Internalization Of Nanoparticles Research Articles

Non-equilibrium transport of nanoparticles across the lipid membrane.

Development of effective strategies for the internalization of nanoparticles is essential in many applications, such as drug delivery. Most, if not all, previous studies are based on equilibrium considerations. In this work, inspired by the recent development of a pro-drug delivery strategy based on reversible esterification, we consider a non-equilibrium transport mechanism for nanoparticles of a 6 nm diameter across the lipid membrane. We divide the transport process into insertion and ejection steps, which are studied with coarse-grained models using free energy and reactive Monte Carlo simulations, respectively. The simulations show that the non-equilibrium transport efficiency is relatively insensitive to the fraction of reactive surface ligands once a modest threshold is surpassed, while the distribution pattern of different (hydrophilic, reactive and permanent hydrophobic) ligands on the nanoparticle surface has a notable impact on both the insertion and ejection steps. Our study thus supports a novel avenue for designing nanoparticles that are able to be efficiently internalized and provides a set of relevant guidelines for surface functionalization.

Selective killing of cancer cells by silica nanoparticles due to increased nanoparticle internalization and cellular sensitivity to oxidative stress

Selective killing of cancer cells by silica nanoparticles due to increased nanoparticle internalization and cellular sensitivity to oxidative stress

Study of Cytotoxicity and Internalization of Redox-Responsive Iron Oxide Nanoparticles on PC-3 and 4T1 Cancer Cell Lines.

Redox-responsive and magnetic nanomaterials are widely used in tumor treatment separately, and while the application of their combined functionalities is perspective, exactly how such synergistic effects can be implemented is still unclear. This report investigates the internalization dynamics of magnetic redox-responsive nanoparticles (MNP-SS) and their cytotoxicity toward PC-3 and 4T1 cell lines. It is shown that MNP-SS synthesized by covalent grafting of polyethylene glycol (PEG) on the magnetic nanoparticle (MNP) surface via SS-bonds lose their colloidal stability and aggregate fully in a solution containing DTT, and partially in conditioned media, whereas the PEGylated MNP (MNP-PEG) without S-S linker control remains stable under the same conditions. Internalized MNP-SS lose the PEG shell more quickly, causing enhanced magnetic core dissolution and thus increased toxicity. This was confirmed by fluorescence microscopy using MNP-SS dual-labeled by Cy3 via labile disulfide, and Cy5 via a rigid linker. The dyes demonstrated a significant difference in fluorescence dynamics and intensity. Additionally, MNP-SS demonstrate quicker cellular uptake compared to MNP-PEG, as confirmed by TEM analysis. The combination of disulfide bonds, leading to faster dissolution of the iron oxide core, and the high-oxidative potential Fe3+ ions can synergically enhance oxidative stress in comparison with more stable coating without SS-bonds in the case of MNP-PEG. It decreases the cancer cell viability, especially for the 4T1, which is known for being sensitive to ferroptosis-triggering factors. In this work, we have shown the effect of redox-responsive grafting of the MNP surface as a key factor affecting MNP-internalization rate and dissolution with the release of iron ions inside cancer cells. This kind of synergistic effect is described for the first time and can be used not only in combination with drug delivery, but also in treatment of tumors responsive to ferroptosis.

Microfluidic-Assisted Production of Gastro-Resistant Active-Targeted Diatomite Nanoparticles for the Local Release of Galunisertib in Metastatic Colorectal Cancer Cells.

The oral route is highly desirable for colorectal cancer (CRC) treatment because it allows concentrating the drug in the colon and achieving a localized effect. However, orally administered drugs are often metabolized in the liver, resulting in reduced efficacy and the need for higher doses. Nanoparticle-based drug delivery systems can be engineered to prevent the diffusion of the drug in the stomach, addressing the release at the target site, and enhancing the efficacy of the delivered drug. Here, an orally administrable galunisertib delivery system is developed with gelatin-covered diatomite nanoparticles targeting the ligand 1-cell adhesion molecule (L1-CAM) on metastatic cells, and further encapsulated in an enteric matrix by microfluidics. The gastro-resistant polymer protects the nanoparticles from the action of the digestive enzymes and allows for a sustained release of galunisertib at the intestinal pH. The efficacy of antibody-antigen interactions to drive the internalization of nanoparticles in the targeted cells is investigated in CRC cells expressing abnormal (SW620) or basal levels (Caco-2, HT29-MTX) of L1-CAM. The combination of local drug release and active targeting enhances the effect of the delivered galunisertib, which inhibits the migration of the SW620 cells with greater efficiency compared to the free drug.

