Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in Plasmodium falciparum parasites.

Reduced susceptibility to ART, the first-line treatment against malaria, is common in South East Asia (SEA). It is associated with point mutations, mostly in kelch13 (k13) but also in other genes, like ubp1. K13 and its compartment neighbors (KICs), including UBP1, are involved in endocytosis of host cell cytosol. We tested 135 mutations in KICs but none conferred ART resistance. Double mutations of k13C580Y with k13R539T or k13C580Y with ubp1R3138H, did also not increase resistance. In contrast, k13C580Y parasites subjected to consecutive RSAs did, but the k13 sequence was not altered. Using isogenic parasites with different k13 mutations, we found correlations between K13 protein amount, resistance, and fitness cost. Titration of K13 and KIC7 indicated that the cellular levels of these proteins determined resistance through the rate of endocytosis. While fitness cost of k13 mutations correlated with ART resistance, ubp1R3138H caused a disproportionately higher fitness cost. IMPORTANCE: Parasites with lowered sensitivity to artemisinin-based drugs are becoming widespread. However, even in these "resistant" parasites not all parasites survive treatment. We found that the proportion of surviving parasites correlates with the fitness cost of resistance-inducing mutations which might indicate that the growth disadvantages prevents resistance levels where all parasites survive treatment. We also found that combining two common resistance mutations did not increase resistance levels. However, selection through repeated ART-exposure did, even-though the known resistance genes, including k13, were not further altered, suggesting other causes of increased resistance. We also observed a disproportionally high fitness cost of a resistance mutation in resistance gene ubp1. Such high fitness costs may explain why mutations in ubp1 and other genes functioning in the same pathway as k13 are rare. This highlights that k13 mutations are unique in their ability to cause resistance at a comparably low fitness cost.

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium.

Endocytosis allows cells to internalise a wide range of molecules from their environment and to maintain their plasma membrane composition. It is vital during development and for maintenance of tissue homeostasis. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualise endocytosis in the neuroepithelium of living zebrafish embryos. Injection of fluorescent tracers into the brain ventricles followed by live imaging was used to study fluid-phase or receptor-mediated endocytosis, for which we used receptor-associated protein (RAP, encoded by Lrpap1) as a ligand for low-density lipoprotein receptor-related protein (LRP) receptors. Using dual-colour imaging combined with expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also found that the RAP-binding receptor Lrp2 (encoded by lrp2a) appears to contribute only partially to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo and support a role for Ocrl in this process. This article has an associated First Person interview with the first author of the paper.

Methods for assessment of membrane protrusion dynamics.

Membrane protrusions are a critical facet of cell function. Mediating fundamental processes such as cell migration, cell-cell interactions, phagocytosis, as well as assessment and remodeling of the cell environment. Different protrusion types and morphologies can promote different cellular functions and occur downstream of distinct signaling pathways. As such, techniques to quantify and understand the inner workings of protrusion dynamics are critical for a comprehensive understanding of cell biology. In this chapter, we describe approaches to analyze cellular protrusions and correlate physical changes in cell morphology with biochemical signaling processes. We address methods to quantify and characterize protrusion types and velocity, mathematical approaches to predictive models of cytoskeletal changes, and implementation of protein engineering and biosensor design to dissect cell signaling driving protrusive activity. Combining these approaches allows cell biologists to develop a comprehensive understanding of the dynamics of membrane protrusions.

Modeling the dynamic behaviors of the COPI vesicle formation regulators, the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes.

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.

Quantitative Assessment of Endosomal Escape of Various Endocytosed Polymer-Encapsulated Molecular Cargos upon Photothermal Heating.

