pH-dependent Manner Research Articles

O-Acetyl Migration within the Sialic Acid Side Chain: A Mechanistic Study Using the Ab Initio Nanoreactor.

Many disease-causing viruses target sialic acids on the surface of host cells. Some viruses bind preferentially to sialic acids with O-acetyl modification at the hydroxyl group of C7, C8, or C9 on the glycerol-like side chain. Studies of proteins binding to sialosides containing O-acetylated sialic acids are crucial in understanding the related diseases but experimentally difficult due to the lability of the ester group. We recently showed that O-acetyl migration among hydroxyl groups of C7, C8, and C9 in sialic acids occurs in all directions in a pH-dependent manner. In the current study, we elucidate a full mechanistic pathway for the migration of O-acetyl among C7, C8, and C9. We used an ab initio nanoreactor to explore potential reaction pathways and density functional theory, pKa calculations, and umbrella sampling to investigate elementary steps of interest. We found that when a base is present, migration is easy in any direction and involves three key steps: deprotonation of the hydroxyl group, cyclization between the two carbons, and the migration of the O-acetyl group. This dynamic equilibrium may play a defensive role against pathogens that evolve to gain entry to the cell by binding selectively to one acetylation state.

Development and characteristics of layered EGCG/Montmorillonite hybrid: An oral controlled-release formulation of EGCG

An oral controlled-release formulation of epigallocatechin-3-gallate (EGCG) was developed by intercalating EGCG into the interlayer space of Food and Drug Administration-approved mucoadhesive montmorillonite (Mt) nanolayers, and characterized by ultraviolet–visible, X-ray diffraction, Fourier transform infrared, and thermogravimetry. The EGCG intercalation was only possible at acidic pH, and occurred through electrostatic and hydrogen bonding interactions allowing restacking of exfoliated Mt nanolayers with EGCG molecules. It was inferred that EGCG molecules were adsorbed flat on the interlayer surface of Mt in a monolayer and formed two-sided hydrogen bonds with the Mt faces, leading to a ∼2-fold increase in the EGCG decomposition temperature. A maximum EGCG loading of ∼40 wt% was achieved at pH 3, reaction time 1 h, and EGCG:Mt ratio 40:10 mg/mL. The EGCG/Mt hybrid showed a sustained release of EGCG in simulated gastric and intestinal fluids in a pH-dependent manner. Deprotonation of EGCG was responsible for further EGCG release at pH 7.4 compared to pH 1.2. The release profiles were fitted to the parabolic diffusion and Bhaskar models, implying that the release was controlled by a diffusion process. These results suggest that Mt with a high loading capacity can be used as the controlled-release nanocarrier of EGCG in oral administration.

Members of Venezuelan Equine Encephalitis complex entry into host cells by clathrin-mediated endocytosis in a pH-dependent manner

Pixuna virus (PIXV) and Río Negro virus (RNV) are mosquito-borne alphaviruses belonging to the Venezuelan Equine Encephalitis (VEE) complex, which includes pathogenic epizootic and enzootic subtypes responsible for life-threatening diseases in equines. Considering that the first steps in viral infection are crucial for the efficient production of new progeny, the aim of this study was to elucidate the early events of the replication cycle of these two viruses. To this end, we used chemical inhibitors and the expression of dominant-negative constructs to study the dependence of clathrin and endosomal pH on PIXV and RNV internalization mechanisms. We demonstrated that both viruses are internalized primarily via clathrin-mediated endocytosis, where the low pH in endosomes is crucial for viral replication. Contributing knowledge regarding the entry route of VEE complex members is important to understand the pathogenesis of these viruses and also to develop new antiviral strategies.