Influences of repeated exposure to low levels of TiO2 nanoparticles on Kruppel-like factors in 3D Caco-2 spheroids and mouse intestines.

Kruppel-like factors (KLFs) are a set of transcription factors (TFs) involved in the regulation of many basic biological processes, and recent studies suggested that nanoparticles (NPs) were capable to change KLFs in different models even at non-cytotoxic concentrations. In this study, we repeatedly exposed 3D Caco-2 spheroids and mice to TiO2 NPs, one of the most frequently used metal oxide NPs, and investigated the changes of KLF-signaling pathways based on RNA-sequencing. Although the internalization of TiO2 NPs did not induce cytotoxicity in vitro, repeated exposure (three times within 7 days) to 15.7 ng/ml TiO2 NPs increased KLF4 but decreased KLF6. Consistently, KLF4/KLF6-regulated gene ontology terms were altered, including those involved in the regulation of gene expression. We further verified that repeated exposure to 15.7 ng/ml TiO2 NPs increased the expression of KLF4 and proto-oncogene, bHLH transcription factor (MYC), but decreased the expression of KLF6 and activating transcription factor 3 (ATF3). But with the increase of NP concentrations, the expression of these genes was decreased. In mice following intragastrical exposure to 4.39 and 43.9 mg/kg TiO2 NPs (once a day for 5 continuous days), we observed increased expression of klf4, klf6, myc, and atf3, along with morphological changes of intestines. We concluded that repeated exposure to low levels of TiO2 NPs altered KLF-signaling pathways in intestinal cells both in vitro and in vivo.

Thiolated β-cyclodextrin modified iron oxide nanoparticles for effective targeted cancer therapy

Inspired by the mitochondria iron-sulfur protein, direct functionalization of thiolated β-cyclodextrin (β-CD-SH) on iron oxide nanoparticles (IONPs) through Fe-S bond was done. The resulting system had an average size of 14 nm with an ellipsoidal shape. The X-Ray photoelectron spectroscopy (XPS) confirmed the formation of the Fe-S bond. Doxorubicin (DOX) was chosen as a model drug, about 12.45 µM mg -1 entrapped in β-CD-SH coated IONPs (TβCD-IONPs). The hybrid nanocarrier possessed high stability and drug loading efficiency. The invitro release data revealed an overall sustainable release profile without initial bust. The Higuchi kinetic model best fits the release mechanism, based on diffusion action in dosages proportional to the square root of time. The surface coating and particle size have a crucial role in the cellular responses and effective toxic mechanisms. The cellular internalization of drug-loaded nanoparticles (NPs) into the breast cancer cell line MCF-07 was done using MTT assay and confocal imaging. The prepared system shows high performance with an IC 50 value at 67 nM of nanoparticle concentration. The prepared nanoparticles are promising candidates for the effective targeted delivery of hydrophobic drugs with enhanced theragnostic activity.

Bridging the In Vitro to In Vivo gap: Using the Chick Embryo Model to Accelerate Nanoparticle Validation and Qualification for In Vivo studies.

We postulate that nanoparticles (NPs) for use in therapeutic applications have largely not realized their clinical potential due to an overall inability to use in vitro results to predict NP performance in vivo. The avian embryo and associated chorioallantoic membrane (CAM) has emerged as an in vivo preclinical model that bridges the gap between in vitro and in vivo, enabling rapid screening of NP behavior under physiologically relevant conditions and providing a rapid, accessible, economical, and more ethical means of qualifying nanoparticles for in vivo use. The CAM is highly vascularized and mimics the diverging/converging vasculature of the liver, spleen, and lungs that serve as nanoparticle traps. Intravital imaging of fluorescently labeled NPs injected into the CAM vasculature enables immediate assessment and quantification of nano-bio interactions at the individual NP scale in any tissue of interest that is perfused with a microvasculature. In this review, we highlight how utilization of the avian embryo and its CAM as a preclinical model can be used to understand NP stability in blood and tissues, extravasation, biocompatibility, and NP distribution over time, thereby serving to identify a subset of NPs with the requisite stability and performance to introduce into rodent models and enabling the development of structure-property relationships and NP optimization without the sacrifice of large populations of mice or other rodents. We then review how the chicken embryo and CAM model systems have been used to accelerate the development of NP delivery and imaging agents by allowing direct visualization of targeted (active) and nontargeted (passive) NP binding, internalization, and cargo delivery to individual cells (of relevance for the treatment of leukemia and metastatic cancer) and cellular ensembles (e.g., cancer xenografts of interest for treatment or imaging of cancer tumors). We conclude by showcasing emerging techniques for the utilization of the CAM in future nano-bio studies.

