Lymphatic network drainage resolves cerebral edema and facilitates recovery from experimental cerebral malaria.

While brain swelling, associated with fluid accumulation, is a known feature of pediatric cerebral malaria (CM), how fluid and macromolecules are drained from the brain during recovery from CM is unknown. Using the experimental CM (ECM) model, we show that fluid accumulation in the brain during CM is driven by vasogenic edema and not by perivascular cerebrospinal fluid (CSF) influx. We identify that fluid and molecules are removed from the brain extremely quickly in mice with ECM to the deep cervical lymph nodes (dcLNs), predominantly through basal routes and across the cribriform plate and the nasal lymphatics. In agreement, we demonstrate that ligation of the afferent lymphatic vessels draining to the dcLNs significantly impairs fluid drainage from the brain and lowers anti-malarial drug recovery from the ECM syndrome. Collectively, our results provide insight into the pathways that coordinate recovery from CM.

Selective brain entry of lipid nanoparticles in haemorrhagic stroke is linked to biphasic blood-brain barrier disruption.

Haemorrhagic stroke represents a significant public health burden, yet our knowledge and ability to treat this type of stroke are lacking. Previously we showed that we can target ischaemic-stroke lesions by selective translocation of lipid nanoparticles through the site of blood-brain barrier (BBB) disruption. The data we presented in this study provide compelling evidence that haemorrhagic stroke in mice induces BBB injury that mimics key features of the human pathology and, more importantly, provides a gate for entry of lipid nanoparticles-based therapeutics selectively to the bleeding site. Methods: Haemorrhagic stroke was induced in mice by intra-striatal collagenase injection. lipid nanoparticles were injected intravenously at 3 h, 24 h & 48 h post-haemorrhagic stroke and accumulation in the brain studied using in-vivo optical imaging and histology. BBB integrity, brain water content and iron accumulation were characterised using dynamic contrast-enhanced MRI, quantitative T1 mapping, and gradient echo MRI. Results: Using in-vivo SPECT/CT imaging and optical imaging revealed biphasic lipid nanoparticles entry into the bleeding site, with an early phase of increased uptake at 3-24 h post-haemorrhagic stroke, followed by a second phase at 48-72 h. Lipid nanoparticles entry into the brain post-haemorrhage showed an identical entry pattern to the trans-BBB leakage rate (Ktrans [min-1]) of Gd-DOTA, a biomarker for BBB disruption, measured using dynamic contrast-enhanced MRI. Discussion: Our findings suggest that selective accumulation of liposomes into the lesion site is linked to a biphasic pattern of BBB hyper-permeability. This approach provides a unique opportunity to selectively and efficiently deliver therapeutic molecules across the BBB, an approach that has not been utilised for haemorrhagic stroke therapy and is not achievable using free small drug molecules.

The impact of graphene oxide sheet lateral dimensions on their pharmacokinetic and tissue distribution profiles in mice.

Although the use of graphene and 2-dimensional (2D) materials in biomedicine has been explored for over a decade now, there are still significant knowledge gaps regarding the fate of these materials upon interaction with living systems. Here, the pharmacokinetic profile of graphene oxide (GO) sheets of three different lateral dimensions was studied. The GO materials were functionalized with a PEGylated DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), a radiometal chelating agent for radioisotope attachment for single photon emission computed tomography (SPECT/CT) imaging. Our results revealed that GO materials with three distinct size distributions, large (l-GO-DOTA), small (s-GO-DOTA) and ultra-small (us-GO-DOTA), were sequestered by the spleen and liver. Significant accumulation of the large material (l-GO-DOTA) in the lungs was also observed, unlike the other two materials. Interestingly, there was extensive urinary excretion of all three GO nanomaterials indicating that urinary excretion of these structures was not affected by lateral dimensions. Comparing with previous studies, we believe that the thickness of layered nanomaterials is the predominant factor that governs their excretion rather than lateral size. However, the rate of urinary excretion was affected by lateral size, with large GO excreting at slower rates. This study provides better understanding of 2D materials in vivo behaviour with varying structural features.

Intracerebral Injection of Graphene Oxide Nanosheets Mitigates Microglial Activation Without Inducing Acute Neurotoxicity: A Pilot Comparison to Other Nanomaterials.

Carbon-based nanomaterials (CNMs) are being explored for neurological applications. However, systematic in vivo studies investigating the effects of CNM nanocarriers in the brain and how brain cells respond to such nanomaterials are scarce. To address this, functionalized multiwalled carbon nanotubes and graphene oxide (GO) sheets are injected in mice brain and compared with charged liposomes. The induction of acute neuroinflammatory and neurotoxic effects locally and in brain structures distant from the injection site are assessed up to 1 week postadministration. While significant neuronal cell loss and sustained microglial cell activation are observed after injection of cationic liposomes, none of the tested CNMs induces either neurodegeneration or microglial activation. Among the candidate nanocarriers tested, GO sheets appear to elicit the least deleterious neuroinflammatory profile. At molecular level, GO induces moderate activation of proinflammatory markers compared to vehicle control. At histological level, brain response to GO is lower than after vehicle control injection, suggesting some capacity for GO to reduce the impact of stereotactic injection on brain. While these findings are encouraging and valuable in the selection and design of nanomaterial-based brain delivery systems, they warrant further investigations to better understand the mechanisms underlying GO immunomodulatory properties in brain.

