Overview of Caffeine Effects on Human Health and Emerging Delivery Strategies

Caffeine is a naturally occurring alkaloid found in various plants. It acts as a stimulant, antioxidant, anti-inflammatory, and even an aid in pain management, and is found in several over-the-counter medications. This naturally derived bioactive compound is the best-known ingredient in coffee and other beverages, such as tea, soft drinks, and energy drinks, and is widely consumed worldwide. Therefore, it is extremely important to research the effects of this substance on the human body. With this in mind, caffeine and its derivatives have been extensively studied to evaluate its ability to prevent diseases and exert anti-aging and neuroprotective effects. This review is intended to provide an overview of caffeine’s effects on cancer and cardiovascular, immunological, inflammatory, and neurological diseases, among others. The heavily researched area of caffeine in sports will also be discussed. Finally, recent advances in the development of novel nanocarrier-based formulations, to enhance the bioavailability of caffeine and its beneficial effects will be discussed.


Introduction
Currently, special attention is being paid to natural molecules and their putative therapeutic effects to delay, or even prevent, the occurrence of many diseases and improve the health status of the population [1]. Indeed, their ingestion is widely believed to have fewer or no adverse effects on humans than most synthetic molecules, and they are also cheaper and easier to obtain [2][3][4]. Caffeine, in particular, has been the subject of intense and in-depth research on the human organism regarding its health-promoting effects and possible beneficial effects on the performance of athletes, especially through its ability to improve anaerobic and aerobic performance, muscle efficiency, and speed, and to reduce fatigue [5][6][7][8][9]. Caffeine is probably the most commonly ingested psychoactive substance in the world, found mainly in coffee, soft drinks, tea, cocoa and chocolate-like products, yerba matte leaves, guarana berries, and some pharmaceuticals [10]. It is rapidly absorbed and distributed in all human tissues, reaching maximum plasma concentrations 30-120 min after oral intake [9].
As far as we know, in vivo studies have already reported that caffeine stimulates the central nervous system by acting as an antagonist of A1 and A2 adenosine receptors, promotes adrenaline release, increases dopamine, noradrenalin, and glutamate levels, blood circulation and respiratory rate, mobilizes intracellular calcium stores, and alters

Benefits of Caffeine on Health
For our search, we used Web of Science. The search restrictions were based on language (English), year of publication from 2018 to present, and type of publication set to journal. The keywords used for the search were "caffeine" in combination with any of these other keywords: "cancer", "anticancer", "antitumor", "anti-tumor", anti-cancer", "neurodegenerative diseases", "autoimmune diseases", "immunological", "immunomodulatory", "immune system", "anti-inflammatory", and "cardiovascular". In the following subsections, we provide an overview of the latest research regarding the impact of caffeine in different illnesses such as cancer, autoimmune diseases, immunomodulation, and ocular, respiratory, neurodegenerative, and cardiovascular diseases.

