Extracellular vesicles (EVs) are lipid membrane bound-cell-derived structures that are a key player in intercellular communication and facilitate numerous cellular functions such as tumor growth, metastasis, immunosuppression, and angiogenesis. They can be used as a drug delivery platform because they can protect drugs from degradation and target specific cells or tissues. With the advancement in the technologies and methods in EV research, EV-therapeutics are one of the fast-growing domains in the human health sector. Therapeutic translation of EVs in clinics requires assessing the quality, safety, and efficacy of the EVs, in which pharmacokinetics is very crucial. We report here the application of physiologically based pharmacokinetic (PBPK) modeling as a principal tool for the prediction of absorption, distribution, metabolism, and excretion of EVs. To create a PBPK model of EVs, researchers would need to gather data on the size, shape, and composition of the EVs, as well as the physiological processes that affect their behavior in the body. The PBPK model would then be used to predict the pharmacokinetics of drugs delivered via EVs, such as the rate at which the drug is absorbed and distributed throughout the body, the rate at which it is metabolized and eliminated, and the maximum concentration of the drug in the body. This information can be used to optimize the design of EV-based drug delivery systems, including the size and composition of the EVs, the route of administration, and the dose of the drug. There has not been any dedicated review article that describes the PBPK modeling of EV. This review provides an overview of the absorption, distribution, metabolism, and excretion (ADME) phenomena of EVs. In addition, we will briefly describe the different computer-based modeling approaches that may help in the future of EV-based therapeutic research.

Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.

TSC renal cystic disease is poorly understood and has no approved treatment. In a new principal cell-targeted murine model of Tsc cystic disease, the renal cystic epithelium is mostly composed of type A intercalated cells with an intact Tsc2 gene confirmed by sequencing, although these cells exhibit a Tsc-mutant disease phenotype. We used a newly derived targeted murine model in lineage tracing and extracellular vesicle (EV) characterization experiments and a cell culture model in EV characterization and cellular induction experiments to understand TSC cystogenesis. Using lineage tracing experiments, we found principal cells undergo clonal expansion but contribute very few cells to the cyst. We determined that cystic kidneys contain more interstitial EVs than noncystic kidneys, excrete fewer EVs in urine, and contain EVs in cyst fluid. Moreover, the loss of Tsc2 gene in EV-producing cells greatly changes the effect of EVs on renal tubular epithelium, such that the epithelium develops increased secretory and proliferative pathway activity. We demonstate that the mTORC1 pathway activity is independent form the EV production, and that the EV effects for a single cell line can vary significantly. TSC cystogenesis involves significant contribution from genetically intact cells conscripted to the mutant phenotype by mutant cell derived EVs.

In tuberous sclerosis complex (TSC), Tsc2 mutations are associated with more severe disease manifestations than Tsc1 mutations and the role of extracellular vesicles (EVs) in this context is not yet studied. We report a comparative analysis of EVs derived from isogenic renal cells except for Tsc1 or Tsc2 gene status and hypothesized that in spite of having similar physical characteristics, EVs modulate signaling pathways differently, thus leading to TSC heterogenicity. We used mouse inner medullary collecting duct (mIMCD3) cells with the Tsc1 (T1G cells) or Tsc2 (T2J cells) gene disrupted by CRISPR/CAS9. EVs were isolated from the cell culture media by size-exclusion column chromatography followed by detailed physical and chemical characterization. Physical characterization of EVs was accessed by tunable resistive pulse sensing and dynamic light scattering, revealing similar average sizes and zeta potentials (at pH 7.4) for EVs from mIMCD3 (123.5 ± 5.7 nm and −16.3 ± 2.1 mV), T1G cells (131.5 ± 8.3 nm and −19.8 ± 2.7 mV), and T2J cells (127.3 ± 4.9 nm and −20.2 ± 2.1 mV). EVs derived from parental mIMCD3 cells and both mutated cell lines were heterogeneous (>90% of EVs < 150 nm) in nature. Immunoblotting detected cilial Hedgehog signaling protein Arl13b; intercellular proteins TSG101 and Alix; and transmembrane proteins CD63, CD9, and CD81. Compared to Tsc2 deletion, Tsc1 deletion cells had reduced EV production and release rates. EVs from Tsc1 mutant cells altered mTORC1, autophagy, and β-catenin pathways differently than EVs from Tsc2-mutated cells. Quantitative PCR analysis revealed the down regulation of miR-212a-3p and miR-99a-5p in EVs from Tsc2-mutated cells compared to EVs from Tsc1-mutant cells. Thus, EV-derived miR-212-3p and mIR-99a-5p axes may represent therapeutic targets or biomarkers for TSC disease.

