Bimodal brush-functionalized nanoparticles selective to receptor surface density.

Nanoparticles or drug carriers which can selectively bind to cells expressing receptors above a certain threshold surface density are very promising for targeting cells overexpressing specific receptors under pathological conditions. Simulations and theoretical studies have suggested that such selectivity can be enhanced by functionalizing nanoparticles with a bimodal polymer monolayer (BM) containing shorter ligated chains and longer inert protective chains. However, a systematic study of the effect of these parameters under tightly controlled conditions is still missing. Here, we develop well-defined and highly specific platforms mimicking particle-cell interface using surface chemistry to provide a experimental proof of such selectivity. Using surface plasmon resonance and atomic force microscopy, we report the selective adsorption of BM-functionalized nanoparticles, and especially, a significant enhanced selective behavior by using a BM with longer protective chains. Furthermore, a model is also developed to describe the repulsive contribution of the protective brush to nanoparticle adsorption. This model is combined with super-selectivity theory to support experimental findings and shows that the observed selectivity is due to the steric energy barrier which requires a high number of ligand-receptor bonds to allow nanoparticle adsorption. Finally, the results show how the relative length and molar ratio of two chains can be tuned to target a threshold surface density of receptors and thus lay the foundation for the rational design of BM-functionalized nanoparticles for selective targeting.

Caveolin-3 negatively regulates endocytic recycling of cardiac KATP channels.

Sarcolemmal ATP-sensitive potassium (KATP) channels play a vital role in cardioprotection. Cardiac KATP channels are enriched in caveolae and physically interact with the caveolae structural protein caveolin-3 (Cav3). Disrupting caveolae impairs the regulation of KATP channels through several signaling pathways. However, the direct functional effect of Cav3 on KATP channels is still poorly understood. Here, we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and showed that Cav3 greatly reduced KATP channel surface density and current amplitude in a caveolae-independent manner. A screen of Cav3 functional domains revealed that a 25 amino acid region in the membrane attachment domain of Cav3 is the minimal effective segment (MAD1). The peptide corresponding to the MAD1 segment decreased KATP channel current in a concentration-dependent manner with an IC50 of ∼5 µM. The MAD1 segment prevented KATP channel recycling, thus decreasing KATP channel surface density and abolishing the cardioprotective effect of ischemic preconditioning. Our research identified the Cav3 MAD1 segment as a novel negative regulator of KATP channel recycling, providing pharmacological potential in the treatment of diseases with KATP channel trafficking defects.NEW & NOTEWORTHY Cardiac KATP channels physically interact with caveolin-3 in caveolae. In this study, we investigated the functional effect of caveolin-3 on KATP channel activity and identified a novel segment (MAD1) in the C-terminus domain of Caveolin-3 that negatively regulates KATP channel surface density and current amplitude by impairing KATP channel recycling. The peptide corresponding to the MAD1 segment abolished the cardioprotective effect of ischemic preconditioning.

Effects of chronic stress on bladder morphology of rats and impact of comfort food diet as an ameliorating agent.

OBJECTIVES: To investigate the effects of chronic stress on bladder morphology and the impact of food preference (standard or comfort foods) on the bladder of stressed rats. METHODS: In total, 32 Wistar male rats (3 months old) were divided into four groups: control (C), stressed (S), control + comfort food (C + CF), and stressed + comfort food (S + CF). Groups C and C + CF were maintained under normal conditions, while groups S and S + CF were subjected to chronic stress by the restraint method. Groups C and S received standard rat chow, while groups C + CF and S + CF received comfort food (Froot Loops®) and standard chow. The stress stimuli were induced daily for 2 h over 8 weeks. After 8 weeks, all animals were killed, and the bladders were removed and used for histomorphometric analysis. RESULTS: Body mass was similar among the groups. Stress did not promote differences regarding food intake, but animals receiving comfort food showed higher calories intake (in kcal/Kg) than animals receiving only standard chow. The C + CF and S + CF groups preferred comfort food over the standard chow; this preference was higher in the S + CF than in the C + CF group. The surface density of smooth muscle was reduced in stressed animals, while connective tissue and elastic system fiber content were increased in stressed groups. Further, epithelial height was increased in rats submitted to chronic stress. The surface density of elastic system fibers was decreased by the consumption of comfort food. CONCLUSIONS: Chronic stress induces morphological modifications on the bladder wall and epithelium. These modifications may be related to lower urinary tract symptoms. Additionally, chronic stress caused a higher preference for comfort food intake which did not ameliorate or aggravate the stress-induced bladder alterations.