Cancer
Cancer is one of the leading causes of death worldwide. It was estimated that in 2020, there were 19.3 million cancer cases, which resulted in 10.0 million cancer deaths [46,47]. By 2030, it is estimated that over 22 million people will develop cancer [47,48]. In addition, cancer is responsible for a significant economic burden on both the health care system and patients [48].
As early as 2000, Hanahan and Weinberg defined the key features (i.e., "hallmarks of cancer") that describe the characteristics necessary to promote cancer growth and metastasis. These hallmarks are self-sufficiency in growth signals, insensitivity to antiproliferative signals, resistance to apoptosis, limitless replicative potential, the induction of angiogenesis, and the activation of tissue invasion and metastasis [49]. In 2011, the authors revised the original hallmarks and added two more cancer-promoting features (genomic instability and tumor-promoting inflammation) and two more hallmarks (deregulation of cellular energetics and avoidance of immune destruction) [50]. As the understanding of cancer underlying mechanisms of progression has grown, as have the available experimental and computational tools; early in 2022, Hanahan reviewed the previously discussed features and included new additional features of cancer, namely, (i) phenotypic plasticity, (ii) non mutational epigenic reprogramming, (iii) polymorphic microbiomes, and (iv) senescent cells [51].
The role of coffee components in suppressing some of the cancer hallmarks defined by Hanahan and Weinberg [52,53] has been reviewed by Gaascht et al. and Cadóna et al., while other authors have fully elucidated the effect of caffeine on the cell cycle [54]. Caffeine anticancer activity has been widely studied [55], and the below-stated findings demonstrate the capacity of caffeine to overcome some of the cancer-promoting hallmarks, such as resistance to cell death and cellular senescence, that play an important role in cancer progression [51]. Further, several works state that caffeine may induce apoptosis through numerous pathways, such as p-53-dependent and -independent, phosphatase and tensin homolog, PI3K/protein kinase B (AKT), and mammalian target of rapamycin (mTOR) pathways [56].
El Far et al. studied the effect of caffeine and other natural substances on the senescent cells of colon and breast cancers. After inducing senescence with doxorubicin, the cells were treated with various doses of caffeine (0, 5,10,15,20,30,40,50, and 60 mM). The IC 50 of caffeine against doxorubicin-treated HCT116 and MCF7 cells was 13.36 ± 2.29 mM and 17.67 ± 3.98 mM, respectively. The authors also examined caffeine-induced apoptosis in both senescent and proliferative cells. At concentrations of 10 and 15 mM, caffeine induced a significant increase in apoptosis in senescent HTC116 cells, and at concentrations of 5, 10, and 15 mM in senescent MCF7 cells compared with proliferative cells [56]. In another study, Machado et al. evaluated the effect of caffeine on two breast cancer cell lines (MCF-7 and MDA-MB-231). The results showed that caffeine at a concentration of 2.5 mM and 5 mM for MCF-7 and MDA-MB-231, respectively, reduced cell viability and induced apoptosis [57]. The antitumoral effects of caffeine were studied in diverse cancer in vitro models, such as glioblastoma, melanoma, and pancreatic and lung cancers [58][59][60].
The antitumoral effects of caffeine have also been evaluated in in vivo tumor models. Venkata Charan Tej and collaborators investigated the effect of caffeine on the carcinogeninduced tumor model of fibrosarcoma. After 250 days of 3-MCA inoculation, there was a dose-dependent decrease in the tumor incidence and growth rate in the groups treated with caffeine (1.030, 2.060, and 4.120 mM) [61]. The anti-tumoral effect of caffeine was related to its action on cytotoxic T lymphocytes. On one hand, caffeine led to a higher percentage of cytotoxic T cells in the tumor, and on the other hand, it decreased the expression of programmed cell death protein 1 (PD-1) on these cells. In addition, it also increased the levels of pro-inflammatory cytokines such as TNF-α and IFN-γ. These results are in line with the previously known inhibitory effect of caffeine on the adenosine-A2a receptor pathway [62], which is one of the immunosuppressive pathways involved in cancer [63,64]. This capacity of caffeine to modulate the immune system in the tumor surroundings alters another important hallmark (i.e., the ability to avoid immune destruction). The modulation of the PD-1, an important immune checkpoint, and consequent enhancement of the T cell responses can exert an antitumor effect. In fact, the inhibitors of this protein are one of the immunotherapies approved by the FDA [65].
The therapeutic effect of caffeine was also demonstrated for renal carcinoma. Xu et al. showed, through in silico studies, that caffeine is able to bind to glucose-6-phosphate dehydrogenase (G6PDH), which is considered a biomarker and potential therapeutic target for this type of cancer. Consistent with the above results, in this study, the use of caffeine at concentrations of up to 0.016 mM for in vitro studies and 60 and 120 mg/kg/day for in vivo studies decreased the viability and proliferation of ACHN and 786-O cancer cells both in vitro and in vivo [64]. G6PDH is an important target in cancer given that is normally Pharmaceuticals 2023, 16, 1067 6 of 35 upregulated in different cancers and its dysregulation can provide valuable conditions for cancer progression [66]. Further, it also has an important role in maintaining the redox balance and biosynthesis of nucleotides and lipids, which is part of another cancer hallmark (i.e., reprogramming cellular metabolism) [67].
As previously mentioned, caffeine has also been tested in combination with other drugs in order to potentiate the antitumoral effect [68][69][70][71]. Higuchi et al. evaluated the efficacy of oral recombinant methioninase (o-rMETase) in combination with caffeine and doxorubicin in an orthotopic xenograft mouse model of synovial sarcoma. After two weeks of treatment, the group treated with the combinatorial treatment was able to induce tumor regression. According to the authors, this can be explained by the ability of caffeine to induce mitotic catastrophe [72]. Other examples of caffeine combination with different drugs are depicted in Table 2.  The results showed the ability of caffeine to reduce the viability of COLO829 and C32 cells by 5-35% and 1-16%, respectively. In addition, it also led to a decrease in thiol degradation and pro-apoptotic effects and did not affect normal melanocytes cells.
Understanding the effects of caffeine on cancer and the mechanisms underlying this effect is of extreme importance. Table 2 summarizes the most recent (from 2018) works on this topic. These studies also contribute to determining the necessary caffeine quantities to achieve a therapeutic effect and to ensure the safe use of caffeine.

