Impact of Targeting Moiety Type and Protein Corona Formation on the Uptake of Fn14-Targeted Nanoparticles by Cancer Cells.

The TWEAK receptor, Fn14, is a promising candidate for active targeting of cancer nanotherapeutics to many solid tumor types, including metastatic breast and primary brain cancers. Targeting of therapeutic nanoparticles (NPs) has been accomplished using a range of targeting moieties including monoclonal antibodies and related fragments, peptides, and small molecules. Here, we investigated a full-length Fn14-specific monoclonal antibody, ITEM4, or an ITEM4-Fab fragment as a targeting moiety to guide the development of a clinical formulation. We formulated NPs with varying densities of the targeting moieties while maintaining the decreased nonspecific adhesivity with receptor targeting (DART) characteristics. To model the conditions that NPs experience following intravenous infusion, we investigated the impact of serum exposure in relation to the targeting moiety type and surface density. To further evaluate performance at the cancer cell level, we performed experiments to assess differences in cellular uptake and trafficking in several cancer cell lines using confocal microscopy, imaging flow cytometry, and total internal reflection fluorescence microscopy. We observed that Fn14-targeted NPs exhibit enhanced cellular uptake in Fn14-high compared to Fn14-low cancer cells and that in both cell lines uptake levels were greater than observed with control, nontargeted NPs. We found that serum exposure increased Fn14-targeted NP specificity while simultaneously reducing the total NP uptake. Importantly, serum exposure caused a larger reduction in cancer cell uptake over time when the targeting moiety was an antibody fragment (Fab region of the monoclonal antibody) compared with the full-length monoclonal antibody targeting moiety. Lastly, we uncovered that full monoclonal antibody-targeted NPs enter cancer cells via clathrin-mediated endocytosis and traffic through the endolysosomal pathway. Taken together, these results support a pathway for developing a clinical formulation using a full-length Fn14 monoclonal antibody as the targeting moiety for a DART cancer nanotherapeutic agent.

Fn14-Directed DART Nanoparticles Selectively Target Neoplastic Cells in Preclinical Models of Triple-Negative Breast Cancer Brain Metastasis.

Triple-negative breast cancer (TNBC) patients with brain metastasis (BM) face dismal prognosis due to the limited therapeutic efficacy of the currently available treatment options. We previously demonstrated that paclitaxel-loaded PLGA-PEG nanoparticles (NPs) directed to the Fn14 receptor, termed "DARTs", are more efficacious than Abraxane─an FDA-approved paclitaxel nanoformulation─following intravenous delivery in a mouse model of TNBC BM. However, the precise basis for this difference was not investigated. Here, we further examine the utility of the DART drug delivery platform in complementary xenograft and syngeneic TNBC BM models. First, we demonstrated that, in comparison to nontargeted NPs, DART NPs exhibit preferential association with Fn14-positive human and murine TNBC cell lines cultured in vitro. We next identified tumor cells as the predominant source of Fn14 expression in the TNBC BM-immune microenvironment with minimal expression by microglia, infiltrating macrophages, monocytes, or lymphocytes. We then show that despite similar accumulation in brains harboring TNBC tumors, Fn14-targeted DARTs exhibit significant and specific association with Fn14-positive TNBC cells compared to nontargeted NPs or Abraxane. Together, these results indicate that Fn14 expression primarily by tumor cells in TNBC BMs enables selective DART NP delivery to these cells, likely driving the significantly improved therapeutic efficacy observed in our prior work.

Nanoparticle-assisted, image-guided laser interstitial thermal therapy for cancer treatment.