Exposure to MMVF in residential and commercial buildings: A literature review and quantitative synthesis.

Man-made vitreous fibers (MMVF) are a class of inorganic fibrous materials that include glass and mineral wools, continuous glass filaments, and refractory ceramic fibers valued for their insulative properties in high temperature applications. Potential health effects from occupational exposure to MMVF have been investigated since the 1970s, with focus on incidence of respiratory tract cancer among MMVF-exposed production workers. The general population may experience exposure to MMVF in residential and/or commercial buildings due to deterioration, construction, or other disruption of materials containing these fibers. Numerous studies have characterized potential exposures that may occur during material disruption or installation; however, fewer have aimed to measure background MMVF concentrations in residential and commercial spaces (i.e., non-production settings) to which the general population may be exposed. In this study, we reviewed and synthesized peer-reviewed studies that evaluated respirable MMVF exposure levels in non-production, indoor environments. Among studies that analyzed airborne respirable MMVF concentrations, 110-fold and 1.5-fold differences in estimated concentrations were observed for those studies utilizing phase contrast optical microscopy (PCOM) versus transmission electron microscopy (TEM) and scanning electron microscopy (SEM), respectively. A positive correlation was observed between respirable air concentrations of MMVF and total surface concentrations of MMVF in seldom-cleaned areas. Ultimately, available evidence suggests that both ambient air and surface concentrations of MMVF in indoor environments are consistently lower than exposure limits developed to prevent negative health outcomes among sensitive populations.

Analysis of Lidar Actuator System Influence on the Quality of Dense 3D Point Cloud Obtained with SLAM.

Mobile mapping technologies, based on techniques such as simultaneous localization and mapping (SLAM) and surface-from-motion (SfM), are being vigorously developed both in the scientific community and in industry. They are crucial concepts for automated 3D surveying and autonomous vehicles. For various applications, rotating multiline scanners, manufactured, for example, by Velodyne and Ouster, are utilized as the main sensor of the mapping hardware system. However, their principle of operation has a substantial drawback, as their scanning pattern creates natural gaps between the scanning lines. In some models, the vertical lidar field of view can also be severely limited. To overcome these issues, more sensors could be employed, which would significantly increase the cost of the mapping system. Instead, some investigators have added a tilting or rotating motor to the lidar. Although the effectiveness of such a solution is usually clearly visible, its impact on the quality of the acquired 3D data has not yet been investigated. This paper presents an adjustable mapping system, which allows for switching between a stable, tilting or fully rotating lidar position. A simple experiment in a building corridor was performed, simulating the conditions of a mobile robot passing through a narrow tunnel: a common setting for applications, such as mining surveying or industrial facility inspection. A SLAM algorithm is utilized to create a coherent 3D point cloud of the mapped corridor for three settings of the sensor movement. The extent of improvement in the 3D data quality when using the tilting and rotating lidar, compared to keeping a stable position, is quantified. Different metrics are proposed to account for different aspects of the 3D data quality, such as completeness, density and geometry coherence. The ability of SLAM algorithms to faithfully represent selected objects appearing in the mapped scene is also examined. The results show that the fully rotating solution is optimal in terms of most of the metrics analyzed. However, the improvement observed from a horizontally mounted sensor to a tilting sensor was the most significant.

Covalently Grafted Peptides to Decellularized Pericardium: Modulation of Surface Density.

The covalent functionalization of synthetic peptides allows the modification of different biomaterials (metallic, polymeric, and ceramic), which are enriched with biologically active sequences to guide cell behavior. Recently, this strategy has also been applied to decellularized biological matrices. In this study, the covalent anchorage of a synthetic peptide (REDV) to a pericardial matrix decellularized via Schiff base is realized starting from concentrated peptide solutions (10-4 M and 10-3 M). The use of a labeled peptide demonstrated that as the concentration of the working solution increased, the surface density of the anchored peptide increased as well. These data are essential to pinpointing the concentration window in which the peptide promotes the desired cellular activity. The matrices were extensively characterized by Water Contact Angle (WCA) analysis, Differential Scanning Calorimetry (DSC) analysis, geometric feature evaluation, biomechanical tests, and preliminary in vitro bioassays.