Autoimmune Diseases and Immunomodulation
Inflammation is usually caused by infection or damage to a tissue [84]. Caffeine has the ability to exert modulation on the immune system. The immune response can be divided into two types: (i) innate and (ii) adaptive immunity [85]. Acute inflammation is a mechanism of innate immunity, whereas chronic inflammation usually contributes to the development of various diseases, such as metabolic disorders, neurodegenerative diseases, and even cancers [86,87]. The effect of caffeine on the innate immune system is related to the reduction in macrophage, neutrophil, and monocyte chemotaxis [88,89]. As for adaptive immunity, the effect of caffeine is due to the inhibition of Th1 and Th2 cell proliferation, as well as to the alteration of B cell function and the consequent reduction in antibody production [89][90][91][92]. Several authors, such as Horrigan et al., Açıkalın et al., and Al Reef et al., already reviewed, in depth, the impact of caffeine on the immune system and its capacity to alleviate autoimmune diseases [88,93,94].
Considering the immunomodulatory effects of caffeine, Wang et al. evaluated its effects on multiple sclerosis. Experimental autoimmune encephalomyelitis is the standard animal model for multiple sclerosis. After inducing the disease in C57BL/6 mice, these were treated with caffeine (10, 20, or 30 mg/kg/day) in drinking water. The results showed that caffeine could reduce inflammatory cell infiltration, the degree of demyelination, and microglial in vivo. It also reduced NLRP3 and p62 protein levels. In vitro assays indicated that caffeine promoted autophagy [95]. In another study, Ghaffary et al. evaluated the potential of mesenchymal stem cells to reduce the severity of rheumatoid arthritis. Wistar rats were treated with mesenchymal stem cells that had previously been incubated with various concentrations of caffeine. The results showed that the rats treated with mesenchymal stem cells, previously treated with 0.5 mM of caffeine, presented decreased disease severity and serum levels of C-reactive protein, nitric oxide, myeloperoxidase, and TNF-α. In addition, the IL-10 serum levels and the weight of the treated rats increased [96].

Ocular Diseases
Adenosine receptors are also expressed by retinal endothelial and retinal pigment epithelial (RPE) cells, as well as choroid and choroidal cells [97]. Therefore, caffeine may also have beneficial effects in ocular diseases, such as choroidal neovascularization and retinal inflammation.
Retinal inflammation is involved in ocular diseases as age-related macular degeneration (AMD) and diabetic retinopathy (DR), among others. For example, AMD is characterized by elevated vitreous levels of IL-1β [98] and plasmatic tumor necrosis receptor 2 (TNF-R2) and low levels of brain-derived neurotrophic factor (BDNF) in the aqueous humor, which negatively affect photoreceptor and retinal ganglion cells' survival [99]. Conti et al. demonstrated that caffeine has an anti-inflammatory effect in RPE cells, decreasing the expression of IL-1β, IL-6, and TNF-α, as well as the nuclear translocation of nuclear factor kappa B (NF-κB). In addition, the topical instillation of caffeine in an ischemia-reperfusion injury mice model was shown to restore physiological BDNF levels and reduce the mRNA levels of IL-6 in the retina, demonstrating its potential for the treatment of retinal inflammation and degeneration [100]. The effect of caffeine on choroidal adenosine receptors, the reduction in cell migration to the injured area, and angiogenesis demonstrate the importance of caffeine in attenuating choroidal neovascularization [97]. Despite the potential of caffeine in the management of such ocular conditions, the available studies are still scarce.

Respiratory Diseases
Currently, there are respiratory diseases for which caffeine is used as a clinical treatment, namely, premature infant diseases such as apnea and bronchopulmonary dysplasia (BPD). BPD is a common neonatal pulmonary complication with a prevalence of 45% in preterm infants [101]. BPD is associated with a nonspecific inflammatory response involving the activation of Toll-like receptors (TLRs), NOD-like receptors (NLRs), and increased levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-18, TNFα) [102]. In addition, NLR3 (NOD-, LRR-, and pyrin domain-containing protein 3), a key player in the pathogenesis of BPD, is responsible for the release of pro-inflammatory cytokines (IL-1β and IL-18) and alveolar cell death through various mechanisms [103,104]. Caffeine is the most commonly used medication for extreme prematurity (less than 28 weeks) and is also very commonly prescribed for very early preterm birth (28 to 32 weeks) [105]. As clinically shown, the early initiation of caffeine treatment (5 and 10 mg/kg/day) is important to achieve a successful outcome. Early treatment significantly reduced BPD incidence and mortality in low-birth-weight neonates [106]. Despite the use of caffeine and its clear benefits, the mechanisms behind the clinical benefits in these diseases are not fully understood.
In vitro studies showed that the treatment of lipopolysaccharide (LPS)-induced macrophages with caffeine caused a reduction in caspase-1 expression and the inhibition of the NLRP3 inflammasome, demonstrating its potential effect on this important target. Moreover, in vivo, the treatment of newborn mice with hypoxia-induced lung injury with caffeine was shown to significantly increase A2a receptor expression and inhibit the NLRP3 inflammasome protein and NF-κB pathway in the lung. The effect of caffeine on these key regulators attenuated inflammatory infiltration, reduced oxidative stress, decreased alveolar cell death, and promoted alveolar development [107]. Similar results were also observed in another study; specifically, caffeine caused a decrease in NF-κB and pro-inflammatory factor levels, increased the expression of A1, A2a, and A2b receptors, and decreased cell death in the lung [108]. Table 3 summarizes recent research findings on the anti-inflammatory effects of caffeine and its effects on autoimmune diseases. Caffeine decreased clinical score, inflammatory cell infiltration degree of the demyelination, and microglia stimulation in mice. In addition, it increased LC3-II/LC3-I levels and decreased NLRP3 and P62 levels.

