3.2.1. Alcohol, Acetaminophen and EV CYP2E1

Circulating CYPs likely play a role in extrahepatic metabolism. Once secreted from the liver, EVs can deliver CYP enzymes to distant sites, where they then affect the target cells by influencing the metabolism of pharmaceutical drugs, drugs of abuse, and other xenobiotics [23]. CYP2E1 is mainly found in the liver, where it is known to be a major metabolizer of alcohol and acetaminophen (APAP) [12]. Importantly, CYP2E1-mediated metabolism produces reactive oxygen species (ROS) responsible for oxidative liver damage and cellular toxicity [12]. While alcohol and APAP misuse are significant contributors to liver damage, they also affect extrahepatic tissues [93]. Moreover, alcohol induces EV release from hepatocytes in association with ROS [94]. Our study has also shown that alcohol-exposure to mice induces the level of EV CYP2E1 in the plasma [87]. We have further demonstrated that when plasma EVs containing increased levels of CYP2E1 are exposed to hepatic and monocytic cells, they exacerbate alcohol- and APAP-induced toxicity [87]. We have also shown that the toxicity is mainly caused by CYP2E1, as a CYP2E1-selective inhibitor significantly reduced EV-exacerbated toxicity by both alcohol and APAP. Together, the results suggest that EVs containing CYP2E1 can cause both intraand intercellular communication. Thus, CYP2E1's presence extrahepatically and its role in cell–cell communication suggests that alcohol and APAP are also being metabolized at different locations throughout the body. It is likely that plasma EVs transmit CYP2E1 from the liver to targets throughout the body, e.g., the brain, where 2E1 can then participate in xenobiotic metabolism and bioactivation of toxic metabolites. EVs carrying CYP2E1 to distant cells may help explain alcohol-induced cellular injury occurring outside the liver.

#### 3.2.2. Tobacco Smoking and EV CYP2A6, 1B1, 1A1

CYP2A6, CYP1B1, and CYP1A1, all of which play a vital role in the metabolism and bioactivation of tobacco/cigarette smoke constituents, were also detected in EVs [23]. Although CYP2A6 and 1B1 are mainly expressed in the respiratory system, they can also be found in liver cells. Thus, EV CYP2A6 and 1B1 may originate from either organ. Both produce toxic metabolites in association with their roles in metabolizing nicotine and PAHs, respectively [10]. Our recent studies suggest that EVs may also play a defensive role, specifically in protecting against smoking-induced HIV-1 pathogenesis [92,95]. CYP-mediated elevations in oxidative stress that accompany tobacco smoking, also promote HIV-1 replication [13,14,50,51]. Additionally, cigarette smoking is associated with EV release in smokers and in various cell types in vitro [96]. Our study revealed that EVs from CSC-treated cells were found to alter their antioxidant capacity and packaging—showing a protective effect against toxicity and viral replication in the early stages of HIV-1 replication [92].

#### 3.2.3. Drug Metabolism and EV CYP3A4

Importantly, we also detected metabolically active CYP3A4 enzyme in plasma exosomes [23]. Being the major drug-metabolizing CYP enzyme, the presence of CYP3A4 in EVs has clinical significance in terms of therapeutics. During drug development, the focus is traditionally on hepatic drug metabolism; however, failing to account for circulating CYPs may result in unintended drug–drug interactions or toxicity. Furthermore, EV CYP3A4 can be used as a biological marker, specifically in examining the metabolism of pharmacological or illicit drugs. Rowland et al. demonstrated a strong relationship between EV CYP3A4 and drug clearance in patients, which suggests that EVs can be a potential tool for identifying variability in drug exposure [86]. The study also found that EV CYP3A4 exhibits comparable kinetics to microsomes taken from liver samples [86]. The circulation of EVs in bodily fluids allows for greater accessibility in terms of isolating these biomarkers. Sometimes called "liquid biopsy," this form of sample collection does not require the use of invasive techniques such as tissue biopsy or liver resection. Rather, to assess the expression of CYP3A4 mRNAs, they can simply be isolated from a blood sample. These findings suggest that EV CYPs may provide a new and easier way to explore variability in pharmaceutical drug metabolism and exposure.

### 3.2.4. Biological and Clinical Significance of CYP Packaging/Circulation in Plasma EVs

Although it is recognized that EVs can envelope functionally active CYP enzymes, the specific mechanistic pathway of this differential packaging is still under investigation. Circulating EVs with metabolically active CYP enzymes may have a considerable impact on neighboring and distant cells and tissue systems. For example, CYP enzymes carried within EVs might influence the metabolism of endogenous and xenobiotic compounds. EVs could be way of removing unwanted CYP enzymes from the cells. Further investigations are warranted to fully appreciate the impact that these modified EVs may have in the body [83]. EVs derived from patient hepatocytes can be utilized as a non-invasive tool to characterize variability in drug response—one way in which EVs may be used as potential biomarkers [83]. Further, EVs are already under investigation to be used as drug delivery systems, designed to contain specific content for transport to different cell types [97]. Thus, EVs might be a useful tool in combating xenobiotic-induced toxicity by controlled alteration of their contents.

