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= Graphene Drug Delivery = Graphene and its derivatives are unique materials that have opened new possibilities in many fields, including drug delivery. There are numerous different types of graphene based drug delivery systems, however, a proper understanding of how they impact the body and its immune system, how to functionalize them, and what other polymers they can be combined with, enables them to be utilized to their full potential. Graphene based systems are being used to treat many different disease states with cancer—breast, oral, and brain cancers to name a few—being the primary target, offering a new tool by which to attack this disease harnessing the unique physio-chemical properties of this relatively new material.

Pristine Graphene and Functionalized Graphene as Candidates for Drug Delivery
When talking about graphene there are two major classes to consider: Pristine graphene and functionalized graphene. Pristine graphene refers to the purest form as first isolated by Andre Geim and Kostya Novoselov in 2004: a single layer of carbon atoms in a lattice network. This form has been primarily used for applications in electronics, physics, chemistry, etc and not within drug delivery due to solubility, purity and toxicity concerns (See section on toxicity for more) as well as manufacturing limitations. The other main class of graphene is functionalized graphene which is effectively a catch-all category for any graphene derived structure with the most common forms being graphene oxide (GO) and reduced graphene oxide (RGO). GO appends oxygen as well as epoxy and hydroxyl groups into the carbon lattice with a higher degree of substitution whereas RGO incorporates significantly fewer oxygen, epoxide, and hydroxyl groups. Figure 1a-c depicts the chemical structure of pristine, GO and RGO.



These functionalized graphene forms provided significant advantages over pristine graphene in that they have more hydrophilic moieties to increase solubility in the body’s aqueous environments as well as having more chemically modifiable groups for drug conjugation. Despite these modifications, GO and RGO both retain the key properties of graphene that make it an exciting material for drug delivery: large surface area for non-covalent modifications afforded by its 2-D structure, extremely high thermal conductivity and photo-adsorption enabling differing triggering strategies, and high structural stability. The chemical modifications of functionalized graphene enable the unique properties to be harnessed in order to create novel drug delivery strategies.

Immunogenicity and toxicity
In order for any drug delivery system to be efficacious, it needs to at a minimum bio-tolerable and non-toxic—not causing harm to the body at the local or systemic level by simply being present. This toxicity has long been an issue with graphene-based delivery systems. After its initial isolation, many research groups attempted to explore using pristine graphene for drug delivery only to find overwhelmingly that it is cytotoxic--disrupting cellular membranes, generating reactive oxygen species--,hemolytic while in systemic circulation, accumulates in major organs and promotes inflammatory responses. The unique 2-dimensional structure that gives rise to its intrinsic properties also enables it to induce these cytotoxic effects. This prompted a rapid switch to GO based strategies.

Early studies concluded that GO and RGO were rather safe since they did not induce cell death in many human based cell lines ranging from fibroblast to stem cells in vitro. More thorough investigation, however, discovered a concentration dependent cytotoxicity. GO doses below 20mg/L did not impact cell viability, adhesion, or proliferation but at a concentration of 50mg/L and above the same fibroblast cell lines experienced significant cytotoxicity as characterized by lose of in vitro adhesion, interruption of mitochondrial respiration, disruption of DNA and induction of apoptosis. Similar results were found during in vivo studies. High doses (>1mg/kg) of GO in mice were associated with shortened survival over a 2-week period, chronic inflammation, accumulation of graphene deposits in lungs and spleen, and histologically relevant damage to major organs. Moreover, at the higher doses, the graphene was unable to be cleared through the kidneys and accumulated in the blood due to it forming aggregates and also binding with plasma proteins. Lower doses (0.1mg/kg and 0.25mg/kg) did not shown the same level of systemic toxicity when injected IV into mice and compared against these higher doses, again highlighting the dose dependent toxic nature of graphene. Despite not inducing the same level of toxicity, lower doses of graphene still have been shown to induce reactive oxygen species formation both in vitro and in vivo, putting exposed cells and tissues under a higher oxidative stress.

Additional surface modifications to GO have been investigated to lower GO’s toxic profile. The two modifications that have shown the most success have been pegylation and amination. These additional functionalizations have been shown to lead to less apoptotic cell death, less reactive oxygen species production, and less accumulation in major organs when given systemically. Pegylated GO has been used up to 100mg/L in vitro with no significant toxicity and up to 20mg/kg in vivo in mice with results similar to that of low dose GO. Dextran and chitosan functionalizations have also been explored, showing increased biocompatibility compared to GO. Graphene-based systems have numerous biocompatibility issues to be cognizant of before using for a drug delivery system. GO can be used in low concentrations, however, additionally surface modifications are necessary if higher concentrations are needed. No long-term studies have been completed to date to assess far reaching toxicity of GO and long-lasting impact on biological systems.

