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Enzyme-based therapy has garnered significant attention for its current applications in various diseases. Despite the notable advantages associated with the use of enzymes as therapeutic agents, that could have high selectivity, affinity, and specificity for the target, their application faces challenges linked to physico-chemical and pharmacological properties. These limitations can be addressed through the encapsulation of enzymes in nanoplatforms as a comprehensive solution to mitigate their degradation, loss of activity, off-target accumulation, and immunogenicity, thus enhancing bioavailability, therapeutic efficacy, and circulation time, thereby reducing the number of administrations, and ameliorating patient compliance. The exploration of novel nanomedicine-based enzyme therapeutics for the treatment of challenging diseases stands as a paramount goal in the contemporary scientific landscape, but even then it is often not enough. Combining an enzyme with another therapeutic (e.g., a small molecule, another enzyme or protein, a monoclonal antibody, or a nucleic acid) within a single nanocarrier provides innovative multidrug-integrated therapy and ensures that both the actives arrive at the target site and exert their therapeutic effect, leading to synergistic action and superior therapeutic efficacy. Moreover, this strategic approach could be extended to gene therapy, a field that nowadays has gained increasing attention, as enzymes acting at genomic level and nucleic acids may be combined for synergistic therapy. This multicomponent therapeutic approach opens opportunities for promising future developments. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies., (© 2024 Wiley Periodicals LLC.)

Purpose: In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity., Methods: The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical "post-formulation" DNA addition vs "pre" addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake., Results: Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 µg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 µg/mL of pDNA with the "post" method, while maintaining their characteristics up to 100 µg/mL of pDNA with the "pre" addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 µg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes., Conclusion: In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Ottonelli et al.)

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors., Competing Interests: Declaration of Competing Interest The authors report no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Spinal cord injury (SCI) is characterized by a cascade of events that lead to sensory and motor disabilities. To date, this condition is irreversible, and no cure exists. To improve myelin repair and limit secondary degeneration, we developed a multitherapy based on nanomedicines (NMeds) loaded with the promyelinating agent triiodothyronine (T3), used in combination with systemic ibuprofen and mouse nerve growth factor (mNGF). Poly-L-lactic-co-glycolic acid (PLGA) NMeds were optimized and loaded with T3 to promote sustained release. In vitro experiments confirmed the efficacy of T3-NMeds to differentiate oligodendrocyte precursor cells. In vivo rat experiments were performed in contusion SCI to explore the NMed biodistribution and efficacy of combo drugs at short- and long-term post-lesion. A strong anti-inflammatory effect was observed in the short term with a reduction of type M1 microglia and glutamate levels, but with a subsequent increase of TREM2. In the long term, an improvement of myelination in NG2-IR, an increase in MBP content, and a reduction of the demyelination area were observed. These data demonstrated that NMeds can successfully be used to obtain more controlled local drug delivery and that this multiple treatment could be effective in improving the outcome of SCIs.

Curcumin is a well-known antioxidant used as traditional medicine in China and India since ages to treat variety of inflammatory ailments as a food supplement. Curcumin has antitumor properties with neuroprotective effects in Alzheimer's disease. Curcumin elevates brain-derived neurotrophic factor (BDNF) and dopamine (DA) levels in the brain indicating its role in substance abuse. Methamphetamine (METH) is one of the most abused substances in the world that induces profound neurotoxicity by inducing breakdown of the blood-brain barrier (BBB), vasogenic edema and cellular injuries. However, influence of curcumin on METH-induced neurotoxicity is still not well investigated. In this investigation, METH neurotoxicity and neuroprotective effects of curcumin nanodelivery were examined in a rat model. METH (20 mg/kg, i.p.) neurotoxicity is evident 4 h after its administration exhibiting breakdown of BBB to Evans blue albumin in the cerebral cortex, hippocampus, cerebellum, thalamus and hypothalamus associated with vasogenic brain edema as seen measured using water content in all these regions. Nissl attaining exhibited profound neuronal injuries in the regions of BBB damage. Normal curcumin (50 mg/kg, i.v.) 30 min after METH administration was able to reduce BBB breakdown and brain edema partially in some of the above brain regions. However, TiO 2 nanowired delivery of curcumin (25 mg/kg, i.v.) significantly attenuated brain edema, neuronal injuries and the BBB leakage in all the brain areas. BDNF level showed a significant higher level in METH-treated rats as compared to saline-treated METH group. Significantly enhanced DA levels in METH-treated rats were also observed with nanowired delivery of curcumin. Normal curcumin was able to slightly elevate DA and BDNF levels in the selected brain regions. Taken together, our observations are the first to show that nanodelivery of curcumin induces superior neuroprotection in METH neurotoxicity probable by enhancing BDNF and DA levels in the brain, not reported earlier., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

A drawback in the development of treatments that can reach the retina is the presence of barriers in the eye that restrain compounds from reaching the target. Intravitreal injections hold promise for retinal delivery, but the natural defenses in the vitreous can rapidly degrade or eliminate therapeutic molecules. Injectable hydrogel implants, which act as a reservoir, can allow for long-term drug delivery with a single injection into the eye, but still suffer due to the fast clearance of the released drugs when traversing the vitreous and random diffusion that leads to lower pharmaceutic efficacy. A combination with HA-covered nanoparticles, which can be released from the gel and more readily pass through the vitreous to increase the delivery of therapeutic agents to the retina, represents an advanced and elegant way to overcome some of the limitations in eye drug delivery. In this article, we developed hybrid PLGA-Dotap NPs that, due to their hyaluronic acid coating, can improve in vivo distribution throughout the vitreous and delivery to retinal cells. Moreover, a hydrogel implant was developed to act as a depot for the hybrid NPs to better control and slow their release. These results are a first step to improve the treatment of retinal diseases by protecting and transporting the therapeutic treatment across the vitreous and to improve treatment options by creating a depot system for long-term treatments.

