δ-Tocopherol Effect on Endocytosis and Its Combination with Enzyme Replacement Therapy for Lysosomal Disorders: A New Type of Drug Interaction?

Induction of lysosomal exocytosis alleviates lysosomal storage of undigested metabolites in cell models of lysosomal disorders (LDs). However, whether this strategy affects other vesicular compartments, e.g., those involved in endocytosis, is unknown. This is important both to predict side effects and to use this strategy in combination with therapies that require endocytosis for intracellular delivery, such as lysosomal enzyme replacement therapy (ERT). We investigated this using δ-tocopherol as a model previously shown to induce lysosomal exocytosis and cell models of type A Niemann-Pick disease, a LD characterized by acid sphingomyelinase (ASM) deficiency and sphingomyelin storage. δ-Tocopherol and derivative CF3-T reduced net accumulation of fluid phase, ligands, and polymer particles via phagocytic, caveolae-, clathrin-, and cell adhesion molecule (CAM)-mediated pathways, yet the latter route was less affected due to receptor overexpression. In agreement, δ-tocopherol lowered uptake of recombinant ASM by deficient cells (known to occur via the clathrin pathway) and via targeting intercellular adhesion molecule-1 (associated to the CAM pathway). However, the net enzyme activity delivered and lysosomal storage attenuation were greater via the latter route. Data suggest stimulation of exocytosis by tocopherols is not specific of lysosomes and affects endocytic cargo. However, this effect was transient and became unnoticeable several hours after tocopherol removal. Therefore, induction of exocytosis in combination with therapies requiring endocytic uptake, such as ERT, may represent a new type of drug interaction, yet this strategy could be valuable if properly timed for minimal interference.

A Comparative Study on the Alterations of Endocytic Pathways in Multiple Lysosomal Storage Disorders.

Many cellular activities and pharmaceutical interventions involve endocytosis and delivery to lysosomes for processing. Hence, lysosomal processing defects can cause cell and tissue damage, as in lysosomal storage diseases (LSDs) characterized by lysosomal accumulation of undegraded materials. This storage causes endocytic and trafficking alterations, which exacerbate disease and hinder treatment. However, there have been no systematic studies comparing different endocytic routes in LSDs. Here, we used genetic and pharmacological models of four LSDs (type A Niemann-Pick, type C Niemann-Pick, Fabry, and Gaucher diseases) and evaluated the pinocytic and receptor-mediated activity of the clathrin-, caveolae-, and macropinocytic routes. Bulk pinocytosis was diminished in all diseases, suggesting a generic endocytic alteration linked to lysosomal storage. Fluid-phase (dextran) and ligand (transferrin) uptake via the clathrin route were lower for all LSDs. Fluid-phase and ligand (cholera toxin B) uptake via the caveolar route were both affected but less acutely in Fabry or Gaucher diseases. Epidermal growth factor-induced macropinocytosis was altered in Niemann-Pick cells but not other LSDs. Intracellular trafficking of ligands was also distorted in LSD versus wild-type cells. The extent of these endocytic alterations paralleled the level of cholesterol storage in disease cell lines. Confirming this, pharmacological induction of cholesterol storage in wild-type cells disrupted endocytosis, and model therapeutics restored uptake in proportion to their efficacy in attenuating storage. This suggests a proportional and reversible relationship between endocytosis and lipid (cholesterol) storage. By analogy, the accumulation of biological material in other diseases, or foreign material from drugs or their carriers, may cause similar deficits, warranting further investigation.

How Carrier Size and Valency Modulate Receptor-Mediated Signaling: Understanding the Link between Binding and Endocytosis of ICAM-1-Targeted Carriers.

Targeting of drug carriers to endocytic cell receptors facilitates intracellular drug delivery. Carrier size and number of targeting moieties (valency) influence cell binding and uptake. However, how these parameters influence receptor-mediated cell signaling (the link between binding and uptake) remains uncharacterized. We studied this using polymer carriers of different sizes and valencies, targeted to endothelial intercellular adhesion molecule-1 (ICAM-1), a marker overexpressed in many pathologies. Unexpectedly, induction of cell signals (ceramide and protein kinase C (PKC) enrichment and activation) and uptake, were independent of carrier avidity, total number of carriers bound per cell, cumulative cell surface area occupied by carriers, number of targeting antibodies at the carrier-cell contact, and cumulative receptor engagement by all bound carriers. Instead, "valency density" (number of antibodies per carrier surface area) ruled signaling, and carrier size independently influenced uptake. These results are key to understanding the interplay between carrier design parameters and receptor-mediated signaling conducive to endocytosis, paramount for intracellular drug delivery.

Altered Clathrin-Independent Endocytosis in Type A Niemann-Pick Disease Cells and Rescue by ICAM-1-Targeted Enzyme Delivery.