Laser interstitial thermal therapy (LITT) guided by magnetic resonance imaging (MRI) is a new treatment option for patients with brain and non-central nervous system (non-CNS) tumors. MRI guidance allows for precise placement of optical fiber in the tumor, while MR thermometry provides real-time monitoring and assessment of thermal doses during the procedure. Despite promising clinical results, LITT complications relating to brain tumor procedures, such as hemorrhage, edema, seizures, and thermal injury to nearby healthy tissues, remain a significant concern. To address these complications, nanoparticles offer unique prospects for precise interstitial hyperthermia applications that increase heat transport within the tumor while reducing thermal impacts on neighboring healthy tissues. Furthermore, nanoparticles permit the co-delivery of therapeutic compounds that not only synergize with LITT, but can also improve overall effectiveness and safety. In addition, efficient heat-generating nanoparticles with unique optical properties can enhance LITT treatments through improved real-time imaging and thermal sensing. This review will focus on (1) types of inorganic and organic nanoparticles for LITT; (2) in vitro, in silico, and ex vivo studies that investigate nanoparticles' effect on light-tissue interactions; and (3) the role of nanoparticle formulations in advancing clinically relevant image-guided technologies for LITT. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Implantable Materials and Surgical Technologies > Nanoscale Tools and Techniques in Surgery.

Harnessing nanomedicine for enhanced immunotherapy for breast cancer brain metastases.

Brain metastases (BMs) are the most common type of brain tumor, and the incidence among breast cancer (BC) patients has been steadily increasing over the past two decades. Indeed, ~ 30% of all patients with metastatic BC will develop BMs, and due to few effective treatments, many will succumb to the disease within a year. Historically, patients with BMs have been largely excluded from clinical trials investigating systemic therapies including immunotherapies (ITs) due to limited brain penetration of systemically administered drugs combined with previous assumptions that BMs are poorly immunogenic. It is now understood that the central nervous system (CNS) is an immunologically distinct site and there is increasing evidence that enhancing immune responses to BCBMs will improve patient outcomes and the efficacy of current treatment regimens. Progress in IT for BCBMs, however, has been slow due to several intrinsic limitations to drug delivery within the brain, substantial safety concerns, and few known targets for BCBM IT. Emerging studies demonstrate that nanomedicine may be a powerful approach to overcome such limitations, and has the potential to greatly improve IT strategies for BMs specifically. This review summarizes the evidence for IT as an effective strategy for BCBM treatment and focuses on the nanotherapeutic strategies currently being explored for BCBMs including targeting the blood-brain/tumor barrier (BBB/BTB), tumor cells, and tumor-supporting immune cells for concentrated drug release within BCBMs, as well as use of nanoparticles (NPs) for delivering immunomodulatory agents, for inducing immunogenic cell death, or for potentiating anti-tumor T cell responses.

Preoperative Delirium Nursing Model Initiatives to Determine the Incidence of Postoperative Delirium Among Elderly Orthopaedic Patients.

Delirium is a common neurocognitive disorder prevalent in hospitalized older adults. The development of delirium is associated with adverse health outcomes, including functional decline and mortality. This study aimed to investigate the incidence and risk factors of postoperative delirium in older adults (≥60 years) who underwent orthopaedic surgery. Elderly orthopaedic patients were recruited from a large urban hospital over 12 months. Patients were preoperatively screened for delirium and followed up at least once daily postoperatively until discharge. Of the 124 patients in the sample, 21 (16.9%) had postoperative delirium. There were no significant differences in any of the baseline characteristics between the delirious and nondelirious patients. Patients using antidepressants were more likely to develop postoperative delirium compared with those not using antidepressants (odds ratio: 2.72, p = .05). Postoperative delirium was common in this sample of older adults who underwent orthopaedic surgery. Aiming prevention strategies toward patients using antidepressants may help reduce the incidence of delirium in this population.

Olfactory bulb-targeted quantum dot (QD) bioconjugate and Kv1.3 blocking peptide improve metabolic health in obese male mice.