Nanoparticles (NPs) are paving the way for improved treatments for difficult to treat diseases diseases; however, much is unknown about their fate in the body. One important factor is the interaction between NPs and blood proteins leading to the formation known as the "protein corona" (PC). The PC, consisting of the Hard (HC) and Soft Corona (SC), varies greatly based on the NP composition, size, and surface properties. This highlights the need for specific studies to differentiate the PC formation for each individual NP system. This work focused on comparing the HC and SC of three NPs with different matrix compositions: a) polymeric NPs based on poly(lactic- co -glycolic) acid (PLGA), b) hybrid NPs consisting of PLGA and Cholesterol, and c) lipidic NPs made only of Cholesterol. NPs were formulated and characterized for their physico-chemical characteristics and composition, and then were incubated in human plasma. In-depth purification, identification, and statistical analysis were then performed to identify the HC and SC components. Finally, similar investigations demonstrated whether the presence of a targeting ligand on the NP surface would affect the PC makeup. These results highlighted the different PC fingerprints of these NPs, which will be critical to better understand the biological influences of the PC and improve future NP designs., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

Doxorubicin hydrochloride (DOX) is currently used to treat orthotropic and metastatic breast cancer. Because of its side effects, the use of DOX in cancer patients is sometimes limited; for this reason, several scientists tried designing drug delivery systems which can improve drug therapeutic efficacy and decrease its side effects. In this study, we designed, prepared, and physiochemically characterized nonionic surfactant vesicles (NSVs) which are obtained by self-assembling different combinations of hydrophilic (Tween 20) and hydrophobic (Span 20) surfactants, with cholesterol. DOX was loaded in NSVs using a passive and pH gradient remote loading procedure, which increased drug loading from ∼1 to ∼45%. NSVs were analyzed in terms of size, shape, size distribution, zeta potential, long-term stability, entrapment efficiency, and release kinetics, and nanocarriers having the best physiochemical parameters were selected for further in vitro tests. NSVs with and without DOX were stable and showed a sustained drug release up to 72 h. In vitro studies, with MCF-7 and MDA MB 468 cells, demonstrated that NSVs, containing Span 20, were better internalized in MCF-7 and MDA MB 468 cells than NSVs with Tween 20. NSVs increased the anticancer effect of DOX in MCF-7 and MDA MB 468 cells, and this effect is time and dose dependent. In vitro studies using metastatic and nonmetastatic breast cancer cells also demonstrated that NSVs, containing Span 20, had higher cytotoxicity than NSVs with Tween 20. The resulting data suggested that DOX-loaded NSVs could be a promising nanocarrier for the potential treatment of metastatic breast cancer., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Central nervous system (CNS) compartments remain one of the most difficult districts for drug delivery. This is due to the presence of the blood-brain barrier (BBB) that hampers 90% of drug passage, dramatically requiring non-invasive treatment strategies. Here, for the first time, the use of opioid-derived deltorphin-derivative peptides to drive biodegradable and biocompatible polymeric (i.e. poly-lactide-co-glycolide, PLGA) nanomedicines delivery across the BBB was described. Opioid-derived peptides were covalently conjugated to furnish activated polymers which were further used for fluorescently tagged nanoformulations. Beyond reporting production, formulation methodology and full physico-chemical characterization, in vivo tests generated clear proof of BBB crossing and CNS targeting by engineered nanomedicines opening the research to further applications of drug delivery and targeting in CNS disease models., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is the most frequent and aggressive primary tumor of the brain and averages a life expectancy in diagnosed patients of only 15 months. Hence, more effective therapies against this malignancy are urgently needed. Several diseases, including cancer, are featured by high levels of reactive oxygen species (ROS), which are possible GBM hallmarks to target or benefit from. Therefore, the covalent linkage of drugs to ROS-responsive molecules can be exploited aiming for a selective drug release within relevant pathological environments. In this work, we designed a new ROS-responsive prodrug by using Melphalan (MPH) covalently coupled with methoxy polyethylene glycol (mPEG) through a ROS-cleavable group thioketal (TK), demonstrating the capacity to self-assembly into nanosized micelles. Full chemical-physical characterization was conducted on the polymeric-prodrug and proper controls, along with in vitro cytotoxicity assayed on different GBM cell lines and "healthy" astrocyte cells confirming the absence of any cytotoxicity of the prodrug on healthy cells (i.e. astrocytes). These results were compared with the non-ROS responsive counterpart, underlining the anti-tumoral activity of ROS-responsive compared to the non-ROS-responsive prodrug on GBM cells expressing high levels of ROS. On the other hand, the combination treatment with this ROS-responsive prodrug and X-ray irradiation on human GBM cells resulted in an increase of the antitumoral effect, and this might be connected to radiotherapy. Hence, these results represent a starting point for a rationale design of innovative and tailored ROS-responsive prodrugs to be used in GBM therapy and in combination with radiotherapy., (Copyright © 2020 Oddone, Boury, Garcion, Grabrucker, Martinez, Da Ros, Janaszewska, Forni, Vandelli, Tosi, Ruozi and Duskey.)

