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Purpose: Apolipoprotein E2 (ApoE2) gene therapy is a potential disease-modifying therapy for Alzheimer's disease (AD). We investigated the potential of plasmid encoding ApoE2 loaded brain-targeted functionalized-liposomes for treatment of AD. This was achieved via systemic administration of liposomes entrapping therapeutic gene targeting the brain of mice., Methods: Targeting and transfection efficiency of designed liposomes were determined in bEnd.3, primary glial and primary neuronal cells. The ability of liposomal formulations to translocate across in vitro blood-brain barrier (BBB) and, thereafter, transfect primary neuronal cells was investigated using in vitro triple co-culture BBB model. We quantified ApoE expression in the brain of mice after single intravenous injection of brain-targeted liposomes loaded with plasmid ApoE2., Results: Dual surface modification enhanced the in vitro transfection efficiency of designed liposomes. Successful delivery of therapeutic gene overcoming BBB by Transferrin-Penetratin- modified liposomes was demonstrated both in vitro and in vivo. Significant (p < 0.05) increase in ApoE levels in the brain of mice was observed after intravenous administration of Tf-Pen-liposomes encasing plasmid ApoE2., Conclusion: The results indicate that dual-ligand based liposomal gene delivery systems had both enhanced brain targeting and gene delivery efficiencies. Transferrin-Penetratin modified liposomes for delivery of plasmid ApoE2 has great potential for AD treatment.

Background: The potential of gene therapy for treatment of neurological disorders can be explored using designed lipid-based nanoparticles such as liposomes, which have demonstrated ability to deliver nucleic acid to brain cells. We synthesized liposomes conjugated to cell-penetrating peptides (CPPs) (vascular endothelial-cadherin-derived peptide [pVec], pentapeptide QLPVM and HIV-1 trans-activating protein [TAT]) and transferrin (Tf) ligand, and examined the influence of surface modifications on the liposome delivery capacity and transfection efficiency of encapsulated plasmid DNA. The design of liposomes was based on targeting molecular recognition of transferrin receptor overexpressed on the blood-brain barrier (BBB) with enhanced internalization ability of CPPs., Methods: CPP-Tf-liposomes were characterized by particle size distribution, zeta potential, protection of encapsulated plasmid DNA, uptake mechanisms and transfection efficiencies. An in vitro triple co-culture BBB model selected the liposomal formulations that were able to cross the in vitro BBB and subsequently, transfect primary neuronal cells. The in vivo biodistribution and biocompatibility of selected formulations were also investigated in mice., Results: Liposomal formulations were able to protect the encapsulated plasmid DNA against enzymatic degradation and presented low hemolytic potential and low cytotoxicity at 100 nM phospholipid concentration. Cellular internalization of nanoparticles occurred via multiple endocytosis pathways. CPP-Tf-conjugated liposomes mediated robust transfection of brain endothelial (bEnd.3), primary glial and primary neuronal cells. Liposomes modified with Tf and TAT demonstrated superior ability to cross the barrier layer and subsequently, transfect neuronal cells compared to other formulations. Quantification of fluorescently labeled liposomes and in vivo imaging demonstrated that this system could efficiently overcome the BBB and penetrate the brain of mice (7.7% penetration of injected dose)., Conclusion: In vitro screening platforms are important tools to enhance the success of brain-targeted gene delivery systems. The potential of TAT-Tf-liposomes as efficient brain-targeted gene carriers in vitro and in vivo was suggested to be related to the presence of selected moieties on the nanoparticle surface., Competing Interests: B.S.R. is supported by a doctoral fellowship from The Brazilian National Council for Scientific and Technological Development (CNPq, Brazil) with a scholarship for B.S.R (Full Doctorate Fellowship (GDE): 221327/2014-2). The authors report no other conflicts of interest in this work., (© 2019 dos Santos Rodrigues et al.)