#### **4. Potential Applications of EVs Containing CYP Enzymes**

#### *4.1. Circulating CYP Enzymes as Biological Markers of Drug-Induced Toxicity*

The current gold standard biomarkers for hepatic injury are based on the measurements of hepatic enzymes levels, including ALT, AST, etc., in plasma or serum. However, the ALT levels do not always correlate with various stages of liver disease due to its relatively short half-life [98]. Therefore, specific components in circulating EVs may have great utility as non-invasive biomarkers for diagnosis and during treatment of hepatic injury. For example, alcohol use increases CYP2E1 expression in plasma EVs [88] and there is a correlation between increased CYP2E1 level and alcohol-induced liver injury [99]. Furthermore, alcohol exposure increases EV release into the circulation [88], making circulatory EVs a potential source of biomarkers in the setting of drug-induced liver injury [84,100].

Recent studies show that much like alcohol's effect on the liver, alcohol can also alter EV cargo [93]. Furthermore, EVs derived from alcohol-treated cells have been shown to exacerbate disease progression through the delivery of altered cellular material to target cells. We previously observed that EVs collected from mouse and human plasma aggravated alcohol and APAP-induced toxicity [87]. In a similar study, Cho et al. demonstrated that CYP2E1-rich EVs from alcohol-exposed rats and patients induced hepatic cell death [88]. These findings further highlight the potential value of CYP2E1-containing EVs as noninvasive, diagnostic biomarkers in alcoholism and microsomal stress [101].

Similarly, smoking induces CYP1A1 [58], CYP1B1 [102], and the activity of these enzymes can exacerbate smoking-related toxicity by providing additional oxidative stress [58]. Since these CYPs are present in EVs [23,86], EVs can serve as markers to diagnose smoking-induced tissue toxicity. Similarly, a strong relationship between EV-derived CYP3A4 and drug clearance in patients [86] suggests that EV CYP3A4 can be used as a biological marker, specifically in examining the metabolism of pharmacological or illicit drugs.

#### *4.2. Use of EV CYPs in Synthetic Biology*

CYPs can catalyze the specific addition of oxygen atoms to chemical scaffolds, which would be very challenging and expensive by traditional methods. Several CYPs and engineered variants are now used to synthesize and produce various compounds on a larger scale and for diverse purposes, including drug discovery and development [1]. For instance, artemisinic acid, a precursor for the *Artemisia annua*-derived antimalarial drug, artemisinin, has been synthesized using an engineered form of the plant's CYP71AV1 enzyme [103]. Furthermore, engineered CYPs have utility in statin synthesis. Compactin is a naturally-occurring HMG-CoA reductase inhibitor originally isolated from *Penicillium citrinium* by Endo et al., 1976 [104]. Using an engineered version of *Amycolatopsis orientalis*-derived CYP105AS1 in *Penicillium chrysogenum* fungi, researchers are now able to synthesize pravastatin from compactin [105]. Moreover, as the proteins packaged in EVs are stable and protected from degradation [106], engineered CYPs can be loaded in EVs (Figure 2), a step that would improve their stability and subsequent activity in the production of various therapeutic molecules.

**Figure 2.** Potential applications of extracellular vesicles containing CYP enzymes include drug metabolism, prodrug activation, supplementation of CYP to subjects with genetic polymorphisms, and industrial synthesis of biomolecules.

#### *4.3. Targeted Delivery of EV CYPs for Prodrug Activation*

The ability of EVs to package and transport a variety of biological cargos has prompted investigators to examine the possibility of loading EVs with specific therapeutic content [107]. Several methods of EV loading have been developed, including electroporation, transfection, and incubation, among others [108]. Utilizing one of these methods, it is conceivable that EVs may be loaded with CYP enzyme, along with CPR. The EV-loaded CYP and prodrugs can be directly administered to the site of disease. For example, in the case of solid cancer, EV CYP can activate anticancer prodrugs at the disease site, reducing toxicity in healthy cells caused by anticancer drugs. Engineering such a delivery system could enhance the efficacy and bioavailability of certain prodrugs, cancer treatments (including brain cancer), and neurological disease therapies.