Effect of abiotic factors on the stability of chosen oligopeptides isolated from the freshwater cyanobacterium Woronichinia naegeliana

Cyanobacterial blooms have a significant impact on water quality. Implementing appropriate treatment methods to remove cyanobacterial secondary metabolites requires assessing their stability. In contrast to cyanotoxins, the effect of abiotic factors on cyanopeptides has been poorly studied. The present study analysed the impact of pH, temperature, visible and ultraviolet (UV) radiation on the stability of chosen oligopeptides found in a freshwater cyanobacterium Woronichinia naegeliana bloom that frequently appears in drinking water reservoirs worldwide. The tested cyanopeptolin 1081 (CYA-1081) and anabaenopeptin 899 (ANB-899) were relatively stable at room temperature for 12 weeks regardless of pH. However, boiling (100 °C) for one hour affected the partial decomposition of the compounds in a pH-dependent manner; the highest decrease in the initial content of CYA-1081 to 47.0% was recorded at pH 9, while for ANB-899 to 42.4% at pH 3. The tested cyanopeptolin was resistant to visible radiation, but UV radiation in an acidic condition caused its degradation by 32.3%. Treatment of ANB-899 with visible or UV radiation for 3 h caused its partial decomposition with a maximum reduction of 40.4 and 70.8%, respectively, at acidic pH. The presented data provided information on factors affecting the cyanopeptides persistence and may be useful in the search for and development of effective methods of removing cyanobacterial metabolites.

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

Fatty acids produced by the gut microbiota dampen host inflammatory responses by modulating intestinal SUMOylation

ABSTRACT The gut microbiota produces a wide variety of metabolites, which interact with intestinal cells and contribute to host physiology. The effect of gut commensal bacteria on host protein SUMOylation, an essential ubiquitin-like modification involved in various intestinal functions, remains, however, unknown. Here, we show that short chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) produced by the gut microbiota increase protein SUMOylation in intestinal cells in a pH-dependent manner. We demonstrate that these metabolites inactivate intestinal deSUMOylases and promote the hyperSUMOylation of nuclear matrix-associated proteins. We further show that BCFAs inhibit the NF-κB pathway, decrease pro-inflammatory cytokine expression, and promote intestinal epithelial integrity. Together, our results reveal that fatty acids produced by gut commensal bacteria regulate intestinal physiology by modulating SUMOylation and illustrate a new mechanism of dampening of host inflammatory responses triggered by the gut microbiota.

Requirement of Peptidyl-Prolyl Cis/Trans isomerases and chaperones for cellular uptake of bacterial AB-type toxins.

Bacterial AB-type toxins are proteins released by the producing bacteria and are the causative agents for several severe diseases including cholera, whooping cough, diphtheria or enteric diseases. Their unique AB-type structure enables their uptake into mammalian cells via sophisticated mechanisms exploiting cellular uptake and transport pathways. The binding/translocation B-subunit facilitates binding of the toxin to a specific receptor on the cell surface. This is followed by receptor-mediated endocytosis. Then the enzymatically active A-subunit either escapes from endosomes in a pH-dependent manner or the toxin is further transported through the Golgi to the endoplasmic reticulum from where the A-subunit translocates into the cytosol. In the cytosol, the A-subunits enzymatically modify a specific substrate which leads to cellular reactions resulting in clinical symptoms that can be life-threatening. Both intracellular uptake routes require the A-subunit to unfold to either fit through a pore formed by the B-subunit into the endosomal membrane or to be recognized by the ER-associated degradation pathway. This led to the hypothesis that folding helper enzymes such as chaperones and peptidyl-prolyl cis/trans isomerases are required to assist the translocation of the A-subunit into the cytosol and/or facilitate their refolding into an enzymatically active conformation. This review article gives an overview about the role of heat shock proteins Hsp90 and Hsp70 as well as of peptidyl-prolyl cis/trans isomerases of the cyclophilin and FK506 binding protein families during uptake of bacterial AB-type toxins with a focus on clostridial binary toxins Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridioides difficile CDT toxin, as well as diphtheria toxin, pertussis toxin and cholera toxin.

A Zn2+-triggered two-step mechanism of CLIC1 membrane insertion and activation into chloride channels.

ABSTRACTThe chloride intracellular channel (CLIC) protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains under investigation. Here, cellular Cl− efflux assays and immunofluorescence microscopy studies have identified intracellular Zn2+ release as the trigger for CLIC1 activation and membrane insertion. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing Cl− efflux in a pH-dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH-mediated activation of Cl− efflux.

Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein.

Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (CY) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus. In vivo results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.

Intestinal alkaline phosphatase (IAP, IAP Enhancer) attenuates intestinal inflammation and alleviates insulin resistance.