Neurodegenerative Diseases
By 2050, the number of dementia cases worldwide is estimated to be 36.5 million [127]. There are several neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and multiple sclerosis [128,129]. For example, Parkinson's disease is triggered by the loss of neurons, which leads to a decrease in dopamine levels. In Alzheimer's disease, there is a deposition of extracellular deposits of amyloid-beta peptides and neurofibrillary tangles [130,131].
Caffeine is considered the most widely consumed psychoactive stimulant in the world. This natural compound is able to cross the blood-brain barrier [132,133] and, according to the literature, may exert a stimulant effect on the central nervous system by modulating several molecular targets, such as the (i) antagonism of adenosine receptors, (ii) promotion of intracellular calcium mobilization, (iii) inhibition of phosphodiesterase, and (iv) inhibition of GABA A receptors. However, except for the blockade of adenosine receptors and consequent inhibition of neurotransmitter-induced signaling pathways, the other mechanisms only exert their effects at toxic concentrations of caffeine [132,[134][135][136]. Recently, Ruggiero et al. reviewed the available literature on the protective effects of caffeine in various neurodegenerative diseases [137]. Among these studies, some emphasized the neuroprotective role of caffeine. For example, Manolo et al. showed that caffeine, at a concentration of 10 mM, is able to protect 96% of the dopaminergic neurons. The coadministration of olanzapine and caffeine did not result in neuroprotection, implying that both dopamine D2-like and A2a receptors are required for neuroprotection [138]. In an in silico study of Parkinson's disease, the authors demonstrated that caffeine has the ability to bind to both wild-type and mutant parkin protein [139]. The mutation of parkin protein is the most common cause of Parkinson's disease, as is the abnormal secretion and accumulation of α-synuclein [140,141]. This last part was detected in the following in vivo studies. Luan et al. investigated whether caffeine could protect against mutant αsynuclein-induced toxicity. Exposing mice to 1 g/L of caffeine in drinking water attenuated apoptotic neuronal cell death as well as microglia and astroglia reactivation, culminating in synucleinopathy [142]. In a similar study, Yan et al. investigated synergetic neuroprotection between caffeine and eicosanoyl-5-hydroxytryptamide. Both compounds are present in coffee and showed no effect at subtherapeutic doses, whereas their combination reduced the accumulation of phosphorylated α-synuclein, and maintained neuronal integrity and function [143]. Table 4 summarizes the most recent research on the neuroprotective effects of caffeine in neurodegenerative diseases and other conditions. In the presence of caffeine, the level of ADAM10 protein increased to 138.5 ± 9.2%, and the levels of APP protein level and ROS decreased to 85.4 ± 3.6% and 48.8 ± 3.2%, respectively. [146] Alzheimer's In vitro HEK293 cells 0.1-10 mM Caffeine induces conformational changes in muscle nicotinic acetylcholine receptors, which are molecular targets of Alzheimer's disease.  Pre-treatment with caffeine reduced brain infarct after hypoxia ischemia and also restored brain activity. [150] Acetaminopheninduced neurotoxicity

In vivo
Swiss albino mice 20 mg/kg IP injected 30 min after treatment with acetaminophen Treatment with caffeine and acetaminophen reduced the formation of ROS compared with the acetaminophen group. In addition, the survival time of caffeine-treated mice increased by 33%. [151] Parkinson's In vivo C57BL/6 mice with motor behavioral deficit induced by 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine 20 mg/kg/day, 7 days before MPTP-induced neurodegeneration and 7 days after Caffeine improved behavioral and neurotransmitter recovery against the induced toxicity. It was also able to restore antioxidant levels and suppress neuroinflammation. [152]

Hypoxic ischemia In vivo
Wild-type C57/bl6 specific pathogen-free mice 5 mg/kg IP injected (120 days) Caffeine administration after hypoxic ischemic brain injury reduced lesions in the gray and white matter and the number of amoeboid microglia and apoptotic cells. The expression of pro-inflammatory cytokines also decreased. [153]

Apnea of prematurity
In vivo

Infection-free pregnant Sprague
Dawley rats 20 mg/kg 1 day followed by 5 mg/kg/day over 14 days or 80 mg/kg 1 day followed by 20 mg/kg/day over 14 days, IP injected Caffeine administration in normoxia reduced oxidative stress and hypermyelination, and increased Golgi bodies. Caffeine at standard and high doses could provide neuroprotective effects. [154] Parkinson's In vivo C57BL/6 male mice 5.1 mM in drinking water Caffeine protected against synucleinopathy by modulating α-syn-induced apoptosis, microglial, and astrocytic activation in the striatum.

