Endocytosis of Nanomedicines: The Case of Glycopeptide Engineered PLGA Nanoparticles.
ABSTRACT: 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.
Project description:Lysosomal Storage Disorders (LSDs) are a group of metabolic syndromes, each one due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and overall about 75% of the patients present neurological impairment. Enzyme Replacement Therapy, although determining some systemic clinical improvements, is ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB). With the aim to deliver the therapeutic enzymes across the BBB, we here assayed biodegradable and biocompatible PLGA-nanoparticles (NPs) in two murine models for LSDs, Mucopolysaccharidosis type I and II (MPS I and MPS II). PLGA-NPs were modified with a 7-aminoacid glycopeptide (g7), yet demonstrated to be able to deliver low molecular weight (MW) molecules across the BBB in rodents. We specifically investigated, for the first time, the g7-NPs ability to transfer a model drug (FITC-albumin) with a high MW, comparable to the enzymes to be delivered for LSDs brain therapy. In vivo experiments, conducted on wild-type mice and knockout mouse models for MPS I and II, also included a whole series of control injections to obtain a broad preliminary view of the procedure efficiency. Results clearly showed efficient BBB crossing of albumin in all injected mice, underlying the ability of NPs to deliver high MW molecules to the brain. These results encourage successful experiments with enzyme-loaded g7-NPs to deliver sufficient amounts of the drug to the brain district on LSDs, where exerting a corrective effect on the pathological phenotype.
Project description: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.
Project description:Rationale: The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. Sonopermeation, which relies on the combination of ultrasound and microbubbles, has emerged as a powerful tool to permeate the BBB, enabling the extravasation of drugs and drug delivery systems (DDS) to and into the central nervous system (CNS). When aiming to improve the treatment of high medical need brain disorders, it is important to systematically study nanomedicine translocation across the sonopermeated BBB. To this end, we here employed multimodal and multiscale optical imaging to investigate the impact of DDS size on brain accumulation, extravasation and penetration upon sonopermeation. Methods: Two prototypic DDS, i.e. 10 nm-sized pHPMA polymers and 100 nm-sized PEGylated liposomes, were labeled with fluorophores and intravenously injected in healthy CD-1 nude mice. Upon sonopermeation, computed tomography-fluorescence molecular tomography, fluorescence reflectance imaging, fluorescence microscopy, confocal microscopy and stimulated emission depletion nanoscopy were used to study the effect of DDS size on their translocation across the BBB. Results: Sonopermeation treatment enabled safe and efficient opening of the BBB, which was confirmed by staining extravasated endogenous IgG. No micro-hemorrhages, edema and necrosis were detected in H&E stainings. Multimodal and multiscale optical imaging showed that sonopermeation promoted the accumulation of nanocarriers in mouse brains, and that 10 nm-sized polymeric DDS accumulated more strongly and penetrated deeper into the brain than 100 nm-sized liposomes. Conclusions: BBB opening via sonopermeation enables safe and efficient delivery of nanomedicine formulations to and into the brain. When looking at accumulation and penetration (and when neglecting issues such as drug loading capacity and therapeutic efficacy) smaller-sized DDS are found to be more suitable for drug delivery across the BBB than larger-sized DDS. These findings are valuable for better understanding and further developing nanomedicine-based strategies for the treatment of CNS disorders.
Project description:Homeopathic remedies (HRs) contain odorant molecules such as flavonoids or terpenes and can lose their efficiency in presence of some competitive odors. Such similarities, along with extreme sensitivity of the olfactory system, widespread presence of olfactory receptors over all organic tissues (where they have metabolic roles besides perception of odors), and potential direct access to the brain through olfactory nerves (ONs) and trigeminal nerves, may suggest the olfactory system as target for HRs. Recent works highlighted that HRs exist in a dual form, that is, a still molecular form at low dilution and a nanoparticulate form at high dilution, and that remnants of source remedy persist in extremely high dilutions. From the literature, both odorants and nanoparticles (NPs) can enter the body through inhalation, digestive absorption, or through the skin, especially, NPs or viruses can directly reach the brain through axons of nerves. Assuming that HRs are recognized by olfactory receptors, their information could be transmitted to numerous tissues through receptor-ligand interaction, or to the brain by either activating the axon potential of ONs and trigeminal nerves or, in their nanoparticulate form, by translocating through axons of these nerves. Moreover, the nanoparticulate form may activate the immune system at multiple levels, induce systemic various biological responses through the pituitary axis and inflammation factors, or modulate gene expression at the cellular level. As immunity, inflammation, pituitary axis, and olfactory system are closely linked together, their permanent interaction triggered by olfactory receptors may thus ensure homeostasis.
Project description:The therapeutic efficiency of active targeting nanoparticulate drug delivery systems (nano-DDS) is highly compromised by the plasma proteins adsorption on nanoparticles (NPs) surface, which significantly hinders cell membrane receptors to recognize the designed ligands, and provokes the off-target toxicity and rapid clearance of NPs in vivo. Herein, we report a novel dihydroartemisinin (DHA)-decorating nano-DDS that in situ specifically recruits endogenous apolipoprotein E (apoE) on the NPs surface. The apoE-anchored corona is able to prolong PLGA-PEG2000-DHA (PPD) NPs circulation capability in blood, facilitate NPs accumulating in tumor cells by the passive enhanced permeability and retention (EPR) effect and low-density lipoprotein receptor (LDLr)-mediated target transport, and ultimately improve the in vivo antitumor activity. Our findings demonstrate that the strategy of in situ regulated apoE-enriched corona ensures NPs an efficient LDLr-mediated tumor-homing chemotherapy.