Poly-adenine-mediated spherical nucleic acids for interfacial recognition of kanamycin.

Kanamycin fluorescence aptasensors were created using a series of di-block oligonucleotide modified gold nanoparticles with various lengths of poly-adenine. In the presence of kanamycin, the double strand structure of the aptamer-reporter strand complex is disrupted, and the dye-labelled reporter strand detaches from the surface of gold nanoparticles, resulting in fluorescence recovery (Ex/Em = 485/520 nm). By adjusting the number of consecutive adenines, the programable aptamer density can be implemented on the gold nanoparticle surface, and the conformation of nucleic acid changed from lying-down to up-right. The apparent binding constant, binding kinetics, and limit of detection of the prepared aptasensors were carefully examined to explore the influence of surface density. Under the optimum condition, the aptasensor had a tenfold lower limit of detection than the thiolated aptamer modified one, as low as 23.6 nM, when a di-block oligonucleotide with twenty consecutive adenines tailed. In addition, satisfactory recoveries ranging from 96.33 to 99.47% were achieved in spiked milk samples with relative standard deviation of 1.2-6.9% (n = 3). This surface density regulation strategy holds great promise in other aptamer-based interfacial recognition and sensing. Schematic presentation of di-block oligonucleotide modified gold nanoparticle with different surface densities and its kanamycin sensing application.

Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol.

The sensing properties of immunosensors are determined not only by the amount of immobilized antibodies but also by the number of effective antigen-binding sites of the immobilized antibody. Protein A (PA) exhibits a high degree of affinity with the Fc part of IgG antibody to feasibly produce oriented antibody immobilization. This work proposes a simple method to control the PA surface density on gold nanostructure (AuNS)-deposited screen-printed carbon electrodes (SPCEs) by mixing concentration-varied PA and bovine serum albumin (BSA), and to explore the effect of PA density on the affinity attachment of anti-salbutamol (SAL) antibodies by electrochemical impedance spectroscopy. A concentration of 100 µg/mL PA and 100 µg/mL BSA can obtain a saturated coverage on the 3-mercaptoproponic acid (MPA)/AuNS/SPCEs and exhibit a 50% PA density to adsorb the amount of anti-SAL, more than other concentration-varied PA/BSA-modified electrodes. Compared with the randomly immobilized anti-SAL/MPA/AuNS/SPCEs and the anti-SAL/PA(100 µg/mL):BSA(0 µg/mL)/MPA/AuNS/SPCE, the anti-SAL/PA(100 µg/mL): BSA(100 µg/mL)/MPA/AuNS/SPCE-based immunosensors have better sensing properties for SAL detection, with an extremely low detection limit of 0.2 fg/mL and high reproducibility (<2.5% relative standard deviation). The mixture of PA(100 µg/mL):BSA(100 µg/mL) for the modification of AuNS/SPCEs has great promise for forming an optimal protein layer for the oriented adsorption of IgG antibodies to construct ultrasensitive SAL immunosensors.

Effect of Glucosamine Conjugate-Functionalized Liposomes on Glioma Cell and Healthy Brain: An Insight for Future Application in Brain Infusion.

Conjugation of D-glucosamine with lipophilic moiety can ease its application in surface modification of liposomes. Interestingly, although D-glucosamine is safe, studies have shed light on "toxic effect" of its conjugates on cancer cells and highlighted its application in targeting glioma. However, understanding the safety of such conjugates for local delivery to the brain is unavailable. Herein, after successful synthesis of D-glucosamine conjugate (GC), the toxicity of functionalized liposome was evaluated both in vitro and in vivo. The study revealed a significant effect on cytotoxicity and apoptosis in vitro as assessed on grade IV-resistant glioma cell lines, SF268, U87MG, using MTT assay and PI staining. Additionally, this effect was not observed on normal human erythrocytes in the hemolysis assay. Furthermore, we demonstrated that GC liposomes were non-toxic to the normal brain tissues of healthy Sprague-Dawley rats. Successful functionalization yielded liposome with uniform particle size, stability, and cellular uptake. With < 10% hemolysis, all the liposomal formulations demonstrated hemato-compatibility but led to high glioma cytotoxicity. The surface density of conjugate played an important role in tumor toxicity (0.5 < 1.0 ≤ 2.0% molar ratio). PI staining revealed that compared to control cell, functionalization led 26-fold increase in induction of apoptosis in glioma cells. Absence of histological and behavioral changes along with the absence of caspase-3 in brain tissue confirmed the suitability of the system for direct infusion in the brain. Thus, this study will aid the future development of clinically useful local chemotherapeutic without "add-in" side effects.