Drug Carrying and Release Strategies
The 2-D structure of graphene derivatives provide a variety of different methods for conjugating and coordinating therapeutics to graphene, subsequently enabling a host of different release strategies at the target site. The main goal of strategy is to increase local delivery and the bioavailability of the drugs of interest while also decreasing systemic toxicity associated with the drug. Any graphene-based system has a tremendous advantage in this realm due to its innate 2-D structure that provides it with two distinct faces to which therapeutics can be conjugated, giving it a comparatively large surface area for its size. Functionalized graphene has the additional benefit of being able to accommodate both hydrophilic and hydrophobic drugs simultaneously. The carbon-ring backbone structure provides the ability for hydrophobic small molecules, such as doxorubicin, to be non-covalently conjugated to GO through van Der walls interactions and pi-pi stacking interactions between the carbon rings. Additionally, since GO has numerous oxygen atoms, it has a sufficient density of hydrogen bond donors and acceptors by which more hydrophilic molecules can be loaded on to it. Most systems rely on primarily on these non-covalent interactions to coordinate drug loading onto GO due to the reversible nature of these interactions.

The reversibility of the interactions enables focused release strategies as well from the GO systems. One of the most common strategies for release from GO is a pH sensitive system. At more neutral pHs, GO can be easily loaded with large amount of therapeutic by balancing the availability of hydrogen bond donors and acceptors between the therapeutic and GO. When administered, the therapeutic remains coordinated throughout the circulation due to the relatively neutral pH of the body and is then released only in the acidic microenvironment of the tumors of interest. The changing pH changes the hydrogen bonding network of the GO and therapeutic causing it to be released, increasing local presentation of the drug while not requiring intensive processes to initially load the drug onto GO. An additional release method that has received significant interest is a thermosensitive trigger. Since GO readily conducts heat, areas of interest can be treated with NIR radiation when a GO drug conjugate is administered. At the treatment site, the GO converts the NIR into heat, triggering the release of the therapeutic only at the site of NIR stimulation.

Cancer Targeting
The most common disease that graphene drug delivery strategies have targeted to date is cancer. Many cancer therapeutics suffer from solubility issues. Several studies have demonstrated that utilizing graphene-based carriers have increased the bioavailability of these drugs due to increasing the solubility and overall stability of the chemotherapeutics in solution. Cancer has been a common target for graphene drug delivery due to its prevalence but also the acidic microenvironments that tumors create that play nicely into the innate pH dependent release mechanisms many graphene delivery strategies leverage. The most common chemotherapeutics that have been used with GO are doxorubicin, cisplatin, and camptothecin. Graphene is a unique material that not only enables multiple types of drugs to be loaded with high efficiencies but also but able to be combined in unique ways with different treatment modalities to produce a highly efficacious treatment.

Hybrid Systems
Because of graphene’s physiochemical properties, there are opportunities for a unique hybrid drug delivery system in which release of drugs can be coupled with an additional treatment or imaging modality within one system. One example of this is photothermal chemotherapy. Since graphene is both an excellent thermal conductor and photo-absorber, some research group leverage graphene nanoparticles to deliver chemotherapeutic and then irradiate the tumor with IR, causing the GO particles to heat up and thermally treat the tumor as well. Hu et al demonstrated that using bortezomib non-covalently conjugated to GO nanocarriers and NIR radiation, they could significantly decrease tumor cell viability compared to bortezomib-GO or NIR along controls. Their system was possible because of GO’s ability to solubilize bortezomib, deliver it to the cells, and also convert the NIR into localize thermal therapy to increase total efficacy of the treatment.

An additional combination uses of GO is combining drug delivery with fluorescent imaging. GO has an inherent fluorescence in the visible/near-infrared spectrum that changes its emission spectrum in relation to the pH. At smaller flake sizes, GO can be more efficiently internalized within cells. These two characteristics enable GO drug delivery systems to be used to not only deliver therapeutics to a specific region but to be able to image where GO went and released the payload while providing a color-coded pH map in tissues.

Conclusion
Graphene is a relative newcomer as a biomaterial and present many unique physiochemical properties that can be leveraged to create novel drug delivery strategies. The field has rapidly developed to leverage these qualities in an attempt to treat pressing disease states. In order to move forward and potentially be translated into the clinic, more robust toxicity data in larger scale and longer timepoint testing needs to be completed to generate a more well-round view of the potential long-term issues graphene and its derivatives can have on the major organ systems.