Encapsulated molecular cargos are efficiently endocytosed by cells. For cytosolic delivery, understanding the dynamic process of cargos release from the carrier vehicles used for encapsulation and the lysosomes where the carrier vehicles are trapped (which in general is the bottleneck), followed by diffusion in the cytosol is important for improving drug/gene delivery strategies. A methodology is reported to image this process on a millisecond scale and to quantitatively analyze the data. Polyelectrolyte capsules with embedded gold nanostars to encapsulate 43 fluorescent molecular cargos with diverse properties, ranging from small fluorophores to fluorescently labeled proteins, siRNA, etc., are used. By short laser irradiation intracellular release of the molecular cargos from endocytosed capsules into the cytosol is triggered, and their intracellular spreading is imaged. Most of the released molecular cargos evenly distribute inside the entire cell, while others are enriched in certain cell compartments. The time the different molecular cargos take to distribute within cells, i.e., the spreading time, is used as a quantifier. Quantitative analysis reveals that intracellular spread cannot be described by free diffusion, but is determined by interaction of the molecular cargo with intracellular components.

Assessment of Gold-Coated Iron Oxide Nanoparticles as Negative T2 Contrast Agent in Small Animal MRI Studies.

PURPOSE: Magnetic resonance imaging (MRI) contrast agents are pharmaceuticals that enable a better visualization of internal body structures. In this study, we present the synthesis, MRI signal enhancement capabilities, in vitro as well as in vivo cytotoxicity results of gold-coated iron oxide nanoparticles (Fe3O4@AuNPs) as potential contrast agents. METHODS: Fe3O4@AuNPs were obtained by synthesizing iron oxide nanoparticles and gradually coating them with gold. The obtained Fe3O4@AuNPs were characterized by spectroscopies, transmission electron microscopy (TEM) and energy dispersive X-ray diffraction. The effect of the nanoparticles on the MRI signal was tested using a 7T Bruker PharmaScan system. Cytotoxicity tests were made in vitro on Fe3O4@AuNP-treated retinal pigment epithelium cells by WST-1 tests and in vivo by following histopathological changes in rats after injection of Fe3O4@AuNPs. RESULTS: Stable Fe3O4@AuNPs were successfully prepared following a simple and fast protocol (<1h worktime) and identified using TEM. The cytotoxicity tests on cells have shown biocompatibility of Fe3O4@AuNPs at small concentrations of Fe (<1.95×10-8 mg/cell). Whereas, at higher Fe concentrations (eg 7.5×10-8 mg/cell), cell viability decreased to 80.88±5.03%, showing a mild cytotoxic effect. MRI tests on rats showed an optimal Fe3O4@AuNPs concentration of 6mg/100g body weight to obtain high-quality images. The histopathological studies revealed significant transient inflammatory responses in the time range from 2 hours to 14 days after injection and focal cellular alterations in several organs, with the lung being the most affected organ. These results were confirmed by hyperspectral microscopic imaging of the same, but unstained tissues. In most organs, the inflammatory responses and sublethal cellular damage appeared to be transitory, except for the kidneys, where the glomerular damage indicated progression towards glomerular sclerosis. CONCLUSION: The obtained stable, gold covered, iron oxide nanoparticles with reduced cytotoxicity, gave a negative T2 signal in the MRI, which makes them suitable for candidates as contrast agent in small animal MRI applications.

Cytotoxicity Assessment of Gallium- and Indium-Based Nanoparticles Toward Human Bronchial Epithelial Cells Using an Impedance-Based Real-Time Cell Analyzer.

The semiconductor manufacturing sector plans to introduce III/V film structures (eg, gallium arsenide (GaAs), indium arsenide (InAs) onto silicon wafers due to their high electron mobility and low power consumption. Aqueous solutions generated during chemical and mechanical planarization of silicon wafers can contain a mixture of metal oxide nanoparticles (NPs) and soluble indium, gallium, and arsenic. In this work, the cytotoxicity induced by Ga- and In-based NPs (GaAs, InAs, Ga2O3, In2O3) and soluble III-V salts on human bronchial epithelial cells (16HBE14o-) was evaluated using a cell impedance real-time cell analysis (RTCA) system. The RTCA system provided inhibition data at different concentrations for multiple time points, for example, GaAs (25 mg/L) caused 60% inhibition after 8 hours of exposure and 100% growth inhibition after 24 hours. Direct testing of As(III) and As(V) demonstrated significant cytotoxicity with 50% growth inhibition concentrations after 16-hour exposure (IC50) of 2.4 and 4.5 mg/L, respectively. Cell signaling with rapid rise and decrease in signal was unique to arsenic cytotoxicity, a precursor of strong cytotoxicity over the longer term. In contrast with arsenic, soluble gallium(III) and indium(III) were less toxic. Whereas the oxide NPs caused low cytotoxicity, the arsenide compounds were highly inhibitory (IC50 of GaAs and InAs = 6.2 and 68 mg/L, respectively). Dissolution experiments over 7 days revealed that arsenic was fully leached from GaAs NPs, whereas only 10% of the arsenic was leached out of InAs NPs. These results indicate that the cytotoxicity of GaAs and InAs NPs is largely due to the dissolution of toxic arsenic species.