The olfactory system is a driver of feeding behavior, whereby olfactory acuity is modulated by the metabolic state of the individual. The excitability of the major output neurons of the olfactory bulb (OB) can be modulated through targeting a voltage-dependent potassium channel, Kv1.3, which responds to changes in metabolic factors such as insulin, glucose, and glucagon-like peptide-1. Because gene-targeted deletion or inhibition of Kv1.3 in the periphery has been found to increase energy metabolism and decrease body weight, we hypothesized that inhibition of Kv1.3 selectively in the OB could enhance excitability of the output neurons to evoke changes in energy homeostasis. We thereby employed metal-histidine coordination to self-assemble the Kv1.3 inhibitor margatoxin (MgTx) to fluorescent quantum dots (QDMgTx) as a means to label cells in vivo and test changes in neuronal excitability and metabolism when delivered to the OB. Using patch-clamp electrophysiology to measure Kv1.3 properties in heterologously expressed cells and native mitral cells in OB slices, we found that QDMgTx had a fast rate of inhibition, but with a reduced IC50, and increased action potential firing frequency. QDMgTx was capable of labeling cloned Kv1.3 channels but was not visible when delivered to native Kv1.3 in the OB. Diet-induced obese mice were observed to reduce body weight and clear glucose more quickly following osmotic mini-pump delivery of QDMgTx/MgTx to the OB, and following MgTx delivery, they increased the use of fats as fuels (reduced respiratory exchange ratio). These results suggest that enhanced excitability of bulbar output neurons can drive metabolic responses.

The dual-function of lipoic acid groups as surface anchors and sulfhydryl reactive sites on polymer-stabilized QDs and Au nanocolloids.

Coating inorganic nanocrystals [e.g., quantum dots (QDs) and gold nanoparticles] with polymer ligands presenting multiple lipoic acid anchoring groups provides nanocolloids with remarkable long-term colloidal and photophysical stability. Here, we show that the natural swelling of macromolecules leaves a fraction of the lipoic acid groups in the surface coating free, which are targeted for activation and conjugation to target molecules, using the reliable sulfhydryl-to-maleimide reaction. This implies that simple and efficient functionalization of the nanocrystals can be achieved without introducing additional reactive groups in the coating. We apply a photomediated ligand exchange strategy to luminescent QDs and AuNPs and react the resulting nanocrystals with maleimide Cy3 dye. We then use optical absorption and resonance energy transfer measurements applied to QD-Cy3 and AuNP-Cy3 conjugates to extract estimates for the fraction of accessible lipoic acid groups per QD or AuNP. In addition, we demonstrate the potential utility of this approach by constructing a ratiometric pH sensor made of QD-SNARF conjugates. Our ligand design combined with the photoligation strategy yield colloidally stable dispersions of QDs and AuNPs that present accessible reactive thiols, without introducing new functionalities or requiring disulfide reducing reagents, making them useful for potential use in applications such as biological sensing and imaging.

Intracellular Delivery of Gold Nanocolloids Promoted by a Chemically Conjugated Anticancer Peptide.

We report on the ability of a chemically synthesized anticancer peptide, SVS-1, to promote the rapid uptake of gold nanorods (AuNRs) and gold nanoparticles (AuNPs) by live HeLa cells. For this, AuNPs and AuNRs, surface ligated with a multicoordinating polymer that presents several amine groups per ligand, are simultaneously reacted with SVS-1 and Texas-Red dye; the latter allows fluorescence visualization of the nanocrystals. Using epifluorescence microscopy, we find that incubation of the SVS-1-conjugated AuNPs and AuNRs with a model cancer cell line yields extended staining throughout the cell cytoplasm, even at low conjugate concentrations (∼0.1 nM). Furthermore, uptake is specific to the SVS-1-conjugated nanocrystals. Additional endocytosis inhibition experiments, where cells have been incubated with the conjugates at 4 °C or in the presence of endocytic inhibitors, show that significant levels of conjugate uptake persist. These results combined indicate an uptake mechanism that does not necessarily rely on endocytosis, a promising finding with implications for the use of nanomaterials in the field of biology and nanomedicine.

