimized. The step from in vitro to in vivo treatment of these drugs often requires development of a method of effective delivery for clinical translation.This review covers epigenetic mechanisms such as DNA methylation, chromatin remodeling and small-RNA-mediated gene regulation. There is a section in the review with examples of diseases where epigenetic alterations lead to impaired pathways, with an emphasis on cancer. Epigenetic drugs, their targets and clinical status are presented. Advantages of using a delivery method for epigenetic drugs as well as examples of current advancements and challenges are also discussed.Epigenetic drugs have the potential to be very effective therapy against a number of diseases, especially cancers and neurological disorders. As with many chemotherapeutics, undesired side effects need to be minimized. Finding a suitable delivery method means reducing side effects and achieving a higher therapeutic index. Each drug may require a unique delivery method exploiting the drug's chemistry or other physical characteristic requiring interdisciplinary participation and would benefit from a better understanding of the mechanisms of action.

In this study, we formulated polyplexes with different compositions for co-delivery of DNA and small-interfering RNA (siRNA). Since DNA and siRNA have distinctive and complementary morphological characteristics (DNA is long and winding and siRNA is short and rigid), we hypothesized that their co-delivery using polyplex would enhance each other's transfection. To test this hypothesis, cationic polymer branched polyethylenimine (bPEI) as a standard transfecting agent and its derivative arginine-rich oligopeptide-grafted bPEI modified with polyethylene glycol (P(SiDAAr)5P3), synthesized in our laboratory, were used as carriers for transfection. Polyplexes at different nucleic acid to polymer weight ratios were characterized for transfection in breast cancer sensitive (MCF-7) and resistant (MCF-7/Adr) cell lines. Gene silencing effect of polyplexes was determined in MDA-MB-231-luc-D3H2LN cell line. The results demonstrated that the polyplexes formed with derivative P(SiDAAr)5P3 show significantly lower toxicity compared to polyplexes formed using bPEI. Further, co-delivery resulted in 20-fold higher DNA transfection and 2-fold higher siRNA transfection as compared to the respective single nucleotide delivery. DNA transfection was ∼100 fold lower in resistant MCF-7/Adr cells than in sensitive MCF-7 cells. Confocal imaging and flow cytometry data demonstrated that enhanced transfection does not solely depend on DNA's cellular uptake, suggesting that other mechanisms contribute to increased transfection. DNA-co-siRNA delivery could be a promising therapeutic approach to achieve synergistic effects, because it can simultaneously target and interfere with multiple regulatory levels in a cell to halt and reverse disease progression.