Stimuli-Responsive Multifunctional Nanomedicine for Enhanced Glioblastoma Chemotherapy Augments Multistage Blood-to-Brain Trafficking and Tumor Targeting.

Minimal therapeutic advances have been achieved over the past two decades for glioblastoma (GBM), which remains an unmet clinical need. Here, hypothesis-driven stimuli-responsive nanoparticles (NPs) for docetaxel (DTX) delivery to GBM are reported, with multifunctional features that circumvent insufficient blood-brain barrier (BBB) trafficking and lack of GBM targeting-two major hurdles for anti-GBM therapies. NPs are dual-surface tailored with a i) brain-targeted acid-responsive Angiopep-2 moiety that triggers NP structural rearrangement within BBB endosomal vesicles, and ii) L-Histidine moiety that provides NP preferential accumulation into GBM cells post-BBB crossing. In tumor invasive margin patient cells, the stimuli-responsive multifunctional NPs target GBM cells, enhance cell uptake by 12-fold, and induce three times higher cytotoxicity in 2D and 3D cell models. Moreover, the in vitro BBB permeability is increased by threefold. A biodistribution in vivo trial confirms a threefold enhancement of NP accumulation into the brain. Last, the in vivo antitumor efficacy is validated in GBM orthotopic models following intratumoral and intravenous administration. Median survival and number of long-term survivors are increased by 50%. Altogether, a preclinical proof of concept supports these stimuli-responsive multifunctional NPs as an effective anti-GBM multistage chemotherapeutic strategy, with ability to respond to multiple fronts of the GBM microenvironment.

Combination of local immunogenic cell death-inducing chemotherapy and DNA vaccine increases the survival of glioblastoma-bearing mice.

Immunotherapy efficacy as monotherapy is negligible for glioblastoma (GBM). We hypothesized that combining therapeutic vaccination using a plasmid encoding an epitope derived from GBM-associated antigen (pTOP) with local delivery of immunogenic chemotherapy using mitoxantrone-loaded PEGylated PLGA-based nanoparticles (NP-MTX) would improve the survival of GBM-bearing mice by stimulating an antitumor immune response. We first proved that MTX retained its ability to induce cytotoxicity and immunogenic cell death of GBM cells after encapsulation. Intratumoral delivery of MTX or NP-MTX increased the frequency of IFN-γ-secreting CD8 T cells. NP-MTX mixed with free MTX in combination with pTOP DNA vaccine increased the median survival of GL261-bearing mice and increased M1-like macrophages in the brain. The addition of CpG to this combination abolished the survival benefit but led to increased M1 to M2 macrophage ratio and IFN-γ-secreting CD4 T cell frequency. These results highlight the benefits of combination strategies to potentiate immunotherapy and improve GBM outcome.

Layer-by-Layer Nanoarchitectonics Using Protein-Polyelectrolyte Complexes toward a Generalizable Tool for Protein Surface Immobilization.

Layer-by-layer (LbL) self-assembly is an attractive method for the immobilization of macromolecules at interfaces. Integrating proteins in LbL thin films is however challenging due to their polyampholyte nature. Recently, we developed a method to integrate lysozyme into multilayers using protein-polyelectrolytes complexes (PPCs). In this work, we extended this method to a wide range of protein-polyelectrolyte combinations. We demonstrated the robustness and versatility of PPCs as building blocks. LL-37, insulin, lysozyme, and glucose oxidase were complexed with alginate, poly(styrenesulfonate), heparin, and poly(allylamine hydrochloride). The resulting PPCs were then LbL self-assembled with chitosan, PAH, and heparin. We demonstrated that multilayers built with PPCs are thicker compared to the LbL self-assembly of bare protein molecules. This is attributed to the higher mass of protein in the multilayers and/or the more hydrated state of the assemblies. PPCs enabled the self-assembly of proteins that could otherwise not be LbL assembled with a PE or with another protein. Furthermore, the results also show that LbL with PPCs enabled the construction of multilayers combining different proteins, highlighting the formation of multifunctional films. Importantly, we show that the adsorption behavior and thus the multilayer growth strongly depend on the nature of the protein and polyelectrolyte used. In this work, we elaborated a rationale to help and guide the use of PPCs for protein LbL assembly. It will therefore be beneficial to the many scientific communities willing to modify interfaces with hard-to-immobilize proteins and peptides.

