Glycyrrhetinic Acid Functionalized Graphene Oxide

Introduction

Graphene and graphene oxide Graphene is an ultra-thin single layer of carbon where the carbon atoms are arranged in a hexagonal lattice. Graphene most common structures are carbon nanotubes (rolled up graphene sheets) and graphite (graphene layers are stacked and held together by weak forces, mainly Van der Vaals) which are becoming more used day by day in all sorts of different applications such as energy storage, thermal applications, mechanical strength and it is starting to develop in biomedicine, like graphene oxide nanoparticles (GONPs) in nanobiotechnology?1?.

Graphene oxides (GO) are a relatively recently discovered carbon nano-structures made from graphite oxidation where carbon atoms are arranged in single layer hexagonal pattern as graphene, but laced with oxygen-containing groups on nanoparticles edges such as carboxyl, hydroxyl, carbonyl or epoxy?2?. Due to GO group’s hydrophilicity, they can be dissolved in water or other solvents allowing the formation of thin films (single carbon atomic layer), making GO potentially useful for micro-electronics?3?.

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GONPs hydrophilicity and easy modifiable and specific surface allows GONPs functionalization with different biocompatible polymers such as polyethylene glycol (PEG) or chitosan amongst others. Many current applications of this GONPs in nanomedicine are emerging, especially the ones related to antimicrobialthera pies, drug delivery and cancer therapies?4?.

Drug delivery in cancer In the field of nanobiotechnology, one of the main branches of study is drug delivery. There are plenty of investigations targeted especially to cancer treatment. For instance, using functionalized graphene oxide.

An ideal design for cancer treatment would be through mitochondria-mediated apoptosis (MMA). Mitochondria are highly important in cellular processes, either essential or pathological. In cancer, cells acquire the ability of not responding to the apoptotic signals of mitochondria, therefore they are able to avoid apoptosis. If mitochondria cell-suicide factors are stimulated, apoptosis will be triggered. Hence, the interest in targeting mitochondria for drug delivery against these kind of pathologies.

However, there is a major challenge, mitochondria permeability is quite low. A way to overcome this limitation could be using ligands targeted to mitochondria. Conjugating our drug of interest to the ligand, we could achieve a proper delivery and presumably, develop a treatment. The widest and most diverse source of such ligands is nature, more specifically, plants.

In the study, Glycyrrhetinic acid (GA) was used due previous studies demonstrating its antitumor activity. In hepatocellular carcinoma cells, protein kinase C ? (PKC ?) is overexpressed and it happens to be the main binding site of GA.

Also, GA interacts with the mitochondrial respiratory chain stimulating the production of hydrogen peroxide. This chemical compound oxidizes pyridine nucleotides and thiol groups, which causes the switch of permeability, opening the mitochondrial pores. In this study they functionalized GO with GA in order to act as a nanocarrier for doxorubicin (DOX), a common drug used in chemotherapy treatments ?6?.

Functionalization

GO’s good biocompatibility and drug loading capacity were critical points taken into account to prepare the GA-functionalized GO. The reaction between –NH2 group of pegylated GA and –COOH group of GO using a cross-linking reagent allows GA-GO conjugation (figure 3), was demonstrated by Fourier transformed infrared (FTIR) spectroscopy. –COOH absorbance peak of GO group (1730 cm?-1?) disappeared in GA-GO, as a new absorbance peak of 1620 cm?-1 corresponding to the –NH2 group of peglated GA appeared confirming the GA-GO conjugation.

GA-GO lateral size of 200 nm and a thickness of less than 10 nm was measured by atomic force microscopy (AFM). Zeta potential measured value was -37.6 mV for GA-GO, significantly more negative than GO zeta potential value(-11.3 mV) at pH 7.4.

In vivo results

The in vivo assay was performed on HepG2-bearing nude mice (HepG2 is a strain of well-differentiated hepatocellular carcinoma cells, non-carcinogenic in nude mice). Bax is a protein that is found in higher proportions in the cytosolic side, but when inserted in the mitochondrial membrane it triggers cytochrome-c release into cytosol. Bcl-2 is an anti-apoptotic protein and acts inhibiting the pro-apoptotic molecules such as Bax. Expression of Bax and Bcl-2 ratio was observed by IHC and the following results were obtained: They performed TUNEL in order to confirm apoptosis and there were indeed stronger signals in those that GA-GO@DOX was administered.

Biodistribution

They observed where DOX concentration was higher, and conclude that DOX was elevated mainly in the liver and tumor-site in those administered GA-GO@DOX, and also higher concentration respect to the other administration forms, DOX·HCl and GO@DOX. It was also tested that this kind of administration decreased tumor weight, stating its apoptotic effect in tumoral cells. This was also confirmed by HxE and Ki67 stainings.

Safety Evaluation

As doxorubicin in its free form is cardiotoxic, they emphasized if it would produce the same effect when given as GA-GO@DOX. They tested ?in vitro ?its toxicity in 2 cell lines: H9c2 cardiomyocytes and L02 hepatic cells. The inhibitory effect was significantly lower at several doses (2.0-16?g/mL) on those injected GA-GO@DOX but high in tumoral cells.

Blood chemistry analysis (including liver markers ALT, AAT & alkaline phosphatase) were performed 21d after injection of GA-GO@DOX and they weren’t different from the control sample only administered saline solution. This indicates that the NP doesn’t give rise to hepatic or systemic toxicity, and that the liver function is not damaged.

Conclusions

Combining inorganic materials with biological ligands and features is a strategy that presents a wide variety of applications. Through nanobiotechnological advances and techniques we can achieve important goals such as a proper drug delivery, as seen through this assay in which functionalizing graphene oxide with the glycyrrhetinic acid ligand allowed accurate target of mitochondria. This proposal could potentially be a future therapeutic cancer treatment and also an inspirational approach for similar unsolved problems.