Uncloaking cell-impermeant gold nanorods via tumor microenvironmental cathepsin B facilitates cancer cell penetration and potent radiosensitization

Major impediments to conveyance of intravenously administered drugs to tumors are biofouling, opsonization, and rapid clearance from the circulation by macrophages and reticuloendothelial phagocytes. Cloaking nanoparticles with stealth epilayers partly overcomes these hurdles but it also foils interactions with tumor cells. Here, we describe the synthesis, characterization, and validation of smart gold nanorods (GNRs) that spontaneously transform from inert passengers in the blood stream to active cell-penetrating nanoparticles within tumors to potently sensitize tumors to radiation therapy. Intrinsically cationic and cell-penetrating GNRs were shielded from phagocytosis with a cloaking polyethylene glycol epilayer containing an intervening cleavable peptide. In the absence of an external trigger, this epilayer is clipped off by the tumor microenvironmental protease, cathepsin B, in colorectal cancers to uncloak and expose the free-circulating native unPEGylated GNR that is readily internalized by cancer cells and turn into immovable small clusters of GNRs. Selective uncloaking of GNRs in the tumor reduced off-target toxicity confirmed by hematologic, biochemical, and histopathological analysis of blood, serum, and normal organs, respectively. Subsequent irradiation led to significant tumor growth delay and improved survival of mice. By addressing multiple barriers to efficient transport and cellular internalization of nanoparticles, our results demonstrate that clinically meaningful radiosensitization can be achieved with rationally designed GNRs.

On predicting heterogeneity in nanoparticle dosage

Nanoparticles are increasingly employed as a vehicle for the targeted delivery of therapeutics to specific cell types. However, much remains to be discovered about the fundamental biology that dictates the interactions between nanoparticles and cells. Accordingly, few nanoparticle-based targeted therapeutics have succeeded in clinical trials. One element that hinders our understanding of nanoparticle–cell interactions is the presence of heterogeneity in nanoparticle dosage data obtained from standard experiments. It is difficult to distinguish between heterogeneity that arises from stochasticity in nanoparticle-cell interactions, and that which arises from heterogeneity in the cell population. Mathematical investigations have revealed that both sources of heterogeneity contribute meaningfully to the heterogeneity in nanoparticle dosage. However, these investigations have relied on simplified models of nanoparticle internalisation. Here we present a stochastic mathematical model of nanoparticle internalisation that incorporates a suite of relevant biological phenomena such as multistage internalisation, cell division, asymmetric nanoparticle inheritance and nanoparticle saturation. Critically, our model provides information about nanoparticle dosage at an individual cell level. We perform model simulations to examine the influence of specific biological phenomena on the heterogeneity in nanoparticle dosage in the absence of heterogeneity in the cell population. Under certain modelling assumptions, we derive analytic approximations of the nanoparticle dosage distribution. We demonstrate that the analytic approximations are accurate, and show that nanoparticle dosage can be described by a Poisson mixture distribution with rate parameters that are a function of Beta-distributed random variables. We discuss the implications of the analytic results with respect to parameter estimation and model identifiability from standard experimental data. Finally, we highlight extensions and directions for future research.