Improvement of biomolecular analysis in thin films using in situ matrix enhanced secondary ion mass spectrometry.

Sensitivity to molecular ions remains a limiting factor for high resolution imaging mass spectrometry of organic and biological materials. Here, we investigate a variant of matrix-enhanced secondary ion mass spectrometry in which the transfer of matrix molecules to the analyte sample is carried out in situ (in situ ME-SIMS). This approach is therefore compatible with both 2D and 3D imaging by SIMS. In this exploratory study, nanoscale matrix layers were sputter-transferred inside our time-of-flight (ToF)-SIMS to a series of thin films of biomolecules (proteins, sugars, lipids) adsorbed on silicon, and the resulting layers were analyzed and depth-profiled. For this purpose, matrix molecules were desorbed from a coated target (obtained by drop-casting or sublimation) using 10 keV Ar3000+ ion beam sputtering, followed by redeposition on a collector carrying the sample to be analyzed. After evaluating the quality of the transfer of six different matrices on bare Si collectors, α-cyano-4-hydroxycinnamic acid (CHCA) was selected for further experiments. The mass spectra and depth profiles obtained from the organic layer prior to and after the sputter-transfer of CHCA were compared, along with those obtained from regular ME-SIMS samples (dried droplets) and, finally, with MALDI data for the same matrix-analyte combinations. Signal amplification factors were calculated by dividing the integrated molecular intensities obtained with or without matrix transfer. While the amplification factors are between 0.5 and 2 for molecules already detected with high intensities in SIMS, such as cholesterol or human angiotensin, other compounds show very large integrated signal amplification, even above two orders of magnitude. This is the case for D-glucose and cardiolipin, for which the molecular ion intensity is low (or very low) under normal SIMS analysis conditions. For such low ionization probability compounds, the beneficial effect of the matrix is unquestionable. Test experiments on mouse brain tissue sections also indicate signal enhancement with the matrix, especially for high mass lipid ions.

Nanoparticle impact on innate immune cell pattern-recognition receptors and inflammasomes activation.

Nanotechnology-based strategies can dramatically impact the treatment, prevention and diagnosis of a wide range of diseases. Despite the unprecedented success achieved with the use of nanomaterials to address unmet biomedical needs and their particular suitability for the effective application of a personalized medicine, the clinical translation of those nanoparticulate systems has still been impaired by the limited understanding on their interaction with complex biological systems. As a result, unexpected effects due to unpredicted interactions at biomaterial and biological interfaces have been underlying the biosafety concerns raised by the use of nanomaterials. This review explores the current knowledge on how nanoparticle (NP) physicochemical and surface properties determine their interactions with innate immune cells, with particular attention on the activation of pattern-recognition receptors and inflammasome. A critical perspective will additionally address the impact of biological systems on the effect of NP on immune cell activity at the molecular level. We will discuss how the understanding of the NP-innate immune cell interactions can significantly add into the clinical translation by guiding the design of nanomedicines with particular effect on targeted cells, thus improving their clinical efficacy while minimizing undesired but predictable toxicological effects.

Thiol-ene Reaction: An Efficient Tool to Design Lipophilic Polyphosphoesters for Drug Delivery Systems.

Poly(ethylene glycol)-b-polyphosphoester (PEG-b-PPE) block copolymer nanoparticles are promising carriers for poorly water soluble drugs. To enhance the drug loading capacity and efficiency of such micelles, a strategy was investigated for increasing the lipophilicity of the PPE block of these PEG-b-PPE amphiphilic copolymers. A PEG-b-PPE copolymer bearing pendant vinyl groups along the PPE block was synthesized and then modified by thiol-ene click reaction with thiols bearing either a long linear alkyl chain (dodecyl) or a tocopherol moiety. Ketoconazole was used as model for hydrophobic drugs. Comparison of the drug loading with PEG-b-PPE bearing shorter pendant groups is reported evidencing the key role of the structure of the pendant group on the PPE backbone. Finally, a first evidence of the biocompatibility of these novel PEG-b-PPE copolymers was achieved by performing cytotoxicity tests. The PEG-b-PPE derived by tocopherol was evidenced as particularly promising as delivery system of poorly water-soluble drugs.