Enhanced Uptake of Luminescent Quantum Dots by Live Cells Mediated by a Membrane-Active Peptide.

The steady progress made over the past three decades in growing a variety of inorganic nanomaterials, with discreet control over their size and photophysical properties, has been exploited to develop several imaging and sensing applications. However, full integration of these materials into biology has been hampered by the complexity of delivering them into cells. In this report, we demonstrate the effectiveness of a chemically synthesized anticancer peptide to facilitate the rapid delivery of luminescent quantum dots (QDs) into live cells. We combine fluorescence imaging microscopy, flow cytometry, and specific endocytosis inhibition experiments to probe QD-peptide conjugate uptake by different cell lines. We consistently find that a sizable fraction of the internalized conjugates does not co-localize with endosomes or the nuclei. These findings are extremely promising for the potential integration of various nanomaterials into biological systems.

Serological, genetic and clinical associations with increased health-care resource utilization in inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel diseases (IBD) are associated with significant morbidity and economic burden. The variable course of IBD creates a need for predictors of clinical outcomes and health resource utilization (HRU) to guide treatment decisions. We aimed to identify clinical, serological or genetic markers associated with inpatient resource utilization in patients with ulcerative colitis (UC) and Crohn's disease (CD). METHODS: Patients with IBD with available genetic and serological data who had at least one emergency department visit or hospitalization in a 3-year period were included. The primary outcome measure was HRU, as measured by the All Patient Refined Diagnosis Related Group classification system. Univariate and multivariate linear and logistic regression models were used to identify the associations with HRU. RESULTS: Altogether 858 (562 CD and 296 UC) patients were included. Anti-CBir1 seropositivity (P = 0.002, effect size [ES]: 0.762, 95% confidence interval [CI] 0.512-1.012) and low socioeconomic status (P = 0.005, ES: 1.620 [95% CI 1.091-2.149]) were independently associated with a high HRU. CD diagnosis (P = 0.006, ES: -0.701 [95% CI -0.959 to -0.443]) was independently associated with a low inpatient HRU. CONCLUSION: In patients with IBD who required at least one emergency department visit or hospitalization, anti-CBir1 antibody status may be a useful biomarker of HRU when formulating management strategies to reduce disease complications and resource utilization.

Self-Assembled Gold Nanoparticle-Fluorescent Protein Conjugates as Platforms for Sensing Thiolate Compounds via Modulation of Energy Transfer Quenching.

The ability of Au and other metal nanostructures to strongly quench the fluorescence of proximal fluorophores (dyes and fluorescent proteins) has made AuNP conjugates attractive for use as platforms for sensor development based on energy transfer interactions. In this study, we first characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal-histidine coordination, where parameters such as NP size and number of attached proteins are varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ∼95-97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further exploited these findings to develop a solution phase sensing platform targeting thiolate compounds. Energy transfer (ET) was employed as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. Our results show that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction, and type of thiol derivatives used. Further analysis of the PL recovery data provides a measure for the equilibrium dissociation constant (Kd-1) for these compounds. These findings combined indicate that the AuNP-fluorescent protein conjugates may offer a potentially useful platform for thiol sensing both in solution and in cell cultures.

Intracellular Delivery of Luminescent Quantum Dots Mediated by a Virus-Derived Lytic Peptide.

We describe a new quantum dot (QD)-conjugate prepared with a lytic peptide, derived from a nonenveloped virus capsid protein, capable of bypassing the endocytotic pathways and delivering large amounts of QDs to living cells. The polypeptide, derived from the Nudaurelia capensis Omega virus, was fused onto the C-terminus of maltose binding protein that contained a hexa-HIS tag at its N-terminus, allowing spontaneous self-assembly of controlled numbers of the fusion protein per QD via metal-HIS interactions. We found that the efficacy of uptake by several mammalian cell lines was substantial even for small concentrations (10-100 nM). Upon internalization the QDs were primarily distributed outside the endosomes/lysosomes. Moreover, when cells were incubated with the conjugates at 4 °C, or in the presence of chemical endocytic inhibitors, significant intracellular uptake continued to occur. These findings indicate an entry mechanism that does not involve endocytosis, but rather the perforation of the cell membrane by the lytic peptide on the QD surfaces.