Project description:Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement.
Project description:We have shown that nanoparticles (NPs) conjugated to trans-activating transcriptor (TAT) peptide bypass the efflux action of P-glycoprotein and increase the transport of the encapsulated ritonavir, a protease inhibitor (PI), across the blood-brain-barrier (BBB) to the central nervous system (CNS). A steady increase in the drug parenchyma/capillary ratio over time without disrupting the BBB integrity suggests that TAT-conjugated NPs are first immobilized in the brain vasculature prior to their transport into parenchyma. Localization of NPs in the brain parenchyma was further confirmed with histological analysis of the brain sections. The brain drug level with conjugated NPs was 800-fold higher than that with drug in solution at two weeks. Drug clearance was seen within four weeks. In conclusion, TAT-conjugated NPs enhanced the CNS bioavailability of the encapsulated PI and maintained therapeutic drug levels in the brain for a sustained period that could be effective in reducing the viral load in the CNS, which acts as a reservoir for the replicating HIV-1 virus.
Project description:Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs). AXL, a putative ZIKV cellular entry receptor, was also highly expressed in ZIKV-susceptible i-BEC and i-NPs. This iPSC-derived BBB model can help elucidate the mechanism by which ZIKV can infect BECs, cross the BBB and gain access to the CNS.
Project description:Even with an efficient combination of antiretroviral therapy (ART), which significantly decreases viral load in human immunodeficiency virus type 1 (HIV-1)-positive individuals, the occurrence of HIV-1-associated neurocognitive disorders (HAND) still exists. Microglia have been shown to have a significant role in HIV-1 replication in the brain and in subsequent HAND pathogenesis. However, due to the limited ability of ART drugs to cross the blood-brain barrier (BBB) after systemic administration, in addition to efflux transporter expression on microglia, the efficacy of ART drugs for viral suppression in microglia is suboptimal. Previously, we developed novel poly (lactic-co-glycolic acid) (PLGA)-based elvitegravir nanoparticles (PLGA-EVG NPs), which showed improved BBB penetration in vitro and improved viral suppression in HIV-1-infected primary macrophages, after crossing an in vitro BBB model. Our objective in the current study was to evaluate the efficacy of our PLGA-EVG NPs in an important central nervous system (CNS) HIV-1 reservoir, i.e., microglia. In this study, we evaluated the cyto-compatibility of the PLGA-EVG NPs in microglia, using an XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay and cellular morphology observation. We also studied the endocytosis pathway and the subcellular localization of PLGA NPs in microglia, using various endocytosis inhibitors and subcellular localization markers. We determined the ability of PLGA-EVG NPs to suppress HIV-1 replication in microglia, after crossing an in vitro BBB model. We also studied the drug levels in mouse plasma and brain tissue, using immunodeficient NOD scid gamma (NSG) mice, and performed a pilot study, to evaluate the efficacy of PLGA-EVG NPs on viral suppression in the CNS, using an HIV-1 encephalitic (HIVE) mouse model. From our results, the PLGA-EVG NPs showed ~100% biocompatibility with microglia, as compared to control cells. The internalization of PLGA NPs in microglia occurred through caveolae-/clathrin-mediated endocytosis. PLGA NPs can also escape from endo-lysosomal compartments and deliver the therapeutics to cells efficiently. More importantly, the PLGA-EVG NPs were able to show ~25% more viral suppression in HIV-1-infected human-monocyte-derived microglia-like cells after crossing the in vitro BBB compared to the EVG native drug, without altering BBB integrity. PLGA-EVG NPs also showed a ~two-fold higher level in mouse brain and a trend of decreasing CNS HIV-1 viral load in HIV-1-infected mice. Overall, these results help us to create a safe and efficient drug delivery method to target HIV-1 reservoirs in the CNS, for potential clinical use.
Project description:Treatment of Central Nervous System (CNS) disorders still remains a major clinical challenge. The Blood-Brain Barrier (BBB), known as the major hindrance, greatly limits therapeutics penetration into the brain. Moreover, even though some therapeutics can cross BBB based on their intrinsic properties or via the use of proper nanoscale delivery vehicles, their therapeutic efficacy is still often limited without the specific uptake of drugs by the cancer or disease-associated cells. As more studies have started to elucidate the pathological roles of major cells in the CNS (for example, microglia, neurons, and astrocytes) for different disorders, nanomedicines that can enable targeting of specific cells in these diseases may provide great potential to boost efficacy. In this review, we aim to briefly cover the pathological roles of endothelial cells, microglia, tumor-associated microglia/macrophage, neurons, astrocytes, and glioma in CNS disorders and to highlight the recent advances in nanomedicines that can target specific disease-associated cells. Furthermore, we summarized some strategies employed in nanomedicine to achieve specific cell targeting or to enhance the drug neuroprotective effects in the CNS. The specific targeting at the cellular level by nanotherapy can be a more precise and effective means not only to enhance the drug availability but also to reduce side effects.