Splenic Capture and In Vivo Intracellular Biodegradation of Biological-Grade Graphene Oxide Sheets.

Carbon nanomaterials, including 2D graphene-based materials, have shown promising applicability to drug delivery, tissue engineering, diagnostics, and various other biomedical areas. However, to exploit the benefits of these materials in some of the areas mentioned, it is necessary to understand their possible toxicological implications and long-term fate in vivo. We previously demonstrated that following intravenous administration, 2D graphene oxide (GO) nanosheets were largely excreted via the kidneys; however, a small but significant portion of the material was sequestered in the spleen. Herein, we interrogate the potential consequences of this accumulation and the fate of the spleen-residing GO over a period of nine months. We show that our thoroughly characterized GO materials are not associated with any detectable pathological consequences in the spleen. Using confocal Raman mapping of tissue sections, we determine the sub-organ biodistribution of GO at various time points after administration. The cells largely responsible for taking up the material are confirmed using immunohistochemistry coupled with Raman spectroscopy, and transmission electron microscopy (TEM). This combination of techniques identified cells of the splenic marginal zone as the main site of GO bioaccumulation. In addition, through analyses using both bright-field TEM coupled with electron diffraction and Raman spectroscopy, we reveal direct evidence of in vivo intracellular biodegradation of GO sheets with ultrastructural precision. This work offers critical information about biological processing and degradation of thin GO sheets by normal mammalian tissue, indicating that further development and exploitation of GO in biomedicine would be possible.

Immunological impact of graphene oxide sheets in the abdominal cavity is governed by surface reactivity.

Graphene oxide (GO) is an oxidised form of graphene that has attracted commercial interest in multiple applications, including inks, printed electronics and spray coatings, which all raise health concerns due to potential creation of inhalable aerosols. Although a number of studies have discussed the toxicity of GO sheets, the in vivo impact of their lateral dimensions is still not clear. Here, we compared the effects of large GO sheets (l-GO, 1-20 µm) with those of small GO sheets (s-GO, < 1 µm) in terms of mesothelial damage and peritoneal inflammation, after intraperitoneal (i.p.) injection in mice. To benchmark the outcomes, long and rigid multi-walled carbon nanotubes (MWCNTs) that were shown to be associated with asbestos-like pathogenicity on the mesothelium were also tested. Our aim was to assess whether lateral dimensions can be a predictor of inflammogenicity for GO sheets in a similar fashion as length is for MWCNTs. While long MWCNTs dispersed in 0.5% BSA induced a granulomatous response on the diaphragmatic mesothelium and immune cell recruitment to the peritoneal cavity, GO sheets dispersed under similar conditions did not cause any response, regardless of their lateral dimensions. We further interrogated whether tuning the surface reactivity of GO by testing different dispersions (5% dextrose instead of 0.5% BSA) may change the biological outcome. Although the change of dispersion did not alter the impact of GO on the mesothelium (i.e. no granuloma), we observed that, when dispersed in protein-free 5% dextrose solution, s-GO elicited a greater recruitment of monocytic cells to the peritoneal cavity than l-GO, or when dispersed in protein-containing solution. Such recruitment coincided with the greater ability of s-GO to interact in vivo with peritoneal macrophages and was associated with a greater surface reactivity in comparison to l-GO. In conclusion, large dimension was not a determining factor of the immunological impact of GO sheets after i.p. administration. For an equal dose, GO sheets with lateral dimensions similar to the length of long MWCNTs were less pathogenic than the MWCNTs. On the other hand, surface reactivity and the ability of some smaller GO sheets to interact more readily with immune cells seem to be key parameters that can be tuned to improve the safety profile of GO. In particular, the choice of dispersion modality, which affected these two parameters, was found to be of crucial importance in the assessment of GO impact in this model. Overall, these findings are essential for a better understanding of the parameters governing GO toxicity and inflammation, and the rational design of safe GO-based formulations for various applications, including biomedicine.

The Effects of Extensive Glomerular Filtration of Thin Graphene Oxide Sheets on Kidney Physiology.