Ex vivo assessment of in vivo DC-targeted antibodies in pre-clinical models.

APCs play a key role at initiating adaptive immune responses by presenting antigens to lymphocytes and DCs are professional APCs. It is critical to understand the differential antigen capture and presentation ability of different DC subsets, which is important for DC-targeted immunotherapy. In this section, we give a brief introduction to different antigen presentation pathways and introduce the key concept of cross-presentation, the major antigen presentation pathway used for anti-viral and anti-tumoral immune responses. CD205, a DC restricted receptor, is highly expressed on certain DCs subsets. We find CD205-mediated antigen uptake to be a useful model for studying antigen uptake and defects. These methods provide an introduction to CD205-mediated pre-clinical delivery of antigens to cross-presenting DCs, which can be adapted to the study of targeting to multiple receptors and other C-type lectins. This is a promising strategy to detect the antigen capture capacity and to study the key players orchestrating tolerance and immunity ex vivo.

Technical note: Assessment of neutrophil endocytosis and proteolytic degradation and its relationship with phagocytosis and oxidative burst in dairy cows.

Conventional assays of polymorphonuclear cell (PMN, neutrophil) function such as oxidative burst (OB) and phagocytosis (PC) are widely used to evaluate innate immunity in the transition period of dairy cows. Oxidative burst is commonly evaluated by measuring PMN median fluorescence intensity (MFI) involving the release of reactive oxygen species after in vitro stimulation. Phagocytosis can be measured by engulfment of fluorescent beads by PMN. DQ-ovalbumin (DQ-OVA) is a molecule suitable for the assessment of intracellular proteolytic degradation, so it might be informative about an additional pathway of pathogen handling by PMN. In this study, we evaluated the use of the DQ-OVA assay for the assessment of PMN function and the relationships among OB, PC, and DQ-OVA results in PMN isolated from blood of dairy cows between 5 and 21 d post partum. Results of the DQ-OVA validation assay were assessed with mixed linear regression models. Pearson correlation tests and kappa values for agreement were used to associate the MFI between each PMN function assay (OB, PC, and DQ-OVA). For the validation assay (9 cows in 3 replicates), PMN incubated with DQ-OVA were stimulated with IFN-γ or inhibited with cytochalasin D, and fluorescence was compared with untreated PMN. Stimulated and inhibited PMN had greater (970 ± 160 units) and lesser (593 ± 55 units) MFI relative to untreated PMN (791 ± 154 units), respectively, indicating that DQ-OVA fluorescence reflected enhanced or reduced endocytic and proteolytic function. To associate the MFI outcomes among OB, PC, and DQ-OVA, 153 samples from 40 cows were analyzed. Results showed significant, although weak association between DQ-OVA and PC MFI (Pearson r = 0.16). When values of MFI were categorized according to the first ("high" PMN functionality), second and third ("moderate" PMN functionality), or fourth ("low" PMN functionality) quartiles, agreement beyond chance (κ) was moderate: κ = 0.38 for DQ-OVA and OB, κ = 0.43 for DQ-OVA and PC, and κ = 0.43 for OB and PC. The DQ-OVA assay may complement traditional PMN functional assays because it provides additional information regarding the combination of endocytosis and proteolytic degradation, but it is not a substitute for assessment of OB or PC.

