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Particle-based systems have become a state-of-the-art method for in vitro expanding cytotoxic T cells by tailoring their surface with activating molecules. However, commonly used methods utilize facile carbodiimide chemistry leading to uncontrolled orientation of the immobilized antibodies on the particle surface that can lead to poor binding to target cells. To address this, selective coupling strategies utilizing regioselective chemical groups such as disulfide bridges offer a simple approach. In this work we present a set of methods to investigate the effect of polymeric nanoparticles, conjugated with either regioselective- or randomly-immobilized antiCD3 and antiCD28 antibodies, on the activation potential, expansion and expression of activation markers in T cells. We show that nanoparticles with well-oriented monovalent antibodies conjugated via maleimide require fewer ligands on the surface to efficiently expand T cells compared to bivalent antibodies randomly-immobilized via carbodiimide conjugation. Analysis of the T cell expression markers reveal that the T cell phenotype can be fine-tuned by adjusting the surface density of well-oriented antibodies, while randomly immobilized antibodies showed no differences despite their ligand density. Both conjugation techniques induced cytotoxic T cells, evidenced by analyzing their Granzyme B secretion. Furthermore, antibody orientation affects the immunological synapse and T cell activation by changing the calcium influx profile upon activation. Nanoparticles with well-oriented antibodies showed lower calcium influx compared to their bivalent randomly-immobilized counterparts. These results highlight the importance of controlling the antibody density and orientation on the nanoparticle surface via controlled coupling chemistries, helping to develop improved particle-based expansion protocols to enhance T cell therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The transfusion of donor red blood cells (RBCs) is seriously hampered by important drawbacks that include limited availability and portability, the requirement of being stored in refrigerated conditions, a short shelf life or the need for RBC group typing and crossmatching. Thus, hemoglobin (Hb)-based oxygen (O 2 ) carriers (HBOCs) which make use of the main component of RBCs and the responsible protein for O 2 transport, hold a lot of promise in modern transfusion and emergency medicine. Despite the great progress achieved, it is still difficult to create HBOCs with a high Hb content to attain the high O 2 demands of our body. Herein a metal-phenolic self-assembly approach that can be conducted in water and in one step to prepare nanoparticles (NPs) fully made of Hb (Hb-NPs) is presented. In particular, by combining Hb with polyethylene glycol, tannic acid (TA) and manganese ions, spherical Hb-NPs with a uniform size around 350-525 nm are obtained. The functionality of the Hb-NPs is preserved as shown by their ability to bind and release O 2 over multiple rounds. The binding mechanism of TA and Hb is thoroughly investigated by UV-vis absorption and fluorescence spectroscopy. The binding site number, apparent binding constant at two different temperatures and the corresponding thermodynamic parameters are identified. The results demonstrate that the TA-Hb interaction takes place through a static mechanism in a spontaneous process as shown by the decrease in Gibbs free energy. The associated increase in entropy suggests that the TA-Hb binding is dominated by hydrophobic interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Whereas adoptive T cell therapy has been extensively studied for cancer treatment, the response is still limited primarily due to immune dysfunction related to poor cell engraftment, tumor infiltration and engagement, and lack of a target. In addition, the modification of therapeutic T cells often suffers from being complex and expensive. Here, we present a strategy to load T cells with SHP099, an allosteric SHP2 inhibitor, to enhance the therapeutic efficacy of the T cells. Remote-loading of SHP099 into lipid nanoparticles decorated with triarginine motifs resulted in nanocrystal formation of SHP099 inside the lipid vesicles and allowed high loading efficiency and prolonged retention of SHP099 nanocrystals within T cells. Cell-loaded SHP099 enabled sustained inhibition of the PD-1/PD-L1 signaling and increased cytolytic activity of the T cells. We show in a mouse model that tumor-homing T cells can circulate with the cargos, improving their tumor accumulation compared to systemically administered lipid nanoparticles. On an established solid tumor model, adoptively transferred SHP099 loaded T cells induced complete tumor eradication and durable immune memory against tumor rechallenging on all treated mice by effectively inhibiting the PD-1/PD-L1 checkpoint signal. We demonstrate that the combination of T cell therapy with SHP2 inhibition is a promising therapeutic strategy, and the lipid nanocrystal platform could be generalized as a promising approach for T cell loading of immunomodulatory drugs.