Aim: We report here the development of tenofovir- and curcumin-loaded lactoferrin nanoparticles (TCNPs) as an HIV-microbicide. Materials & methods: TCNPs were subjected to various physicochemical characterization experiments, followed by in vitro and in vivo experiments to assess their efficacy. Results: TCNPs had a diameter of 74.31 ± 2.56 nm with a gross encapsulation of more than 61% for each drug. Nanoparticles were effective against HIV-1 replication, with an IC50 of 1.75 μM for curcumin and 2.8 μM for tenofovir. TCNPs provided drug release at the application site for up to 8-12 h, with minimal leakage into the systemic circulation. TCNPs showed spermicidal activity at ≥200 μM and induced minimal cytotoxicity and inflammation in the vaginal epithelium as revealed by histopathological and ELISA studies. Conclusion: We demonstrated that TCNPs could serve as a novel anti-HIV microbicidal agent in rats. [Formula: see text].

The infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and resultant coronavirus diseases-19 (COVID-19) disproportionally affects minorities, especially African Americans (AA) compared to the Caucasian population. The AA population is disproportionally affected by COVID-19, in part, because they have high prevalence of underlying conditions such as obesity, diabetes, and hypertension, which are known to exacerbate not only kidney diseases, but also COVID-19. Further, a decreased adherence to COVID-19 guidelines among tobacco smokers could result in increased infection, inflammation, reduced immune response, and lungs damage, leading to more severe form of COVID-19. As a result of high prevalence of underlying conditions that cause kidney diseases in the AA population coupled with tobacco smoking make the AA population vulnerable to severe form of both COVID-19 and kidney diseases. In this review, we describe how tobacco smoking interact with SARS-CoV-2 and exacerbates SARS-CoV-2-induced kidney diseases including renal failure, especially in the AA population. We also explore the role of extracellular vesicles (EVs) in COVID-19 patients who smoke tobacco. EVs, which play important role in tobacco-mediated pathogenesis in infectious diseases, have also shown to be important in COVID-19 pathogenesis and organ injuries including kidney. Further, we explore the potential role of EVs in biomarker discovery and therapeutics, which may help to develop early diagnosis and treatment of tobacco-induced renal injury in COVID-19 patients, respectively.

Tuberous sclerosis complex (TSC) is associated with TSC1 or TSC2 gene mutations resulting in hyperactivation of the mTORC1 pathway. This mTORC1 activation is associated with abnormal tissue development and proliferation such that in the kidney there are both solid tumors and cystic lesions. This review summarizes recent advances in tuberous sclerosis complex nephrology and focuses on the genetics and cell biology of tuberous sclerosis complex renal disease, highlighting a role of extracellular vesicles and the innate immune system in disease pathogenesis.

The diagnosis of neurocognitive disorders associated with HIV infection, alcohol, and tobacco using CSF or neuroimaging are invasive or expensive methods, respectively. Therefore, extracellular vesicles (EVs) can serve as reliable noninvasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. Hence, our objective was to investigate the expression of astrocytic (GFAP) and neuronal (L1CAM) specific proteins in EVs circulated in the plasma of HIV subjects, with and without a history of alcohol consumption and tobacco smoking. The protein expression of GFAP (p < 0.01) was significantly enhanced in plasma EVs obtained from HIV-positive subjects and alcohol users compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. The L1CAM expression was found to be significantly elevated in cigarette smokers (p < 0.05). However, its expression was not found to be significant in HIV subjects and alcohol users. Both GFAP and L1CAM levels were not further elevated in HIV-positive alcohol or tobacco users compared to HIV-positive nonsubstance users. Taken together, our data demonstrate that the astrocytic and neuronal-specific markers (GFAP and L1CAM) can be packaged in EVs and circulate in plasma, which is further elevated in the presence of HIV infection, alcohol, and/or tobacco. Thus, the astroglial marker GFAP and neuronal marker L1CAM may represent potential biomarkers targeting neurological dysfunction upon HIV infection and/or alcohol/tobacco consumption.

The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100–250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies.