Assessment of Histiotrophic Nutrition Using Fluorescent Probes.

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin). The methods are simple and can provide complimentary functional and structural information in studies of the effects of embryotoxicants on visceral yolk sac function.

GRP75 modulates oncogenic Dbl-driven endocytosis derailed via the CHIP-mediated ubiquitin degradation pathway.

Chaperone-assisted proteasome degradation of oncogenic protein acts as an upstream signal controlling tumorigenesis and progression. The understanding of the co-regulation of chaperone and oncoprotein of endocytosis pathways is extremely limited. In this study, we showed for the first time that proto-Dbl (dbl proto-oncogene product) is co-enriched with mitochondrial chaperone GRP75 in endocytosis vesicles from ovarian cancer cells. onco-Dbl, produced by oncogenic mutation/degradation of proto-Dbl, markedly enhanced cellular macropinocytosis but suppressed clathrin-mediated endocytosis and clathrin-independent endocytosis pathways, presenting a derailed endocytosis phenotype. GRP75 was associated with proto-Dbl inside cells and modulated Dbl-driven endocytosis derailed by a co-regulatory mode. In spite of not being a component of the Hsc70/Hsp90/proto-Dbl complex, the degradation of proto-Dbl was promoted by GRP75 through the CHIP-mediated ubiquitin-proteasome pathway, of which GRP75 acts as a cooperator with CHIP but also acts as a competitor to Hsc70 and Hsp90 in the multiple chaperones-assisted pro-folding/pro-degradation machinery. Knockdown or inhibition of GRP75 attenuated proto-Dbl degradation and reduced the onco-Dbl level, which differentially impaired Rho GTPases activation and therefore shifted the endocytosis-derailed phenotype. Our data uncovered a novel GRP75-Dbl endocytosis regulatory axis and provided an alternative using chaperone inhibitor to shut down the oncoprotein-driven endocytosis derailment mechanism.

Bioinspired Cell-Derived Nanovesicles versus Exosomes as Drug Delivery Systems: a Cost-Effective Alternative.

Cell Derived Nanovesicles (CDNs) have been developed from the rapidly expanding field of exosomes, representing a class of bioinspired Drug Delivery Systems (DDS). However, translation to clinical applications is limited by the low yield and multi-step approach in isolating naturally secreted exosomes. Here, we show the first demonstration of a simple and rapid production method of CDNs using spin cups via a cell shearing approach, which offers clear advantages in terms of yield and cost-effectiveness over both traditional exosomes isolation, and also existing CDNs fabrication techniques. The CDNs obtained were of a higher protein yield and showed similarities in terms of physical characterization, protein and lipid analysis to both exosomes and CDNs previously reported in the literature. In addition, we investigated the mechanisms of cellular uptake of CDNs in vitro and their biodistribution in an in vivo mouse tumour model. Colocalization of the CDNs at the tumour site in a cancer mouse model was demonstrated, highlighting the potential for CDNs as anti-cancer strategy. Taken together, the results suggest that CDNs could provide a cost-effective alternative to exosomes as an ideal drug nanocarrier.

Biocompatibility assessment of silk nanoparticles: hemocompatibility and internalization by human blood cells.

Many nanoparticles are designed for use as potential nanomedicines for parenteral administration. However, emerging evidence suggests that hemocompatibility is important, but is highly particle- and test-bed dependent. Thus, knowledge of bulk material properties does not predict the hemocompatibility of uncharacterized nanoparticles, including silk nanoparticles. This study compares the hemocompatibility of silk versus silica nanoparticles, using whole human blood under quasi-static and flow conditions. Substantial hemocompatibility differences are noted for some nanoparticles in quasi-static versus dynamic studies; i.e., the inflammatory response to silk nanoparticles is significantly lower under flow versus quasi-static conditions. Silk nanoparticles also have very low coagulant properties - an observation that scales from the macro- to the nano-level. These nanoparticle hemocompatibility studies are complemented by preliminary live cell measurements to evaluate the endocytosis and trafficking of nanoparticles in human blood cells. Overall, this study demonstrates that nanoparticle hemocompatibility is affected by several factors, including the test bed design.

