Intravenous Injection In Mice Research Articles

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Quantification of Poly(ethylene glycol) Crowding on Nanodiamonds toward Quantum Biosensor for Improved Prevention Effects on Protein Adsorption and Lung Accumulation.

Nanometer-sized diamonds (NDs) containing nitrogen vacancy centers have garnered significant attention as potential quantum sensors for reading various types of physicochemical information in vitro and in vivo. However, NDs intrinsically aggregate when placed in biological environments, hampering their sensing capacities. To address this issue, the grafting of hydrophilic polymers onto the surface of NDs has been demonstrated considering their excellent ability to prevent protein adsorption. To this end, crowding of the grafted chains plays a crucial role because it is directly associated with the antiadsorption effect of proteins; however, its quantitative evaluation has not been reported previously. In this study, we graft poly(ethylene glycol) (PEG) with various molecular weights onto NDs, determine their crowding using a gas adsorption technique, and disclose the cross-correlation between the pH in the grafting reaction, crowding density, molecular weight, and the prevention effect on protein adsorption. PEG-grafted NDs exhibit a pronounced effect on the prevention of lung accumulation after intravenous injection in mice. PEG crowding was compared to that calculated by using a diameter determined by dynamic light scattering (DLS) assuming a sphere.

Abstract LB438: Preclinical characterization of ONM-412, an ultra-pH sensitive nanoparticle encapsulated IL-12 fusion protein

Abstract Background: Interleukin-12 is a potent pleiotropic cytokine, but its clinical translation has been hindered by toxicities. To deliver IL-12 to tumors with high spatial and temporal precision while minimizing off-tumor effects, we have developed an ultra-pH sensitive nanoparticle platform - ON-BOARD - that shields payloads from systemic exposure and targets solid tumors by responding to tumor acidity. Herein, we report the preclinical efficacy and safety characterization of ONM-412, an ON-BOARD nanoparticle encapsulated IL-12Fc fusion protein, and muONM-412, an encapsulated murine IL-12Fc. Methods: Properties and stability profiles of ONM-412 were characterized. pH-specific bioactivity was determined in cell-based reporter and IFNγ induction assays while the activation of ONM-412 was compared to a protease-cleavable IL-12 prodrug. Efficacy and tolerability of muONM-412 were studied in vivo after intravenous injection in mice. Pharmacodynamic response was evaluated by measuring cytokine levels in plasma and tissue, and the changes in tumor immune microenvironment were characterized. Toxicity was measured by body weight loss, systemic cytokine levels and clinical chemistry. Anti-tumor efficacy of muONM-412 was evaluated in MC38 tumor-bearing mice. The safety and tolerability of ONM-412 is also undergoing evaluation in a toxicology study with cynomolgus monkeys. Body weight, hematology, clinical chemistry and urine analysis were performed. Results: ONM-412 showed high encapsulation efficiency (>85%) in uniformly distributed particles (Dh<50nm) with 6-month on-going shelf-life stability. pH-specific release was confirmed in vitro with a >100-fold activation window between the acid-activated and intact formulations by an HEK reporter assay and an IFNγ induction assay. ONM-412 showed rapid and complete recovery of IL-12 bioactivity in <10 minutes after acid-activation while MMP demasking of a prodrug required overnight incubation and compromised potency. In vivo, mice treated with muONM-412 demonstrated significantly improved tolerability compared to unencapsulated IL-12Fc: body weight loss was reduced, no liver toxicity was observed, and plasma IFNγ levels were >1,000-fold lower. muONM-412 induced tumor immune remodeling, with increased infiltration by IFNγ+ and GzmB+ CD8 T and NK cells, and demonstrated strong dose-responsive anti-tumor efficacy in MC38 tumor-bearing animals. Preliminary results from an on-going study in cynomolgus monkeys suggest ONM-412 is well-tolerated at equivalent doses to those that induced maximum antitumor response in mice. Conclusions: ONM-412 significantly improved tolerability over unencapsulated IL-12Fc and showed potent anti-tumor efficacy in mice. It was well-tolerated in mice and non-human primates, showing potential for clinical translation. Citation Format: Qingtai Su, Stephen Gutowski, Austin Burcham, Bhargavi Allu, Zirong Chen, Fiona Stavros, Ruolan Han, Jason B. Miller, Tian Zhao. Preclinical characterization of ONM-412, an ultra-pH sensitive nanoparticle encapsulated IL-12 fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB438.