Characterization of mesenchymal stem cells with augmented internalization of magnetic nanoparticles: The implication of therapeutic potential

Mesenchymal stem cells (MSCs) possess the potential to repair damaged tissue and regenerate the cells with a specific function. MSCs dispersed in the circulatory system tend to enter pulmonary tissue, arming the ability to change the lung microenvironment, prevent pulmonary fibrosis, and treat pulmonary injuries. It has been demonstrated that magnetic nanoparticles (MNPs) not only can be used as the vector for drug delivery, which can improve the specificity and control the drug released; but also act as the contract media for imaging, possessing the potential of being tracer and conducting differentiation to comply repairment. Epigallocatechin gallate (EGCG) has been known to greatly enhance MNP uptake by tumor cells under the influence of magnetic fields. However, whether EGCG significantly improves the MNP uptake by MSCs in the magnetic fields and whether MSCs maintain the characteristics of MSCs with such treatment remains unknown. In this study, we demonstrated that EGCG significantly enhanced the MNP uptake by MSCs in the magnetic field. With 30 µM EGCG in the magnetic field for 2 h, the cell-associated MNPs are increased 7.1 and 3.4 times by mouse and human MSCs without a distinguished influence on cell morphology, respectively. Furthermore, we evaluated the osteogenic differentiation potential of human MSCs after MNP uptake by assessing the relative gene expressions of osteogenic markers Runt-related transcription factor 2 (RUNX2), Osterix (Sp7), and Alkaline phosphatase (ALPL). There is no definite evidence showing that the expressions of these osteogenic marker genes, upon MNPs uptake, are significantly different from those of the control group. Surface antigen expression assay by flow cytometry revealed a similar expression population of human MSC surface markers CD73 and CD90. The results suggest that the stemness and differentiation capacity of MSCs are not affected after the MNP uptake. Our study demonstrates that combining EGCG and magnetic fields is an effective method to enhance MNP uptake by MSCs. The study serves as the foundation of the research related to the clinical application of combining MNPs and MSCs. We anticipate that the therapeutic potential of MSCs can be explored further to treat new coronary pneumonia and other diseases through this research platform.

Toxicity of nanomixtures to human macrophages: Joint action of silver and polystyrene nanoparticles

Increasing use of nano-enabled products provides many benefits in various industrial processes and medical applications, but it also raises concern about release of nanoparticles (NPs) into the environment and subsequent human exposure. While potential toxicity of individual NPs types has been well described in scientific literature, exposure and health-related effects of nanomixtures has been poorly described. This study aimed to evaluate the combined effect of silver (AgNP) and polystyrene NPs (PSNP) on the human macrophages. AgNP are one of the most commercialized NPs due to efficient antimicrobial activity, while PSNP are ubiquitous in terrestrial and aquatic environments due to plastic pollution and degradation of polystyrene-based products. Differentiated monocytic cell line THP-1 were used as an in vitro model of human macrophages. Multiple aspects of cellular response to AgNP-PSNP nanomixture were analyzed including cell death, induction of apoptosis, oxidative stress response, expression of pro- and anti-inflammatory cytokines, and nanomechanical properties of cells. NPs uptake was visualized by confocal microscopy and quantified using flow cytometry. Results show that nanomixture increased apoptosis and cell death, expression of IL-6, IL-8 and TNFa, oxidative stress and mitochondrial dysfunction in cells compared to AgNP and PSNP applied as single treatments, indicating mixture additive action. Anti-inflammatory cytokines IL1b, IL-4 and IL-10 were not affected by combined exposure compared to single NPs. Visualization of NPs uptake and internalization showed that AgNP and PSNP were localized mostly in cytoplasm, with small fraction of AgNP translocated into cell nuclei, which explain increased number of double-stranded DNA breaks following exposure of cells to AgNPs alone or in the mixture. Study outcomes represent clear warnings on the human co-exposure to AgNP and PSNP that needs to be implemented in risk assessment approaches towards toxic-free environment.

Theranostic Hyaluronan Coated EDTA Modified Magnetic Mesoporous Silica Nanoparticles for Targeted Delivery of Cisplatin

