In vivo toxicological evaluation of peptide conjugated gold nanoparticles for potential application in colorectal cancer diagnosis

Colorectal cancer (CRC) is among the leading cause of cancer-related deaths in South Africa and worldwide. Efforts are being made at finding improved diagnostic tools, as early detection (before metastasis) is a major factor in CRC treatment. Colonoscopy is the most reliable detection method, but is a specialised and expensive procedure, which is invasive, not readily available and not patient-friendly. There is a risk of developing interval cancers, as colonoscopies are performed every 10 years after the age of 40. The development of non-invasive, cost efficient and readily available diagnostic tools to CRC, which can be performed at more regular intervals, using tumour-targeting molecular imaging agents, is of urgent attention. Gold nanoparticles (AuNPs) possess several physicochemical properties, including ease of synthesis, biocompatibility, and the ability to be conjugated by ligands or biomolecules such as polyethylene glycol (PEG) and peptides for improved stability, tissue targeting and selectivity. These factors potentiate the role in biomedical applications, including cancer theranostics. Conjugation of AuNPs with a targeting molecule (e.g. antibody or peptide) is directed against cancer cell receptors. The peptides, p.C, p.L, and p.14, bind to CRC cells in vitro. Conjugation of AuNPs with these peptides should be investigated for CRC diagnosis in vivo, as it is hypothesised to allow examinations at shorter intervals through imaging techniques. This could reduce the risk of interval cancers, but before developing this novel tool, in vivo toxicity evaluations are essential. This study was therefore aimed at investigating the short- and long-term toxicological effects of a single intravenous injection of peptides (p.C, p.L, and p.14) conjugated to AuNPs in a healthy rat model. Citrate-capped AuNPs were synthesised by the citrate-reduction method, and conjugated with each peptide (biotinylated) using a combination of PEG (99% PEG-OH and 1% PEG-biotin) as a stabilising agent and linker, via biotin-streptavidin interaction. Healthy male Wistar rats were intravenously injected with 14 nm citrate-AuNPs, PEG-, p.C-PEG, p.L-PEG, and p.14-PEG-AuNPs (100 μg/kg body weight), and the control rats were injected with phosphate buffered saline. The animals were monitored for behavioural, physiological, biochemical, haematological and histological changes, as well as inflammatory responses. Phase 1 rats were sacrificed 2 weeks post-injection to determine the immediate or acute toxicity of the AuNPs, while phase 2 animals were sacrificed 12 weeks post-injection, to investigate the delayed or persistence toxicity of the AuNPs. Results revealed no significant toxicities with the citrate, PEG-, p.C-PEG and p.14-PEG-AuNPs over 12 weeks post-exposure, as evidenced by biochemical assays such as serum marker enzymes, liver and kidney function markers, and cholestatic indicators; haematological parameters; oxidative stress markers; and histopathological examinations. P.L-PEG-AuNPs, however, caused significant toxicity (p<0.05) to rats, as evidenced by increased relative liver weight, increased malondialdehyde levels, and total white blood cell counts 2 weeks post-exposure when compared to the control group. This was, however, reversed during the 12 weeks post-exposure. Further, there were no evidence of inflammatory responses, using pro-inflammatory markers including phospho interleukin 18 (IL-18) and interferon-γ (IFN-γ), as indicated by immunohistochemical staining of the liver, spleen, kidney and colon of rats 2 weeks post-injection of AuNPs. Citrate, PEG-, p.C-PEG, and p.14-PEG-AuNPs did not induce immediate, acute or persistent toxicity, while p.L-PEG-AuNPs induced a transient acute toxicity. It can be concluded that 14 nm spherical citrate-AuNPs at 100 μg/kg body weight is a good candidate for biomedical applications, and as a suitable carrier for diagnostic and/or therapeutic molecules. Combination of 99% PEG-OH and 1% PEG-biotin is an appropriate option for stabilising AuNPs in biological environment, and conjugating secondary diagnostic or therapeutic biomolecules or agents to citrate-capped AuNPs. Peptide-conjugated AuNPs are suitable for the development into a diagnostic tool for CRC in vivo

In vivo toxicological evaluation of peptide conjugated gold nanoparticles for potential application in colorectal cancer diagnosis