Visualization of the hepatic and renal cell uptake and trafficking of tetrahedral DNA origami in tumour.

DNA nanostructures, known for their programmability, ease of modification, and favourable biocompatibility, have gained widespread application in the biomedical field. Among them, Tetrahedral DNA Origami (TDOs), as a novel DNA nanostructure, possesses well-defined structures, multiple modification sites, and large cavities, making it a promising drug carrier. However, current understanding of TDOs' interactions with biological systems, particularly with target cells and organs, remains unexplored, limiting its further applications in biomedicine. In this work, we prepared TDOs with an average particle size of 40 nm and labelled them with Cy5 fluorescent molecules. Following intravenous injection in mice, the uptake of TDOs by different types of liver and kidney cells was observed. Results indicated that TDOs accumulate in renal tubules and are metabolized by Kupffer cells, epithelial cells, and hepatocytes in the liver. Additionally, in a tumour-bearing mouse model, TDOs passively targeted tumour tissues and exhibited excellent tumour penetration and retention after rapid metabolism in hepatocytes. Our findings provide crucial insights for the development of TDO-based drug delivery systems.

Variations in the inner core affect the pharmacokinetics of indomethacin-encapsulated polymeric micelles

Hydrophobic ion pairing (HIP) is a drug encapsulation technology that uses electrostatic interactions between a drug and an additive. However, although polymeric micelles can encapsulate hydrophobic drugs in the core, the encapsulated drug often leaks. Therefore, we designed polymeric micelles with HIP functionalized in a hydrophobic inner core using three diblock copolymers comprising polypeptides with different ratios of polar and hydrophobic amino acids and polyethylene glycol (PEG) to encapsulate indomethacin (IND). The three IND-encapsulated HIP micelles showed different area under the curve (AUC) values as an index of blood retention after intravenous injection in mice. Despite having the same PEG shell, IND-PEG-poly(H/F)n showed a 1.56-fold higher AUC than IND-PEG-poly(D/F)n. PEG interface morphologies were evaluated to determine the differences in pharmacokinetic parameters caused by changes in inner core HIP patterns. The micellarized diblock copolymer was desorbed from IND-PEG-poly(D/F)n due to electrostatic repulsion between IND and the diblock copolymer comprising aspartic acid. Our results suggest that changes in the HIP patterns of the micelle inner core affected the PEG interface morphologies, such as PEG density and diblock copolymer desorption from micelles. These phenomena might lead to changes in the interaction of plasma proteins and drug dispositions.

Nogo-A neutralization in the central nervous system with a blood-brain barrier-penetrating antibody

The poor penetration of monoclonal antibodies (mAb) across the blood-brain barrier (BBB) impedes the development of regenerative therapies for neurological diseases. For example, Nogo-A is a myelin-associated protein highly expressed in the central nervous system (CNS) whose inhibitory effects on neuronal plasticity can be neutralized with direct administration of 11C7 mAb in CNS tissues/fluids, but not with peripheral administrations such as intravenous injections. Therefore, in the present study, we engineered a CNS-penetrating antibody against Nogo-A by combining 11C7 mAb and the single-chain variable fragment (scFv) of 8D3, a rat antibody binding transferrin receptor 1 (TfR) and mediating BBB transcytosis (11C7-scFv8D3). The binding of 11C7-scFv8D3 to Nogo-A and to TfR/CD71 was validated by capture ELISA and Biolayer Interferometry. After intravenous injection in mice, capture ELISA measurements revealed fast plasma clearance of 11C7-scFv8D3 concomitantly with brain and spinal cord accumulation at levels up to 19 fold as high as those of original 11C7 mAb. 11C7-scFv8D3 detection in the parenchyma indicated effective blood-to-CNS transfer. A single dose of 11C7-scFv8D3 induced stronger activation of the growth-promoting AkT/mTOR/S6 signaling pathway than 11C7 mAb or control antibody. Taken together, our results show that BBB-crossing 11C7-scFv8D3 engages Nogo-A in the mouse CNS and stimulates neuronal growth mechanisms.