Cardiovascular Diseases
Cardiovascular disease (CVD), the leading cause of mortality, accounted for 17.8 million deaths worldwide between 1980 and 2017 [160]. By 2030, an estimated 23.6 million people per year will die due to CVD. Caffeine intake, particularly through the consumption of coffee, tea, and other products, has shown various cardiovascular effects. Turnbull et al. reviewed more than 300 studies regarding the effects of caffeine on cardiovascular health, published from the late 1980s to 2017. Overall, the results suggest that caffeine consumption does not increase the risk of CVD and may have a protective effect against this group of diseases [161]. However, recent studies on this topic have shown that high caffeine consumption may have the opposite effect.
A study of 347,077 people (UK Biobank) concluded that coffee consumption may modestly increase the risk of cardiovascular disease. A nonlinear association was found between long-term coffee consumption and cardiovascular disease. Individuals who consumed coffee in high doses (>6 cups/day, >450 mg caffeine/day) were more likely to develop cardiovascular disease (22%) than those who consumed less coffee (1-2 cups/day or 75-150 mg caffeine/day) [162]. In addition, the authors examined the association between coffee consumption, plasma lipids, and CVD risk in 362,571 individuals (UK Biobank). The results showed that high coffee consumption (>6 cups/day) may increase CVD risk by increasing the levels of low-density lipoprotein cholesterol (LDL-C), total cholesterol (total-C), and apolipoprotein B (ApoB) [163].
However, other studies have reported the potential beneficial effects of moderate coffee consumption, in line with Turnbull et al.'s literature review [161]. For instance, a study involving 20,487 Italian participants concluded that moderate coffee consumption (3-4 cups/day) was associated with a low risk of CVD-related mortality. In addition, an inverse correlation was found between NT-proBNP levels (N-terminal fragment of the B-type natriuretic peptide, which is associated with higher stroke risk) and coffee consumption [164]. Similarly, a study of more than 500,000 participants in England reported that a caffeine intake of 121-182 mg/day from coffee (2-3 cups/day) or tea (4-6 cups/day) was associated with a low risk of coronary artery disease [165]. In addition, a US follow-up study of 23,878 participants over 16 years found that the daily caffeine consumption of about 100-200 mg or >200 mg is associated with a lower risk of CVD mortality [166]. An inverse association between coffee consumption and CVD risk factors (blood pressure and arterial stiffness) was also observed in another study, showing the beneficial effect of moderate coffee consumption [167]. A similar association was observed concerning coffee consumption and hypertension risk [168].
Therefore, despite some studies linking high coffee or caffeine consumption to CVD risk, most studies have reported that its moderate consumption has potentially beneficial and even protective effects on CVD. Table 5 summarizes the recent research on the effects of caffeine on CVD. Moderate coffee consumption (3-4 cups/day) has been associated with lower CVD mortality. [164] Prospective >500,000 individuals (40-69 years old) The consumption of 2-3 cups of coffee per day (121-182 mg caffeine/day) was associated with a low risk of coronary artery disease.
[165] In vitro, caffeine (2 mM) induced autophagy by inhibiting mTOR signaling and decreased proliferation of VMCs by inhibiting WNT signaling. In vivo, caffeine at 2.57 mM (in drinking water, 2 weeks before and after injury) decreased vascular restenosis. [171] In vivo Zebrafish Caffeine (128 and 334 µM in zebrafish culture water) caused a similar decrease in HR. [172] HR, heart rate; HF-HRV, heart rate variability; mTOR, mammalian target of rapamycin; VSMCs, vascular smooth muscle cells.

Caffeine Impact on Sports Performance
Coffee's best-known constituent, caffeine, is the most widely consumed psychotropic drug in the world, with an estimated daily intake of up to 4 mg/kg body weight in American adults [173][174][175][176]. It is a psychostimulant that can lead to physical dependence [177]. Caffeine intake is widespread among inactive individuals and high-performance athletes, especially since 2004, when it was removed from the World Anti-Doping Agency's list of banned substances for competition [178]. It is also readily available in various forms such as capsules, powders, caffeinated beverages, and energy drinks [173].
However, while there is evidence that caffeine improves athletic performance [173][174][175][176][177][178][179][180][181], due to particular protocols and study designs, some research seems conflicting. Some studies show ergogenic effects on aerobic endurance (>90 min), high-intensity efforts (20-60 min), muscular endurance, sprint performance and maximal strength (0 to 5 min), and ultra-endurance (>240 min) and endurance races with prolonged intermittent sprints (team sports), while others report no evidence for its administration [180][181][182]. We assume that an ergogenic substance is a substance used with the aim of improving athletic performance and promoting recovery after exercise by delaying fatigue. The word is of Greek origin: ergo (work) and gen (generation). As a result, it is commonly consumed by athletes, and research suggests that 75 to 90% of athletes consume caffeine before or during athletic competition [181]. In an analysis of 20,686 urine samples from elite athletes, 73.8% of the samples contained caffeine at concentrations greater than 0.1 µg/mL, suggesting that three out of four athletes consume caffeine before or during competition [175].
It should be recalled that the consumption of caffeine is not prohibited for athletes, with the maximum allowable concentration being 12 mg/L of urine (International Olympic Committee). The fact that caffeine affects the nervous system, adipose tissue, and skeletal muscle originally led to the hypothesis that caffeine might affect athletic performance. For example, caffeine may increase skeletal muscle contractile force at submaximal contraction and increase the athlete's pain threshold or perceived exertion, which could lead to longer training sessions [180,181].
However, it should be remembered that caffeine intake has several side effects. Blood pressure increases both at rest and during exercise and heart rate increases, and it may impair recovery and sleep patterns, most likely in athletes who do not regularly consume caffeine [180]. In addition, Martins et al. demonstrated that high doses of caffeine have side effects. In a recent study using a caffeine dose of 12 mg/kg, almost all participants reported side effects such as tachycardia and palpitations, anxiety or nervousness, headache, and insomnia [175].
However, according to our research, it seems important to us to better evaluate certain aspects to achieve better scientific clarification with implications for practice, such as the ideal dosage, time of intake, abstinence, training time vs. caffeine consumption, physiological factors, gender, and caffeine users or not.

