**1. Introduction**

Volcanic obsidian was widely used in ancient times for stone tools, with its highly glassy nature making it sharper than other lithics for cutting purposes. In Europe and the Mediterranean, there are just several island sources in Italy and Greece, and a few in the inland Carpathian region, all having been used by the beginning of the Neolithic period, ca. 6000 BCE. Maritime transport was necessary for access to the island sources, while other materials would have been moved in opposite directions. Obsidian was likely a small part of this exchange system. The location and geological studies for each of the sources goes back to the early 20th century, while the ability to chemically identify specific sources began in the 1960s. The development of non-destructive and especially portable X-ray fluorescence spectrometers has revolutionized the number of artifacts tested since 2010, providing new interpretations of obsidian selection and use, which changed over time.


Deng et al. designed liposomes triggered by X-ray irradiation. In this work, a liposomal formulation was embedded with a photosensitizer (verteporfin) and gold nanoparticles. Irradiation induces 1O2 generation by the photosensitizer. Singlet oxygen acts by oxidizing unsaturated lipids, leading to liposomal membrane destabilization and cargo release. The gold nanoparticles interact with X-rays acting as a radiosensitizer. These liposomes were used to encapsulate DXR and evaluated for its activity in xenograft mouse model bearing HCT 116 colorectal cancer. Animals were divided into groups to receive PBS, liposome only, irradiation only (4 Gy), or liposome plus irradiation (4 Gy), and the ability of these treatments to control tumor growth was evaluated. Two weeks post-treatment, the tumor volume increase was verified for animals receiving PBS (3.0-fold), liposome only (2.9-fold), and irradiation only (3.4-fold). On the other hand, treatment with liposome plus irradiation allowed for tumor size reduction (74%) as compared to the PBS control group [72].

Zu et al. developed liposomes encapsulating hemoglobin (Hb) and DXR. In this formulation, Hb alleviates hypoxia, and DXR release is radio-triggered by the action of ROS in the lipid membrane. When evaluated for ROS generation, this was higher in B16 (murine melanoma) tumors after treatment with the formulation plus irradiation as compared to either formulation alone or irradiation alone. Regarding the triggered release of DXR, 10 min after 2 Gy irradiation, up to 6% of content was released. When the irradiation dose was enhanced to 8 Gy, up to 20% of DXR was released. For the nonirradiated formulation, only ~1% of DXR was released in the same time. As expected, when evaluated for antitumor efficacy in a highly radioresistant tumor model (C57BL/6 mice bearing B16), RT alone (8 Gy) was not able to significantly inhibit tumor growth. Combining RT to the formulation co-encapsulating Hb and DXR allowed for significant tumor growth inhibition. This was superior to the combination of RT plus liposomes encapsulating Hb only or RT plus liposomes encapsulating DXR only, confirming the synergistic effects of the combination [73].

Two liposomal formulations designed to be irradiation-triggered were patented by Fologea et al. One formulation consists of pH-sensitive liposomes designed to encapsulate an agent and an organic halogen (such as chloral hydrate, fluoral hydrate, or bromal hydrate). The triggering mechanism relies on the reaction of the organic halogen with water in the vesicle's aqueous interior upon irradiation. This reaction releases H+, leading to a drop in the internal pH and disruption of the pH-sensitive bilayer, releasing the encapsulated content [112]. The other liposomal formulation was designed to contain a nanoscintillator responsive to irradiation, an enzyme capable of hydrolyzing the liposomal membrane, and an enzyme activator in a photolabile molecular cage. Triggered release occurs in a series of events that take place upon irradiation. When the nanoscintillator is irradiated, light is emitted, and the photolabile molecular cage with an enzyme activator is disrupted. The enzyme is then activated to disrupt the liposomal membrane releasing the encapsulated agent [113]. Another patent by Bondurant et al. relates to liposomes composed of a stable forming lipid, an ionizing radiation polymerizable colipid, and a chain transfer agent. Upon irradiation, radical species produced in water can initiate radical chain polymerizations in the polymerizable colipid (e.g., polymerizable phosphatidylcholines), leading to membrane destabilization and content release. The transfer of irradiationgenerated free radical ions by the chain transfer agent (such as thiols or esters) increases the polymerization of the radiation polymerizable colipid, enhancing the trigger capacity [114].

#### **7. Microbeam and Minibeam Radiation Therapy to Modulate the EPR Effect**

Aiming at the reduction of radiation-induced side effects, many novel RT treatment strategies are arising. Spatially fractionated radiotherapy such as microbeam radiation therapy (MRT) and minibeam radiation therapy (MBRT) have attracted interest in the last years. Figure 3 represents commonly used beam widths for MRT and MBRT.