In this study, we investigated the effects of intestinal alkaline phosphatase (IAP) in controlled intestinal inflammation and alleviated associated insulin resistance (IR). We also explored the possible underlying molecular mechanisms, showed the preventive effect of IAP on IR in vivo, and verified the dephosphorylation of IAP for the inhibition of intestinal inflammation in vitro. Furthermore, we examined the preventive role of IAP in IR induced by a high-fat diet in mice. We found that an IAP + IAP enhancer significantly ameliorated blood glucose, insulin, low-density lipoprotein, gut barrier function, inflammatory markers, and lipopolysaccharide (LPS) in serum. IAP could dephosphorylate LPS and nucleoside triphosphate in a pH-dependent manner in vitro. Firstly, LPS is inactivated by IAP and IAP reduces LPS-induced inflammation. Secondly, adenosine, a dephosphorylated product of adenosine triphosphate, elicited anti-inflammatory effects by binding to the A2A receptor, which inhibits NF-κB, TNF, and PI3K-Akt signalling pathways. Hence, IAP can be used as a natural anti-inflammatory agent to reduce intestinal inflammation-induced IR.

Targeted co-delivery of doxorubicin and methotrexate to breast cancer cells by a pH-sensitive biocompatible polymeric system based on β-cyclodextrin crosslinked glycodendrimer with magnetic ZnO core

Nowadays, co-delivery of multiple therapeutics and combinational therapies using a delivery system is a promising approach to attain a synergistic anticancer effect. Hence, in the present study, we designed and synthesized a new pH-sensitive and biocompatible glycodendrimer immobilized on ZnO coated magnetic nanocomposite (Fe3O4@ZnO@CAMAD-β-CD) with antioxidant properties for co-delivery of doxorubicin (DOX) and methotrexate (MTX) to breast cancer cells. The structural properties of the new synthesized system were analyzed using various techniques. The drug loading results revealed that the Fe3O4@ZnO@CAMAD-β-CD nanocomposite displayed encapsulation efficiency (EE) of 96.08 % for DOX and 68.03 % for MTX. In vitro drug release study at pHs 5, 6.8, and 7.4 also showed that the release process was noticeably controlled, and pH-dependent manner with tumor target release behavior. Furthermore, the results of MTT cytotoxicity assessment, DAPI staining, confocal microscopy, and flow cytometry analysis against MDA-MB-231 breast cancer cell lines illustrated that the prepared Fe3O4@ZnO@CAMAD-β-CD has good biocompatibility and could easily enter the cells. The flow-cytometry analysis also showed that the apoptotic effects of DOX-MTX- Fe3O4@ZnO@CAMAD-β-CD have higher due to the synergistic effect in comparison to free drugs. The DPPH test for the study of antioxidant activity of Fe3O4@ZnO@CAMAD-β-CD nanocomposite showed that the carrier has excellent antioxidant activity (81.3 %). According to the results of this study, the prepared Fe3O4@ZnO@CAMAD-β-CD as a biocompatible carrier could be successfully used in the targeted co-delivery of DOX and MTX to cancer cells.

Design and Fabrication of pH-Sensitive Curcumin Coated Gold Nanorods as Anti-Angiogenic Targeting Nanostructures

Curcumin's limited bioavailability, caused by its hydrophobic nature, is a significant stumbling block to its application. This paper describes the development of a pH-responsive targetable carrier that overcomes the challenges associated with using curcumin. The use of gold nanorods in the creation of curcumin nanocarriers aids the combination of plasmonic phototherapy with biotherapy. This study used the pH-sensitive release and receptor targeting nanocarriers poly-L-histidine and ATWLPPR. The carrier's components are confirmed through synthesis and characterization. At a concentration of 0.66 g/mL), the functionalized gold nanorod nanocarrier had a 32 - 43 nm size range. The gold nanorod level is increased to 1.33 g/mL by crosslinking them with amine-functionalized PEG. Curcumin was released in a pH-dependent manner by the nanocarrier. Furthermore, in vitro tests to corroborate the efficiency of this therapeutic model show that when gold nanorods are routed through the peptide, the intake of gold nanorods is doubled.