Novel strategy for oral peptide delivery in incretin-based diabetes treatment.

OBJECTIVE: To fulfil an unmet therapeutic need for treating type 2 diabetes by developing an innovative oral drug delivery nanosystem increasing the production of glucagon-like peptide-1 (GLP-1) and the absorption of peptides into the circulation. DESIGN: We developed a nanocarrier for the oral delivery of peptides using lipid-based nanocapsules. We encapsulated the GLP-1 analogue exenatide within nanocapsules and investigated in vitro in human L-cells (NCl-H716) and murine L-cells (GLUTag cells) the ability of the nanosystem to trigger GLP-1 secretion. The therapeutic relevance of the nanosystem in vivo was tested in high-fat diet (HFD)-induced diabetic mice following acute (one administration) or chronic treatment (5 weeks) in obese and diabetic mice. RESULTS: We demonstrated that this innovative nanosystem triggers GLP-1 secretion in both human and murine cells as well as in vivo in mice. This strategy increases the endogenous secretion of GLP-1 and the oral bioavailability of the GLP-1 analogue exenatide (4% bioavailability with our nanosystem).The nanosystem synergizes its own biological effect with the encapsulated GLP-1 analogue leading to a marked improvement of glucose tolerance and insulin resistance (acute and chronic). The chronic treatment decreased diet-induced obesity, fat mass, hepatic steatosis, together with lower infiltration and recruitment of immune cell populations and inflammation. CONCLUSION: We developed a novel nanosystem compatible with human use that synergizes its own biological effect with the effects of increasing the bioavailability of a GLP-1 analogue. The effects of the formulation were comparable to the results observed for the marketed subcutaneous formulation. This nanocarrier-based strategy represents a novel promising approach for oral peptide delivery in incretin-based diabetes treatment.

EPR monitoring of wound oxygenation as a biomarker of response to gene therapy encoding hCAP-18/LL37 peptide.

PURPOSE: To investigate the value of electron paramagnetic resonance oximetry to follow oxygenation in wounds treated by a plasmid-encoding host defense peptide hCAP-18/LL37. METHODS: Flaps were created on diabetic mice (7- or 12-week-old db/db mice) presenting different levels of microangiopathy. The hCAP-18/LL37-encoding plasmids were administered in wounds by electroporation. Low-frequency electron paramagnetic resonance oximetry using lithium phthalocyanine as the oxygen sensor was used to monitor wound oxygenation in flaps during the healing process. Flaps were analyzed by immunohistochemistry to assess hypoxia and cell proliferation. Kinetics of closure was also assessed in excisional skin wounds. RESULTS: A reoxygenation of the flap was observed during the healing process in the 7-week-old db/db treated mice, but not in the untreated mice and the 12-week-old mice. Histological studies demonstrated less hypoxic regions and higher proportion of proliferating cells in hCAP-18/LL37-treated flaps in the 7-week-old db/db treated mice compared with untreated mice. Consistently, the kinetics of excisional wound closure was improved by hCAP-18/LL37 treatment in the 7-week-old db/db but not in the 12-week-old mice. CONCLUSIONS: Oxygenation measured by electron paramagnetic resonance oximetry is a promising biomarker of response to treatments designed to modulate wound oxygenation. Magn Reson Med 79:3267-3273, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).

Size Effect on Lipid Nanocapsule-Mediated GLP-1 Secretion from Enteroendocrine L Cells.