Optimal multivalent targeting of membranes with many distinct receptors.

Cells can often be recognized by the concentrations of receptors expressed on their surface. For better (targeted drug treatment) or worse (targeted infection by pathogens), it is clearly important to be able to target cells selectively. A good targeting strategy would result in strong binding to cells with the desired receptor profile and barely binding to other cells. Using a simple model, we formulate optimal design rules for multivalent particles that allow them to distinguish target cells based on their receptor profile. We find the following: (i) It is not a good idea to aim for very strong binding between the individual ligands on the guest (delivery vehicle) and the receptors on the host (cell). Rather, one should exploit multivalency: High sensitivity to the receptor density on the host can be achieved by coating the guest with many ligands that bind only weakly to the receptors on the cell surface. (ii) The concentration profile of the ligands on the guest should closely match the composition of the cognate membrane receptors on the target surface. And (iii) irrespective of all details, the effective strength of the ligand-receptor interaction should be of the order of the thermal energy [Formula: see text], where [Formula: see text] is the absolute temperature and [Formula: see text] is Boltzmann's constant. We present simulations that support the theoretical predictions. We speculate that, using the above design rules, it should be possible to achieve targeted drug delivery with a greatly reduced incidence of side effects.

Quantitative Co-Localization and Pattern Analysis of Endo-Lysosomal Cargo in Subcellular Image Cytometry and Validation on Synthetic Image Sets.

Late endosomes and lysosomes (LE/LYSs) play a central role in trafficking of endocytic cargo, secretion of exosomes, and hydrolysis of ingested proteins and lipids. Failure in such processes can lead to lysosomal storage disorders in which a particular metabolite accumulates within LE/LYSs. Analysis of endocytic trafficking relies heavily on quantitative fluorescence microscopy, but evaluation of the huge image data sets is challenging and demands computer-assisted statistical tools. Here, we describe how to use SpatTrack ( www.sdu.dk/bmb/spattrack ), an imaging toolbox, which we developed for quantification of the distribution and dynamics of endo-lysosomal cargo from fluorescence images of living cells. First, we explain how to analyze experimental images of endocytic processes in Niemann Pick C2 disease fibroblasts using SpatTrack. We demonstrate how to quantify the location of the sterol-binding protein NPC2 in LE/LYSs relative to cholesterol -rich lysosomal storage organelles (LSOs) stained with filipin. Second, we show how to simulate realistic vesicle patterns in the cell geometry using Markov Chain Monte Carlo and suitable inter-vesicle and cell-vesicle interaction potentials. Finally, we use such synthetic vesicle patterns as "ground truth" for validation of two-channel analysis tools in SpatTrack, revealing their high reliability. An improved version of SpatTrack for microscopy-based quantification of cargo transport through the endo-lysosomal system accompanies this protocol.

Integrin Targeting and Toxicological Assessment of Peptide-Conjugated Liposome Delivery Systems to Activated Endothelial Cells.

Utilization of functionalized liposomes as the means of targeted delivery of therapeutics may enhance specific transport of biologically active drugs to target tissues, while avoiding or reducing undesired side effects. In the present investigation, peptide-conjugated cationic liposomes were constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells and macrophages. There was unaltered secretion of cytokines after exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5ß1) were more effective in targeting of activated endothelial cells, as compared to a liposome with a peptide that targeted both the fibronectin and vitronectin receptors, as well as liposomes with a control peptide. The liposome targeted to the fibronectin receptor also displayed uptake in endothelial cells in co-culture with activated macrophages. Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells.

Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles.

Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)-pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.

Physiologically-based pharmacokinetic modeling to predict the clinical pharmacokinetics of monoclonal antibodies.