Although retroviral therapy (ART) has changed the HIV infection from a fatal event to a chronic disease, treated HIV patients demonstrate high prevalence of HIV associated comorbidities including cardio/cerebrovascular diseases. The incidence of stroke in HIV infected subjects is three times higher than that of uninfected controls. Several clinical and postmortem studies have documented the higher incidence of ischemic stroke in HIV infected patients. The etiology of stroke in HIV infected patients remains unknown; however, several factors such as coagulopathies, opportunistic infections, vascular abnormalities, atherosclerosis and diabetes can contribute to the pathogenesis of stroke. In addition, chronic administration of ART contributes to the increased risk of stroke in HIV infected patients. Concurrently, experimental studies in murine model of ischemic stroke demonstrated that HIV infection worsens stroke outcome, increases blood brain barrier permeability and increases neuroinflammation. Additionally, residual HIV viral proteins, such as Trans-Activator of Transcription, glycoprotein 120 and Negative regulatory factor, contribute to the pathogenesis. This review presents comprehensive information detailing the risk factors contributing to ischemic stroke in HIV infected patients. It also outlines experimental evidence demonstrating the impact of HIV infection on stroke outcomes, in addition to possible novel therapeutic approaches to improve these outcomes.

Smoking, which is highly prevalent in HIV-infected populations, has been shown to exacerbate HIV replication, in part via the cytochrome P450 (CYP)-induced oxidative stress pathway. Recently, we have shown that extracellular vesicles (EVs), derived from tobacco- and/or HIV-exposed macrophages, alter HIV replication in macrophages by cell-cell interactions. We hypothesize that cigarette smoke condensate (CSC) and/or HIV-exposed macrophagederived EVs carry relatively high levels of pro-oxidant and pro-inflammatory cargos and/or low levels of antioxidant and anti-inflammatory cargos, which are key mediators for HIV pathogenesis. Therefore, in this study, we investigated differential packaging of pro- and anti-inflammatory cytokines/chemokines and pro- and anti-oxidant contents in EVs after CSC exposure to myeloid cells (uninfected U937 and HIV-infected U1 cells). Our results showed that relatively long to short exposures with CSC increased the expression of cytokines in EVs isolated from HIV-infected U1 macrophages. Importantly, pro-inflammatory cytokines, especially IL-6, were highly packaged in EVs isolated from HIV-infected U1 macrophages upon both long and short-term CSC exposures. In general, anti-inflammatory cytokines, particularly IL-10, had a lower packaging in EVs, while packaging of chemokines was mostly increased in EVs upon CSC exposure in both HIV-infected U1 and uninfected U937 macrophages. Moreover, we observed higher expression of CYPs (1A1 and 1B1) and lower expression of antioxidant enzymes (SOD-1 and catalase) in EVs from HIV-infected U1 macrophages than in uninfected U937 macrophages. Together, they are expected to increase oxidative stress factors in EVs derived from HIV-infected U1 cells. Taken together, our results suggest packaging of increased level of oxidative stress and inflammatory elements in the EVs upon exposure to tobacco constituents and/or HIV to myeloid cells, which would ultimately enhance HIV replication in macrophages via cell-cell interactions.

To enhance efficacy, bioavailability and reduce toxicity of first-line highly active anti-retroviral regimen, zidovudine + efavirenz + lamivudine loaded lactoferrin nanoparticles were prepared (FLART-NP) and characterized for physicochemical properties, bioactivity and pharmacokinetic profile.

Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or “slow” substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors.

We report that a combination of anti-HIV-1 drug efavirenz (EFV), anti-microbial-spermicidal curcumin (Cur) and lactoferrin nanoparticles (ECNPs) act as MPT formulation. These nanoparticles are of well dispersed spherical shape with 40–70 nm size, with encapsulation efficiency of 63 ± 1.9% of Cur & 61.5% ± 1.6 of EFV, significantly higher than that of single drug nanoparticles (Cur, 59 ± 1.34%; EFV: 58.4 ± 1.79). ECNPs were found to be sensitive at pH 5 and 6 and have not effected viability of vaginal micro-flora, Lactobacillus. Studies in rats showed that ECNPs delivers 88–124% more drugs in vaginal lavage as compared to its soluble form, either as single or combination of EFV and Cur. The ECNPs also shows 1.39–4.73 fold lower concentration of absorption in vaginal tissue and plasma compared to soluble EFV + Cur. Furthermore, ECNPs show significant reduction in inflammatory responses by 1.6–3.0 fold in terms of IL-6 and TNF-α in vaginal tissue and plasma compared to soluble EFV + Cur. ECNPs showed improved pharmacokinetics profiles in vaginal lavage with more than 50% of enhancement in AUC, AUMC, Cmax and t1/2 suggesting longer exposure of Cur and EFV in vaginal lavage compared to soluble EFV + Cur. Histopathological analysis of vaginal tissue shows remarkably lower toxicity of ECNPs compared to soluble EFV + Cur. In conclusion, ECNPs are significantly safe and exhibit higher bioavailability thus constitute an effective MPT against HIV.