Glioblastoma is a hostile brain tumor associated with high infiltration leading to poor prognosis. Anti-cancer chemotherapeutic agents have limited access into the brain due to the presence of the blood brain barrier (BBB). In this study, we designed a dual functionalized liposomal delivery system, surface modified with transferrin (Tf) for receptor mediated transcytosis and a cell penetrating peptide-penetratin (Pen) for enhanced cell penetration. We loaded doxorubicin and erlotinib into liposomes to enhance their translocation across the BBB to glioblastoma tumor. In vitro cytotoxicity and hemocompatibility studies demonstrated excellent biocompatibility for in vivo administration. Co-delivery of doxorubicin and erlotinib loaded Tf-Pen liposomes revealed significantly (p < 0.05) higher translocation (~15%) across the co-culture endothelial barrier resulting in regression of tumor in the in vitro brain tumor model. The biodistribution of Tf-Pen liposomes demonstrated ~12 and 3.3 fold increase in doxorubicin and erlotinib accumulation in mice brain, respectively compared to free drugs. In addition, Tf-Pen liposomes showed excellent antitumor efficacy by regressing ~90% of tumor in mice brain with significant increase in the median survival time (36 days) along with no toxicity. Thus, we believe that this study would have high impact for treating patients with glioblastoma., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Liposome based delivery systems provide a promising strategy for treatment of neurodegenerative diseases. A rational design of brain-targeted liposomes can support the development of more efficient treatments with drugs and gene materials. Here, we characterized surface modified liposomes with transferrin (Tf) protein and penetratin (Pen), a cell-penetrating peptide, for efficient and targeted gene delivery to brain cells. PenTf-liposomes efficiently encapsulated plasmid DNA, protected them against enzymatic degradation and exhibited a sustained in vitro release kinetics. The formulation demonstrated low cytotoxicity and was non-hemolytic. Liposomes were internalized into cells mainly through energy-dependent pathways especially clathrin-mediated endocytosis. Reporter gene transfection and consequent protein expression in different cell lines were significantly higher using PenTf-liposomes compared to unmodified liposomes. The ability of these liposomes to escape from endosomes can be an important factor which may have likely contributed to the high transfection efficiency observed. Rationally designed bifunctional targeted-liposomes provide an efficient tool for improving the targetability and efficacy of synthesized delivery systems. This investigation of liposomal properties attempted to address cell differences, as well as, vector differences, in gene transfectability. The findings indicate that PenTf-liposomes can be a safe and non-invasive approach to transfect neuronal cells through multiple endocytosis pathways., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: The purpose of this work was the development of a multicompartimental nanocarrier for the simultaneous encapsulation of paclitaxel (PTX) and genistein (GEN), associating antiangiogenic and cytotoxic properties in order to potentiate antitumoral activity., Method: Polymeric nanocapsules containing PTX were obtained by interfacial deposition of preformed polymer and coated with a phospholipid bilayer entrapping GEN. Physical-chemical and morphological characteristics were characterized, including size and size distribution, drug entrapment efficiency and drug release profile. In vivo studies were performed in EAT bearing Swiss mice., Results: Entrapment efficiency for both drugs in the nanoparticles was approximately 98%. Average particle diameter was 150 nm with a monomodal distribution. In vitro assays showed distinct temporal drug release profiles for each drug. The dose of 0.2 mg/kg/day of PTX resulted in 11% tumor inhibition, however the association of 12 mg/kg/day of GEN promoted 44% tumor inhibition and a 58% decrease in VEGF levels., Conclusions: Nanoparticles containing GEN and PTX with a temporal pattern of drug release indicated that the combined effect of cytotoxic and antiangiogenic drugs present in the formulation contributed to the overall enhanced antitumor activity of the nanomedicine.

The limitations imposed on brain therapy by the blood–brain barrier (BBB) have warranted the development of carriers that can overcome and deliver therapeutic agents into the brain. We strategically designed liposomal nanoparticles encasing plasmid DNA for efficient transfection and translocation across the in vitro BBB model as well as in vivo brain-targeted delivery. Liposomes were surface modified with two ligands, cell-penetrating peptide (PFVYLI or R9F2) for enhanced internalization into cells and transferrin (Tf) ligand for targeting transferrin-receptor expressed on brain capillary endothelial cells. Dual-modified liposomes encapsulating pDNA demonstrated significantly (P < 0.05) higher in vitro transfection efficiency compared to single-modified nanoparticles. R9F2Tf-liposomes showed superior ability to cross in vitro BBB and, subsequently, transfect primary neurons. Additionally, these nanoparticles crossed in vivo BBB and reached brain parenchyma of mice (6.6%) without causing tissue damage. Transferrin receptor-targeting with enhanced cell penetration is a relevant strategy for efficient brain-targeted delivery of genes. Dual-modified liposomes surface modified with cell-penetrating peptides (PFVYLI and R9F2) and transferrin ligand were designed to overcome the blood brain barrier and efficiently deliver a plasmid DNA into the brain of mice. Our results showed the superior ability of R9F2Tf-liposomes to translocate across the in vitro blood brain barrier model and transfect neuronal cells as well as enhanced in vivo brain-targeted delivery properties. Thus, R9F2Tf-liposomes have potential as a carrier for brain-targeted gene delivery. Unlabelled Image [ABSTRACT FROM AUTHOR]