Previously, gene-directed enzyme prodrug therapy (GDEPT), which utilizes gene transfer of CYP enzyme and cytochrome CPR within a viral vector, has been proposed as a novel way to increase therapeutic efficacy and decrease systemic side effects of anticancer prodrugs, e.g., cyclophosphamide (CPA) and ifosfamide (IFA) [1]. The purpose of CYP-based GDEPT is to facilitate local CPA/IFA bioactivation by expressing CYP enzymes directly within tumor cells [109]. While initial trials of CYP-based GDEPT systems have demonstrated safety and enhanced chemosensitivity to tumors, no GDEPT products are currently on the market [110,111]. As EVs are already under investigation as potential delivery systems [97], it is possible that EVs loaded with CYP and CPR could replace the viral vector in CYP-based GDEPT systems (Figure 2). The approach of loading EVs with CYP and CPR would be safe and economical due to the biological origin of EVs. Moreover, several therapeutics fail to achieve optimal concentrations in the CNS due to their inability to cross the BBB. In such instances, EVs can be engineered to target CNS cells and deliver their contents. For example, in delivering a prodrug along with its activating CYP enzyme to microglial cells, EVs could be conjugated with anti-TEME119 antibody [112], which is specific to microglia, to target and deliver EV cargo to these cells. Further, bacterial CYP enzymes have been expressed and engineered to activate prodrugs [1]. Loading of these CYPs in EVs targeted to a particular tissue can increase their stability and further promote their prodrug-converting activity in target sites.

#### *4.4. Delivery of EV CYPs to Supplement Naturally Inactive CYPs*

EV-loaded CYPs can also be administered to subjects with loss of function polymorphisms for particular CYP enzymes (Figure 2). Genetic polymorphisms of drug-metabolizing enzymes can result in either decreased, increased, or complete lack of activity of an enzyme, leading to disease susceptibility [113,114] or variability in drug response [115–117]. CYP2D6, which is known to metabolize approximately 20% of drugs, is the most polymorphic CYP enzyme in many ethnic populations and varies from 1–50% [118]. Several CYP2D6 variants cause very low to no activity with several drugs [119]. Similarly, CYP3A5 contributes significantly to drug metabolism in humans and is not expressed in 90% of Caucasians [120]. Thus, certain drugs, e.g., tacrolimus and sirolimus, that are metabolized by CYP3A5, tend to accumulate and cause toxicity in most Caucasians [120]. Thus, administrating EVs loaded with CYP3A5 to Caucasians could be helpful in metabolizing 3A5 substrates and decreasing their respective toxicities.

#### *4.5. Current Challenges Associated with Using EVs as Therapeutics*

Although EVs have advantageous properties over synthetic delivery systems in terms of their biological source and ability to deliver functional cargo, clinical translation of EVs as diagnostic or prognostic markers of pathological states remains a challenge due to various reasons. One reason might be the lack of uniformity in isolation, characterization and analysis methods of EVs. This can lead to variations in EV counts and phenotypes between different laboratories, making data analysis and clinical translation difficult. Furthermore, the half-life of exosomes in athymic nude mice was reported to be 30 min, and clearance was estimated to be 6 h after intravenous injections [121]. However, due to compartmental changes as EVs travel throughout the human body, it is difficult to estimate the half-life of EVs in blood. Moreover, EVs from different cells and EVs with different sizes possess different biodistribution profiles. In addition to all these concerns, most studies regarding the physiological or pathological effects of EVs have been done in cell culture models. However, cells under in vivo conditions are under a constant steady-state exposure to EVs. Therefore, the extent to which controlled EV exposures under in vitro conditions corresponds to the in vivo environment remains unclear.

The purification and detection of EVs is improving with the help of technological advancements. Moreover, International Society for Extracellular Vesicles (ISEV) attempts to provide guidelines to isolate and characterize EVs in order to improve reproducibility and to avoid ambiguity in the identification of EVs [122,123]. EVs can be engineered in order to increase their circulation time and improve their delivery to target tissues. For example, EVs can be coated with polyethylene glycol, which is known to increase the half-life of nanoparticles [124]. Increased expression of CD47 on the EV surface can also improve the circulation time of EVs by opposing the actions of phosphatidylserine, which promotes the initiation of phagocytosis and subsequent removal from the circulation by macrophages [125,126]. Therefore, exploring EV circulation kinetics, targeting, internalization, and cell–cell trafficking routes will be useful in engineering EVs for therapeutic purposes.

#### **5. Conclusions**

Considering the profound contribution of CYP enzymes in mediating xenobiotic metabolism and bioactivation of toxicants, the presence of CYP enzymes in EVs and their biological significance cannot be ignored. As EVs circulate throughout the body via biological fluids and participate in cellular communication, they may be clinically useful as biomarkers for drug-induced toxicity, synthesis of drug/metabolite synthesis, and targeted prodrug activation. Thus, further investigating the roles of circulating CYPs in extrahepatic cells would help generate novel treatment options for neurological diseases, cancer, and more.

**Funding:** The authors acknowledge the funding from the National Institutes of Health Grant DA047178 to S.Ku. **Conflicts of Interest:** The authors declare no conflict of interest.