Accurate prediction of the clinical pharmacokinetics of new therapeutic entities facilitates decision making during drug discovery, and increases the probability of success for early clinical trials. Standard strategies employed for predicting the pharmacokinetics of small-molecule drugs (e.g., allometric scaling) are often not useful for predicting the disposition monoclonal antibodies (mAbs), as mAbs frequently demonstrate species-specific non-linear pharmacokinetics that is related to mAb-target binding (i.e., target-mediated drug disposition, TMDD). The saturable kinetics of TMDD are known to be influenced by a variety of factors, including the sites of target expression (which determines the accessibility of target to mAb), the extent of target expression, the rate of target turnover, and the fate of mAb-target complexes. In most cases, quantitative information on the determinants of TMDD is not available during early phases of drug discovery, and this has complicated attempts to employ mechanistic mathematical models to predict the clinical pharmacokinetics of mAbs. In this report, we introduce a simple strategy, employing physiologically-based modeling, to predict mAb disposition in humans. The approach employs estimates of inter-antibody variability in rate processes of extravasation in tissues and fluid-phase endocytosis, estimates for target concentrations in tissues derived through use of categorical immunohistochemical scores, and in vitro measures of the turnover of target and target-mAb complexes. Monte Carlo simulations were performed for four mAbs (cetuximab, figitumumab, dalotuzumab, trastuzumab) directed against three targets (epidermal growth factor receptor, insulin-like growth factor receptor 1, human epidermal growth factor receptor 2). The proposed modeling strategy was able to predict well the pharmacokinetics of cetuximab, dalotuzumab, and trastuzumab at a range of doses, but trended towards underprediction of figitumumab concentrations, particularly at high doses. The general agreement between model predictions and experimental observations suggests that PBPK modeling may be useful for the a priori prediction of the clinical pharmacokinetics of mAb therapeutics.

Ligand-targeted bacterial minicells: Futuristic nano-sized drug delivery system for the efficient and cost effective delivery of shRNA to cancer cells.

In this study, shRNA against VEGFA was packaged in bacterial minicells and surface of minicells was modified with folic acid. Analysis of cellular internalization revealed that folic acid conjugated minicells internalized through receptor mediated endocytosis in folate and PSMA receptor positive KB and LNCaP cells, respectively. In contrast, A549 (folate receptor negative) cells showed minute internalization. In vitro delivery of FAminicellsVEGFA significantly reduced the expression of VEGFA mRNA in KB and LNCaP cells whereas expression of VEGFA remained unaltered in A549 cells. FAminicellsVEGFA significantly reduced tumor volume in mice bearing KB and LNCaP xenograft. On contrary, gradual increase in the tumor volume was recorded in A549 xenograft. FAminicellsVEGFA significantly silenced the VEGFA mRNA in KB and LNCaP xenograft. Expression of VEGFA remained same in FAminicellsVEGFA delivered A549 xenograft. In vivo biodistribution study showed that majority of FAminicellsVEGFA were localized in the tumor followed by intravenous administration.

In-vitro cytotoxicity assessment of carbon-nanodot-conjugated Fe-aminoclay (CD-FeAC) and its bio-imaging applications.

We have investigated the cytotoxic assay of Fe-aminoclay (FeAC) nanoparticles (NPs) and simultaneous imaging in HeLa cells by photoluminescent carbon nanodots (CD) conjugation. Non-cytotoxic, photostable, and CD NPs are conjugated with cationic FeAC NPs where CD NPs play a role in bio-imaging and FeAC NPs act as a substrate for CD conjugation and help to uptake of NPs into cancer cells due to positively charged surface of FeAC NPs in physiological media. As increase of CD-FeAC NPs loading in HeLa cell in vitro, it showed slight cytotoxicity at 1000 µg/mL but no cytotoxicity for normal cells up to concentration of 1000 µg/mL confirmed by two 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR) assays, with further observations by 4',6-diamidino-2-phenylindole (DAPI) stained confocal microscopy images, possessing that CD-FeAC NPs can be used as potential drug delivery platforms in cancer cells with simultaneous imaging. Graphical abstract CD conjugation with organo-building blocks of delaminated FeAC NPs.