**Figure 3.** Schematic representation of commonly used widths for microbeam, minibeam, and broadbeam radiotherapy preclinical studies. Reproduced from Brönnimann et al., Nature Publishing Group, 2016 [115].

#### *7.1. Microbeam Radiation Therapy*

MRT was proposed by Slatkin and coworkers in 1992 [116]. In MRT, a multislit collimator is used in order to shape an X-ray beam so that the dose deposition profile consists of areas of peaks and valleys, spatially modulated on the micrometer scale (beam width ranging from 25 to 100 μm). On the peak areas, doses might consist of several hundred Grays, while valley regions present doses below the level of tissue tolerance. This distinct dose deposition enables a substantial reduction on toxicity to the normal tissue combined with a tumor control rate similar to that observed with conventional RT. The increased therapeutic index that can be obtained with MRT is of high importance for improved cancer treatment with radiation [117,118]. The mechanisms that underlie the increased antitumor efficacy combined with the sparing of surrounding normal tissues are not yet fully understood. One of the suggested biological mechanisms consists of the fact that MRT damages preferentially the tumor microvasculature in comparison to normal tissue microvasculature.

With this in mind, MRT can be used to modulate the EPR effect. It is true that the EPR effect has been validated in both experimental animal models and different human tumors [3]. However, the importance and extent of the EPR effect in patients is still the focus of intense debate. The EPR effect is known to be more pronounced in small animal models, usually used to evaluate the preclinical efficacy of nanocarriers, as compared to human tumors. This is one explanation for the disbalance between the high amount of promising preclinical nanocarriers and the relative small number of these carriers in the clinical setting [122]. Therefore, a strategy to enhance tumor vessel permeability optimizing the EPR effect is highly desirable.

Sabatasso et al. demonstrated in mice bearing U-87 human glioblastoma that tumor irradiation with MRT (synchrotron generated parallel X-rays in a range of 25–50 μm in width) led to FITC-dextran (~27 nm) extravasation in this area. In nonirradiated animals, administered FITC-dextran remained intravascular with no extravasation to the control tumors. When animals were treated with MRT (150 Gy peak dose), a twofold increase in FITC-dextran transpermeability was observed 45 min postirradiation, as compared to the

control. A study with FluoSpheresTM (100 nm microspheres) showed that they remained constrained to MRT paths. This indicates, as expected, a size-dependent transpermeability following MRT. It was possible to conclude from this study that the transient vascular permeability induced by MRT can be a strategy to improve the delivery of nanocarriers to the tumor site [123].

#### *7.2. Minibeam Radiation Therapy*

Despite promising preclinical studies for MRT, its production depends on large synchrotron facilities, of which there are currently only a few around the world. In addition to the difficulty to access these facilities, specialized hospitals are rarely close to them, hindering the clinical application of MRT [124]. In order to overcome this problem, efforts are put into using nonsynchrotron irradiators (such as a conventional X-ray tube) to produce microbeams [125]. Another strategy consists of using slightly wider 'minibeams' (beam widths ~500–700 μm), which are more easily produced with custom multislit collimators placed in small animal irradiators, as compared to microbeams [124,126,127]. Minibeam radiation therapy (MBRT) leads to similar radiobiological effects as compared to MRT and is also hypothesized to improve the delivery of nanocarriers. This idea was evaluated by Price et al. on a study of the effects of MBRT on the delivery of liposomal DXR. An in vivo study was performed on mice with syngeneic orthotopic triple-negative breast cancer. Animals were irradiated with either MBRT (28 Gy) or broadbeam radiation therapy (7 Gy) and 24 h later received the liposomal DXR. The delivery of liposomal DXR to the tumor was increased by 7.1-fold for MBRT and 2.7-fold for broadbeam radiation therapy compared to liposomal DXR alone (sham RT) [127].

#### **8. Limitations and Future Directions**

Besides the clear advantages of combining nanocarrier-assisted delivery of molecules and RT, which were presented in this review, there are also limitations that must be considered. Among limitations that come from the nanocarriers, it is important to remember that none of them are completely safe and nontoxic. This is affected by the lack of knowledge regarding their distribution and action, controlled by characteristics such as size, charge, degradation, and cancer stage and type, among others [128,129]. The commercial viability and availability of nanocarriers also have to be considered. Some of the most important challenges are: Possible scale-up problems, low production rate, difficulties in separating by-products, lack of quality control system, inconsistencies in reproducibility, and the high cost [130]. These limitations help us understand why the impact of nanocarrier-assisted drug delivery on human patients and clinical outcome has so far been insufficient [131]. For their future wide application, collaborative research efforts with more in vivo studies and clinical trials are necessary [128]. If the developed products are novel, safe, and meet the medical needs with cost effective benefits, the investment in nanocarrier development is likely to increase [130].