One of the eminent capabilities of nanoparticles in drug delivery is active targeting. Mesoporous silica nanoparticles (MSNs) are among the well-known nanocarriers, which have been utilized for gene and drug deliveries due to their particular physical and chemical properties. The present study aimed to introduce an engineered type of EDTA-modified Magnetic Mesoporous Silica Nanoparticles (MMSNs) with hyaluronan coating as a theranostic agent for delivery of an anti-cancer drug (cisplatin). EDTA was first applied as a ligand for platinum-based drug loading. The nanoparticles were synthesized in several steps. The properties of each step were characterized by various techniques such as Fourier transform infrared (FT-IR), X-Ray diffraction (XRD), vibrating sample magnetometer (VSM), and N2 adsorption/desorption. Morphology was also checked by field emission scanning electron microscopy (FE-SEM) and Transmission electron microscopy (TEM). In addition, loading capacity (LC) and loading efficiency, and in vivo release of cisplatin were analyzed through ICP-OES. Eventually, the plasma concentration profile and cisplatin pharmacokinetics parameters after intravenous administration of EDTA-MMSN @ HA were evaluated. The average particle diameters of prepared structure were approximate range of 70–100 nm. The profile release of cisplatin in acidic medium was faster in 72 h, which confirmed the pH-responsive behavior of the nanoparticles. As the results of cytotoxicity showed, coating with HA can improve the internalization of nanoparticles in cancer cells compared to normal cells. Finally, EDTA-MMSN @ HA can be introduced as a new favor biocompatible and biodegradable nanoplatform for targeted drug delivery which can improve pharmacokinetic parameters. Overall, this study successfully synthesized a novel multifunctional pH-responsive MSN for efficient drug delivery and magnetic resonance imaging.

Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.

In vitro toxicity and internalization of gold nanoparticles (AuNPs) in human epithelial colorectal adenocarcinoma (Caco-2) cells and the human skin keratinocyte (HaCaT) cells

Ingestion and transdermal delivery are two common routes of nanoparticle (NP) exposure. In this study, the intracellular uptake, cytotoxicity and genotoxicity of 14 nm and 20 nm citrate-stabilized gold nanoparticles (AuNPs), 14 nm polyethylene glycol (PEG)-liganded carboxyl AuNPs, 14 nm PEG-liganded hydroxyl AuNPs and 14 nm PEG-liganded amine AuNPs were assessed on human epithelial colorectal adenocarcinoma (Caco-2) cells and the human skin keratinocyte (HaCaT) cells. The uptake of AuNPs in the cells was confirmed through darkfield microscopy and hyperspectral imaging followed by spectral angle mapping (SAM). A high level of citrate AuNPs was found in both cell lines whilst uptake of PEGylated AuNPs was low, irrespective of their functional groups. Cytotoxicity assessed by cell impedance was only observed for the 14 nm citrate-stabilized AuNPs. Enhanced cell proliferation was also observed in 14 nm PEG-liganded hydroxyl and 14 nm PEG-liganded amine AuNP-treated Caco-2 and HaCaT cells. For the assessment of genotoxicity, the in vitro micronucleus assay was used. Dose-dependent genotoxicity was observed in both Caco-2 and HaCaT cells, with all the AuNPs inducing genotoxicity. In conclusion, the entry of NPs into the cells as well as toxicity was dependent on their physicochemical properties such as surface coating and different chemical functional groups.

Differential near-infrared imaging of heterocysts using single-walled carbon nanotubes

The internalization of near-infrared (NIR) optical nanoprobes in photosynthetic microbes can be exploited for applications ranging from energy conversion to biomolecule delivery. However, the intrinsic, species-dependent properties of microbial cell walls, including their surface charge density, composition, thickness, and elasticity, can severely impact nanoprobe uptake and affect the cellular response. An examination of the interaction of the optical nanoprobe in various species and its impact on cell viability is, therefore, imperative for the development of new imaging technologies. Herein, we extend the technology recently developed for internalizing fluorescent single-walled carbon nanotubes (SWCNTs) in prokaryotes, specifically unicellular Synechocystis sp. PCC 6803, to a filamentous cyanobacterial strain, Nostoc punctiforme. Using a combination of NIR fluorescence, scanning electron microscopy (SEM), and Raman spectroscopy, we investigate uptake in vegetative cells as well as differentiated heterocysts. We demonstrate a strong dependence of long-term cell integrity, activity, and viability on SWCNT surface functionalization. We further show differential uptake of SWCNTs across a single filament, with positively charged functionalized SWCNTs preferentially localizing within the heterocysts of the filament. This cell dependency of the nanoparticle internalization motivates the use of SWCNTs as a NIR stain for monitoring cell differentiation.Graphical abstract

A multi-biomarker approach to assess the sublethal effects of settleable atmospheric particulate matter from an industrial area on Nile tilapia (Oreochromis niloticus)

