Blending of Designer Synthetic Polymers to a Dual Targeted Nanoformulation for SARS-CoV-2 Associated Kidney Damage.

As the COVID-19 pandemic has continued to spread, studies have shown that hospitalized COVID-19 patients are at significant risk for developing acute kidney injury (AKI), which can cause increased morbidity, the need for dialysis treatment, chronic kidney diseases, and even death. In this paper, we present a proof-of-concept study for the utilization of combination therapeutic-loaded dual-targeted biodegradable nanoparticles (NPs) to treat concurrent AKI and COVID-19 in patients by delivering the therapeutics across the gut epithelial barrier and to the kidney, in order to lower the viral load as well as reduce the symptoms of AKI. Despite recent vaccination efforts and the end of the COVID-19 pandemic in sight, problems related to the long-term effects of COVID-19 will continue to persist, including impacts on patients suffering from AKI and other chronic renal conditions. Therefore, the dual-targeted blended polymeric NP developed in this study to treat concurrent COVID-19 infection and AKI is a useful proof-of-concept nanoplatform for future treatments of these complications.

Leishmania donovani Exploits Macrophage Heme Oxygenase-1 To Neutralize Oxidative Burst and TLR Signaling-Dependent Host Defense.

Suppression of host oxidative burst is essential for survival of the intracellular parasite Leishmania donovani Screening of macrophage antioxidant enzymes during infection revealed marked upregulation of the heme-degrading enzyme, heme oxygenase-1 (HO-1). Moreover, HO-1-silenced RAW macrophages depicted increased superoxide production and decreased parasite survival. HO-1 induction decreased cellular heme content, thereby inhibiting the heme-dependent maturation of gp91phox, a catalytic component of major reactive oxygen species-producing enzyme NAD(P)H oxidase. Decreased gp91phox expression resulted in reduced stability of p22phox, another component of the catalytic center of NAD(P)H oxidase. Replenishing infected cells with exogenous heme reversed these effects and restored NAD(P)H oxidase activity. Persistent HO-1 expression at late hour of infection prompted us to investigate its effect on other host defense parameters, and inhibition study revealed a reciprocal relationship of HO-1 with host proinflammatory responses. Among all the HO-1-mediated heme degradation products (CO, Fe, and biliverdin), only CO documented potent anti-inflammatory effects. Quenching of CO during infection increased the production of disease-resolving cytokines IL-12 and TNF-α. Coimmunoprecipitation experiments revealed that CO inhibited the interaction of TLR4 with MyD88 and TIR domain-containing adapter-inducing IFN-ß, thereby dampening the activation of NF-κB and IFN regulatory factor 3-mediated production of proinflammatory cytokines. Administration of HO-1 inhibitor tin protoporphyrin IX dichloride in infected BALB/c mice led to a decrease in liver and spleen parasite burden along with increased production of IL-12 and TNF-α. These results suggest that HO-1 on one hand inhibits reactive oxygen species generation and on the other hand downregulates host favorable cytokine responses, thereby facilitating intramacrophage parasite survival.

Lipid Metabolism Modulatory Cisplatin Prodrug Sensitizes Resistant Prostate Cancer toward Androgen Deprivation Therapy.

Mainstream treatment modalities which dominate the therapeutic landscape of prostate cancer (PCa) are prostatectomy, radiation therapy, and androgen deprivation therapy (ADT) or castration. These therapeutic options can extend the life expectancy of the patients but eventually fail to completely cure the disease. Despite undergoing ADT, patients still experience disease recurrence. One of the reasons for this recurrence is the binding of the basal androgens present in blood plasma to the androgen receptor (AR). At this stage, the disease becomes castration-resistant prostate cancer (CRPC) showing resistance to ADT promoting progression, and there is no effective treatment available. Although another male cancer such as testicular cancer responds to cisplatin-based therapy very well, PCa is resistant to cisplatin. In our continued effort to find the pathways that are important for such resistance, we link in this report, tumor metabolism driven androgen regulation and PCa resistance toward cisplatin-based therapy. To delve deeper into understanding how metabolic modulatory cisplatin prodrugs can be used to target the ADT resistant population, we demonstrate that metabolic inhibition by a cisplatin prodrug, Platin-L has the potential to modulate AR activity and resensitize ADT resistant cells toward cisplatin-based chemotherapy as well as ADT. The mode of action for Platin-L is inhibition of fatty acid oxidation (FAO) of prostate cancer cells. We demonstrated that FAO inhibition by Platin-L in PCa cells contribute to AR regulation resulting in altered tumorigenicity of androgen sensitive prostate cancer.

Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

Inhibition of Mac-1 allows human macrophages to migrate against the direction of shear flow on ICAM-1.

All immune cells must transit from the blood to distal sites such as the lymph nodes, bone marrow, or sites of infection. Blood borne monocytes traffic to the site of inflammation by adhering to the endothelial surface and migrating along endothelial intracellular adhesion molecule 1 (ICAM-1) by their ligand's macrophage 1 antigen (Mac-1) and lymphocyte functional antigen 1 (LFA-1) to transmigrate through the endothelium. Poor patient prognoses in chronic inflammation and tumors have been attributed to the hyper recruitment of certain types of macrophages. Therefore, targeting the binding of ICAM-1 to its respective ligands provides a novel approach to targeting the recruitment of macrophages. To that end, we determined whether the loss of Mac-1 expression could induce this upstream migration behavior by using blocking antibodies against Mac-1 to examine the effects of hydrodynamic flow on the migration of the human macrophage cell line U-937 on ICAM-1 surfaces. Blocking Mac-1 on U-937 cells led to upstream migration against the direction of shear flow on ICAM-1 surfaces. In sum, the ability of macrophages to migrate upstream when Mac-1 is blocked represents a new avenue to precisely control the differentiation, migration, and trafficking of macrophages.