Colorectal cancer (CRC) is among the leading cause of cancer-related deaths in South Africa and worldwide. Efforts are being made at finding improved diagnostic tools, as early detection (before metastasis) is a major factor in CRC treatment. Colonoscopy is the most reliable detection method, but is a specialised and expensive procedure, which is invasive, not readily available and not patient-friendly. There is a risk of developing interval cancers, as colonoscopies are performed every 10 years after the age of 40. The development of non-invasive, cost efficient and readily available diagnostic tools to CRC, which can be performed at more regular intervals, using tumour-targeting molecular imaging agents, is of urgent attention. Gold nanoparticles (AuNPs) possess several physicochemical properties, including ease of synthesis, biocompatibility, and the ability to be conjugated by ligands or biomolecules such as polyethylene glycol (PEG) and peptides for improved stability, tissue targeting and selectivity. These factors potentiate the role in biomedical applications, including cancer theranostics. Conjugation of AuNPs with a targeting molecule (e.g. antibody or peptide) is directed against cancer cell receptors. The peptides, p.C, p.L, and p.14, bind to CRC cells in vitro. Conjugation of AuNPs with these peptides should be investigated for CRC diagnosis in vivo, as it is hypothesised to allow examinations at shorter intervals through imaging techniques. This could reduce the risk of interval cancers, but before developing this novel tool, in vivo toxicity evaluations are essential. This study was therefore aimed at investigating the short- and long-term toxicological effects of a single intravenous injection of peptides (p.C, p.L, and p.14) conjugated to AuNPs in a healthy rat model. Citrate-capped AuNPs were synthesised by the citrate-reduction method, and conjugated with each peptide (biotinylated) using a combination of PEG (99% PEG-OH and 1% PEG-biotin) as a stabilising agent and linker, via biotin-streptavidin interaction. Healthy male Wistar rats were intravenously injected with 14 nm citrate-AuNPs, PEG-, p.C-PEG, p.L-PEG, and p.14-PEG-AuNPs (100 μg/kg body weight), and the control rats were injected with phosphate buffered saline. The animals were monitored for behavioural, physiological, biochemical, haematological and histological changes, as well as inflammatory responses. Phase 1 rats were sacrificed 2 weeks post-injection to determine the immediate or acute toxicity of the AuNPs, while phase 2 animals were sacrificed 12 weeks post-injection, to investigate the delayed or persistence toxicity of the AuNPs. Results revealed no significant toxicities with the citrate, PEG-, p.C-PEG and p.14-PEG-AuNPs over 12 weeks post-exposure, as evidenced by biochemical assays such as serum marker enzymes, liver and kidney function markers, and cholestatic indicators; haematological parameters; oxidative stress markers; and histopathological examinations. P.L-PEG-AuNPs, however, caused significant toxicity (p<0.05) to rats, as evidenced by increased relative liver weight, increased malondialdehyde levels, and total white blood cell counts 2 weeks post-exposure when compared to the control group. This was, however, reversed during the 12 weeks post-exposure. Further, there were no evidence of inflammatory responses, using pro-inflammatory markers including phospho interleukin 18 (IL-18) and interferon-γ (IFN-γ), as indicated by immunohistochemical staining of the liver, spleen, kidney and colon of rats 2 weeks post-injection of AuNPs. Citrate, PEG-, p.C-PEG, and p.14-PEG-AuNPs did not induce immediate, acute or persistent toxicity, while p.L-PEG-AuNPs induced a transient acute toxicity. It can be concluded that 14 nm spherical citrate-AuNPs at 100 μg/kg body weight is a good candidate for biomedical applications, and as a suitable carrier for diagnostic and/or therapeutic molecules. Combination of 99% PEG-OH and 1% PEG-biotin is an appropriate option for stabilising AuNPs in biological environment, and conjugating secondary diagnostic or therapeutic biomolecules or agents to citrate-capped AuNPs. Peptide-conjugated AuNPs are suitable for the development into a diagnostic tool for CRC in vivo

Prevention of Fe2+ induced lipid peroxidation by aqueous extract of Garcinia kola leaf in some rat tissues. Innovations in Pharmaceuticals and pharmacotherapy

Cell injury in aerobic organism subjected to oxidative stress has been caused by lipid peroxidation. The ability of aqueous extract of Garcina kola leaf (3.3-33.3µg/ml) to prevent 60µM Fe2+ induced lipid peroxidation in rat liver and brain were assessed respectively using TBARS (Thiobarbituric acid reactive species. Fe2+ - chelating ability of the extract was also determined. The result of the study revealed that incubating the liver and brain in the presence of iron exhibited high percentage inhibition against thiobarbituric acid reactive species (TBARS) induced by iron (ii) sulphate (60µM) with IC50 value of 72.58±29.16µg/ml and 89.36µg/ml respectively, while the extract shows strong iron chelating ability of 79.93% at concentration (2.3µg/ml) with an EC value of 62.0µg/ml. The inhibitory effect of aqueous extract of Garcinia kola shown in TBARS and Iron chelation assays were concentration dependent. The results however suggest that Garcinia kola is beneficial in the treatment of various cellular damages due to its ability to reduce lipid peroxidation

Investigation of bioactive compounds in Crassocephalum rubens leaf and in vitro anticancer activity of its biosynthesized gold nanoparticles