L cells are enteroendocrine cells located throughout the gastrointestinal tract that secrete physiologically important peptides. The most characterized peptides secreted by L cells are the peptide YY (PYY) and the glucagon-like peptides 1 (GLP-1) and 2 (GLP-2). These peptides are released rapidly into the circulation after oral nutrient ingestion. Recently, lipid-based nanoparticles (NP) have been described as triggers for GLP-1 secretion by L cells. NP physicochemical properties play a key role in the NP-cell interaction, and drive NP cell internalization. We herein hypothesize that lipid-based NP with appropriate size would not only be able to deliver drugs into blood circulation but also act like endogenous ligands to stimulate GLP-1 secretion. We tested five different size (25, 50, 100, 150, and 200 nm) lipid nanocapsules (LNC) on murine L cells in vitro to confirm this hypothesis. Our study showed that GLP-1 secretion was induced only by the 200 nm size LNC, highlighting the importance of LNC particle size on the secretion of GLP-1 by L cells. The different formulations did not affect proglucagon mRNA expression, suggesting that there was not an increased GLP-1 synthesis. As a proof of concept, we further demonstrated in normoglycemic mice that 200 nm LNC administration increases GLP-1 levels by 4- and 3-fold compared to untreated control mice 60 and 180 min after the administration, respectively. Our study suggests that 200 nm LNC as a nanocarrier to encapsulate drug candidates and as a ligand to induce endogenous GLP-1 secretion might represent a promising strategy for type 2 diabetes mellitus treatment.

On glioblastoma and the search for a cure: where do we stand?

Although brain tumours have been documented and recorded since the nineteenth century, 2016 marked 90 years since Percival Bailey and Harvey Cushing coined the term "glioblastoma multiforme". Since that time, although extensive developments in diagnosis and treatment have been made, relatively little improvement on prognosis has been achieved. The resilience of GBM thus makes treating this tumour one of the biggest challenges currently faced by neuro-oncology. Aggressive and robust development, coupled with difficulties of complete resection, drug delivery and therapeutic resistance to treatment are some of the main issues that this nemesis presents today. Current treatments are far from satisfactory with poor prognosis, and focus on palliative management rather than curative intervention. However, therapeutic research leading to developments in novel treatment stratagems show promise in combating this disease. Here we present a review on GBM, looking at the history and advances which have shaped neurosurgery over the last century that cumulate to the present day management of GBM, while also exploring future perspectives in treatment options that could lead to new treatments on the road to a cure.

Coadministration of a Plasmid Encoding HIV-1 Gag Enhances the Efficacy of Cancer DNA Vaccines.

DNA vaccination holds great promise for the prevention and treatment of cancer and infectious diseases. However, the clinical ability of DNA vaccines is still controversial due to the limited immune response initially observed in humans. We hypothesized that electroporation of a plasmid encoding the HIV-1 Gag viral capsid protein would enhance cancer DNA vaccine potency. DNA electroporation used to deliver plasmids in vivo, induced type I interferons, thereby supporting the activation of innate immunity. The coadministration of ovalbumin (OVA) and HIV-1 Gag encoding plasmids modulated the adaptive immune response. This strategy favored antigen-specific Th1 immunity, delayed B16F10-OVA tumor growth and improved mouse survival in both prophylactic and therapeutic vaccination approaches. Similarly, a prophylactic DNA immunization against the melanoma-associated antigen gp100 was enhanced by the codelivery of the HIV-1 Gag plasmid. The adjuvant effect was not driven by the formation of HIV-1 Gag virus-like particles. This work highlights the ability of both electroporation and the HIV-1 Gag plasmid to stimulate innate immunity for enhancing cancer DNA vaccine immunogenicity and demonstrates interesting tracks for the design of new translational genetic adjuvants to overcome the current limitations of DNA vaccines in humans.

Pentacyclic Triterpene Bioavailability: An Overview of In Vitro and In Vivo Studies.

Pentacyclic triterpenes are naturally found in a great variety of fruits, vegetables and medicinal plants and are therefore part of the human diet. The beneficial health effects of edible and medicinal plants have partly been associated with their triterpene content, but the in vivo efficacy in humans depends on many factors, including absorption and metabolism. This review presents an overview of in vitro and in vivo studies that were carried out to determine the bioavailability of pentacyclic triterpenes and highlights the efforts that have been performed to improve the dissolution properties and absorption of these compounds. As plant matrices play a critical role in triterpene bioaccessibility, this review covers literature data on the bioavailability of pentacyclic triterpenes ingested either from foods and medicinal plants or in their free form.

A Mechanistic Study on Nanoparticle-Mediated Glucagon-Like Peptide-1 (GLP-1) Secretion from Enteroendocrine L Cells.