The treatment of diseases that affect the central nervous system (CNS) represents a great research challenge due to the restriction imposed by the blood-brain barrier (BBB) to allow the passage of drugs into the brain. However, the use of modified nanomedicines engineered with different ligands that can be recognized by receptors expressed in the BBB offers a favorable alternative for this purpose. In this work, a BBB-penetrating peptide, angiopep-2 (Ang-2), was conjugated to poly(lactic- co -glycolic acid) (PLGA)-based nanoparticles through pre- and post-formulation strategies. Then, their ability to cross the BBB was qualitatively assessed on an animal model. Proof-of-concept studies with fluorescent and confocal microscopy studies highlighted that the brain-targeted PLGA nanoparticles were able to cross the BBB and accumulated in neuronal cells, thus showing a promising brain drug delivery system.

Congenital disorder of glycosylation (CDG) type Ia is a multisystem disorder that occurs due to mutations in the phosphomannomutase 2 (PMM2) gene, which encodes for an enzyme involved in the N‑glycosylation pathway. Mutated PMM2 leads to the reduced conversion of mannose‑6‑P to mannose‑1‑P, which results in low concentration levels of guanosine 5'‑diphospho‑D‑mannose (GDP‑Man), a nucleotide‑activated sugar essential for the construction of protein oligosaccharide chains. In the present study, an in vitro therapeutic approach was used, based on GDP‑Man‑loaded poly (D,L‑lactide‑co‑glycolide) (PLGA) nanoparticles (NPs), which were used to treat CDG‑Ia fibroblast cultures, thus bypassing the glycosylation pathway reaction catalysed by PMM2. To assess the degree of hypoglycosylation in vitro, the present study examined the activities of α‑mannosidase, β‑glucoronidase and β‑galactosidase in defective and normal fibroblasts. GDP‑Man (30 µg/ml GDP‑Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of α‑mannosidase and β‑galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of β‑glucoronidase increased from 6.5 to 32.5% and was significantly higher compared with that noted in the untreated CDG‑Ia fibroblasts. The glycosylation process of fibroblasts was also analysed by two‑dimensional electrophoresis. The results demonstrated that treatment caused the reappearance of several glycosylated proteins. The data in vitro showed that GDP‑Man PLGA NPs have desirable efficacy and warrant further evaluation in a preclinical validation animal model.

Trans-resveratrol (RSV) was microencapsulated in Eudragit ® RS100 and RL100 resin blends. Lyophilized microspheres were characterized in the solid state for their micromeritic properties and drug loading. FT-IR, PXRD, and DSC analyzes suggested that RSV formed an intimate microcrystalline dispersion within the polymer network, also confirmed by SEM analysis. This produced a reduced degradation of RSV after storage at 40 °C, compared to the neat drug, and a protection of the drug from UV light-induced trans-cis isomerization (60% intact drug was found after 60 s irradiation at 350 nm, compared to 37% for the pure drug). Solubility and in vitro dissolution studies indicated that microencapsulation did not improve the dissolution pattern of RSV in simulated gastric and intestinal aqueous fluids. Evaluation of the in vitro antioxidant activity showed that, compared to the neat drug in aqueous solution, RSV loaded in the microspheres retained for a longer time, up to 22 days of incubation, the initial ORAC capacity. The present study thus demonstrated that Eudragit ® Retard resins can be used to easily produce micro-sized solid dispersions with RSV, for potential oral administration, contributing to ameliorate the physico-chemical stability and antioxidant activity of this compound.

The treatment of Alzheimer's disease (AD) is up to today one of the most unsuccessful examples of biomedical science. Despite the high number of literature evidences detailing the multifactorial and complex etiopathology of AD, no cure is yet present on the market and the available treatments are only symptomatic. The reasons could be ascribed on two main factors: (i) lack of ability of the majority of drugs to cross the blood-brain barrier (BBB), thus excluding the brain for any successful therapy; (ii) lack of selectivity and specificity of drugs, decreasing the efficacy of even potent anti-AD drugs. The exploitation of specifically engineered nanomedicines planned to cross the BBB and to target the most "hot" site of action (i.e., β-amyloid) is one of the most interesting innovations in drug delivery and could reasonably represent an promising choice for possible treatments and even early-diagnosis of AD. In this chapter, we therefore outline the most talented approaches in AD treatment with a specific focus on the main advantages/drawbacks and future possible translation to clinic application., (© 2019 Elsevier B.V. All rights reserved.)