The development of cancer therapies has become difficult due to high metastasis, and lack of tissue selectivity, which in most cases affects normal cells. Demand for anticancer therapy is therefore increasing on daily basis. Gold nanoparticles (AuNPs) have many applications in biomedical field. Biological synthesis of AuNPs using aqueous extract of Crassocephalum rubens (AECR) was designed to investigate the in vitro anticancer potential. The synthesized AuNPs were characterized by UV–vis spectroscopy, high-resolution transmission electron microscopy, and Fourier transform infrared spectroscopy. The characterization results showed the formation of green AuNPs of wavelength 538 nm, and mostly spherical AuNPs with 20 5 nm size. Significant anticancer activity of the AECRAuNPs on MCF-7 and Caco-2 cells was noted at higher concentrations (125 and 250mg/mL) during 24 and at all concentrations tested during 48 h. It can therefore be concluded that AECR leaves can mediate stable AuNPs with anticancer properties

Hepatoprotective Effect of Aqueous Extract of Solanum macrocarponLeavesagainst Carbon tetrachloride-Induced Liver Damage in Rats

Liver damage is a growing concern of today’s modern society. The increasing incidence of exposure to toxic agents has contributed to liver diseases. There is therefore need for hepatoprotective agents. This study was aimed at investigating the protective effect of aqueous extract of the leaves of Solanum macrocarponagainst CCl4-induced liver damage in rats. Six groups of four animals each were used for the investigation. Group 1 served as control, groups 2, 3 and 4 animals were pre-treated with leaf extract of Solanum macrocarponat 250mg/kg, 500mg/kg and 750mg/kg body weight respectively for 14 days prior to a single intraperitoneal administration of CCl4. Animals in groups 5 and 6 received only the extract at a dose of 750mg/kg body weight and CCl4respectively. All animals were sacrificed 24 h after the administration of CCl4.The liver functions tests were performed in addition to their histopathological evaluation.Results obtained showed significant adverse changes in the levels of all measured parameters in CCl4treated rats. However, pre-treatment with aqueous extract of S. macrocarponprevented the adverse changes. Our findings suggest that S. macrocarponprotects the liver against CCl4-induced damage. This could be attributed to the presence of phytochemical compounds in the plant

Neuronal alterations in the prefrontal cortex of rats with carbon tetrachloride (CCl4) induced hepatic damage

In cirrhosis, some toxic substances accumulate in the brain and modify its functional integrity. In this study, we investigate the impacts of liver damage on the neuronal profile of the prefrontal cortex (PFC) in a rat model of hepatic damage induced with carbon tetrachloride (CCl4). This study also evaluated the possible role of liver dysfunction in the etiology of neurodegenerative characteristics associated with the PFC. Ten male Wistar rats weighing 120 to 190 g body weight were used for this study. The rats were divided into 2 groups (A and B) of 5 rats each. The rats in group A (control group) were treated with phosphate buffered saline (PBS) solution only while the rats in group B (treatment group) were treated with carbon tetrachloride (CCl4). The prefrontal cortices of the rats were excised from skulls of the rats, fixed in formol calcium, while the livers were excised from the abdomen of the rats and were fixed in formol saline for cytoarchitectural study using Cresyl fast violet and hematoxylin and eosin stains respectively. The main neuropathological findings observed in this study include cortical necrosis, uneven neuronal loss with varying range of vacuolations in the prefrontal cortices of the CCl4 treated rats when compared with the PBS treated rats. It was observed that the administration of CCl4 induces changes in hepatocellular morphology of the treated rats and these include moderate vascular congestion and extensive cytoplasmic damage in the hepatocytes. These results could be due to loss of hepatic functions

PROTECTIVE EFFECT OF ETHANOLIC EXTRACT OF CRASSOCEPHALUM RUBENSLEAVES ON CARBON TETRACHLORIDE -INDUCED LIVER DAMAGE IN RATS

Natural products, most especially from plant origin, possess antioxidant propertieswhich are known toplaycrucial roles in preventing and treating various pathological conditions occasioned by free radicals. Crassocephalum rubens is aone of those plants, and this study investigated the protective properties of ethanolic extract of C.rubens(EECR) leaves against hepatic damageinduced by carbon tetrachloride (CCl4). Thirty rats divided into 6 groups (n=5) were used for the investigation. Group 1 served as normal control while groups 2, 3 and 4 were pretreated for 21 days with EECR leaves at 150 mg/kg, 300 mg/kg and 450 mg/kg b.w.respectively, prior to a single intraperitoneal administration of CCl4. Animals in groups 5 received only the extract at a dose of 450 mg/kg body weight whileanimals in group 6 were given only CCl4. All animals were sacrificed 24 hafter the administration of CCl4. CCl4significantly (p < 0.05) induced marked hepatic damage as revealed by increased activities of plasma ALT, AST, GGT and ALP. Also, plasma total protein and albumin were significantly decreased in CCl4-treated animals relativeto normal control. Analyses of antioxidant statusshowed that CCl4elicited a significant decrease in the activities of antioxidant enzymes, with an increase in malondialdehyde levelsin liver. Pre-treatment with the EECR leavesat all doses testedhowever,significantly (p<0.05) reduced the observed biochemical lesions. The hepatoprotective effect of the EECR may be traceable to the presence of phytochemicals inherent in the plant