Curcumin-loaded multifunctional chitosan gold nanoparticles: An enhanced PDT/PTT dual-modal phototherapeutic and pH-responsive antimicrobial agent

Overuse of antibiotics has led to the emergence of multidrug resistant (MDR) bacteria.. Photothermal (PTT) and photodynamic therapy (PDT) have may be effective alternatives for antibiotics in the treatment of bacterial infections. In this study, based on chitosan (CS)-coated gold nanoparticles, a pH stimulus-responsive drug delivery system was developed, which can anchor to the cell membrane for photodynamic therapy and photothermal therapy, and enhance the therapeutic potential of curcumin (Cur). Release experiments showed that AuNPs/CS-Cur nanocomposites released curcumin in a pH-dependent manner, which may facilitate the drug to be delivered to the acidic bacterial infection environment. CS as the outer layer covered on gold nanoparticles could improve the dispersibility of Cur in aqueous solution, gold nanoparticles prevent rapid photobleaching of curcumin, thus ensuring the yield of singlet oxygen under irradiation, and enhance the electrostatic binding with bacteria cell membrane. Under light conditions, AuNPs/CS-Cur can produce a large amount of reactive oxygen species and heat to kill S. aureus and E. coli. Compared with free Cur-mediated PDT, the complex significantly improved the synergistic PTT/PDT photoinactivation ability against S. aureus and E. coli. In addition, AuNPs/CS-Cur had good biocompatibility. Therefore, AuNPs/CS-Cur possessed the characteristics of electrostatic targeting, photodynamic and photothermal antibacterial therapy, which would become an efficient and safe antibacterial nano-platform and provide new ideas for the treatment of bacterial infection.

Formulation and evaluation of a smart drug delivery system of 5-fluorouracil for pH-sensitive chemotherapy

The conventional chemotherapeutic drugs have many side effects due to their non-selective tissue distribution, reduced drug concentration of the drug at the tumor site, and the drug resistance. To overcome these problems the chemotherapeutic agent should selectively accumulate the tumor site and stays there for a prolonged period of time releasing the payloads in a controlled manner. This can be achieved by the administration of a smart drug delivery system (SDDS) loaded with the active drug molecules. In this work, 5-fluorouracil (5-FU) is loaded into amine functionalised hollow mesoporous silica nanoparticles (HMSN-NH2) and then coated with a biocompatible polydopamine (PDA) to formulate SSDS for 5-FU for pH-sensitive drug release. The physiochemical properties were characterised; the structural morphology was observed by using optical microscope, scanning electron microscope and transmission electron microscope, chemical interaction between the drug and excipients were characterised from Fourier transform infrared spectroscopy, the entrapment efficiency of loaded drug and the pH-dependent drug release rate were evaluated using UV-visible spectroscopy. It was observed that, the drug is compatible with excipients by retaining all the characteristics peaks of 5-FU with negligible changes in the position in all physical mixtures. The PDA coated 5-FU loaded HMSN-NH2 also exhibits a nearly spherical and non-aggregated morphology. The release rate was showed to increase with increase in concentration of structure-directing agent (Triton X 100) in the rate of a maximum release at the end of 72 h in pH 4. The prepared novel PDA coated 5-FU HMSN-NH2 was found to be capable of delivering the anti-cancer drug 5-FU specifically at the tumor site in a pH-dependent stimuli-responsive manner. It also showed a controlled release for a period of 72 h. The enhanced cytotoxicity against HeLa cell line were found for the formulated SSD form.

Modulating Fingolimod (FTY720) Anti-SARS-CoV-2 Activity Using a PLGA-Based Drug Delivery System.

COVID-19 has resulted in more than 490 million people being infected worldwide, with over 6 million deaths by April 05th, 2022. Even though the development of safe vaccine options is an important step to reduce viral transmission and disease progression, COVID-19 cases will continue to occur, and for those cases, efficient treatment remains to be developed. Here, a drug repurposing strategy using nanotechnology is explored to develop a therapy for COVID-19 treatment. Nanoparticles (NPs) based on PLGA for fingolimod (FTY720) encapsulation show a size of ∼150 nm and high drug entrapment (∼90%). The NP (NP@FTY720) can control FTY720 release in a pH-dependent manner. Cytotoxicity assays using different cell lines show that NP@FTY720 displays less toxicity than the free drug. Flow cytometry and confocal microscopy reveal that NPs are actively internalized mostly through caveolin-mediated endocytosis and macropinocytosis pathways and co-localized with lysosomes. Finally, NP@FTY720 not only exhibits anti-SARS-CoV-2 activity at non-cytotoxic concentrations, but its biological potential for viral infection inhibition is nearly 70 times higher than that of free drug treatment. Based on these findings, the combination of drug repurposing and nanotechnology as NP@FTY720 is presented for the first time and represents a promising frontline in the fight against COVID-19.