L cells have attracted particular interest because of the pleiotropic effects of their secreted peptides (i.e., glucagon-like peptide (GLP) 1 and 2, peptide YY (PYY)). L cells express different G-protein-coupled receptors (GPCRs) that can be activated by endogenous ligands found in the gut lumen. We herein hypothesized that lipid-based nanoparticles could mimic endogenous ligands and thus activate GLP-1 secretion in type 2 diabetes mellitus treatment. To assess this hypothesis, lipid-based nanoparticles (nanostructured lipid carriers (NLC), lipid nanocapsules (LNC), and liposomes) and PLGA nanoparticles were added to the L cells and GLP-1 secretion was quantified. Among these nanoparticles, only NLC resulted effective at inducing GLP-1 secretion in both murine and human L cells in vitro. The mRNA expression of proglucagon showed that this effect was due to an increased GLP-1 secretion and not to an increased GLP-1 synthesis. The mechanism by which NLC triggered GLP-1 secretion by L cells revealed an extracellular interaction of NLC, exerting a physiological GLP-1 secretion. We herein demonstrate that nanomedicine can be used to induce GLP-1 secretion from murine and human L cells.

Synthesis and In Vitro Evaluation of Polyethylene Glycol-Paclitaxel Conjugates for Lung Cancer Therapy.

PURPOSE: Pulmonary drug delivery is considered an attractive route of drug administration for lung cancer chemotherapy. However, fast clearance mechanisms result in short residence time of small molecule drugs in the lung. Therefore, achieving a sustained presence of chemotherapeutics in the lung is very challenging. In this study, we synthesized two different polyethylene glycol-paclitaxel ester conjugates with molecular weights of 6 and 20 kDa in order to achieve sustained release of paclitaxel in the lung. METHODS: One structure was synthesized with azide linker using "click" chemistry and the other structure was synthesized with a succinic spacer. The physicochemical and biological properties of the conjugates were characterized in vitro. RESULTS: Conjugation to polyethylene glycol improved the solubility of paclitaxel by up to four orders of magnitude. The conjugates showed good stability in phosphate buffer saline pH 6.9 (half-life ≥72 h) and in bronchoalveolar lavage (half-life of 3 to 9 h) at both molecular weights, but hydrolyzed quickly in mouse serum (half-life of 1 to 3 h). The conjugates showed cytotoxicity to B16-F10 melanoma cells and LL/2 Lewis lung cancer cells but less than free paclitaxel or Taxol, the commercial paclitaxel formulation. CONCLUSIONS: These properties imply that the conjugates have the potential to retain paclitaxel in the lung for a prolonged duration and to sustain its release locally for a better efficacy.

PLGA based drug delivery systems: Promising carriers for wound healing activity.

Wound treatment remains one of the most prevalent and economically burdensome healthcare issues in the world. Current treatment options are limited and require repeated administrations which led to the development of new therapeutics to satisfy the unmet clinical needs. Many potent wound healing agents were discovered but most of them are fragile and/or sensitive to in vivo conditions. Poly(lactic-co-glycolic acid) (PLGA) is a widely used biodegradable polymer approved by food and drug administration and European medicines agency as an excipient for parenteral administrations. It is a well-established drug delivery system in various medical applications. The aim of the current review is to elaborate the applications of PLGA based drug delivery systems carrying different wound healing agents and also present PLGA itself as a wound healing promoter. PLGA carriers encapsulating drugs such as antibiotics, anti-inflammatory drugs, proteins/peptides, and nucleic acids targeting various phases/signaling cycles of wound healing, are discussed with examples. The combined therapeutic effects of PLGA and a loaded drug on wound healing are also mentioned.

Nanostructured lipid carriers: Promising drug delivery systems for future clinics.

During the past decade, the number of studies describing nanostructured lipid carriers (NLCs)-based formulations has been dramatically increased. The raise in NLC exploitation is essentially due to defeated barriers within the technological process of lipid-based nanoparticles' formulation and increased knowledge of the underlying mechanisms of transport of NLCs via different routes of administration. This review article aims to give an overview on the current state of the art of NLC as controlled drug delivery systems for future clinics through novel NLC applications providing examples of successfull outcomes. The reported data clearly illustrate the promise of these nanoparticles for novel treatments in the near future. From the Clinical Editor: The understanding of the nanostructured lipid carriers (NLC)-based formulations has improved with continuing research recently. The result has seen an increase in the use of these in the clinical setting. In this comprehensive review, the authors discussed the current state and major challenges in the use of nanostructured lipid carriers as controlled drug delivery systems.