Optimal Dosage
Higher-than-ideal caffeine doses, 3-6 mg/kg, before exercise do not further improve athletic performance. Additional and higher doses of caffeine may lead to side effects in athletes [180].
Low doses of caffeine (~200 mg) have also been shown to improve attention, alertness, and mood, and cognitive processes during and after strenuous exercise. Thus, the ergogenic effects of low doses of caffeine appear to be due to changes in the central nervous system [180].
The generally accepted dosage of caffeine for performance enhancement is between 3 and 6 mg/kg, 60 min before exercise [175].
Although a meta-analysis reported that caffeine intake can be ergogenic in a variety of physical activities, the "optimal" caffeine dose remains difficult to determine [178].

Timing of Intake
The early ingestion of caffeine prior to physical activity has been shown to enhance performance. For example, caffeine can improve performance during high-intensity sprints when taken 45-60 min before exercise [176].
Because caffeine has so many positive effects on exercise performance, it can-and perhaps should-be taken before or during exercises. For most sports, it is recommended that caffeine be taken about 60 min before the start of the first set of the training session if used before exercise. This period varies depending on the individual, the type of event, and the type of caffeine ingested, with caffeinated mouthwashes and chewing gums generally requiring much less time. For longer training sessions, there is evidence that ingesting caffeine later in the day, and at lower doses, may be effective [181]. Other interesting data refer to the concentration peak that occurs in the first 15 min [183].

Abstinence
It appears that, short-term, caffeine withdrawal before competitions does not enhance the ergogenic effects of caffeine in habitual users. Withdrawal is associated with numerous negative consequences, including headache, fatigue, irritability, muscle pain, sleep disturbance, and nausea. However, these acute withdrawal symptoms, shortly before important competitions, may have a negative impact on the subjective self-confidence and well-being of the athlete [181].

Training Time vs. Caffeine Consumption
Increases in physical performance as a function of training time have been demonstrated in various sports. Studies suggest that anaerobic and aerobic activities may be more powerful due to the diurnal fluctuations of the circadian cycle between 4 and 8 pm. Morning caffeine consumption had a more beneficial effect than afternoon consumption [175].

Physiological Factors
Hypothetically, the potential performance enhancement from caffeine ingestion may be greater in trained individuals than in untrained individuals because trained individuals have an enhanced neuromuscular action potential. Trained individuals have a higher concentration of adenosine A2a receptors than untrained individuals [175,185].
The main finding of this review is that very low doses of caffeine (>1-2 mg/kg, generally taken 60 min before exercise) improve resistance training performance in terms of muscle strength, muscle endurance, and average speed [174].
Aerobic endurance appears to be the sport in which caffeine consumption most consistently produces moderate to significant benefits, although the magnitude of the effect varies among individuals [185].

Gender
Caffeine ingestion positively affects resistance exercise performance in women, and the magnitude of these effects appears to be comparable to those observed in men [184]. Even considering the woman's menstrual cycle, a study showed that caffeine increased peak aerobic cycling power in the early follicular, preovulatory, and mid-luteal phases. Thus, the ingestion of 3 mg of caffeine per kg of body mass might be considered an ergogenic aid for eumenorrheic women during all three phases of the menstrual cycle [186].

Caffeine Consumers or Not
For the first study using a performance test, 17 moderately trained men were recruited, 8 of whom did not routinely consume caffeine (<25 mg/day) and 9 of whom regularly consumed caffeine (>300 mg/day). It was found that there were no differences between the groups in time to exhaustion at any of the doses, suggesting that habitual caffeine consumption does not attenuate the ergogenic effects of caffeine [181]. In another study, on cycling, habitual caffeine intake was found to have no effect on athletic performance, suggesting that habituation to caffeine has no negative effect on caffeine ergogenesis [187].