RT, in turn, has as its major limitation the damage caused to normal surrounding tissues. In this manner, modern RT and imaging techniques are enhancing the applications of radiation oncology. To the best of our knowledge, the nanocarrier-assisted delivery of molecules has only been evaluated in combination with photon RT, as reviewed herein. Photon RT is also the treatment modality applied to most patients worldwide. However, particle therapy, such as proton and carbon ions, is a promising strategy to be evaluated in combination with nanocarrier drug delivery. These therapy modalities are characterized by steep dose falloff with a minimal exit dose beyond a specified target. This allows for a more accurate and precise treatment, reducing healthy tissue exposure and, therefore, the toxicity [132]. Nevertheless, it is important to have in mind that such techniques have a high cost. So far, their use is concentrated in high-income countries that have centers able to deliver these particles [133].

In addition, the combination of nanocarrier drug delivery and RT has only been evaluated with single RT courses. Fractionated and hypofractionated RT should be evaluated in future in vivo studies [132].

Another important point is the heterogeneity in the RT response observed for tumors of the same histology [134]. Response prediction by means of liquid biopsy and circulating biomarkers allows for a refined treatment with patient stratification by biological parameters. Molecularly designed systems to target specific cancer will lead to future personalized cancer care [132,135].

#### **9. Conclusions**

In this review, we present the state of the art of nanocarriers designed to be used in combination with RT. So far, these systems were used to encapsulate one of or a combination of different radiosensitizing molecules such as classic chemotherapeutic drugs, natural products, hypoxic cell radiosensitizers, or DNA repair inhibitors. Nanocarriers have also been successfully designed to encapsulate and deliver oxygen, modulating the hypoxic TME; or to capture and deliver antibodies to antigen-presenting cells, boosting the abscopal effect. X-ray irradiation was shown to be an efficient exogenous trigger to nanoparticles and liposomes, enhancing the therapeutic ratio of these formulations. Finally, we presented how the transient and efficient vascular permeability arising from spatially fractionated RT modalities, such as microbeam and minibeam radiation therapy, can modulate the EPR effect and enhance the passive delivery of nanocarriers to the tumor site. All these different strategies of combining nanocarrier-assisted delivery of molecules and RT were validated in in vitro and/or in vivo studies. Thus, nanotechnology can be used to overcome the limitations of RT and the other way around.

**Author Contributions:** Conceptualization, M.S.F.; design of Figure 2, M.S.F.; writing—original draft preparation, M.S.F. and E.R.G.; writing—review and editing, M.S.F. and E.R.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES for supporting Eliza Rocha Gomes with a scholarship.

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

#### **Abbreviations**

#### **References**

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**3. Results and discussion**

### **3.1 Regression models and analysis for surface roughness**

The multi-linear regression coefficients are summarised in **Table 5**, exhibiting the correlation between the input parameters and the output surface roughness for straight-line, inner and outer arc profiles for material thicknesses of 4, 8 and 12 mm. The values of coefficients for all profiles and thicknesses demonstrate a similar trend, showing that constant and variable *X*1 is positive and variables *X*2 and *X*3 are negative. The coefficient indicates the change in the mean response relating in the variation of the specific term, whilst the other term in the model remains constant. The relationship between a term and response is denoted by the sign of the coefficient [44]. The negative correlation coefficient denotes an inverse relationship between variables and responses; and therefore, if it is positive as the coefficient increases, the response mean value also increases. Therefore, an increasing rate of traverse speed (*X*1) results in an incremental value of surface roughness. Moreover, an increasing rate of abrasive mass flow and waterjet pressure indicates/obtains a decreasing value of surface roughness. The values of R2 , R2 adj and R2 pred for 4, 8 and 12 mm ranged from 94.33– 99.08%, 90.94–98.52% and 88.66–96.17%, respectively. This indicates that regression models denote an acceptable confirmation of the relationship between the independent variables and Ra response, which denotes a high significance of the model. Therefore, the multi-linear model is reliable and can be utilised in the optimisation of process parameters. It can be observed that the R2 , R2 adj and R2 pred obtained from straight-line, inner and outer arcs profiles have a uniform gap of at least 2%, which is comparable for all material thicknesses. Hence, this minimal gap denotes an insignificant difference between the surface roughness achieved from straight and curvature profiles [36].