A local dyshomeostasis of zinc ions in the vicinity of amyloid aggregates has been proposed in Alzheimer's disease (AD) due to the sequestration of zinc in senile plaques. While an increase in zinc levels may promote the aggregation of amyloid beta (Aβ), increased brain zinc might also be beneficial rescuing some pathological alterations caused by local zinc deficiency. For example, increased Aβ degradation by metalloproteinases, and a reduction in inflammation can be hypothesized. In addition, zinc may allow a stabilization of the number of synapses in AD brains. Thus, to evaluate whether altering zinc-levels within the brain is a promising new target for the prevention and treatment of AD, we employed novel zinc loaded nanoparticles able to deliver zinc into the brain across the blood-brain barrier. We performed in vivo studies using wild type (WT) and APP23 mice to assess plaque load, inflammatory status and synapse loss. Furthermore, we performed behavioral analyses. After chronically injecting these nanoparticles for 14 days, our results show a significant reduction in plaque size and effects on the pro-inflammatory cytokines IL-6 and IL-18. On behavioral level we could not detect negative effects of increased brain zinc levels in APP23 mice and treatment with g7-NP-Zn normalized the observed hyperlocomotion of APP23 mice. Therefore, we conclude that a targeted increase in brain zinc levels may have beneficial effects in AD., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Aim: The investigation on protein coronas (PCs) adsorbed onto nanoparticle (NP) surface is representing an open issue due to difficulties in detection and clear isolation of the adsorbed proteins. In this study, we investigated protocols able to isolate the compositions of PCs of three polymeric NPs., Materials & Methods: Unfunctionalized NPs and two functionalized NPs were considered as proof-of-concept for the qualitative and semiquantitative analysis of both the corona levels (stably or weakly adsorbed coronas [SC/WC]) of these different nanocarriers., Results: The protocols applied were able to discriminate between the SC and WC. In particular, experimental results indicated that stably adsorbed coronas are prevalently composed by ApoE, while WC by albumin in all the NPs. Otherwise, some differences in WC could be correlated with surface functionalization., Conclusion: This experimental approach allows characterizing the whole PCs, proposing a protocol for isolation of different types of proteins composing PCs.

Nanoparticles (NPs) represent one of the most promising tools for drug-targeting and drug-delivery. However, a deeper understanding of the complex dynamics that happen after their in vivo administration is required. Particularly, plasma proteins tend to associate to NPs, forming a new surface named the 'protein corona' (PC). This surface is the most exposed as the 'visible side' of NPs and therefore, can have a strong impact on NP biodistribution, targeting efficacy and also toxicity. The PC consists of two poorly delimited layers, known as 'hard corona' (HC) and 'soft corona' (SC), that are affected by the complexity of the environment and the formed protein-surface equilibrium during in vivo blood circulation. The HC corona is formed by proteins strongly associated to the NPs, while the SC is an outer layer consisting of loosely bound proteins. Several studies attempted to investigate the HC, which is easier to be isolated, but yielded poor reproducibility, due to varying experimental conditions. As a consequence, full mapping of the HC for different NPs is still lacking. Moreover, the current knowledge on the SC, which may play a major role in the 'first' interaction of NPs once in vivo, is very limited, mainly due to the difficulties in preserving it after purification. Therefore, multi-disciplinary approaches leading to the obtainment of a major number of information about the PC and its properties is strongly needed to fully understand its impact and to better support a more safety and conscious application of nanotechnology in medicine. WIREs Nanomed Nanobiotechnol 2017, 9:e1467. doi: 10.1002/wnan.1467 For further resources related to this article, please visit the WIREs website., (© 2017 Wiley Periodicals, Inc.)

Brain diseases and injuries are growing to be one of the most deadly and costly medical conditions in the world. Unfortunately, current treatments are incapable of ameliorating the symptoms let alone curing the diseases. Many brain diseases have been linked to a loss of function in a protein or enzyme, increasing research for improving their delivery. This is no easy task due to the delicate nature of proteins and enzymes in biological conditions, as well as the many barriers that exist in the body ranging from those in circulation to the more specific barriers to enter the brain. Several main techniques are being used (physical delivery, protein/enzyme conjugates, and nanoparticle delivery) to overcome these barriers and create new therapeutics. This review will cover recently published data and highlights the benefits and deficits of possible new protein or enzyme therapeutics for brain diseases., (© 2017 Elsevier Inc. All rights reserved.)

While the role of the blood-brain barrier (BBB) is increasingly recognized in the (development of treatments targeting neurodegenerative disorders, to date, few strategies exist that enable drug delivery of non-BBB crossing molecules directly to their site of action, the brain. However, the recent advent of Nanomedicines may provide a potent tool to implement CNS targeted delivery of active compounds. Approaches for BBB crossing are deeply investigated in relation to the pathology: among the main important diseases of the CNS, this review focuses on the application of nanomedicines to neurodegenerative disorders (Alzheimer, Parkinson and Huntington's Disease) and to other brain pathologies as epilepsy, infectious diseases, multiple sclerosis, lysosomal storage disorders, strokes.

The research of efficacious non-invasive therapies for the treatment of brain diseases represents a huge challenge, as people affected by disorders of the central nervous system (CNS) will significantly increase. Moreover, the blood-brain barrier is a key factor in hampering a number of effective drugs to reach the CNS. This review is therefore focusing on possible interventions of nanomedicine-based approaches in selected diseases affecting the CNS. A wide overview of the most outstanding results on preclinical evaluations of the potential of nanomedicine in brain diseases (i.e. brain tumor, Alzheimer, Parkinson, epilepsy and others) is given, with highlights on the data with relevant interest and real possibility in translation from bench-to-bedside. Moreover, a critical evaluation on the rationale in planning nanosystems to target specific brain pathologies is described, opening the path to a more structured and pathology-tailored design of nanocarriers.

Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington's disease (HD), suggesting that locally synthesized, newly formed cholesterol is less available to neurons. This may be detrimental for neuronal function, especially given that locally synthesized cholesterol is implicated in synapse integrity and remodeling. Here, we used biodegradable and biocompatible polymeric nanoparticles (NPs) modified with glycopeptides (g7) and loaded with cholesterol (g7-NPs-Chol), which per se is not blood-brain barrier (BBB) permeable, to obtain high-rate cholesterol delivery into the brain after intraperitoneal injection in HD mice. We report that g7-NPs, in contrast to unmodified NPs, efficiently crossed the BBB and localized in glial and neuronal cells in different brain regions. We also found that repeated systemic delivery of g7-NPs-Chol rescued synaptic and cognitive dysfunction and partially improved global activity in HD mice. These results demonstrate that cholesterol supplementation to the HD brain reverses functional alterations associated with HD and highlight the potential of this new drug-administration route to the diseased brain., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Cerebrolysin is a peptide mixture able to ameliorate symptomatology and delay progression of neurological disorders such as Alzheimer's disease and dementia. The administration of this drug in humans presents several criticisms due to its short half-life, poor stability, and high doses needed to achieve the effect. This paper investigates the potential of polylactic-co-glycolide (PLGA) nanoparticles (NPs) as sustained release systems for iv administration of cerebrolysin in normal and brain injured rats. NPs were prepared by water-in-oil-in-water (w/o/w) double emulsion technique and characterized by light scattering for mean size and zeta potential and by scanning electron microscopy (SEM) for surface morphology. The NPs produced by double sonication under cooling at 60 W for 45 s, 12 mL of 1 % w:v of PVA, and 1:0.6 w:w drug/PLGA ratio (C-NPs4) displayed an adequate loading of drug (24 ± 1 mg/100 mg of NPs), zeta potential value (-13 mV), and average diameters (ranged from 250 to 330 nm) suitable to iv administration. SEM images suggested that cerebrolysin was molecularly dispersed into matricial systems and partially adhered to the NP surface. A biphasic release with an initial burst effect followed by sustained release over 24 h was observed. Long-term stability both at room and at low temperature of freeze-dried NPs was investigated. To gain deeper insight into NP stability after in vivo administration, the stability of the best NP formulation was also tested in serum. These PLGA NPs loaded with cerebrolysin were able to reduce brain pathology following traumatic brain injury. However, the size, the polydispersivity, and the surface properties of sample were significantly affected by the incubation time and the serum concentration.

The success of nanomedicine as a new strategy for drug delivery and targeting prompted the interest in developing approaches toward basic and clinical neuroscience. Despite enormous advances on brain research, central nervous system (CNS) disorders remain the world's leading cause of disability, in part due to the inability of the majority of drugs to reach the brain parenchyma. Many attempts to use nanomedicines as CNS drug delivery systems (DDS) were made; among the various non-invasive approaches, nanoparticulate carriers and, particularly, polymeric nanoparticles (NPs) seem to be the most interesting strategies. In particular, the ability of poly-lactide-co-glycolide NPs (PLGA-NPs) specifically engineered with a glycopeptide (g7), conferring to NPs' ability to cross the blood brain barrier (BBB) in rodents at a concentration of up to 10% of the injected dose, was demonstrated in previous studies using different routes of administrations. Most of the evidence on NP uptake mechanisms reported in the literature about intracellular pathways and processes of cell entry is based on in vitro studies. Therefore, beside the particular attention devoted to increasing the knowledge of the rate of in vivo BBB crossing of nanocarriers, the subsequent exocytosis in the brain compartments, their fate and trafficking in the brain surely represent major topics in this field.

Glycogenosis type II, or Pompe Disease, is a lysosomal storage disease caused by the deficiency of acid alpha-glucosidase (GAA), leading to glycogen accumulation in muscles. A recombinant human GAA (rhGAA, Myozyme®) is currently used for enzyme replacement therapy. Despite its efficacy in most of patients, some of them show a diminished response to the treatment with rapidly progressive clinical deterioration, due to immuno-mediated enzyme inactivation. To demonstrate that Nanoparticles (NPs) could be profitably exploited to carry macromolecules, PLGA NPs loaded with rhGAA (GAA-NPs) were prepared by double emulsion solvent evaporation. Their surface morphology, particle size, zeta-potential and biochemical activity were assessed. "Pulse and chase" experiments were made by administrating GAA-NPs on patients' fibroblasts. Biochemical activity tests showed a more efficient cellular uptake of rhGAA loaded to NPs and a more significant stability of the enzyme (up to 7 days) in vitro, if compared to the same amount of rhGAA free enzyme. This data allows to envision in vivo experiments, in significant animal models, to further characterize lysosomal enzyme loaded-NPs' efficacy and toxicity.

Aim: Drug targeting to the CNS is challenging due to the presence of blood-brain barrier. We investigated chitosan (Cs) nanoparticles (NPs) as drug transporter system across the blood-brain barrier, based on mAb OX26 modified Cs., Materials & Methods: Cs NPs functionalized with PEG, modified and unmodified with OX26 (Cs-PEG-OX26) were prepared and chemico-physically characterized. These NPs were administered (intraperitoneal) in mice to define their ability to reach the brain., Results: Brain uptake of OX26-conjugated NPs is much higher than of unmodified NPs, because: long-circulating abilities (conferred by PEG), interaction between cationic Cs and brain endothelium negative charges and OX26 TfR receptor affinity., Conclusion: Cs-PEG-OX26 NPs are promising drug delivery system to the CNS.