Effects of PEG antibodies on in vivo performance of LNP-mRNA vaccines

Polyethylene glycol (PEG) plays important roles in stabilizing and lengthening circulation time of lipid nanoparticle (LNP) vaccines. Nowadays various levels of PEG antibodies have been detected in human blood, but the impact and mechanism of PEG antibodies on the in vivo performance of LNP vaccines has not been clarified thoroughly. By illustrating the distribution characteristics of PEG antibodies in human, the present study focused on the influence of PEG antibodies on the safety and efficacy of LNP-mRNA vaccine against COVID-19 in animal models. It was found that PEG antibodies led to shortened blood circulation duration, elevated accumulation and mRNA expression in liver and spleen, enhanced expression in macrophage and dendritic cells, while without affecting the production of anti-Spike protein antibodies of COVID-19 LNP vaccine. Noteworthily, PEG antibodies binding on the LNP vaccine increased probability of complement activation in animal as well as in human serum and led to lethal side effect in large dosage via intravenous injection of mice. Our data suggested that PEG antibodies in human was a risky factor of LNP-based vaccines for biosafety concerns but not efficacy.

Efficient delivery of mesenchymal stem/stromal cells to injured liver by surface PEGylation

BackgroundMesenchymal stem/stromal cells (MSCs) have been used in clinical trials for various diseases. These have certain notable functions such as homing to inflammation sites, tissue repair, and immune regulation. In many pre-clinical studies, MSCs administered into peripheral veins demonstrated effective therapeutic outcomes. However, most of the intravenously administered MSCs were entrapped in the lung, and homing to target sites was less than 1%. This occurred mainly because of the adhesion of MSCs to vascular endothelial cells in the lung. To prevent this adhesion, we modified the surface of MSCs with polyethylene glycol (PEG; a biocompatible polymer) using the avidin–biotin complex (ABC) method.MethodsThe surface of MSCs was modified with PEG using the ABC method. Then, the cell adhesion to mouse aortic endothelial cells and the tissue distribution of PEG-modified MSCs were evaluated. Moreover, the homing to the injured liver and therapeutic effect of PEG-modified MSCs were evaluated using carbon tetrachloride-induced acute liver failure model mice.ResultsThe PEG modification significantly suppressed the adhesion of MSCs to cultured mouse aortic endothelial cells as well as the entrapment of MSCs in the lungs after intravenous injection in mice. PEG-modified MSCs efficiently homed to the injured liver of carbon tetrachloride-induced acute liver failure model mice. More importantly, the cells significantly suppressed serum transaminase levels and leukocyte infiltration into the injured liver.ConclusionThese results indicate that PEG modification to the surface of MSCs can suppress the lung entrapment of intravenously administered MSCs and improve their homing to the injured liver.Graphical

Differences in tissue distribution ability of evodiamine and dehydroevodiamine are due to the dihedral angle of the molecule stereo-structure.

Introduction: This researcher focused at the evodiamine and dehydroevodiamine tissue distribution and structure-pharmacokinetics (PK) relationship after intravenous injection in mice. Methods: Using a transmembrane transport experiment, the permeability of evodiamine and dehydroevodiamine on Caco-2 cells was evaluated. The tissue distribution and pharmacokinetics (PK) of evodiamine and dehydroevodiamine in mice were studied. To comprehend the connection between structure and tissue distribution, physicochemical property evaluations and molecular electrostatic potential (MEP) calculations were performed. Results: Dehydroevodiamine's Papp values in vitro were 10-5cm/s, whereas evodiamine's were 10-6cm/s. At a dose of 5mg/kg, the brain concentration of dehydroevodiamine was 6.44 times more than that of evodiamine. By MEP or physicochemical measures, the permeability difference between evodiamine and dehydroevodiamine is unaffected. The dihedral angle of the stereo-structure appears to be the main cause of the difference in tissue distribution ability between evodiamine and dehydroevodiamine. Discussion: Dehydroevodiamine has a dihedral angle of 3.71° compared to 82.34° for evodiamine. Dehydroevodiamine can more easily pass through the phospholipid bilayer than evodiamine because it has a more planar stereo-structure. Dehydroevodiamine is therefore more likely to pass cross the blood-brain barrier and enter the brain in a tissue-specific manner.