Future Directions: Nanotechnology-Based Delivery Strategies
Caffeine is usually consumed through the ingestion of beverages, especially coffee, tea, and pharmaceuticals, which allows for rapid absorption and distribution in all tissues [9]. However, caffeine has a short half-life (3-5 h) [188]. In addition, the oral intake of high concentrations of caffeine may cause gastrointestinal problems [189] and its wide distribution may lead to undesirable side effects, such as the stimulation of the nervous system.
Nanotechnology is a multidisciplinary field that enables the manipulation of matters at the nanoscale (1 to 100 nm) and the creation of novel devices with unique properties [190]. Nanotechnology is frequently explored for drug delivery to a target tissue. Drug delivery systems (DDS) or nanocarriers offer important advantages for caffeine delivery, namely, a high loading capacity, the co-encapsulation of different drugs, controlled and sustained release, a high surface area allowing greater interaction with tissue, and a high ability to permeate through tissues [191]. In addition, other routes of administration besides oral can be used, such as intranasal [192] and dermal [188] (Figure 2). delivery systems (DDS) or nanocarriers offer important advantages for caffeine delivery, namely, a high loading capacity, the co-encapsulation of different drugs, controlled and sustained release, a high surface area allowing greater interaction with tissue, and a high ability to permeate through tissues [191]. In addition, other routes of administration besides oral can be used, such as intranasal [192] and dermal [188] (Figure 2). Nanocarriers' compositions are tailored depending on the drug(s), route of administration, and target tissue. Therefore, different nanocarrier compositions based on lipids, polymers, or metals have been proposed for caffeine delivery, as reported in this section.
Lipidic nanocarriers have been widely explored for topical drug delivery through the skin for cosmetic and pharmaceutical applications [193]. The composition of lipid carriers is an important factor to be considered to improve skin permeation and therapeutic effects. For example, among the various phospholipids (1,2-distearoyl-snglycero-3phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC)) tested for liposome preparation and the topical delivery of caffeine, DPPG was the most promising. Ex vivo studies showed that DPPG was able to enhance the permeation of encapsulated and free caffeine through hairless rat skin by disrupting the lipid barrier of the stratum corneum (SC) [194]. A similar effect was observed for lipid nanocapsules (NCs) in porcine skin. The ability of lipid NCs to increase skin permeation of free caffeine has been attributed to the combination of several factors, namely, the occlusion effect of nanoparticles on the skin surface, accumulation in hair follicles, and the effect on barrier function of SC [195]. On the other hand, the incorporation of propylene glycol into phosphatidyl liposomes has been shown to enhance the permeation of caffeine through the skin, as demonstrated ex vivo in human full-thickness skin [196]. In this sense, the researchers proposed the combination of the lipolytic activity of caffeine with the increased permeation capacity of propylene glycol liposomes as a noninvasive treatment for cellulitis [196]. Amasya et al. also proposed semisolid lipid nanoparticles as a promising treatment for cellulitis because they can penetrate the skin and reach the adipose tissue [197].
Flexible liposomes composed of phosphatidylcholine and higher surfactant content (polysorbate 80 and polysorbate 20) were also proposed for the treatment of alopecia by topical application [198]. The therapeutic potential of caffeine in alopecia is due to its ability to inhibit 5-α-reductase and phosphodiesterase and increase vasodilatation and blood supply to hair follicles [199]. The nanocarriers co-encapsulating minoxidil and caffeine resulted in an increase in hair length comparable to the aqueous solution of the Nanocarriers' compositions are tailored depending on the drug(s), route of administration, and target tissue. Therefore, different nanocarrier compositions based on lipids, polymers, or metals have been proposed for caffeine delivery, as reported in this section.
Lipidic nanocarriers have been widely explored for topical drug delivery through the skin for cosmetic and pharmaceutical applications [193]. The composition of lipid carriers is an important factor to be considered to improve skin permeation and therapeutic effects. For example, among the various phospholipids (1,2-distearoyl-snglycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG), 1,2-dilauroylsn-glycero-3-phosphocholine (DLPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) tested for liposome preparation and the topical delivery of caffeine, DPPG was the most promising. Ex vivo studies showed that DPPG was able to enhance the permeation of encapsulated and free caffeine through hairless rat skin by disrupting the lipid barrier of the stratum corneum (SC) [194]. A similar effect was observed for lipid nanocapsules (NCs) in porcine skin. The ability of lipid NCs to increase skin permeation of free caffeine has been attributed to the combination of several factors, namely, the occlusion effect of nanoparticles on the skin surface, accumulation in hair follicles, and the effect on barrier function of SC [195]. On the other hand, the incorporation of propylene glycol into phosphatidyl liposomes has been shown to enhance the permeation of caffeine through the skin, as demonstrated ex vivo in human full-thickness skin [196]. In this sense, the researchers proposed the combination of the lipolytic activity of caffeine with the increased permeation capacity of propylene glycol liposomes as a noninvasive treatment for cellulitis [196]. Amasya et al. also proposed semisolid lipid nanoparticles as a promising treatment for cellulitis because they can penetrate the skin and reach the adipose tissue [197].