A dyshomeostasis of zinc ions has been reported for many psychiatric and neurodegenerative disorders including schizophrenia, attention deficit hyperactivity disorder, depression, autism, Parkinson's and Alzheimer's disease. Furthermore, alterations in zinc-levels have been associated with seizures and traumatic brain injury. Thus, altering zinclevels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. However, given the restriction of zinc uptake into the brain by the blood-brain barrier, methods for controlled regulation and manipulation of zinc concentrations within the brain are rare. Here, we performed in vivo studies investigating the possibility of brain targeted zinc delivery using zinc-loaded nanoparticles which are able to cross the blood-brain barrier. After injecting these nanoparticles, we analyzed the regional and time-dependent distribution of zinc and nanoparticles within the brain. Moreover, we evaluated whether the presence of zinc-loaded nanoparticles alters the expression of zinc sensitive genes and proteins such as metallothioneins and zinc transporters and quantified possible toxic effects. Our results show that zinc loaded g7 nanoparticles offer a promising approach as a novel non - invasive method to selectively enrich zinc in the brain within a small amount of time.

Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around 200 nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe₃O₄; size, 10-15 nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the 1,700-2,000 cm(-1) region. The precise PLGA IR-signature (1,750 cm(-1) centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs' fate in in vitro studies on cell cultures.

Metronomic chemotherapy supports the idea that long-term, sustained, constant administration of chemotherapeutics, currently not achievable, could be effective against numerous cancers. Particularly appealing are liposomal formulations, used to solubilize hydrophobic therapeutics and minimize side effects, while extending drug circulation time and enabling passive targeting. As liposome alone cannot survive in circulation beyond 48 h, sustaining their constant plasma level for many days is a challenge. To address this, we develop, as a proof of concept, an implantable nanochannel delivery system and ultra-stable PEGylated lapatinib-loaded liposomes, and we demonstrate the release of intact vesicles for over 18 d. Further, we investigate intravasation kinetics of subcutaneously delivered liposomes and verify their biological activity post nanochannel release on BT474 breast cancer cells. The key innovation of this work is the combination of two nanotechnologies to exploit the synergistic effect of liposomes, demonstrated as passive-targeting vectors and nanofluidics to maintain therapeutic constant plasma levels. In principle, this approach could maximize efficacy of metronomic treatments., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: AIMS, MATERIALS & METHODS: The capacity of polymeric nanoparticles (NPs) to reach the target regardless of the administration route is a neglected field of investigation in pharmaceutical nanotechnology. Therefore, after having demonstrated in previous studies that glycopeptide-engineered NPs (g7-NPs) were able to reach the brain after intravenous administrations in rodents, this article aims to evaluate whether they can reach the CNS when administered by different routes., Results & Conclusions: The confocal microphotographs on murine brain sections showed the capability of g7-NPs to reach the target also after intraperitoneal, intranasal and oral administrations. This could open new vistas for the future application of g7-NPs in the therapeutic treatment of CNS diseases.

Aim: In order to increase the knowledge on the use of nanoparticles (NPs) in brain targeting, this article describes the conjugation of the sequence 12-32 (g21) of leptin to poly-lactide-co-glycolide NPs. The capability of these modified NPs to reach the brain was evaluated in rats after intravenous administration., Materials & Methods: The g21 was linked on the surface of NPs labeled with tetramethylrhodamine by means of the Avidin-Biotin technology. The g21-labeled NPs were injected into the tail vein of rats and, after animal sacrifice, the brain localization was evaluated by confocal microscopy, fluorescence microscopy and electron microscopy. Studies to evaluate the biodistribution of the g21-modified NPs in comparison to the unmodified NPs were also carried out. Moreover, to confirm the absence of any anorectic effect of g21 linked on the surface of NPs, appropriate studies were used to assess the rats., Results: After intravenous administration, the g21-modified NPs were able to cross the blood-brain barrier and to enter the brain parenchyma. The biodistribution studies of both unmodified and modified NPs pointed out an uptake at liver and spleen level, whereas only the g21-modified NPs showed brain localization. The food-intake experiments pointed out that the intravenous administration of g21 conjugated to the NP surface did not produce any anorectic effect in the rats., Conclusion: g21-modified NPs were able to cross the blood-brain barrier. These new modified NPs could be effectively considered as useful carrier systems for brain drug delivery.

A series of amphiphilic ion pairs of erythromycin (ERY) with lipoamino acids (LAAs) were produced. The ion pairs were prepared by evaporation of a water/ethanol co-solution of the drug and LAA bearing an alkyl side chain of 10-16 carbon atoms. For the sake of comparison, equimolar physical mixtures were prepared by triturating ERY and the LAA in the absence of any solvent. FTIR spectroscopy confirmed the structure of ion pairs, while differential scanning calorimetry and powder X-ray diffractometry were used to assess the formation of new saline species. The solubility pattern of the coevaporates in different aqueous and organic solvents confirmed their amphiphilic properties. ERY-LAA ion pairs were submitted to an in vitro microbiological assay against different bacterial strains, both susceptible and resistant to macrolides. The presence of the LAA moiety was shown not altering the antibacterial spectrum of activity of the drug. These results can be the basis for a further evaluation of ERY-LAA ion pairs as a mean to improve the penetration of the drug inside bacterial cells and to optimize the loading of ERY in lipid-based nanocarriers.