Antinociceptive effects of Treculia africana decne (african breadfruit) seed lectin in Wistar rats

The aim: The use of synthetic compounds to treat many diseases must be strictly controlled due to their potential health hazards. Hence, there is a need to search for natural products to serve as safe alternatives to synthetic products. This study investigated the antinociceptive effects and anti-inflammatory activities of Treculia africana seed lectin. Materials and methods: Lectins were purified from Treculia africana seeds using ion exchange and size-exclusion chromatography. The antinociceptive activity of the lectin was assessed in Wistar rats using abdominal writhing and paw-licking tests induced by acetic acid and formalin, respectively. Anti-inflammatory activity was assessed using carrageenan-induced paw oedema. Results: Treculia africana seed lectins at 10 mg/kg (p.o.) produced sedation, reduced ambulation, reduced response to touch, analgesia, and decreased defecation in experimental animals. Administration of Treculia africana seed lectin (1 mg/kg and 10 mg/kg) in experimental animals significantly reduced (P < 0.05) acetic acid-induced muscular writhing in a dose-dependent manner with 23.88 and 36.80 per cent inhibition, respectively. Both early and late phases of formalin-induced nociception were significantly inhibited (P < 0.001) by the lectin at all doses (0.1, 1.0 and 10.0 mg/kg), comparably with the standard drug, diclofenac sodium. At 10 mg/kg, T. africana lectin caused a 69.12 % and 65.55 % reduction in both early and late phases of formalin-induced paw licking. Treculia africana lectin also significantly brought about a reduction (P < 0.05) in inflammation induced by sub-plantar injection of carrageenan as measured by a decrease in paw swollenness. Conclusion: The study showed that Treculia africana lectin possesses antinociceptive and anti-inflammatory properties and can potentially be employed therapeutics to ameliorate pain and inflammatio

Evaluation of antioxidant potentials of different solvent-fractions of Dialium indium (African Black velvet tamarind) fruit pulp - in vitro

Plant phytonutrients have been harnessed for their various curative properties both in vitro and in vivo. In this study African black velvet tamarind (ABVT) fruit pulp was evaluated for it antioxidant potentials using chloroform and hexane fractions through different antioxidant parameters. In the results; total phenolic contents quantified in mg GAE/dried sample in chloroform and hexane extracts were; 14.57 &plusmn;5.85 and 9.78 &plusmn;4.61, total flavonoid contents in chloroform and hexane extracts as; 48.58 &plusmn;0.00 and 27.35 &plusmn;0.00 while the FRAP (&micro;g AAE.g-1 dried sample) was lower in chloroform (298.10 &plusmn;0.00) than hexane extracts (1029.81 &plusmn;0.00). More also, ability of varied concentrations of the extracts (with their IC50) to cause inhibition against Fe2+-induced MDA that was determined by TBARS in rat's brain and liver tissue homogenates, Fe2+-chelating ability and other antioxidant assays, showed an appreciable significant (p &lt;0.05) difference. The various antioxidant properties showed by ABVT has indicated that, if the pulp is incorporated in diet, it could serve as an alternative in managing various ROS-induced degenerative ailments as it has been clearly demonstrated in the protection of brain and liver homogenates from Fe2+-induced oxidative stress.&nbsp

Nutrition: A Natural and Promising Option in Colorectal Cancer Intervention

Nutrition: a natural and promising option in colorectal cancer intervention Nutrition plays a significant role in the intervention of colorectal cancer (CRC) by decreasing the risks of colorectal carcinogenesis. Products from both plant and animal origins have been involved in the prevention and/or treatment of CRC. Intake of dietary products including fibre-rich foods, nutraceuticals, wholegrains, dairy products, and limited consumption or avoidance of red/processed meat and alcohol could reduce the risk of CRC. These nutritional compounds, in CRC intervention, could be in form of folklore/alternative medicine or isolated compounds used in the production of many chemotherapeutic agents. Monitoring of individual’s nutritional status could serve as a possible preventive or therapeutic measure against CRC, majorly by interaction with intestinal microbiota, thereby potentiating host anti-cancer immune response and/or interfering with mechanisms of carcinogenesis