Flexible liposomes composed of phosphatidylcholine and higher surfactant content (polysorbate 80 and polysorbate 20) were also proposed for the treatment of alopecia by topical application [198]. The therapeutic potential of caffeine in alopecia is due to its ability to inhibit 5-α-reductase and phosphodiesterase and increase vasodilatation and blood supply to hair follicles [199]. The nanocarriers co-encapsulating minoxidil and caffeine resulted in an increase in hair length comparable to the aqueous solution of the drugs and the commercial alcoholic solution. Nevertheless, liposomes loaded with caffeine and minoxidil led to a significant increase in hair weight, an indicator of healthy and strong hair, demonstrating the potential of liposomes for the treatment of hair loss [198]. Other types of nanosystems, namely, nanoemulsions containing eucalyptol and oleic acid, have been shown to accumulate in hair follicles and increase caffeine retention in these structures, demonstrating the potential of these nanosystems for the treatment of alopecia [200]. Considering that hair follicles are nourished by blood vessels, targeted accumulation in these structures may enhance the permeation of caffeine. Therefore, these approaches can also be used to develop novel therapeutics for diseases of other tissues to avoid systemic or oral delivery.
In this sense, proniosomes have been proposed for the treatment of migraine by the topical application of caffeine. As expected, caffeine-loaded proniosomes, applied topically to Swiss albino mice, were able to penetrate the skin. Moreover, the treatment resulted in a significantly higher caffeine concentration in the blood and brain, as well as prolonged and sustained effects, compared with orally administered caffeine solution [188]. Recently, the co-delivery of caffeine and ergotamine to the brain by intranasal administration (olfactory route) has also been proposed. Hybrid lipid-polymer nanoparticles of lecithin, poly(lactic-co-glycolic acid) (PLGA), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine functionalized with polyethylene glycol (PEGylated DPPC) showed a high encapsulation efficiency (87%) and controlled release over a period of 48 h. In addition, the results showed that the nanoparticles had high targeting accuracy in the brain without causing toxic effects. Furthermore, the synergistic effects of the drugs enhanced the anti-migraine effect [192].
The anti-cancer effect of caffeine has also been enhanced by the use of nanocarriers. Liu et al. prepared lipid-based nanosystems for the co-delivery of caffeine and imiquimod. Caffeine enhanced the therapeutic effect of the immunomodulator imiquimod and radiotherapy in an orthotopic breast cancer model. The authors suggested that this may be due to the modulation of the tumor microenvironment [201]. On the other hand, polymer-based nanocarriers, i.e., gelatin nanoparticles loaded with caffeine, showed the ability to decrease the viability and proliferative capacity of murine melanoma cells (B16F10) without causing significant cytotoxic effects in normal fibroblast cells (L929) [202]. Other studies have reported the combination of caffeine with metallic nanocarriers. For example, silver-caffeine complexes anchored to magnetic nanoparticles were proposed for the treatment of hepatocellular carcinoma [203]. This type of cancer is known to be resistant to radiotherapy and chemotherapy and can be caused by hepatitis-related infections. The most promising nanoparticles showed higher cytotoxicity against the cancer cells (hepatocellular carcinoma cells, HCC) than against the normal cells (normal hepatic cells, WRL-68). On the other hand, the targeted hyperthermia effect of the magnetic nanoparticles can improve the anti-tumor effect of the formulation and avoid the side effects of the commonly used therapeutics. In addition, these silver-caffeine magnetic nanoparticles also showed antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus cereus [203]. Other caffeine-metal nanoparticles have been developed for antibacterial applications. Khan et al. [204] demonstrated the ability of caffeine-gold nanoparticles to inhibit biofilm formation and eliminate mature biofilms. In addition, the nanoparticles showed antibacterial activity against resistant pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes), demonstrating their potential for treating chronic infections.
Overall, the different types of nanocarriers have shown the potential to improve the therapeutic effect of caffeine. Table 6 provides an overview of the recent research on the development of lipid-, polymer-, and metal-based nanocarriers loading caffeine for biomedical applications.    Nanocrystals with a size of 694 nm showed a delayed, but higher and longer delivery of caffeine, being detected in serum for at least 5 days. [209] NLCs, nanostructured lipid carriers; AgNPs, silver nanoparticles; DPPG, 1,2-dipalmitoyl-sn-glycero-3phosphoglycerol, sodium salt; CTAB, cetyltrimethylammonium bromide; EGCG, (-)-epicatechin-3-gallate.

Conclusions
Coffee is the most consumed caffeinated beverage, while caffeine can also be found in tea, soft drinks, and energy beverages. Studies on the associations between coffee consumption and a range of health outcomes have been completed. Epidemiological studies reveal that, for the majority of people, coffee consumption is advantageous and adversely connected with risk for a number of diseases. Numerous researchers have recently conducted studies on the effects of caffeine on diseases such as cancer, cardiovascular, immunological, inflammatory, and neurological disorders, among others, as well as in sports, suggesting that this field of study is expanding quickly. To clarify the link between caffeine consumption and specific diseases and to examine consumption patterns in relation to health outcomes, randomized controlled studies are required because association does not imply causality. Because most studies have focused on adults, little is known about the negative consequences of children and adolescents consuming items with caffeine. On the other hand, several advancements in innovative DDS have been made in order to lessen the adverse effects and boost bioavailability for the treatment of various diseases. Thus, DDS have potential importance for clinical applications in several diseases, potentiating the effect of caffeine. However, the growing volume of articles, meta-analyses, and scientific evidence is not yet sufficient to confirm the quality and quantity of caffeine in the treatment of several disorders and in sports, being an avenue to explore in the future.

Conflicts of Interest:
The authors declare no conflict of interest.