Abstract 522: Selective estrogen receptor a-UPR activators (SERA): a novel class of small molecules that eradicate estrogen receptor positive tumors

Abstract While therapy of estrogen receptor alpha positive (ERα+) breast cancer is often initially successful with ERα modulating or inhibiting drugs, eventual drug resistance is universal and such treatments are rarely curative. To address this considerable medical need, several generations of novel anti-tumor small molecule agents specifically targeting the estrogen-ERα interaction have been synthesized and characterized. We are pursuing an alternative approach, activation of an endogenous cell death pathway downstream of ERα with non-competitive Selective Estrogen Receptor Activators (SERAs). These compounds exhibit their effects by dysregulating calcium homeostasis in ERα+ tumor cells leading to hyper-activation of the anticipatory unfolded protein response (a-UPR) and necrotic cell death, a mechanism distinct from current breast cancer therapeutics. These molecules cause increases in P-EIF2a and P-AMPK, and cleavage of ATF6α, which are all hallmarks of the hyper-activation of a-UPR. SERA molecules cause death of ERα+ breast cancer cells in culture with 24hr IC50 values in the low nanomolar range, including breast cancer cell lines harboring ERα mutations known to contribute to acquired ERα modulator therapy resistance, while IC50 values in ERα-negative cells are orders of magnitude higher. In the MCF-7 ERα+ breast cancer xenograft model, once weekly IV injections of SERA2 at 10mg/kg cause durable, complete tumor regression, and SERA2 also has impressive activity in a challenging-to-treat PDX model that is resistant to fulvestrant and tamoxifen. SERAs are well-tolerated after multiple intravenous injections in mice, rats, and dogs, and clinical trial-compatible formulations have been developed. These findings suggest that SERA molecules can address the important unmet clinical need for more effective therapies for patients with ERα+ tumors who progress on conventional SERM, SERD, and targeted therapies. Citation Format: Michael P. Mulligan, Matthew W. Boudreau, David J. Shapiro, Timothy M. Fan, Paul J. Hergenrother, Kerry Barnhart, David Azorsa, Jeff Kiefer, Spyro Mousses. Selective estrogen receptor a-UPR activators (SERA): a novel class of small molecules that eradicate estrogen receptor positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 522.

CRISPR interference for sequence-specific regulation of fibroblast growth factor receptor A in Schistosoma mansoni.

Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function studies targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A (FGFRA). SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2'-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.

Bone-targeting polyphosphodiesters that promote osteoblastic differentiation.

Polymers for pharmaceutical use have been attractive in medical treatments because of the conjugation of multifunctional components and their long circulation time in the blood stream. Bone-targeted drug delivery systems are also no exceptional, and several polymers have been proposed for the treatment of bone diseases, such as cancer metastasis and osteoporosis. Herein, we report that polyphosphodiesters (PPDEs) have a potential to enhance osteoblastic differentiation, and they have a targeting ability to bone tissues in vivo. Two types of PPDEs, poly (ethylene sodium phosphate) (PEP•Na) and poly (propylene sodium phosphate) (PPP•Na), have been synthesized. Regardless of the alkylene structure in the main chain of PPDEs, the gene expression of osteoblast-specific transcription factors and differentiation markers of mouse osteoblastic-like cells (MC3T3-E1 cells) cultured in a differentiation medium was significantly upregulated by the addition of PPDEs. Moreover, it was also clarified that the signaling pathway related to cytoplasmic calcium ions was activated by PPDEs. The mineralization of MC3T3-E1 cells has a similar trend with its gene expression and is synergistically enhanced by PPDEs with β-glycerophosphate. The biodistribution of fluorescence-labeled PPDEs was also determined after intravenous injection in mice. PPDEs accumulated well in the bone through the blood stream, whereas polyphosphotriesters (PPTEs) tended to be excreted from the kidneys. Hydrophilic PEP•Na showed a superior bone affinity as compared with PPP•Na. PPDEs could be candidate polymers for the restoration of bone remodeling and bone-targeting drug delivery platforms.

ERK signaling is required for nicotine-induced conditional place preference by regulating neuroplasticity genes expression in male mice

Nicotine is an addictive compound that interacts with nicotinic acetylcholine receptors (nAChRs) in the ventral tegmental area (VTA), inducing a release of dopamine in the nucleus accumbens (NAc). When neurons undergo repeated exposure to nicotine, several adaptive changes in neuroplasticity occur. Activation of nAChRs involves numerous intracellular signaling cascades that likely contribute to neuroplasticity and ultimately the establishment of nicotine addiction. Nevertheless, the molecular mechanisms underlying this adaptation remain unclear. To explore the effects of nicotine on neuroplasticity, a stable nicotine-induced conditioned place preference (CPP) model was constructed by intravenous injection in mice. Using a PCR array, we observed significant changes in the expression of synaptic plasticity-related genes in the VTA (16 mRNAs) and NAc (40 mRNAs). When mice were pre-treated with PD98059, an extracellular signal-regulated kinase (ERK) inhibitor, more gene expression changes in the VTA (53 mRNAs) and NAc (60 mRNAs) were found. Moreover, PD98059 pre-treatment blocked the increased p-ERK/ERK and p-CREB/CREB ratios and decreased the expression of synaptic plasticity-related proteins such as SAP102, PSD95, synaptophysin, and BDNF, these changes might contribute to preventing the establishment of nicotine-induced CPP. Furthermore, neurons from the VTA and NAc of nicotine CPP mice had an increased dendritic spine density and complexity of dendritic morphology by Golgi staining. PD98059 also blocked this dynamic. These results demonstrate that repeated exposure to nicotine may remold the expression of neuroplasticity-related genes by activating the ERK signaling pathway in the VTA and NAc, and is related to the establishment of nicotine-induced CPP.