The results detailed in **Table 5** show that the highest value of R2 , R2 adj and R2 pred for 4 and 8 mm material thickness are achieved in Ra3 with the values of 97.26, 94.84 and 92.45%; 98.64, 97.82 and 95.06%; 99.08, 98.52 and 96.17% respectively. Thus, Ra2 achieved the highest percentage of R2 , R2 adj and R2 pred for 12 mm material thickness

 **5.** *Summary of multi-linear regression coefficients for Ra.*


**4. Regular physical activity and exercise**

Exercise is often classified as aerobic, strength (resistance) and flexibility exercise and each of them has their own utility and limitations. The common recommendation (e.g. 150 minutes or more of moderate-to-vigorous intensity physical activity per week, spread over at least 3 days/week, with no more than 2 consecutive days without activity) is applicable to most adults and help to achieve cardiovascular fitness and other benefits. Brisk walking can be regularly included into the daily life. Two to three sessions/week of resistance exercise on non-consecutive days are also recommended [25]. Increased physical activity is effective in preventing diabetes, and the protective benefit is especially pronounced in persons at the highest risk for the disease [26]. At higher BMI, exercise is protective against diabetes and is dose-dependent. The prevention of diabetes and reduction of hyperglycemia in diabetes occur even without significant weight reduction [27]. American Diabetes Association recommends that prolonged sitting should be interrupted every 30 minutes for blood glucose benefits, particularly in adults with type 2 diabetes [25]. Possible physical activity even while seated (e.g. by leg exercise with or without sit down leg exercise machines) may also help. A more intensive physical activity program including at least 275 minutes per week may be needed to assist weight loss and avoid regain [21]. Higher levels of physical activity before pregnancy or in early pregnancy are associated with a significantly lower risk of developing gestational diabetes mellitus [28]. NICE guideline recommends women with gestational diabetes to take regular exercise (such as walking for 30 minutes after a meal) to improve blood glucose control [29]. **Figure 2** summarizes the potential benefits of regular physical activity and exercise in relation to diabetes.

reducing 1 mmol/L of LDL cholesterol and is about four times more effective than lowering 0.9% of glycated hemoglobin (HbA1c), and thus, in diabetes, BP control is more efficacious

**Clinical management perspective:** The current guidelines recommend to treat hypertension in type 2 diabetes to an systolic BP target of 130–140 mmHg and a diastolic BP target of 80 mmHg and to consider the lower targets if the patients are younger or when additional CV risk factors or microvascular diseases are present [21]. In the studies, the number of antihypertensive drugs required to achieve the systolic blood pressure around 130–140 mmHg are often more than two and even more than three in many patients. Treatment of hypertension in patients with diabetes is

**Public health perspective:** In non-industrialized countries, there is urgent need of establishing the network of the State supported rural and particularly urban health centers/clinics with general practitioners (preferably well trained with residential training in General Practice after medical graduation). It is required (i) for the comprehensive longitudinal health care of the people with diabetes, hypertension, CVD and other conditions, (ii) for supervision of healthcare workers to implement various public health programs including that for hypertension and CVDs and (iii) to provide the State supported diagnostic facilities and antidiabetic, antihypertensive and other CVD drugs and antibiotics (then only it may be possible to restrict the over-the-counter sale of antibiotics and other drugs as the

**Research perspective:** Study of the occurrence of CVD complications in the people with pre-hypertension level of BP and impaired glucose intolerance may indicate the different levels of HbA1c and BP to start antihypertensive treatment. The combined presence of the subclinical or borderline hypertension and glucose intolerance (as 'subclinical or borderline syndrome') may be another CVD risk factor, as a residual risk factor of CVDs, at the population level.

The health effects of tobacco use, including on CVD, and various public health measures to control it are well known [2, 10]. Harmful tobacco products also include smokeless tobacco

**Clinical management perspective:** The five A's framework (Ask, Advise, Assess, Assist, Arrange) has been developed to allow physicians to incorporate smoking cessation counseling into busy clinical practices [23]. Others have further added 'Assess again'. There are various pharmacological agents available to help patients quit smoking and tobacco use. However, before such products were available and even now hundreds of thousands of people including health professionals have had stopped tobacco use once they realized the possible complications to them. Physicians' priority, repeated advice and time spent on explaining its importance and its process of quitting play a crucial role for the motivation of the patients to quit tobacco use.  However, the most important wellknown point, to emphasize to the patients repeatedly, is that after stopping tobacco use whether abruptly or slowly,

Exercise is often classified as aerobic, strength (resistance) and flexibility exercise and each of them has their own utility and limitations. The common recommendation (e.g. 150 minutes

like snuff, gutkha, gul, chimo, mawa, nass, pan masala, tambaku and others [22].

and more easy than lowering glucose [20].