Pharmaceutical intervention often requires therapeutics and/or their carriers to enter cells via endocytosis. Therefore, endocytic aberrancies resulting from disease represent a key, yet often overlooked, parameter in designing therapeutic strategies. In the case of lysosomal storage diseases (LSDs), characterized by lysosomal accumulation of undegraded substances, common clinical interventions rely on endocytosis of recombinant enzymes. However, the lysosomal defect in these diseases can affect endocytosis, as we recently demonstrated for clathrin-mediated uptake in patient fibroblasts with type A Niemann-Pick disease (NPD), a disorder characterized by acid sphingomylinase (ASM) deficiency and subsequent sphingomyelin storage. Using similar cells, we have examined if this is also the case for clathrin-independent pathways, including caveolae-mediated endocytosis and macropinocytosis. We observed impaired caveolin-1 enrichment at ligand-binding sites in NPD relative to wild type fibroblasts, corresponding with altered uptake of ligands and fluid-phase markers by both pathways. Similarly, aberrant lysosomal storage of sphingomyelin induced by pharmacological means also diminished uptake. Partial degradation of the lysosomal storage by untargeted recombinant ASM led to partial uptake enhancement, whereas both parameters were restored to wild type levels by ASM delivery using model polymer nanocarriers specifically targeted to intercellular adhesion molecule-1. Carriers also restored caveolin-1 enrichment at ligand-binding sites and uptake through the caveolar and macropinocytic routes. These results demonstrate a link between lysosomal storage in NPD and alterations in clathrin-independent endocytosis, which could apply to other LSDs. Hence, this study shall guide the design of therapeutic approaches using viable endocytic pathways.

Tumor ablation and nanotechnology.

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

Intertwined mechanisms define transport of anti-ICAM nanocarriers across the endothelium and brain delivery of a therapeutic enzyme.

The interaction of drug delivery systems with tissues is key for their application. An example is drug carriers targeted to endothelial barriers, which can be transported to intra-endothelial compartments (lysosomes) or transcellularly released at the tissue side (transcytosis). Although carrier targeting valency influences this process, the mechanism is unknown. We studied this using polymer nanocarriers (NCs) targeted to intercellular adhesion molecule-1 (ICAM-1), an endothelial-surface glycoprotein whose expression is increased in pathologies characterized by inflammation. A bell-shaped relationship was found between NC targeting valency and the rate of transcytosis, where high and low NC valencies rendered less efficient transcytosis rates than an intermediate valency formulation. In contrast, an inverted bell-shape relationship was found for NC valency and lysosomal trafficking rates. Data suggested a model where NC valency plays an opposing role in the two sub-processes involved in transcytosis: NC binding-uptake depended directly on valency and exocytosis-detachment was inversely related to this parameter. This is because the greater the avidity of the NC-receptor interaction the more efficient uptake becomes, but NC-receptor detachment post-transport is more compromised. Cleavage of the receptor at the basolateral side of endothelial cells facilitated NC transcytosis, likely by helping NC detachment post-transport. Since transcytosis encompasses both sets of events, the full process finds an optimum at the intersection of these inverted relationships, explaining the bell-shaped behavior. NCs also trafficked to lysosomes from the apical side and, additionally, from the basolateral side in the case of high valency NCs which are slower at detaching from the receptor. This explains the opposite behavior of NC valency for transcytosis vs. lysosomal transport. Anti-ICAM NCs were verified to traffic into the brain after intravenous injection in mice, and both cellular and in vivo data showed that intermediate valency NCs resulted in higher delivery of a therapeutic enzyme, acid sphingomyelinase, required for types A and B Niemann-Pick disease.

ICAM-1-Targeted Nanocarriers Attenuate Endothelial Release of Soluble ICAM-1, an Inflammatory Regulator.

Targeting of drug nanocarriers (NCs) to intercellular adhesion molecule-1 (ICAM-1), an endothelial-surface protein overexpressed in many pathologies, has shown promise for therapeutic delivery into and across this lining. Yet, due to the role of ICAM-1 in inflammation, the effects of targeting this receptor need investigation. Since ICAM-1 binding by natural ligands (leukocyte integrins) results in release of the "soluble ICAM-1" ectodomain (sICAM-1), an inflammatory regulator, we investigated the influence of targeting ICAM-1 with NCs on this process. For this, sICAM-1 was measured by ELISA from cell-medium supernatants, after incubation of endothelial cell (EC) monolayers in the absence versus presence of anti-ICAM NCs. In the absence of NCs, ECs released sICAM-1 when treated with a pro-inflammatory cytokine (TNFα). This was reduced by inhibiting matrix metalloproteinases MMP-9 or MMP-2, yet inhibiting both did not render additive effects. Release of sICAM-1 mainly occurred at the basolateral versus apical side, and both MMP-9 and MMP-2 influenced apical release, while basolateral release depended on MMP-9. Interestingly, anti-ICAM NCs reduced sICAM-1 to a greater extent than MMP inhibition, both at the apical and basolateral sides. This effect was enhanced with time, although NCs had been removed after binding to cells, ruling out a "trapping" effect of NCs. Instead, inhibiting anti-ICAM NC endocytosis counteracted their inhibition on sICAM-1 release. Hence, anti-ICAM NCs inhibited sICAM-1 release by mobilizing ICAM-1 from the cell-surface into intracellular vesicles. Since elevated levels of sICAM-1 associate with numerous diseases, this effect represents a secondary benefit of using ICAM-1-targeted NCs for drug delivery.