Understanding how two-dimensional (2D) nanomaterials interact with the biological milieu is fundamental for their development toward biomedical applications. When thin, individualized graphene oxide (GO) sheets were administered intravenously in mice, extensive urinary excretion was observed, indicating rapid transit across the glomerular filtration barrier (GFB). A detailed analysis of kidney function, histopathology, and ultrastructure was performed, along with the in vitro responses of two highly specialized GFB cells (glomerular endothelial cells and podocytes) following exposure to GO. We investigated whether these cells preserved their unique barrier function at doses 100 times greater than the dose expected to reach the GFB in vivo. Both serum and urine analyses revealed that there was no impairment of kidney function up to 1 month after injection of GO at escalating doses. Histological examination suggested no damage to the glomerular and tubular regions of the kidneys. Ultrastructural analysis by transmission electron microscopy showed absence of damage, with no change in the size of podocyte slits, endothelial cell fenestra, or the glomerular basement membrane width. The endothelial and podocyte cell cultures regained their full barrier function after >48 h of GO exposure, and cellular uptake was significant in both cell types after 24 h. This study provided a previously unreported understanding of the interaction between thin GO sheets with different components of the GFB in vitro and in vivo to highlight that the glomerular excretion of significant amounts of GO did not induce any signs of acute nephrotoxicity or glomerular barrier dysfunction.

Radiolabeling, whole-body single photon emission computed tomography/computed tomography imaging, and pharmacokinetics of carbon nanohorns in mice.

In this work, we report that the biodistribution and excretion of carbon nanohorns (CNHs) in mice are dependent on their size and functionalization. Small-sized CNHs (30-50 nm; S-CNHs) and large-sized CNHs (80-100 nm; L-CNHs) were chemically functionalized and radiolabeled with [(111)In]-diethylenetriaminepentaacetic acid and intravenously injected into mice. Their tissue distribution profiles at different time points were determined by single photon emission computed tomography/computed tomography. The results showed that the S-CNHs circulated longer in blood, while the L-CNHs accumulated faster in major organs like the liver and spleen. Small amounts of S-CNHs- and L-CNHs were excreted in urine within the first few hours postinjection, followed by excretion of smaller quantities within the next 48 hours in both urine and feces. The kinetics of excretion for S-CNHs were more rapid than for L-CNHs. Both S-CNH and L-CNH material accumulated mainly in the liver and spleen; however, S-CNH accumulation in the spleen was more prominent than in the liver.

Molecular and Genomic Impact of Large and Small Lateral Dimension Graphene Oxide Sheets on Human Immune Cells from Healthy Donors.

Graphene oxide (GO) is attracting great interest in biomedical sciences. The impact of GO on immune cells is one fundamental area of study that is often overlooked, but critical in terms of clinical translation. This work investigates the effects of two types of thoroughly characterized GO sheets, different in their lateral dimension, on human peripheral immune cells provided from healthy donors using a wide range of assays. After evaluation of cell viability, the gene expression was analyzed, following GO exposure on 84 genes related to innate and adaptive immune responses. Exposure to GO small sheets was found to have a more significant impact on immune cells compared to GO large sheets, reflected in the upregulation of critical genes implicated in immune responses and the release of cytokines IL1ß and TNFα. These findings were further confirmed by whole-genome microarray analysis of the impact of small GO sheets on T cells and monocytes. Activation in both cell types was underlined by the overexpression of genes such as CXCL10 and receptor CXCR3. Significant energy-dependent pathway modulation was identified. These findings can potentially pave the foundations for further design of graphene that can be used for immune modulation applications, for example in cancer immunotherapy.

Tissue distribution and urinary excretion of intravenously administered chemically functionalized graphene oxide sheets.

The design of graphene-based materials for biomedical purposes is of great interest. Graphene oxide (GO) sheets represent the most widespread type of graphene materials in biological investigations. In this work, thin GO sheets were synthesized and further chemically functionalized with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), a stable radiometal chelating agent, by an epoxide opening reaction. We report the tissue distribution of the functionalized GO sheets labeled with radioactive indium (111In) after intravenous administration in mice. Whole body single photon emission computed tomography (SPECT/CT) imaging, gamma counting studies, Raman microscopy and histological investigations indicated extensive urinary excretion and predominantly spleen accumulation. Intact GO sheets were detected in the urine of injected mice by Raman spectroscopy, high resolution transmission electron microscopy (HR-TEM) and electron diffraction. These results offer a previously unavailable pharmacological understanding on how chemically functionalized GO sheets transport in the blood stream and interact with physiological barriers that will determine their body excretion and tissue accumulation.

siRNA liposome-gold nanorod vectors for multispectral optoacoustic tomography theranostics.

Therapeutic applications of gene silencing using siRNA have seen increasing interest over the past decade. The optimization of the delivery and biodistribution of siRNA using liposome-gold nanorod (AuNRs) nanoscale carriers can greatly benefit from adept imaging methods that can visualize the time-resolved delivery performance of such vectors. In this work, we describe the effect of AuNR length incorporated with liposomes and show their complexation with siRNA as a novel gene delivery vehicle. We demonstrate the application of multispectral optoacoustic tomography (MSOT) to longitudinally visualize the localisation of siRNA carrying liposome-AuNR hybrids within tumors. Combination of in vivo MSOT with ex vivo fluorescence cryo-slice imaging offers further insight into the siRNA transport and activity obtained.