Aim: Nanoneuroscience, based on the use polymeric nanoparticles (NPs), represents an emerging field of research for achieving an effective therapy for neurodegenerative diseases. In particular, poly-lactide-co-glycolide (PLGA) glyco-heptapetide-conjugated NPs (g7-NPs) were shown to be able to cross the blood-brain barrier (BBB). However, the in vivo mechanisms of the BBB crossing of this kind of NP has not been investigated until now. This article aimed to develop a deep understanding of the mechanism of BBB crossing of the modified NPs., Materials & Methods: Loperamide and rhodamine-123 (model drugs unable to cross the BBB) were loaded into NPs, composed of a mixture of PLGA, differently modified with g7 or with a random sequence of the same aminoamids (random-g7). To study brain targeting of these model drugs, loaded NPs were administered via the tail vein in rats in order to perform both pharmacological studies and biodistribution analysis along with fluorescent, confocal and electron microscopy analysis, in order to achieve the NP BBB crossing mechanism. Computational analysis on the conformation of the g7- and random-g7-NPs of the NP surface was also developed., Results: Only loperamide delivered to the brain with g7-NPs created a high central analgesia, corresponding to the 14% of the injected dose, and data were confirmed by biodistribution studies. Electron photomicrographs showed the ability of g7-NPs in crossing the BBB as evidenced by several endocytotic vesicles and macropinocytotic processes. The computational analysis on g7 and random-g7 showed a different conformation (linear vs globular), thus suggesting a different interaction with the BBB., Conclusion: Taken together, this evidence suggested that g7-NP BBB crossing is enabled by multiple pathways, mainly membrane-membrane interaction and macropinocytosis-like mechanisms. The results of the computational analysis showed the Biousian structure of the g7 peptide, in contrast to random-g7 peptide (globular conformation), suggesting that this difference is pivotal in explaining the BBB crossing and allowing us to hypothesize regarding the mechanism of BBB crossing by g7-NPs.

Aims: To develop an appropriate liposomal formulation for gene delivery against primary effusion lymphoma (PEL), a herpesvirus HHV8-associated B-cell lymphoma., Materials & Methods: Cationic, cationic PEGylated and cationic PEGylated anti-CD138 liposomes (ILp) linking a monoclonal antibody expressed on PEL cells were prepared by a thin layer evaporation method, followed by extrusion technique. The formulations were mixed with a model oligonucleotide to form the lipoplexes and tested on a BCBL-1 cell (a PEL cell line). The transfection efficiency was evaluated by flow cytometry and confocal laser scanning microscopy analysis., Results: Based on antigen-antibody interaction, ILp mediated a specific gene delivery as shown by a significant increase in the transfection rate and a localized internalization of the oligonucleotide, in comparison with cationic liposomes and cationic PEGylated liposomes., Conclusion: ILp could be proposed as effective carriers for oligonucleotide transfer in BCBL-1 cells. In vitro experimental results encourage us to further test the in vivo therapeutic potential of ILp for specific delivery of antitumoral agents.

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical-chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers., (Copyright 2010 Elsevier B.V. All rights reserved.)

This study aims to evaluate the effects of the two most widely used glycosaminoglycans (dermatan sulphate and heparin) on both the structural and biological properties of collagen-based modified membranes (COL/GAGs membranes) designed for tissue engineering. The molecular weight of dermatan sulphate and heparins was correlated with the membrane feasibility and the cell (fibroblasts and keratinocytes) ability to adhere and proliferate on the COL/GAG membranes. Microstructure and physico-chemical properties of COL/GAGs membranes were examined using scanning electron microscopy and differential scanning calorimetry; the free amino group content and the swelling properties were also detected. The morphology, proliferation and growth behaviour of keratinocytes and fibroblasts were investigated using microscopical approach and in vitro colorimetric assay. Both fibroblasts and keratinocytes are able to grow and proliferate on COL/dermatan sulphate membranes. Fibroblasts revealed significantly higher proliferation on the membranes prepared with heparin if compared to the proliferation on the membrane without heparin (COL membrane). Particularly, a combination of the membranes formulated adding high molecular weight dermatan sulphate and high molecular weight heparin could be suitable to be used as biomaterials for epidermal substitute.

The pharmaceutical treatment of central nervous system (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, noninvasive drug delivery systems for CNS are actively studied. The development of these new delivery systems started with the discovery that properly surface-engineered colloidal vectors, and in particular liposomes and polymeric nanoparticles, with a diameter approximately 200nm, were shown to be able to cross the blood-brain barrier (BBB) without apparent damage, and to deliver drugs or genetic materials into the brain. However, even if this ability was confirmed by confocal microscopy and measured by biodistribution experiments or by means of the pharmacological effect exerted by the embedded drugs, a clear understanding of the main characteristics of the colloidal systems that are important for BBB crossing is still lacking. It is also shown that the presence of the drug is able to modify the surface of these systems, with unpredictable results on the colloidal systems biodistribution; thus, the results obtained in the absence of the loaded drug have to be taken cautiously. Moreover, since the loaded drug is only a fraction of the colloidal system that is administered, the presence of the carrier in the body and into CNS, especially in the case of long-term therapies, might cause adverse effects not yet fully understood. Thus, even if promising results have been obtained, and some colloidal systems loaded with a drug are the US Food and Drug Administration (FDA) approved for human use (but not for brain targeting), a long way of research has to be done in order to use these drug delivery systems for the treatment of CNS pathologies., (Copyright 2009 Elsevier B.V. All rights reserved.)