Encapsulation of bioactives within electrosprayed κ-carrageenan nanoparticles

In this study, an electrospray synthesis approach was utilized in which a solution mixture of a sensitive bioactive agent, d-limonene (DL, R-(+)-Limonene), and a nature-inspired polymer, κ-carrageenan (κC) was applied to design DL-κC nanoparticles (NPs) in a one step process. The engineered DL-κC NPs displayed spherical morphology and the maximum encapsulation efficiency of NPs was about 97 % by altering the mass ratio of DL to κC. The developed DL-κC NPs showed a pH-dependent release manner in vitro. Both photostability and thermostability of DL were promoted by increasing the κC concentration, and >85 % of the original DL could be preserved following 120 min of UV-light exposure in the NPs with 0.5 % κC. The results demonstrated that electrosprayed κC NPs are promising candidates for the design of high-loading pH-sensitive NPs for encapsulation of highly sensitive bioactive agents.

Efficient adsorption and reduction of Cr(VI) by a novel polyaniline modified magnetic iron-based waterworks sludge from aqueous solution

As a major solid waste from the waterworks, the iron-based waterworks sludge (IBWS) was currently disposed of in landfills or discharged into water body, causing land consumption and environment contamination. This work investigated a novel reclamation of IBWS to create a composite for aqueous Cr(VI) adsorption and reduction. IBWS was magnetized by calcination and further modified with polyaniline (PANI) to get PANI@MIBWS for enhanced adsorbent separation and Cr(VI) removal. After being characterized using advanced techniques, the composite was employed in aqueous Cr(VI) removal studies. PANI@MIBWS removed Cr(VI) in a pH-dependent manner, with the highest Cr(VI) removal at pH 2. The spontaneous and endothermic Cr(VI) elimination process by the composite was best explained by pseudo-second-order and Langmuir models. In the sequence of Cl-＜SO42-＜PO43-, increasing Cl-, SO42- and PO43- concentrations resulted in declining Cr(VI) elimination. Chemical, electrostatic adsorption, Cr(VI) reduction to Cr (III), and chelation were involved into the Cr(VI) removal from water by PANI@MIBWS. The study found that the novel composite was effective for Cr(VI)-bearing wastewater treatment and had easy regenerability and good reusability, providing not only a fresh insight into the sustainable and eco-friendly disposal of IBWS, but also useful information for Cr(VI)-bearing wastewater treatment.

Liposomal Formulations of a New Zinc(II) Complex Exhibiting High Therapeutic Potential in a Murine Colon Cancer Model.

Colorectal cancer is the second leading cause of cancer-related mortality. Many current therapies rely on chemotherapeutic agents with poor specificity for tumor cells. The clinical success of cisplatin has prompted the research and design of a huge number of metal-based complexes as potential chemotherapeutic agents. In this study, two zinc(II) complexes, [ZnL2] and [ZnL(AcO)], where AcO is acetate and L is an organic compound combining 8-hydroxyquinoline and a benzothiazole moiety, were developed and characterized. Analytical and spectroscopic studies, namely, NMR, FTIR, and UV-Vis allowed us to establish the complexes’ structures, demonstrating the ligand-binding versatility: tetradentate in [ZnL(AcO)] and bidentate in [ZnL2]. Complexes were screened in vitro using murine and human colon cancer cells cultured in 2D and 3D settings. In 2D cells, the IC50 values were <22 µM, while in 3D settings, much higher concentrations were required. [ZnL(AcO)] displayed more suitable antiproliferative properties than [ZnL2] and was chosen for further studies. Moreover, based on the weak selectivity of the zinc-based complex towards cancer cell lines in comparison to the non-tumorigenic cell line, its incorporation in long-blood-circulating liposomes was performed, aiming to improve its targetability. The resultant optimized liposomal nanoformulation presented an I.E. of 76% with a mean size under 130 nm and a neutral surface charge and released the metal complex in a pH-dependent manner. The antiproliferative properties of [ZnL(AcO)] were maintained after liposomal incorporation. Preliminary safety assays were carried out through hemolytic activity that never surpassed 2% for the free and liposomal forms of [ZnL(AcO)]. Finally, in a syngeneic murine colon cancer mouse model, while free [ZnL(AcO)] was not able to impair tumor progression, the respective liposomal nanoformulation was able to reduce the relative tumor volume in the same manner as the positive control 5-fluorouracil but, most importantly, using a dosage that was 3-fold lower. Overall, our results show that liposomes were able to solve the solubility issues of the new metal-based complex and target it to tumor sites.