Curcumin is known to have neuroprotective role and possess antioxidant, anti-inflammatory activities. Rotenone, a flavonoid induced neurotoxicity in dopaminergic cells is being widely studied in Parkinson's Disease (PD) research. In the present study, curcumin loaded lactoferrin nano particles prepared by sol-oil chemistry were used to protect dopaminergic cell line SK-N-SH against rotenone induced neurotoxicity. These curcumin loaded nano particles were of 43-60 nm diameter size and around 100 nm hydrodynamic size as assessed by transmission electron microscopy, atomic force microscopy and dynamic light scattering analysis respectively. The encapsulation efficiency was 61.3% ± 2.4%. Cellular uptake of curcumin through these nano particles was confirmed by confocal imaging and spectrofluorimetric analysis. The curcumin loaded lactoferrin nanoparticles showed greater intracellular drug uptake, sustained retention and greater neuroprotection than soluble counterpart. Neuroprotective activity was characterized through viability assays and by estimating ROS levels. Furthermore rotenone induced PD like features were characterized by decrease in tyrosine hydroxylase expression and increase in α-synuclein expression. Taken together curcumin loaded lactoferrin nanoparticles could be a promising drug delivery strategy against neurotoxicity in dopaminergic neurons.

Efavirenz (EFV), a non‐nucleoside reverse transcriptase inhibitor, is a drug that is frequently included in highly active antiretroviral therapy for treatment of HIV infection. Decreased bioavailability and increased toxicity limit its use. We report a formulation of efavirenz‐loaded lactoferrin nanoparticles (lacto‐EFV‐nano) for oral delivery which exhibited significantly improved pharmacological properties coupled with reduced toxicity compared with its free form.

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.

Legume lectins are the most thoroughly studied group of lectins and have been widely linked to many pathological processes. Their use as immunohistochemistry markers for cell profiling and cancer diagnosis have made these molecules important tools for immunological studies and have stimulated the prospection and characterization of new lectins. The crystal structures of a recombinant seed lectin from Vatairea macrocarpa (rVML) and its complexes with GalNAcα1-O-Ser, GalNAc and α-lactose, have been determined at 1.90, 1.97, 2.70 and 1.83Å resolution, respectively. Small angle X-ray scattering and calorimetry assays have confirmed the same pH stable oligomerization pattern and binding profiles proposed for its wild-type counterpart. In silico analyzes have explored the potential of this recombinant lectin as new tool for cancer research through a comparative profile with other legume lectins widely used for cancer diagnosis and prognosis. The results suggest the recognition of specific epitopes exhibited on different cancer cells as a process that relies on the disposition of hydrophobic clusters and charged regions around the lectin carbohydrate-binding site, favouring the anchorage of different groups in the antigen boundaries, highlighting the different potential of each analyzed lectin. In conclusion, the experimental results and comparative analysis show that rVML is as a promising tool for cancer research, able to bind with high affinity specific tumor-associated antigens, highly stable and easily produced.

Zidovudine (AZT) is one of the most referred antiretroviral drug. In spite of its higher bioavailability (50-75%) the most important reason of its cessation are bone marrow suppression, anemia, neutropenia and various organs related toxicities. This study aims at the improvement of oral delivery of AZT through its encapsulation in lactoferrin nanoparticles (AZT-lactonano). The nanoparticles (NPs) are of 50-60 nm in size and exhibit 67% encapsulation of the AZT. They are stable in simulated gastric and intestinal fluids. Anti-HIV-1 activity of AZT remains unaltered in nanoformulation in acute infection. The bioavailability and tissue distribution of AZT is higher in blood followed by liver and kidney. AZT-lactonano causes the improvement of pharmacokinetic profile as compared to soluble AZT; a more than 4 fold increase in AUC and AUMC in male and female rats. The serum Cmax for AZT-lactonano was increased by 30%. Similarly there was nearly 2-fold increase in Tmax and t1/2. Our in vitro study confirms that, the endosomal pH is ideal for drug release from NPs and shows constant release from up to 96h. Bone marrow micronucleus assay show that nanoformulation exhibits approximately 2fold lower toxicity than soluble form. Histopathological and biochemical analysis further confirms that less or no significant organ toxicities when nanoparticles were used. AZT-lactonano has shown its higher efficacy, low organs related toxicities, improved pharmacokinetics parameter while keeping the antiviral activity intact. Thus, the nanoformulation are safe for the target specific drug delivery.