Local application of radioactive sources as brachytherapy is well established in oncology. This treatment is highly invasive however, due to the insertion of millimeter sized metal seeds. The authors report the development of a new concept for brachytherapy, based on gold-palladium (AuPd) alloy nanoparticles, intrinsically radiolabeled with 103 Pd. These are formulated in a carbohydrate-ester based liquid, capable of forming biodegradable gel-like implants upon injection. This allows for less invasive administration through small-gauge needles. [ 103 Pd]AuPd nanoparticles with sizes around 20 nm are prepared with radiolabeling efficiencies ranging from 79% to >99%. Coating with the hydrophobic polymer poly(N-isopropylacrylamide) leads to nanoparticle diameters below 40 nm. Dispersing the nanoparticles in ethanol with water insoluble carbohydrate esters gives "nanogels", a low viscosity liquid capable of solidifying upon injection into aqueous environments. Both nanoparticles and radioactivity are stably retained in the nanogel over 25 days (>99%) after formation in aqueous buffers. Animals bearing CT26 murine tumors are injected intratumorally with 25 MBq of the 103 Pd-nanogel, and display tumor growth delay and significantly increase median survival times compared with control groups. Excellent retention in the tumor of both the 103 Pd and the nanoparticle matrix itself is observed, demonstrating a potential for replacing currently used brachytherapy seeds., (© 2021 Wiley-VCH GmbH.)

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 10 3 antibodies/μm 2 ), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rationale: The ability to treat invalidating neurological diseases is impeded by the presence of the blood-brain barrier (BBB), which inhibits the transport of most blood-borne substances into the brain parenchyma. Targeting the transferrin receptor (TfR) on the surface of brain capillaries has been a popular strategy to give a preferential accumulation of drugs or nanomedicines, but several aspects of this targeting strategy remain elusive. Here we report that TfR-targeted gold nanoparticles (AuNPs) can accumulate in brain capillaries and further transport across the BBB to enter the brain parenchyma. Methods: We characterized our targeting strategy both in vitro using primary models of the BBB and in vivo using quantitative measurements of gold accumulation by inductively-coupled plasma-mass spectrometry together with morphological assessments using light microscopy after silver enhancement and transmission electron microscopy with energy-dispersive X-ray spectroscopy. Results: We find that the uptake capacity is significantly modulated by the affinity and valency of the AuNP-conjugated antibodies. Specifically, antibodies with high and low affinities mediate a low and intermediate uptake of AuNPs into the brain, respectively, whereas a monovalent (bi-specific) antibody improves the uptake capacity remarkably. Conclusion: Our findings indicate that monovalent ligands may be beneficial for obtaining transcytosis of TfR-targeted nanomedicines across the BBB, which is relevant for future design of nanomedicines for brain drug delivery., Competing Interests: Competing interests: All authors have declared no conflicting interest in relation to the theme and contents of the manuscript. The anti-TfRA, anti-TfRD, and anti-TfRA/BACE1 antibodies were obtained from Genentech, Inc. via a Material Transfer Agreement (MTA A15783). Genentech, Inc. had no influence on study design, experimental procedures, data analysis, or preparation of the manuscript.

Increasing cell survival in stem cell therapy is an important challenge for the field of regenerative medicine. Here, we report theranostic mesoporous silica nanoparticles that can increase cell survival through both diagnostic and therapeutic approaches. First, the nanoparticle offers ultrasound and MRI signal to guide implantation into the peri-infarct zone and away from the most necrotic tissue. Second, the nanoparticle serves as a slow release reservoir of insulin-like growth factor (IGF)-a protein shown to increase cell survival. Mesenchymal stem cells labeled with these nanoparticles had detection limits near 9000 cells with no cytotoxicity at the 250 µg/mL concentration required for labeling. We also studied the degradation of the nanoparticles and showed that they clear from cells in approximately 3 weeks. The presence of IGF increased cell survival up to 40% (p<0.05) versus unlabeled cells under in vitro serum-free culture conditions.