Iron and steel industries discharge a large amount of atmospheric particulate matter (PM) containing metals and metallic nanoparticles (NPs) that contaminate not only the air, but also settle into the aquatic environments. However, the effects of settleable atmospheric particulate matter (SePM) on aquatic fauna are still poorly understood. This study aimed to evaluate the sublethal effects of a short-term exposure to a realistic concentration of SePM on Nile tilapia (Oreochromis niloticus) using a multi-biomarker approach: relative ventricular mass (RVM) and heart function, blood oxidative stress, stress indicators, hemoglobin concentration, metallic NPs internalization, and metal bioaccumulation. Exposed fish exhibited reduced hemoglobin content and elevated plasma cortisol and glucose levels, reflecting stressed states. Furthermore, SePM caused blood oxidative stress increasing lipid and protein oxidation, decreasing glutathione levels, and inhibiting superoxide and glutathione reductase activities. SePM exposure also increased RVM and improved cardiac performance, increasing myocardial contractile force and rates of contraction and relaxation. In the heart tissue there was a significant accumulation of Fe > Zn > > Cr > Cu > Rb > Ni > V > Mn > Se > Mo > As. On the other hand, in the erythrocytes there was significant accumulation of Sn > Zn > > Cr > Ti > Mn = Ni > Nb > As > Bi. The highest bioaccumulation factors were found for Cr, Zn and Ni in both tissues. NPs (Ti, Sn, Al, Fe, Cu, Si, Zn) were also detected in ventricular myocardium of fish exposed and nanocrystallographic analysis revealed a predominance of anatase phase of TiO2-NP, which is regarded to be more cytotoxic. The association between blood oxidative stress and energy expenditure to sustain increased cardiac pumping capacity under stress condition suggests that SePM has negative impacts on fish physiological performance, threatening their survival, growth rate and/or population establishment.

Pro-drug Nanoparticle Loaded Supramolecular Hydrogels for Drug Delivery to IDH1 Wild-Type Glioblastoma

Abstract AIMS A hyaluronic acid-based hydrogel with mechanical properties comparable to human brain tissue is being investigated as an implantable drug-delivery vehicle for post-surgical treatment of IDH1 wild-type glioblastoma (GBM). Hydrogels are to be loaded with polymeric pro-drug nanoparticles to enable controlled drug release and increased drug penetration through brain parenchyma for enhanced treatment of residual disease within and beyond the invasive margin. METHOD A panel of patient-derived glioblastoma cell lines resected from tumour core and invasive margin, have been used to generate tumour spheroids. Chemotherapeutic drug screening has been performed to select lead compounds for formulation and gel loading. Synergies of drug combinations have been studied for potential combination loading. Upcoming work will investigate nanoparticle internalisation, spheroid penetration and prodrug nanoparticle potency. Nanoparticle loaded hydrogels are then to be studied for validation of effective in vitro application. RESULTS Current results demonstrate that patient-derived cell lines can generate robust, reproducible tumour spheroids with diameters approx. 350-400 µm. Patient-to-patient differences in drug potencies were observed, with differences also noted between counterpart core and marginal cells from the same patients, illustrating the importance of using patient-derived cells and distinct regional cells to study heterogenous disease such as GBM. Drug combination experiments indicated synergy of topoisomerase I inhibitor (Irinotecan) with PARP inhibitor (Ola-parib). Interestingly, combination of PARP inhibition with topoisomerase II inhibitors (Doxorubicin, Etoposide) was found not synergistic. CONCLUSION Together, data gathered on single drug and drug-drug combinations will inform upcoming formulation and in vitro application of hydrogels planned, with eventual translation to pre-clinical in vivo models.