Skin electroporation of a plasmid encoding hCAP-18/LL-37 host defense peptide promotes wound healing.

Host defense peptides, in particular LL-37, are emerging as potential therapeutics for promoting wound healing and inhibiting bacterial growth. However, effective delivery of the LL-37 peptide remains limiting. We hypothesized that skin-targeted electroporation of a plasmid encoding hCAP-18/LL-37 would promote the healing of wounds. The plasmid was efficiently delivered to full-thickness skin wounds by electroporation and it induced expression of LL-37 in the epithelium. It significantly accelerated reepithelialization of nondiabetic and diabetic wounds and caused a significant VEGFa and interleukin (IL)-6 induction. IL-6 was involved in LL-37-mediated keratinocyte migration in vitro and IL-6 neutralizing antibodies delivered to mice were able to suppress the wound healing activity of the hCAP-18/LL-37 plasmid. In a hindlimb ischemia model, electroporation of the hCAP-18/LL-37 plasmid increased blood perfusion, reduced muscular atrophy, and upregulated the angiogenic chemokines VEGFa and SDF-1a, and their receptors VEGF-R and CXCR-4. These findings demonstrate that a localized gene therapy with LL-37 is a promising approach for the treatment of wounds.

Combined effects of PLGA and vascular endothelial growth factor promote the healing of non-diabetic and diabetic wounds.

Growth factor therapies to induce angiogenesis and thereby enhance the blood perfusion, hold tremendous potential to address the shortcomings of current impaired wound care modalities. Vascular endothelial growth factor stimulates (VEGF) wound healing via multiple mechanisms. Poly(lactic-co-glycolic acid) (PLGA) supplies lactate that accelerates neovascularization and promotes wound healing. Hence, we hypothesized that the administration of VEGF encapsulated in PLGA nanoparticles (PLGA-VEGF NP) would promote fast healing due to the sustained and combined effects of VEGF and lactate. In a splinted mouse full thickness excision model, compared with untreated, VEGF and PLGA NP, PLGA-VEGF NP treated wounds showed significant granulation tissue formation with higher collagen content, re-epithelialization and angiogenesis. The cellular and molecular studies revealed that PLGA-VEGF NP enhanced the proliferation and migration of keratinocytes and upregulated the expression of VEGFR2 at mRNA level. We demonstrated the combined effects of lactate and VEGF for active healing of non-diabetic and diabetic wounds. FROM THE CLINICAL EDITOR: The study of wound healing has been under a tremendous amount of research over recent years. In diabetic wounds, vasculopathy leading to localized ischemia would often result in delayed wound healing. In this article, the authors encapsulated vascular endothelial growth factor stimulates (VEGF) in PLGA nanoparticles and studies the potential pro-healing effects. It was found that the combination of these two components provided synergistic actions for healing. The encouraging results should provide a basis for combination therapy in the future.

Self-assembling doxorubicin-tocopherol succinate prodrug as a new drug delivery system: synthesis, characterization, and in vitro and in vivo anticancer activity.

Self-assembled prodrugs forming nanoaggregates are a promising approach to enhance the antitumor efficacy and to reduce the toxicity of anticancer drugs. To achieve this goal, doxorubicin was chemically conjugated to d-α-tocopherol succinate through an amide bond to form N-doxorubicin-α-d-tocopherol succinate (N-DOX-TOS). The prodrug self-assembled in water into 250 nm nanostructures when stabilized with d-α-tocopherol poly(ethylene glycol) 2000 succinate. Cryo-TEM analysis revealed the formation of nanoparticles with a highly ordered lamellar inner structure. NMR spectra of the N-DOX-TOS nanoparticles indicated that N-DOX-TOS is located in the core of the nanoparticles while PEG chains and part of the tocopherol are in the corona. High drug loading (34% w/w) and low in vitro drug release were achieved. In vitro biological assessment showed significant anticancer activity and temperature-dependent cellular uptake of N-DOX-TOS nanoparticles. In vivo, these nanoparticles showed a greater antitumor efficacy than free DOX. N-DOX-TOS nanoparticles might have the potential to improve DOX-based chemotherapy.