Succinic anhydride functionalized microcantilevers for protein immobilization and quantification.

Microcantilever-based systems have been proposed as sensing platforms owing to their high sensitivity when used as mass sensors. The controlled immobilization on a surface of biomolecules used as recognition elements is fundamental in order to realize a highly specific and sensitive biosensor. Here, we introduce for the first time the application to a microcantilever-based system of a reliable chemical functionalization consisting of silanization with an aminosilane followed by a modification resulting in a carboxylated thin film. This chemical functionalization was tested for reproducibility of molecule deposition and for its protein grafting ability. Finally, this system was employed for the quantification of grafted proteins on the microcantilever surface. Moreover, a theoretical surface density of immobilized proteins estimated with bioinformatics tools was compared with the experimental surface density data, providing information about the orientation that the biomolecules assumed with respect to the sensing surface.

Effects of the Surface Densities of Glycoclusters on the Determination of Their IC50 and Kd Value Determination by Using a Microarray.

Pseudomonas aeruginosa (PA) is an opportunistic bacterium involved in 10-30% of nosocomial diseases. It causes severe lung injury to cystic fibrosis patients, often leading to patient death. PA strains are multidrug resistant, thus making the design of new therapeutics a challenge for public health. One promising therapeutic option is to design glycoclusters that target the virulence factor of PA. LecA is a galactose-specific lectin that might be involved in adhesion and biofilm formation by PA. The DNA-directed immobilization (DDI) microarray is a powerful tool for screening and understanding of structure-activity relationships between glycoclusters and lectins. High-throughput and multiplexed analysis of lectin-glycocluster interactions on a DDI microarray allows measurement of IC50 and dissociation constant (Kd ) values with minute amounts of material. In order to study the robustness of the DDI microarray in determination of IC50 and Kd values, the impact of glycocluster surface density was investigated. The data obtained show that measured IC50 values were influenced by glycocluster surface density: as the density of glycoclusters increases, the measured IC50 values increase too. In contrast, the measured Kd values were not affected by glycocluster surface density, provided that the experimental conditions allow interaction between glycocluster and lectin at single-molecule level (no surface cluster effect).

Protein engineering and the use of molecular modeling and simulation: the case of heterodimeric Fc engineering.

Computational and structure guided methods can make significant contributions to the development of solutions for difficult protein engineering problems, including the optimization of next generation of engineered antibodies. In this paper, we describe a contemporary industrial antibody engineering program, based on hypothesis-driven in silico protein optimization method. The foundational concepts and methods of computational protein engineering are discussed, and an example of a computational modeling and structure-guided protein engineering workflow is provided for the design of best-in-class heterodimeric Fc with high purity and favorable biophysical properties. We present the engineering rationale as well as structural and functional characterization data on these engineered designs.

Loss of p14 diminishes immunogenicity in melanoma via non-canonical Wnt signaling by reducing the peptide surface density.

Immunotherapy has achieved tremendous success in melanoma. However, only around 50% of advanced melanoma patients benefit from immunotherapy. Cyclin-dependent kinase inhibitor 2A (CDKN2A), encoding the two tumor-suppressor proteins p14ARF and p16INK4a, belongs to the most frequently inactivated gene loci in melanoma and leads to decreased T cell infiltration. While the role of p16INK4a has been extensively investigated, knowledge about p14ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished their recognition and killing by melanoma differentiation antigen (MDA)-specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. Immunopeptidomic analyses revealed that antigen presentation via human leukocyte antigen class I (HLA-I) molecules was enhanced upon p14ARF downregulation in general, but absolute and relative expression of cognate peptides was decreased. However, this phenotype is associated with a favorable outcome for melanoma patients. Limiting Wnt5a signaling reverted this phenotype, suggesting an involvement of non-canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA-specific T cell responses by decreasing both absolute and relative MDA-peptide presentation in melanoma.

Improved physicochemical stability of fish oil nanoemulsion via a dense interfacial layer formed by hyaluronic acid-poly(glyceryl)10-stearate.