Evaluation and manipulation of tissue and cellular distribution of cardiac progenitor cell-derived extracellular vesicles.

Cardiac progenitor cell-derived extracellular vesicles (CPC-EVs) have been successfully applied via different delivery routes for treating post-myocardial infarction injury in several preclinical models. Hence, understanding the in vivo fate of CPC-EVs after systemic or local, i.e. myocardial, delivery is of utmost importance for the further therapeutic application of CPC-EVs in cardiac repair. Here, we studied the tissue- and cell distribution and retention of CPC-EVs after intramyocardial and intravenous injection in mice by employing different EV labeling and imaging techniques. In contrast to progenitor cells, CPC-EVs demonstrated no immediate flush-out from the heart upon intramyocardial injection and displayed limited distribution to other organs over time, as determined by near-infrared imaging in living animals. By employing CUBIC tissue clearing and light-sheet fluorescent microscopy, we observed CPC-EV migration in the interstitial space of the myocardium shortly after EV injection. Moreover, we demonstrated co-localization with cTnI and CD31-positive cells, suggesting their interaction with various cell types present in the heart. On the contrary, after intravenous injection, most EVs accumulated in the liver. To potentiate such a potential systemic cardiac delivery route, targeting the cardiac endothelium could provide openings for directed CPC-EV therapy. We therefore evaluated whether decorating EVs with targeting peptides (TPs) RGD-4C or CRPPR connected to Lamp2b could enhance EV delivery to endothelial cells. Expression of both TPs enhanced CPC-EV uptake under in vitro continuous flow, but did not affect uptake under static cell culture conditions. Together, these data demonstrate that the route of administration influences CPC-EV biodistribution pattern and suggest that specific TPs could be used to target CPC-EVs to the cardiac endothelium. These insights might lead to a better application of CPC-EV therapeutics in the heart.

Delivery of Corn-Derived Nanoparticles with Anticancer Activity to Tumor Tissues by Modification with Polyethylene Glycol for Cancer Therapy.

We recently reported that intratumoral injection of corn-derived nanoparticles (cNPs) affords anticancer activity in tumor-bearing mice. To increase their applicability in cancer therapy, we examined the tissue distribution of cNPs after intravenous injection in mice, modified their surface with polyethylene glycol (PEG) to improve tumor delivery, and examined tissue distribution and anticancer activity of PEG-cNPs in tumor-bearing mice. N-(Carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG) was added to cNPs by sonication to obtain PEG-cNPs, and the ratio of DSPE-PEG to cNPs was optimized by evaluating the modification efficiency. cNPs and PEG-cNPs were labeled with fluorescent dyes DiO or DiR, and their tissue distribution was subsequently examined after intravenous administration to mice. Finally, we determined the anticancer activity and toxicity of PEG-cNPs. No detectable fluorescence intensity was observed in mouse serum after intravenous DiR-cNP injection. DSPE-PEG was successfully modified into cNPs, and a PEG:cNPs ratio of 50 was determined as optimal for preparing PEG-cNPs, based on their size and zeta potential. DiO-PEG-cNPs exhibited significantly higher serum concentrations and lower liver accumulation than DiO-cNPs. Moreover, DiR-PEG-cNPs accumulated in tumor tissues of colon26 tumor-bearing mice. Repeated intravenous PEG-cNP injections significantly retarded tumor growth, with no significant hepatotoxicity or nephrotoxicity. Overall, these results indicate that controlling the tissue distribution of cNPs via PEG modification on their surface can be a valuable strategy for developing intravenously injectable cNPs for cancer therapy.