Margatoxin-bound quantum dots as a novel inhibitor of the voltage-gated ion channel Kv1.3.

Venom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots (QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well-characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx (QD-MgTx) for their ability to inhibit Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells. Our data indicate that MgTx inhibits 79% of the outward current in Kv1.3-transfected cells and that the QD-MgTx conjugate is able to achieve a similar level of block, albeit a slightly reduced efficacy (66%) and at a slower time course (50% block by 10.9 ± 1.1 min, MgTx; vs. 15.3 ± 1.2 min, QD-MgTx). Like the unbound peptide, the QD-MgTx conjugate inhibits both Kv1.3 and Kv1.2 at a 1 nM concentration, whereas it does not inhibit other screened Shaker channels. We tested the ability of QD-MgTx to inhibit native Kv1.3 expressed in the mouse olfactory bulb (OB). In brain slices of the OB, the conjugate acted similarly to MgTx to inhibit Kv1.3, causing an increased action potential firing frequency attributed to decreased intraburst duration rather than interspike interval. Our data demonstrate a retention of known biophysical properties associated with block of the vestibule of Kv1.3 by QD-MgTx conjugate compared to that of MgTx, inferring QDs could provide a useful tool to deliver ion channel inhibitors to targeted tissues in vivo.

Multifunctional and High Affinity Polymer Ligand that Provides Bio-Orthogonal Coating of Quantum Dots.

We detail the design of hydrophilic metal-coordinating ligands and their use for the effective coating of luminescent quantum dots (QDs). The ligand design exploits the specific, reagent-free nucleophilic addition reaction of amine-modified molecules toward maleic anhydride to introduce several lipoic acid metal anchors, hydrophilic zwitterion moieties, and specific reactive groups along a poly(isobutylene-alt-maleic anhydride) (PIMA) chain. Tunable reactive groups tested in this study include azide, biotin, carboxyl, and amine. Cap exchange with these multilipoic acid ligands via a photochemical ligation strategy yields homogeneous QD dispersions that are colloidally stable over several biologically relevant conditions and for extended periods of time. The zwitterionic coating yields compact nanoparticle size and imparts nonsticky surface properties onto the QDs, preventing protein absorption. The introduction of a controllable number of reactive groups allows conjugation of the QDs to biomolecules via bio-orthogonal coupling chemistries including (1) attachment of the neurotransmitter dopamine to QDs via amine-isothiocyanate reaction to produce a platform capable of probing interactions with cysteine in proteins, based on charge transfer interactions; (2) self-assembly of biotinylated QDs with streptavidin-dye; and (3) ligation of azide-functionalized QDs to cyclooctyne-modified transferrin via copper-free click chemistry, used for intracellular delivery. This ligand design strategy can be used to prepare an array of metal-coordinating ligands adapted for coating other inorganic nanoparticles, including magnetic and plasmonic nanomaterials.

Controlling the Architecture, Coordination, and Reactivity of Nanoparticle Coating Utilizing an Amino Acid Central Scaffold.