This study aimed to synthesize a novel emulsifier, hyaluronic acid-poly(glyceryl)10-stearate (HA-PG10-C18), and employ it for the fabrication of nanoemulsions incorporating deep-sea fish oil to improve their apparent solubility and physicochemical stability. 1H NMR and FT-IR analyses indicated successful synthesis of HA-PG10-C18. Nanoemulsions of deep-sea fish oil loaded with HA-PG10-C18 (HA-PG10-C18@NE) were successfully fabricated by ultrasonic emulsification. The fixed aqueous layer thickness (FALT) of PG10-C18@NE and HA-PG10-C18@NE was determined and the FALT of both nanoemulsions was similar, while the surface density of HA-PG10-C18@NE (4.92 × 10-12 ng/nm2) is 60 % higher than that of PG10-C18@NE (3.07 × 10-12 ng/nm2). Notably, HA-PG10-C18@NE demonstrated an exceptional physicochemical stability when exposed to various stressed environmental conditions, especially its freeze-thaw stability. Moreover, after simulated in vitro digestion, the HA-PG10-C18@NE exhibited a comparatively greater liberation of free fatty acids (94.0 ± 1.7 %) when compared to the release observed in PG10-C18@NE (85.5 ± 2.2 %).

A facile strategy for tuning the density of surface-grafted biomolecules for melt extrusion-based additive manufacturing applications.

Melt extrusion-based additive manufacturing (ME-AM) is a promising technique to fabricate porous scaffolds for tissue engineering applications. However, most synthetic semicrystalline polymers do not possess the intrinsic biological activity required to control cell fate. Grafting of biomolecules on polymeric surfaces of AM scaffolds enhances the bioactivity of a construct; however, there are limited strategies available to control the surface density. Here, we report a strategy to tune the surface density of bioactive groups by blending a low molecular weight poly(ε-caprolactone)5k (PCL5k) containing orthogonally reactive azide groups with an unfunctionalized high molecular weight PCL75k at different ratios. Stable porous three-dimensional (3D) scaffolds were then fabricated using a high weight percentage (75 wt.%) of the low molecular weight PCL5k. As a proof-of-concept test, we prepared films of three different mass ratios of low and high molecular weight polymers with a thermopress and reacted with an alkynated fluorescent model compound on the surface, yielding a density of 201-561 pmol/cm2. Subsequently, a bone morphogenetic protein 2 (BMP-2)-derived peptide was grafted onto the films comprising different blend compositions, and the effect of peptide surface density on the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) was assessed. After two weeks of culturing in a basic medium, cells expressed higher levels of BMP receptor II (BMPRII) on films with the conjugated peptide. In addition, we found that alkaline phosphatase activity was only significantly enhanced on films containing the highest peptide density (i.e., 561 pmol/cm2), indicating the importance of the surface density. Taken together, these results emphasize that the density of surface peptides on cell differentiation must be considered at the cell-material interface. Moreover, we have presented a viable strategy for ME-AM community that desires to tune the bulk and surface functionality via blending of (modified) polymers. Furthermore, the use of alkyne-azide "click" chemistry enables spatial control over bioconjugation of many tissue-specific moieties, making this approach a versatile strategy for tissue engineering applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s42242-024-00286-2.

How water stability relates with timing, size, and community successions of harmful algal blooms: A case study in the Three Gorges Reservoir.

Effective management of Harmful Algal Blooms (HABs) requires understanding factors influencing their occurrence. This study explores these dynamics in the Pengxi River, a tributary of the Three Gorges Reservoir, focusing on nutrient stratification and algal blooms. We hypothesized that nutrient levels in eutrophic waters with stable stratification correlate with HAB magnitude and that disruption of stratification triggers blooms due to nutrient shifts. A 38-day sampling campaign in Gaoyang Lake (April 16-May 23, 2022) revealed that consistent weather between April 26 and May 16 led to a surface density layer, restricting nutrient transfer and causing a bloom with 173.0 µg L-1 Chl-a on May 1. After a heavy rain on May 18, a peak bloom on May 20, dominated by Ceratium hirundinella, showed 533 µg L-1 Chl-a. There was a significant negative correlation between Cyanobacteria and C. hirundinella biomasses (r = -0.296, P < 0.01), highlighting nutrient availability and physical stability's roles in regulating HABs.

Positioning of integrin ß1, caveolin-1 and focal adhesion kinase on the adhered membrane of spreading cells.