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue, but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work, we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol-coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time-consuming analytical characterization. We utilized this technique to analyze 243,000 mm³ of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail vein accumulated in the liver, whereas those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation.

Delivery is one of the most critical obstacles confronting nanoparticle use in cancer diagnosis and therapy. For most oncological applications, nanoparticles must extravasate in order to reach tumor cells and perform their designated task. However, little understanding exists regarding the effect of nanoparticle shape on extravasation. Herein we use real-time intravital microscopic imaging to meticulously examine how two different nanoparticles behave across three different murine tumor models. The study quantitatively demonstrates that high-aspect ratio single-walled carbon nanotubes (SWNTs) display extravasational behavior surprisingly different from, and counterintuitive to, spherical nanoparticles although the nanoparticles have similar surface coatings, area, and charge. This work quantitatively indicates that nanoscale extravasational competence is highly dependent on nanoparticle geometry and is heterogeneous.

Raman imaging offers unsurpassed sensitivity and multiplexing capabilities. However, its limited depth of light penetration makes direct clinical translation challenging. Therefore, a more suitable way to harness its attributes in a clinical setting would be to couple Raman spectroscopy with endoscopy. The use of an accessory Raman endoscope in conjunction with topically administered tumor-targeting Raman nanoparticles during a routine colonoscopy could offer a new way to sensitively detect dysplastic lesions while circumventing Raman's limited depth of penetration and avoiding systemic toxicity. In this study, the natural biodistribution of gold surface-enhanced Raman scattering (SERS) nanoparticles is evaluated by radiolabeling them with (64) Cu and imaging their localization over time using micropositron emission tomography (PET). Mice are injected either intravenously (IV) or intrarectally (IR) with approximately 100 microcuries (μCi) (3.7 megabecquerel (MBq)) of (64) Cu-SERS nanoparticles and imaged with microPET at various time points post injection. Quantitative biodistribution data are obtained as % injected dose per gram (%ID g(-1)) from each organ, and the results correlate well with the corresponding microPET images, revealing that IV-injected mice have significantly higher uptake (p < 0.05) in the liver (5 h = 8.96% ID g(-1); 24 h = 8.27% ID g(-1)) than IR-injected mice (5 h = 0.09% ID g(-1); 24 h = 0.08% ID g(-1)). IR-injected mice show localized uptake in the large intestine (5 h = 10.37% ID g(-1); 24 h = 0.42% ID g(-1)) with minimal uptake in other organs. Raman imaging of excised tissues correlate well with biodistribution data. These results suggest that the topical application of SERS nanoparticles in the mouse colon appears to minimize their systemic distribution, thus avoiding potential toxicity and supporting the clinical translation of Raman spectroscopy as an endoscopic imaging tool., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy.

The difficulty in delineating brain tumor margins is a major obstacle in the path toward better outcomes for patients with brain tumors. Current imaging methods are often limited by inadequate sensitivity, specificity and spatial resolution. Here we show that a unique triple-modality magnetic resonance imaging-photoacoustic imaging-Raman imaging nanoparticle (termed here MPR nanoparticle) can accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively. The MPRs were detected by all three modalities with at least a picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to MPR accumulation and retention by the tumors, with no MPR accumulation in the surrounding healthy tissue, allowing for a noninvasive tumor delineation using all three modalities through the intact skull. Raman imaging allowed for guidance of intraoperative tumor resection, and a histological correlation validated that Raman imaging was accurately delineating the brain tumor margins. This new triple-modality-nanoparticle approach has promise for enabling more accurate brain tumor imaging and resection. [ABSTRACT FROM AUTHOR]