Investigation on the self-assembly of the NFL-TBS.40-63 peptide and its interaction with gold nanoparticles as a delivery agent for glioblastoma

NFL-TBS.40-63 peptide is a recently discovered peptide derived from the light neurofilament chain (NFL). In this study, we demonstrated that the Biotinylated-NFL-peptide (BIOT-NFL) can spontaneously self-assemble into well-organized nanofibers (approximately 5 nm width and several micrometers in length) in several solutions, whereas the typical self-assembly was not systematically observed from other peptides with or without coupling. The critical aggregation concentration that allows the BIOT-NFL-peptide to aggregate and auto associate was determined at 10−4 mol/L by surface tension measurements. X-ray scattering of BIOT-NFL-peptide also demonstrated its beta-sheet structure that can facilitate the intermolecular interactions involved in the self-assembly process. The possible disassembly of self-assembled BIOT-NFL-peptide-nanofibers was examined via a dialysis membrane study. We further investigated the interaction between nanofibers formed by BIOT-NFL-peptide and gold nanoparticles. Interestingly, a strong interaction was demonstrated between these nanoparticles and BIOT-NFL-peptide resulted in the formation of BIOT-NFL-peptide-nanofibers grandly decorated by gold nanoparticles. Finally, we investigated the internalization of gold nanoparticles coupled with BIOT-NFL-nanofibers into F98 rat glioblastoma cells, which was increased compared to the non-coupled control gold nanoparticles. All these results indicate that this peptide could be a promising therapeutic agent for targeted delivery.

Effect of graphene oxide nanoparticles on viability of BAP3 hybridoma cells

Graphene oxide (GO) is a promising material, which is likely to find applications in the fields of medicine and biotechnology. However, the current knowledge of its influence on human organism is limited. Even less information is available on the effects of GO on the cell lines widely used in biotechnology. The aim of this work is to describe the interaction between GO nanoparticles and BAP3 hybridoma cells which produce anti-human-PSG1 IgG, in vitro. We studied the effect of GO nanoparticles on cell viability and the intensity of internalization (adhesion) of nanoparticles by the cells. We used GO nanoparticles of different size, with surface being functionalized by linear or branched PEG (GO-PEG). The PEG coating level was 20% (by mass). The following nanoparticle concentrations were used: 5 g/mL and 25 g/mL. The BAP3 cells were cultured in a 48-well cell culture plates in serum-free DCCM-1 media in the presence of GO nanoparticles. The cells were cultured for 24 hours at 37 С and 5% СО2. Cell viability was assessed by a flow cytometer utilizing Zombie Aqua (ZA) staining. Internalization (adhesion) of nanoparticles was monitored using a flow cytometer by GO fluorescense in the samples (ex = 488 nm). Moreover, interactions between hybridoma cells and GO nanoparticles were visualized by EVOS M5000 visualization system, which included an inverted fluorescent microscope.
 We demonstrated that GO nanoparticles possess a cytotoxic effect when applied at high concentration (25 g/mL). The highest cytotoxic effect is caused by GO nanoparticles coated with linear PEG. The degree of nanoparticle internalization (adhesion) was shown to be significantly lower when the particles were present at lower (5 g/mL) concentration. Internalization (adhesion) of nanoparticles of smaller size was more abundant. Furthermore, these nanoparticles were shown to have a stronger cytotoxic effect compared to larger particles. In general, cytotoxicity of GO nanoparticles decreases with increasing size, which is especially evident if the fact that the mean effective diameter of the nanoparticles coated with branched PEG is considered larger than their linear PEG-coated counterparts. The data obtained allow us to draw a correlation between the cytotoxic effect of GO nanoparticles and the level of their internalization (adhesion) by the cells. In general, this work concerns some novel aspects of interaction between GO nanoparticles and hybridoma cells.

CD44-targeted nanoparticles with GSH-responsive activity as powerful therapeutic agents against breast cancer

DOX-loaded nanoparticles able to actively target CD44-receptors and respond to redox stimuli were proposed as non-conventional chemotherapeutic strategy in breast cancer. A covalent conjugate of human serum albumin and hyaluronic acid was prepared and assembled by a GSH-mediated desolvation in disulfide-crosslinked solid nanoparticles with mean diameter of 120 nm ± 3.4. The effective internalization of nanoparticles in cancer cells via CD44-receptors, together with the more efficient intracellular release, resulted in a significant increase of drug efficacy, with IC50 reduced from 0.9959 and 2.516 μg mL−1 to 0.4014 and 0.3094 μg mL−1 for MCF-7 and MDA-MB-231, respectively. Conversely, no enhancement in drug toxicity was recorded in healthy MCF-10A cells. The efficacy of the proposed formulation was further investigated in the different biological steps involved in metastasis process, paving the way for further in vivo experiments.