Cationic solid lipid nanoparticles (SLNs) have recently been suggested for non-viral gene delivery as a promising alternative to the liposomes. The aim of this study was to investigate the possibility to obtain re-dispersible cationic SLNs after a freeze-drying process in the absence of lyo- and/or cryoprotectors. The physical-chemical characteristics of cationic SLNs and their ability to bind gene material were investigated before and after the freeze-drying. To perform this study three samples of cationic SLNs, based on stearic acid, Compritol or cetylpalmitate, were prepared and characterized by PCS (photon correlation spectroscopy) and AFM (atomic force microscopy). The results indicated that solely the re-dispersed sample of stearic acid (SLN-SA) became very similar in terms of size and morphology to the fresh prepared sample, although it displayed a sensible reduction of the zeta potential (from 39.2 to 23.3 mV). By both the DSC (differential scanning calorimetry) and the ESCA (electron spectroscopy for chemical analysis) determinations, the reduction of the zeta potential was ascribed to the loss of the cationic lipids from the particle surface due to the rearrangement of the stearic acid lattice after the freeze-drying. Finally, the gel electrophoresis analysis demonstrated that SLN-SA re-suspended in PBS are unable to complex the DNA, while the SLN-SA re-dispersed in water displayed the same ability to bind DNA as the fresh prepared sample. We can conclude that cationic SLNs, based on stearic acid, retain the ability to complex DNA even after the freeze-drying in the absence of lyo- or cryoprotectors; thus, the powder form of this sample represents an attractive candidate to be investigated as in vivo DNA vector formulation.

The relatively hydrophilic bile acid, ursodeoxycholic acid (UDCA), was used as an additive to DOTAP cationic liposomes to evaluate the effect on the cellular uptake of an oligonucleotide. Nuclear magnetic resonance studies were applied to estimate the relative amount of incorporated UDCA into the lipidic bilayers. DOTAP or DOTAP-UDCA vesicles (MixVes; DOTAP/UDCA molar ratios 1:0.25, 1:0.5, 1:1, and 1:2) formed complexes with 5'-fluorescein conjugated 29-mer phosphorothioate oligonucleotides (PS-ODNs) and studied using gel electrophoresis. In addition, the complexes were tested after transfection to assess the cellular uptake and the localization of the oligo in a HaCaT cell line by the use of cytofluorimetric and confocal microscopic analysis. DOTAP lipid formulated in the presence of a defined amount of UDCA forms more stable, flexible, and active MixVes. In particular, the MixVes at 1:0.25 and 1:0.5 molar ratios increase and modify the cellular uptake of PS-ODNs if compared with DOTAP liposomes 3 hours after the transfection studies. Moreover, the in vitro data suggest that these new formulations are not toxic.

The aim of this work was to develop a novel strategy for the formulation of biodegradable PLA microspheres as delivery systems for proteins or peptides. The strategy is based on the exploitation of the gel-sol transition of the thermoreversible Pluronic F127 gel. The gel allows the formation of the particles without be co-entrapped in the matrix. The microspheres prepared using the novel technique (TG-Ms, or thermoreversible gel-method microspheres) were characterized in vitro (as concerns the size, the morphology, the protein encapsulation, the release and the protein distribution in the polymer matrix), in comparison with microspheres prepared using the classical double emulsion/solvent evaporation method (w/o/w-Ms). Two types of bovine serum albumin (BSA), with different water solubility, were used as model proteins. TG-Ms exhibited small size (7-50 m) and high protein content (8.6%, w/w) regardless of the BSA water solubility, in contrast with w/o/w-Ms, which revealed a size range of 100-130 microm and a protein content related to the BSA water solubility. TG-Ms, in spite of their smaller size respect of the w/o/w-Ms, displayed a reduced initial burst effect and a higher rate in the second release phase that resulted in a quasi-constant profile. The release behavior of the TG-Ms may be attributable to both the localization of the protein in the particle core, as shown by the confocal laser scanning microscopy analysis on labeled-BSA loaded microspheres, and the few pores in the matrix, as shown by the scanning electron microscopy. A working hypothesis about the mechanism of the particle formation was also discussed.

Vegetable cells are unusual findings in Papanicolaou-stained cervical smears; these structures could be wrongly mistaken for abnormal human cells, worm eggs, or spores by a cytologist encountering the possibility of meeting those elements in cytological analysis. Five cervicovaginal smears showing similar vegetable cells have been detected over a 3-yr period (2002-2004) in the course of a population screening program for cancer of the uterine cervix in Modena (Italy) involving 32,500 women. According to the clinical histories of the patients, the vaginal pharmaceutical drugs or appliances used were of different types: vaginal lavages, pessaries, and vaginal creams. Following a careful investigation, the only substance that can lead to vegetal elements has been identified as polysaccharide galactomannan, which is one of the excipient present in the drugs used. The authors have identified the origin of these contaminants and the means of pollution, using cytological and pharmaceutical investigation.

The direct evaluation of the heterogeneity of the particle population of nanometric drug delivery systems as liposomes is difficult to achieve owing to the dimension and the carrier characteristics. The influence of the lipidic ratio and composition on the physical stability of liposomes during their storage was investigated using atomic force microscopy (AFM) and photon correlation spectroscopy (PCS). Liposomes were made by a mixture of different lipids and obtained using distinct methods of preparation. AFM images, acquired immediately after the deposition of the sample on mica surface, clearly showed the spherical shape of the lipidic vesicles. In all the 7 months of the experiment, the average sizes of the different liposomes evaluated using the two techniques were comparable. According to PCS analysis, AFM images confirmed that almost all the diversified vesicular systems tended to form aggregates during their storage; this loss of stability was strengthened by the increase of polydispersity index value. The different behaviours observed were to ascribe to the lipidic composition more than the methods of liposome preparation. In conclusion, AFM technique owing to the relative simplicity cold be useful for the technological control of size distribution profile according to the preparative factors and moreover to the batch-to-batch reproducibility.