Magnetic torque–driven living microrobots for increased tumor infiltration

Biohybrid bacteria-based microrobots are increasingly recognized as promising externally controllable vehicles for targeted cancer therapy. Magnetic fields in particular have been used as a safe means to transfer energy and direct their motion. Thus far, the magnetic control strategies used in this context rely on poorly scalable magnetic field gradients, require active position feedback, or are ill-suited to diffuse distributions within the body. Here, we present a magnetic torque-driven control scheme for enhanced transport through biological barriers that complements the innate taxis toward tumor cores exhibited by a range of bacteria, shown for Magnetospirillum magneticum as a magnetically responsive model organism. This hybrid control strategy is readily scalable, independent of position feedback, and applicable to bacterial microrobots dispersed by the circulatory system. We observed a fourfold increase in translocation of magnetically responsive bacteria across a model of the vascular endothelium and found that the primary mechanism driving increased transport is torque-driven surface exploration at the cell interface. Using spheroids as a three-dimensional tumor model, fluorescently labeled bacteria colonized their core regions with up to 21-fold higher signal in samples exposed to rotating magnetic fields. In addition to enhanced transport, we demonstrated that our control scheme offers further advantages, including the possibility for closed-loop optimization based on inductive detection, as well as spatially selective actuation to reduce off-target effects. Last, after systemic intravenous injection in mice, we showed significantly increased bacterial tumor accumulation, supporting the feasibility of deploying this control scheme clinically for magnetically responsive biohybrid microrobots.

In vivo visualization and molecular targeting of the cardiac conduction system.

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo, providing a proof of principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging, and arrhythmia management.

Binding of adenovirus species C hexon to prothrombin and the influence of hexon on vector properties in vitro and in vivo

The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.

Toxicity Assessment of Mesoporous Silica Nanoparticles upon Intravenous Injection in Mice: Implications for Drug Delivery.

Nanoparticles are popular tools utilized to selectively deliver drugs and contrast agents for identification and treatment of disease. To determine the usefulness and translational potential of mesoporous silica nanoparticles (MSNs), further evaluations of toxicity are required. MSNs are among the most utilized nano-delivery systems due to ease of synthesis, pore structure, and functionalization. This study aims to elucidate toxicity as a result of intravenous injection of 25 nm MSNs coated with chitosan (C) or polyethylene glycol (PEG) in mice. Following acute and chronic injections, blood was evaluated for standard blood chemistry and complete blood count analyses. Blood chemistry results primarily indicated that no abnormalities were present following acute or chronic injections of MSNs, or C/PEG-coated MSNs. After four weekly administered treatments, vital organs showed minor exacerbation of pre-existing lesions in the 35KPEG-MSN and moderate exacerbation of pre-existing lesions in uncoated MSN and 2KPEG-MSN treatment groups. In contrast, C-MSN treatment groups had minimal changes compared to controls. This study suggests 25 nm MSNs coated with chitosan should elicit minimal toxicity when administered as either single or multiple intravenous injections, but MSNs coated with PEG, especially 2KPEG may exacerbate pre-existing vascular conditions. Further studies should evaluate varying sizes and types of nanoparticles to provide a better overall understanding on the relation between nanoparticles and in vivo toxicity.

Molecular Signature of Astrocytes for Gene Delivery by the Synthetic Adeno-Associated Viral Vector rAAV9P1.

Astrocytes have crucial functions in the central nervous system (CNS) and are major players in many CNS diseases. Research on astrocyte‐centered diseases requires efficient and well‐characterized gene transfer vectors. Vectors derived from the Adeno‐associated virus serotype 9 (AAV9) target astrocytes in the brains of rodents and nonhuman primates. A recombinant (r) synthetic peptide‐displaying AAV9 variant, rAAV9P1, that efficiently and selectively transduces cultured human astrocytes, has been described previously. Here, it is shown that rAAV9P1 retains astrocyte‐targeting properties upon intravenous injection in mice. Detailed analysis of putative receptors on human astrocytes shows that rAAV9P1 utilizes integrin subunits αv, β8, and either β3 or β5 as well as the AAV receptor AAVR. This receptor pattern is distinct from that of vectors derived from wildtype AAV2 or AAV9. Furthermore, a CRISPR/Cas9 genome‐wide knockout screening revealed the involvement of several astrocyte‐associated intracellular signaling pathways in the transduction of human astrocytes by rAAV9P1. This study delineates the unique receptor and intracellular pathway signatures utilized by rAAV9P1 for targeting human astrocytes. These results enhance the understanding of the transduction biology of synthetic rAAV vectors for astrocytes and can promote the development of advanced astrocyte‐selective gene delivery vehicles for research and clinical applications.