References

  1. Randviir, E. P., Brownson, D. A. C., & Banks, C. E. (2014). A decade of graphene research: Production, applications and outlook. ?Materials Today?, ?17?(9), 426–432.
  2. Kumar Gupta, D., Singh Rajaura, R., & Sharma, K. (2015). Synthesis and Characterization of Graphene Oxide Nanoparticles and their Antibacterial Activity. Science and Technology?, ?1?(1), 16–24.
  3. Dimiev, A. M., & Tour, J. M. (2014). Mechanism of graphene oxide formation. ?ACS Nano?, ?8?(3), 3060–3068.
  4. Wu, S. Y., An, S. S. A., & Hulme, J. (2015). Current applications of graphene oxide in nanomedicine. ?International Journal of Nanomedicine?, ?10?, 9–24.
  5. Gerani, K., Mortaheb, H. R., & Mokhtarani, B. (2017). Enhancement in Performance of Sulfonated PES Cation-Exchange Membrane by Introducing Pristine and Sulfonated Graphene Oxide Nanosheets Synthesized through Hummers and Staudenmaier Methods. Polymer – Plastics Technology and Engineering. https://doi.org/10.1080/03602559.2016.1233260
  6. Zhang, C., Liu, Z., Zheng, Y., Geng, Y., Han, C., Shi, Y., Kong, L. (2018). Glycyrrhetinic Acid Functionalized Graphene Oxide for Mitochondria Targeting and Cancer Treatment In Vivo. Small. https://doi.org/10.1002/smll.201703306

 

Mahatma Gandhi – Independence Movement For India

“Mahatma Gandhi was a key factor in the independence movement for India. He was a peaceful leader and is known for his civil disobedience that influenced the world. Gandhi was a man who did not believe in violence to achieve greatness. He is named the “Great soul,” and people still talk about him today because of what he did in his life. A huge impact has been made on India and world history because of Gandhi’s successes.

Gandhi was born on October 2, 1869 in Porbandar India. He was many things, including a lawyer, writer, politician, and most importantly a social activist and nationalist. He took much pride in India and used that pride to fight for independence. He became the outstanding leader of the nationalist movement against the British! The British rule of India was against the idea of giving India their independence.

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When India was ruled by Britain, Gandhi knew he could help India in gaining their independence. He led India to their independence by using civil disobedience acts that were non-violent. Since this is what Gandhi believed in, he produced the term “satyagraha,” which means “truth and love against force.” He felt so strongly about this idea that he sincerely believed it could change the world for the better. However, war and violence is almost inevitable even to this day. This did not stop Gandhi because he did everything he could to make that change.

In 1930, Gandhi led a civil disobedience movement called the Salt March. It took from March to April to complete the long journey to the ocean. The salt march was a two hundred and forty mile march to the ocean in order to make salt. He did this along with thousands of followers because the British had just put out a tax on salt. It is known that everyone needs salt to live. This really affected the people of India, especially the poorest of them all. It was not as successful as Gandhi thought it would be, considering he and about six thousand others were arrested. However, it was a way that united the people of India, only making them stronger. Once Gandhi was released from prison, he knew something else had to be done in order to gain independence for India.

Help was provided by Gandhi in many other ways and acts of civil disobedience, such as protests and organizations. In South Africa, he peacefully protested against the discrimination against the Indians. He tried desperately to get rid of the caste system, which was a class system but no one was able to move up or down in it. He was involved in many acts so as to end Britain’s’ discrimination towards India. This was only the beginning. After World War I, Gandhi started to organize more peaceful protests in order to get some relief for the population of India. Soon enough, British troops started gunning down the innocent Indian protesters along with Gandhi himself.

The reputation of Gandhi was growing all over the world. Millions of people were aware of his peaceful protests and other acts of civil disobedience. He knew he had the world’s eyes on him, so he took advantage of that. Gandhi was against the caste system to a very high extent. It seems to be ironic that he was born into a very privileged caste system. However, he knew it was unfair.

One of Gandhi’s tactics in order to free the “untouchables” from isolation was to start fasting. He and his people began fasting in order to influence the British to change their minds on the discrimination towards the untouchables, who are the lowest of the low in the caste system. He continued to fast until there was a change being made. To his great success, the British let go of the isolation law against the untouchables. The British knew how many people knew of Gandhi and his ways of peace and they did not want the pressure of being held responsible for Gandhi and his people dying of starvation.

After all the accomplishments Mahatma Gandhi achieved throughout his entire lifetime, he ended up shot and killed on the 30th of January in 1948. He was assassinated by Nathuram Godse. At the time, Gandhi was 78 and very weak, fragile, and thin from fasting. Nathuram Godse shot him three times in the stomach and chest while Gandhi was on his way to a prayer meeting. It was a devastating death. His followers, along with the rest of the world were deeply affected considering Gandhi was the man who got them through it all. This caused a riot. There were riots and violence all over the Brahmins because that is what Nathuram Godse was. Unfortunately, it would have been very disappointing for Gandhi if he were around to witness what his people were doing; considering he was absolutely against the idea of violence in all.

Gandhi helped India gain their independence without ever firing a single shot! The Indian Independence movement lasted from 1857 all the way to 1947. He inspired civil disobedience movements all around the world and took control over the Indian Nationalist Movement when striving for success. Gandhi reached out to seek possible change wherever he went and however he possibly could. He tried to compromise and find a possible solution to the issues and problems that the British kept feeding him. People still talk about Gandhi to this day and how he was a hero. He got India through one of the toughest times in history and did it without ever attempting any violent acts or protests. Historians say that Gandhi himself, was able to prove that one man really can make a difference.”

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