Synthesizing and modifying peptides for chemoselective ligation and assembly into quantum dot-peptide bioconjugates.

Quantum dots (QDs) are well-established as photoluminescent nanoparticle probes for in vitro or in vivo imaging, sensing, and even drug delivery. A critical component of this research is the need to reliably conjugate peptides, proteins, oligonucleotides, and other biomolecules to QDs in a controlled manner. In this chapter, we describe the conjugation of peptides to CdSe/ZnS QDs using a combination of polyhistidine self-assembly and hydrazone ligation. The former is a high-affinity interaction with the inorganic surface of the QD; the latter is a highly efficient and chemoselective reaction that occurs between 4-formylbenzoyl (4FB) and 2-hydrazinonicotinoyl (HYNIC) moieties. Two methods are presented for modifying peptides with these functional groups: (1) solid phase peptide synthesis; and (2) solution phase modification of pre-synthesized, commercial peptides. We further describe the aniline-catalyzed ligation of 4FB- and HYNIC-modified peptides, in the presence of a fluorescent label on the latter peptide, as well as subsequent assembly of the ligated peptide to water-soluble QDs. Many technical elements of these protocols can be extended to labeling peptides with other small molecule reagents. Overall, the bioconjugate chemistry is robust, selective, and modular, thereby potentiating the controlled conjugation of QDs with a diverse array of biomolecules for various applications.

Active cellular sensing with quantum dots: transitioning from research tool to reality; a review.

The application of luminescent semiconductor quantum dots (QDs) within a wide range of biological imaging and sensing formats is now approaching its 15th year. The unique photophysical properties of these nanomaterials have long been envisioned as having the potential to revolutionize biosensing within cellular studies that rely on fluorescence. However, it is only now that these materials are making the transition towards accomplishing this goal. With the idea of understanding how to actively incorporate QDs into different types of cellular biosensing, we review the progress in many of the areas relevant to achieving this goal. This includes the synthesis of the QDs themselves, with an emphasis on minimizing potential toxicity, along with the general methods for making these nanocrystalline structures stable in aqueous media. We next survey some methods for conjugating QDs to biomolecules to allow them to participate in active biosensing. Lastly, we extensively review many of the applications where QDs have been demonstrated in an active role in cellular biosensing. These formats cover a wide range of possibilities including where the QDs have contributed to: monitoring the cell's interaction with its extracellular environment; elucidating the complex molecular interplay that characterizes the plasma membrane; understanding how cells continuously endocytose and exocytose materials across the cellular membrane; visualizing organelle trafficking; and, perhaps most importantly, monitoring the intracellular presence of target molecules such as nucleic acids, nutrients, cofactors, and ions or, alternatively, intracellular responses to external changes in the environment. We illustrate these processes with examples from the recent literature and focus on what QDs can uniquely contribute along with discussing the benefits and liabilities of each sensing strategy. A perspective on where this field is expected to develop in both the near and long-term is also provided.

Optical imaging and magnetic field targeting of magnetic nanoparticles in tumors.

To address efficacy issues of cancer diagnosis and chemotherapy, we have developed a magnetic nanoparticle (MNP) formulation with combined drug delivery and imaging properties that can potentially be used in image-guided drug therapy. Our MNP consists of an iron-oxide magnetic core coated with oleic acid (OA) and stabilized with an amphiphilic block copolymer. Previously, we reported that our MNP formulation can provide prolonged contrast for tumor magnetic resonance imaging and can be loaded with hydrophobic anticancer agents for sustained drug delivery. In this study, we developed MNPs with optical imaging properties using new near-infrared dyes to quantitatively determine their long-term biodistribution and tumor localization with and without an external magnetic field in mice with xenograft breast tumors. MNPs localized slowly in the tumor, reaching a peak 48 h post-injection before slowly declining over the next 11 days. One hour exposure of the tumor to a magnetic field further enhanced MNP localization to tumors. Our MNPs can be developed with combined drug delivery and multimodal imaging properties to improve cancer diagnosis, provide sustained treatment, and monitor therapeutic effects in tumors over time.