We have investigated the relationship between the spreading of anchorage-dependent cells and the surface-density distribution of plasma membrane adhesion proteins. The surface positioning and density of integrin ß1, caveolin-1 (cav-1), the phosphorylated caveolin-1 (p-cav-1) and the focal adhesion kinase (FAK) located on the adhering cell membrane (ACM) of HUVEC cells was studied. Imaging with TIRF microscopy was used, which enabled us to observe a few-nanometers-thin section of the cell above the plasma membrane in combination with image-based analyses. Integrin ß1 and cav-1 have spatial interdependence on the ACM. Cells treated with substances that act on cell spreading caused changes in the size of the ACM area, as well as a redistribution of several proteins under investigation. Changes to the ACM area correlated positively with those to the surface density of the cav-1. The high integrin ß1 and the low cav-1 surface density, and vice versa, following the treatments show that the presence of one of them not only spatially excludes, but also reduces, the occurrence of the other protein on the ACM, which indicates a regulative mechanism between integrin ß1 and cav-1.

The effect of AS1411 surface density on the tumor targeting properties of PEGylated silver nanotriangles.

Aim: To determine the optimal AS1411 density on polyethylene glycol (PEG)ylated silver nanotriangles (PNTs) for targeting breast cancer cells. Methods: PNTs modified with different AS1411 densities (ANTs) were constructed, characterized and evaluated for their targeting properties in breast cancer cells and a mouse model of breast cancer. Results: AS1411 was successfully conjugated to PNTs. The accumulation and cellular uptake of 10-ANTs were the highest. 10-ANTs plus near-IR laser irradiation displayed the greatest inhibitory effect on cell viability. However, 5-ANTs had the highest accumulation in tumor tissues. When combined with NIR laser, 5-ANTs exhibited the best in vivo photothermal therapy effect. Conclusion: The optimal AS1411 densities at the cellular and animal levels were 10% and 5%, respectively.

Rab35 GTPase positively regulates endocytic recycling of cardiac KATP channels.

ATP-sensitive K+ (KATP) channel couples membrane excitability to intracellular energy metabolism. Maintaining KATP channel surface expression is key to normal insulin secretion, blood pressure and cardioprotection. However, the molecular mechanisms regulating KATP channel internalization and endocytic recycling, which directly affect the surface expression of KATP channels, are poorly understood. Here we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and characterized Rab35 GTPase as a key regulator of KATP channel endocytic recycling. Electrophysiological recordings and surface biotinylation assays showed decreased KATP channel surface density with co-expression of a dominant negative Rab35 mutant (Rab35-DN), but not other recycling-related Rab GTPases, including Rab4, Rab11a and Rab11b. Immunofluorescence images revealed strong colocalization of Rab35-DN with recycling Kir6.2. Rab35-DN minimized the recycling rate of KATP channels. Rab35 also regulated KATP channel current amplitude in isolated adult cardiomyocytes by affecting its surface expression but not channel properties, which validated its physiologic relevance and the potential of pharmacologic target for treating the diseases with KATP channel trafficking defects.

Polyglycerol and Poly(ethylene glycol) exhibit different effects on pharmacokinetics and antibody generation when grafted to nanoparticle surfaces.

Poly(ethylene glycol) (PEG) is widely employed for passivating nanoparticle (NP) surfaces to prolong blood circulation and enhance localization of NPs to target tissue. However, the immune response of PEGylated NPs-including anti-PEG antibody generation, accelerated blood clearance (ABC), and loss of delivery efficacy-is of some concern, especially for treatments that require repeat administrations. Although polyglycerol (PG), which has the same ethylene oxide backbone as PEG, has received attention as an alternative to PEG for NP coatings, the pharmacokinetic and immunogenic impact of PG has not been studied systematically. Here, linear PG, hyperbranched PG (hPG), and PEG-coated polylactide (PLA) NPs with varying surface densities were studied in parallel to determine the pharmacokinetics and immunogenicity of PG and hPG grafting, in comparison with PEG. We found that linear PG imparted the NPs a stealth property comparable to PEG, while hPG-grafted NPs needed a higher surface density to achieve the same pharmacokinetic impact. While linear PG-grafted NPs induced anti-PEG antibody production in mice, they exhibited minimal accelerated blood clearance (ABC) effects due to the poor interaction with anti-PEG immunoglobulin M (IgM). Further, we observed no anti-polymer IgM responses or ABC effects for hPG-grafted NPs.