Cell specific mitochondria targeted metabolic alteration for precision medicine.

Mitochondria play important roles in the maintenance of cellular health. In cancer, these dynamic organelles undergo significant changes in terms of membrane hyperpolarization, altered metabolic functions, fusion-fission balance, and several other parameters. These alterations promote cancer growth, proliferation and spread, and the eventual development of metastatic disease and therapeutic resistance. Thus, routing therapeutics to the mitochondrial compartments can be one of the most promising methodologies for tackling such changes to achieve cancer control. Over the last decade, targeted cancer medicine has experienced tremendous growth, enabling the targeting of mitochondria for greater therapeutic specificity. Here, we demonstrate a feasibility method to specifically target the mitochondria of prostate cancer cells. We achieve such dual targeting by utilizing two functionalized polymers and constructing a single blended nanoparticle (NP). Such a targeting strategy was developed utilizing a polymeric platform that differed in terms of the length of the amphiphilic portions, the linker between the hydrophobic portions, and the attached targeting moieties. In doing this, we demonstrate prostate cancer specific mitochondrial delivery of a chemotherapeutic prodrug to create repair-resistant adducts within mitochondrial DNA promoting cellular death. This article documents the synthetic strategy, optimization of blended NPs for cell specific mitochondria targeting, and the utility of the proof-of-concept design was demonstrated using a combination of analytical and in vitro studies.

Not all (cells) who wander are lost: Upstream migration as a pervasive mode of amoeboid cell motility.

Leukocytes possess the ability to migrate upstream-against the direction of flow-on surfaces of specific chemistry. Upstream migration was first characterized in vitro for T-cells on surfaces comprised of intracellular adhesion molecule-1 (ICAM-1). Upstream migration occurs when the integrin receptor αLß2 (also known as lymphocyte function-associated antigen-1, or LFA-1) binds to ICAM-1. LFA-1/ICAM-1 interactions are ubiquitous and are widely found in leukocyte trafficking. Upstream migration would be employed after cells come to arrest on the apical surface of the endothelium and might confer an advantage for both trans-endothelial migration and tissue surveillance. It has now been shown that several other motile amoeboid cells which have the responsibility of trafficking from blood vessels into tissues, such as Marginal zone B cells, hematopoietic stem cells, and neutrophils (when macrophage-1 antigen, Mac-1, is blocked), can also migrate upstream on ICAM-1 surfaces. This review will summarize what is known about the basic mechanisms of upstream migration, which cells have displayed this phenomenon, and the possible role of upstream migration in physiology and tissue homeostasis.

New Pathway for Cisplatin Prodrug to Utilize Metabolic Substrate Preference to Overcome Cancer Intrinsic Resistance.

Tumor cells adapt to diverse survival strategies defying our pursuit of multimodal cancer therapy. Prostate cancer (PCa) is an example that is resistant to one of the most potent chemotherapeutics, cisplatin. PCa cells survive and proliferate using fatty acid oxidation (FAO), and the dependence on fat utilization increases as the disease progresses toward a resistant form. Using a pool of patient biopsies, we validated the expression of a key enzyme carnitine palmitoyltransferase 1 A (CPT1A) needed for fat metabolism. We then discovered that a cisplatin prodrug, Platin-L, can inhibit the FAO of PCa cells by interacting with CPT1A. Synthesizing additional cisplatin-based prodrugs, we documented that the presence of an available carboxylic acid group near the long chain fatty acid linker on the Pt(IV) center is crucial for CPT1A binding. As a result of fat metabolism disruption by Platin-L, PCa cells transition to an adaptive glucose-dependent chemosensitive state. Potential clinical translation of Platin-L will require a delivery vehicle to direct it to the prostate tumor microenvironment. Thus, we incorporated Platin-L in a biodegradable prostate tumor-targeted orally administrable nanoformulation and demonstrated its safety and efficacy. The distinctive FAO inhibitory property of Platin-L can be of potential clinical relevance as it offers the use of cisplatin for otherwise resistant cancer.

Correction to "New Pathway for Cisplatin Prodrug to Utilize Metabolic Substrate Preference to Overcome Cancer Intrinsic Resistance".

[This corrects the article DOI: 10.1021/acscentsci.3c00286.].

Metabolic Modulation of the Tumor Microenvironment Leads to Multiple Checkpoint Inhibition and Immune Cell Infiltration.

Cancer cells are known to be glycolytic, driving increased glucose consumption and its conversion to lactate. This process modulates the tumor microenvironment (TME). In the TME, glycolytically activated immune cells often become anergic, leading to an increase in immune checkpoint proteins such as programmed cell death protein-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Most glycolytic inhibitors not only inhibit glycolysis of cancer but also of immune cells. Therefore, using a nanoparticle-delivered agent to preferentially inhibit glycolysis in tumor cells, and not in immune cells, has the potential to attenuate the expression of checkpoint proteins. Pyruvate dehydrogenase kinase 1 (PDK1) can be an important target to achieve tumor specific glycolysis inhibition. We report TME modulation by a mitochondrion-targeted nanoparticle (NP) containing a prodrug of dichloroacetate (DCA), a PDK1 inhibitor. We demonstrated that the targeted NP alters the TME which results in increased immunological activation against cancer cells, causing a decrease in mean tumor volume. Here, we also show findings that when Mito-DCA, a prodrug of DCA, was combined with anti-PD-1, a checkpoint inhibitor, results from in vivo syngeneic models showed an upregulation in the number of tumor infiltrating lymphocytes. This work provides a platform to bring therapeutic efficacy by selectively inhibiting glycolysis of cancer cells.