180 Diabetes and Its Complications

**3. Promoting cessation of smoking and tobacco use**

people otherwise would not have any access to such life-saving drugs) [15].

lucidly reviewed in the position paper of American Society of Hypertension [12].

**4. Regular physical activity and exercise**

it should not be used even once; otherwise the habit is likely to be resumed.

**Figure 2.** Potential benefits of regular physical activity and exercise in relation to diabetes.

**Research perspective:** Although type 2 diabetic subjects are insulin resistant, they are not resistant to the stimulatory effects of exercise on glucose utilization [30]. The local cellular and other metabolic adaptations could explain the increase in glucose utilization and improvement in the glucose tolerance in exercise and trained muscles. A major challenge has been to elucidate the molecules or cascade of molecules that act through insulin-independent, exercise-stimulated signaling pathway [30]. If such molecules produced during exercise are secreted in the blood and act like hormones in other tissues, organs, muscles and fat, study of the effect in the resting state by the reuse of serum of blood collected during exercise in the same person may give insight into such actions and the difference in the contents of resting and post-exercise sera may help to elucidate the molecules.

**Research perspective:** Many overweight older people with diabetes have knee osteoarthritis which may prevent them to walk freely. Exercises to strengthen the quadriceps—for example, quadriceps-setting exercise and straightleg raises—are effective in reducing pain and improving function in patients with knee osteoarthritis. However, people often may not adhere to the recommended methods of quadriceps strengthening exercises. Regular exercise with a bicycle ergometer in such patients can have beneficial effects not only for knee osteoarthritis but also for diabetes and CVD [31].


**4. The use of -omics technologies to improve nutritional bioavailability**

Omics is a multidisciplinary study that refers to studies in applied biology that end with -omics, including but not limited to genomics, transcriptomics, proteomics, metabolomics, phenomics, epigenomics, nutrigenomics, vaccinomics, metagenomics and various others. These studies are mainly conducted through the application of several high-throughput technologies that mainly encompass qualitative and/or quantitative detection of novel or known genes (nucleotides), mRNA transcripts or transcription factors, proteins, metabolites and other parameters through genomics, transcriptomics, proteomics and metabolomics, respectively [87, 88].

The molecular data obtained through high-throughput technological applications are quite intense, comprehensive and may be complex in other instances. This could make the integration of omics data quite a challenging task if the experimental analyses were not designed to contribute to downstream data analyses. The integration of omics data would bring about a comprehensive overview of data on various biological variables, thereby allowing researchers to have a comprehensive manner in which they could study relationships among biological variables within a biological system. Thus, it would be possible for researchers to even predict the quantitative and qualitative effect an introduction or deduction that a selected element or compound could have in the gene network or pathway within a biological system. For example, it would be possible to predict what effect could high concentrations of Ca have on the phenotype or the production of a selected phenolic acid in a selected grain compartment (endosperm) [89]. The integration of these technologies would enable researchers to determine which gene region could be targeted to improve the levels of a desirable metabolite without affecting other biological systems. We would then be able to know what is the optimum level of fertilizer required to drive the translocation of minerals into the wheat grain, at which optimum growth stage, etc.

Biological systems are complex in that there are many biological variables that encompass biological processes, thus, making integrative analyses through omics approaches a major challenge due to several technological limits associated with analysing biological processes that entail a large number of variables [90]. Thus, there is still a great need for researchers to form consortiums aimed at integrating research efforts that contribute towards integrating the data obtained from the application of omics technologies to have an integrated biological system that will allow easier manipulation of data. Integrating multiple omics data is still a major challenge in that there are several computational issues associated with integrating a series of multilayer datasets [91, 92].

Phenomics involve the use of high-throughput non-invasive colour imaging, near infrared imaging, far infrared and fluorescence imaging technologies, which are capable of acquiring several physical traits such as the plant structure, phenology, soil water content, canopy/ leaf temperature, physiological state of the photosynthetic machinery as well as automated weighing and water efficiency usage measurement [93]. These technologies have the capability to provide solutions to genomics-enabled crop improvement through the high-throughput platforms that can be integrated with genomics-based platforms [93].