We have developed a versatile strategy to prepare a series of multicoordinating and multifunctional ligands optimized for the surface-functionalization of luminescent quantum dots (QDs) and gold nanoparticles (AuNPs) alike. Our chemical design relies on the modification of l-aspartic acid precursor to controllably combine, through simple peptide coupling chemistry, one or two lipoic acid (LA) groups and poly(ethylene glycol) (PEG) moieties in the same ligand. This route has provided two sets of modular ligands: (i) bis(LA)-PEG, which presents two lipoic acids (higher coordination) appended onto a single end-functionalized PEG, and (ii) LA-(PEG)2 made of two PEG moieties (higher branching, with various end reactive groups) appended onto a single lipoic acid. These ligands are combined with a new photoligation strategy to yield hydrophilic and reactive QDs that are colloidally stable over a broad range of conditions, including storage at nanomolar concentration and under ambient conditions. AuNPs capped with these ligands exhibit excellent stability in various biological conditions and improved resistance against NaCN digestion. This route also provides compact nanocrystals with tunable surface reactivity. As such, we have covalently coupled QDs capped with bis(LA)-PEG-COOH to transferrin to facilitate intracellular uptake. We have also characterized and quantified the coupling of dye-labeled peptides to QD surfaces using fluorescence resonance energy transfer interactions in QD-peptide-dye assemblies.

A multifunctional polymer combining the imidazole and zwitterion motifs as a biocompatible compact coating for quantum dots.

We introduce a set of multicoordinating imidazole- and zwitterion-based ligands suited for surface functionalization of quantum dots (QDs). The polymeric ligands are built using a one-step nucleophilic addition reaction between poly(isobutylene-alt-maleic anhydride) and distinct amine-containing functionalities. This has allowed us to introduce several imidazole anchoring groups along the polymer chain for tight coordination to the QD surface and a controllable number of zwitterion moieties for water solubilization. It has also permitted the introduction of reactive and biomolecular groups for further conjugation and targeting. The QDs capped with these new ligands exhibit excellent long-term colloidal stability over a broad range of pH, toward excess electrolyte, in cell-growth media, and in the presence of natural reducing agents such as glutathione. These QDs are also resistant to the oxidizing agent H2O2. More importantly, by the use of zwitterion moieties as the hydrophilic block, this polymer design provides QDs with a thin coating and compact overall dimensions. These QDs are easily self-assembled with full size proteins expressed with a polyhistidine tag via metal-histidine coordination. Additionally, the incorporation of amine groups allows covalent coupling of the QDs to the neurotransmitter dopamine. This yields redox-active QD platforms that can be used to track pH changes and detect Fe ions and cysteine through charge-transfer interactions. Finally, we found that QDs cap-exchanged with folic acid-functionalized ligands could effectively target cancer cells, where folate-receptor-mediated endocytosis of QDs into living cells was time- and concentration-dependent.

Photoligation of an amphiphilic polymer with mixed coordination provides compact and reactive quantum dots.

We introduce a new set of multicoordinating polymers as ligands that combine two distinct metal-chelating groups, lipoic acid and imidazole, for the surface functionalization of QDs. These ligands combine the benefits of thiol and imidazole coordination to reduce issues of thiol oxidation and weak binding affinity of imidazole. The ligand design relies on the introduction of controllable numbers of lipoic acid and histamine anchors, along with hydrophilic moieties and reactive functionalities, onto a poly(isobutylene-alt-maleic anhydride) chain via a one-step nucleophilic addition reaction. We further demonstrate that this design is fully compatible with a novel and mild photoligation strategy to promote the in situ ligand exchange and phase transfer of hydrophobic QDs to aqueous media under borohydride-free conditions. Ligation with these polymers provides highly fluorescent QDs that exhibit great long-term colloidal stability over a wide range of conditions, including a broad pH range (3-13), storage at nanomolar concentration, under ambient conditions, in 100% growth media, and in the presence of competing agents with strong reducing property. We further show that incorporating reactive groups in the ligands permits covalent conjugation of fluorescent dye and redox-active dopamine to the QDs, producing fluorescent platforms where emission is controlled/tuned by Förster Resonance Energy Transfer (FRET) or pH-dependent charge transfer (CT) interactions. Finally, the polymer-coated QDs have been coupled to cell-penetrating peptides to facilitate intracellular uptake, while subsequent cytotoxicity tests show no apparent decrease in cell viability.