Abstract 1739: Cell-specific cargo delivery using synthetic bacterial spores

Abstract The overexpression of the cell surface receptor “HER-2” is a common marker for many breast and ovarian cancers. New treatments for these cancers involve the use of monoclonal antibodies directed against HER2. SSHELs are synthetic bacterial spore-like particles wherein the spore’s cell surface is partially reconstituted around 1 µm-diameter silica beads. Via a unique cysteine engineered protein, the surface of SSHELs may be covalently decorated with molecules of interest. We showed that SSHELs decorated with anti-HER2 affibodies (SSHELs∝HER2) specifically bound to target cells and were internalized. Doxorubicin loaded SSHELs (Dox-SSHELs∝HER2) were internalized and trafficked to lysosomes, whereupon the cargo was released in a pH-dependent manner. Dox-SSHELs∝HER2 reduce tumor growth with lower toxicity in a mouse tumor xenograft model when compared to the free drug. Further, when compared to an FDA approved liposome-encased doxorubicin (AD), Dox-SSHELs∝HER2 showed similar efficacy in reducing tumor growth, but without any of the typical side effects correlated with AD injections (including weight loss, tumor ulceration, skin descaling). SKOV3 and SKBR3 cells’ targeting by SSHELs∝HER2 was evaluated by flow cytometry and confocal microscopy. Dox-SSHELs∝HER2 anti-tumor effect was evaluated in vitro by flow cytometry and caspase assay. HER2- cells were used as a control. Dox-SSHELs∝HER2’s efficacy was tested in vivo using athymic nude mice injected with SKOV3 cells and then treated for up to 40 days. SSHELs∝HER2 preferentially target cancer cells proportionally to their concentration, as flow cytometry showed. Additional analysis showed the inability of SSHELs∝HER2 to bind HER2+ cells if the receptor was previously blocked. Delivery-mechanism and co-localization analysis showed that SSHELs∝HER2 are preferentially taken up using macropinocytosis and that they undergo a lysosomal pathway where acidic pH contributes to release the cargo. Dox-SSHELs∝HER2 reduce the viability of the treated samples inducing DNA damages and triggering caspases with increase efficiency towards HER2+ cells. In vivo, we demonstrated that Dox-SSHELs∝HER2 can reduce SKOV3 tumor growth up to 75% when compared to the free drug. Additionally, the doxorubicin delivered by SSHELs∝HER2 did not lead to any side effect compared to the free drug or AD. Although new therapies are rising and becoming available for cancer treatments, limited options are still available. We suggest that SSHELs provide several unique benefits and that they may be used as an additional strategy for this purpose. With a facile, reproducible manufacturing process that allows for straightforward targeting towards specific cell types, pH driven cargo release, and lower overall toxicity compared to a leading treatment, we suggest that SSHELs may represent a versatile strategy opening new research avenues for targeted drug delivery treatment of breast and ovarian cancer. Citation Format: Domenico D'Atri, Minsuk Kong, Maria Teresa Bilotta, Kandice Tanner, David J. Fitzgerald, Kumaran S. Ramamurthi. Cell-specific cargo delivery using synthetic bacterial spores [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1739.

Identification of a Subtype-Selective Allosteric Inhibitor of GluN1/GluN3 NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) are Ca2+-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca2+-permeability and lower Mg2+-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC50 value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 μM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.