Science, Irving K. Barber Faculty of (Okanagan)
Non UBC
Computer Science, Mathematics, Physics and Statistics, Department of (Okanagan)
DSpace
Molecules 29 (11): 2438 (2024)
Jackson, Nolan
Cecchi, Daniel
Beckham, Wayne
Chithrani, Devika
Radiotherapy is an essential component of the treatment regimens for many cancer patients. Despite recent technological advancements to improve dose delivery techniques, the dose escalation required to enhance tumor control is limited due to the inevitable toxicity to the surrounding healthy tissue. Therefore, the local enhancement of dosing in tumor sites can provide the necessary means to improve the treatment modality. In recent years, the emergence of nanotechnology has facilitated a unique opportunity to increase the efficacy of radiotherapy treatment. The application of high-atomic-number (Z) nanoparticles (NPs) can augment the effects of radiotherapy by increasing the sensitivity of cells to radiation. High-Z NPs can inherently act as radiosensitizers as well as serve as targeted delivery vehicles for radiosensitizing agents. In this work, the therapeutic benefits of high-Z NPs as radiosensitizers, such as their tumor-targeting capabilities and their mechanisms of sensitization, are discussed. Preclinical data supporting their application in radiotherapy treatment as well as the status of their clinical translation will be presented.
Citation: Jackson, N.; Cecchi, D.;Beckham, W.; Chithrani, D.B.Application of High-Z Nanoparticlesto Enhance Current RadiotherapyTreatment. Molecules 2024, 29, 2438.https://doi.org/10.3390/molecules29112438Academic Editor: Josef JampilekReceived: 29 April 2024Revised: 19 May 2024Accepted: 20 May 2024Published: 22 May 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).moleculesReviewApplication of High-Z Nanoparticles to Enhance CurrentRadiotherapy TreatmentNolan Jackson 1, Daniel Cecchi 1, Wayne Beckham 1,2 and Devika B. Chithrani 1,3,4,5,*1 Department of Physics and Astronomy, University of Victoria, Victoria, BC V8P 5C2, Canada2 British Columbia Cancer-Victoria, Victoria, BC V8R 6V5, Canada3 Centre for Advanced Materials and Related Technologies, Department of Chemistry, University of Victoria,Victoria, BC V8P 5C2, Canada4 Division of Medical Sciences, University of Victoria, Victoria, BC V8P 5C2, Canada5 Department of Computer Science, Mathematics, Physics and Statistics, Okanagan Campus,University of British Columbia, Kelowna, BC V1V 1V7, Canada* Correspondence: devikac@uvic.caAbstract: Radiotherapy is an essential component of the treatment regimens for many cancer patients.Despite recent technological advancements to improve dose delivery techniques, the dose escalationrequired to enhance tumor control is limited due to the inevitable toxicity to the surrounding healthytissue. Therefore, the local enhancement of dosing in tumor sites can provide the necessary means toimprove the treatment modality. In recent years, the emergence of nanotechnology has facilitateda unique opportunity to increase the efficacy of radiotherapy treatment. The application of high-atomic-number (Z) nanoparticles (NPs) can augment the effects of radiotherapy by increasing thesensitivity of cells to radiation. High-Z NPs can inherently act as radiosensitizers as well as serveas targeted delivery vehicles for radiosensitizing agents. In this work, the therapeutic benefits ofhigh-Z NPs as radiosensitizers, such as their tumor-targeting capabilities and their mechanisms ofsensitization, are discussed. Preclinical data supporting their application in radiotherapy treatmentas well as the status of their clinical translation will be presented.Keywords: nanoparticle; radiotherapy; radiosensitizer; cancer; chemotherapy; therapeutics1. IntroductionAlthough massive strides in cancer therapeutics have been made over the years, cancerremains one of the leading causes of death worldwide. This has prompted continuousresearch to advance therapeutic options for this life-threatening disease, causing the land-scape of cancer treatment to constantly evolve to improve patient outcomes. As a result,modern therapeutic cancer treatments, such as immunotherapy, targeted therapy, photody-namic therapy, etc., have emerged recently. However, the three main cornerstone treatmentoptions, radiotherapy, surgery, and chemotherapy, still maintain their indispensable rolein the management of patients’ diseases, continually benefitting from new medical andtechnological advancements to enhance the efficacy of each of the treatment options.Currently, radiotherapy is a pivotal treatment modality used in the management ofcancer. It is suggested that approximately 50% of cancer patients benefit from receivingradiotherapy, either as an independent treatment option or used in conjunction withsurgery and/or chemotherapy, making it an essential component of treatment regimensfor many patients [1]. In radiotherapy, ionizing radiation, such as high-energy X-rays,electrons, protons, neutrons, or light ions, transverse through a patient to locally target anddamage cancer cells, with each type of ionizing radiation varying in its biological effects [2].This offers a strategic advantage in treating deep-seated tumors that would otherwise bechallenging to treat via conventional surgical methods.Molecules 2024, 29, 2438. https://doi.org/10.3390/molecules29112438 https://www.mdpi.com/journal/moleculesMolecules 2024, 29, 2438 2 of 23While radiotherapy has proven to be invaluable due to its precision in targeting andkilling tumor cells, it also presents some challenges. A major limitation in the overallefficacy of the treatment stems from the inevitable normal tissue toxicity that is induced.Despite radiotherapy being a highly localized, the ionizing radiation transverses throughthe patient to reach the target, so a portion of the dose is deposited in healthy tissue. Thiscan lead to adverse effects and prevents the dose escalation that is required to enhancetumor control. Addressing this delicate balance between tumor control and the sparingof healthy tissue is an important problem in radiotherapy, which has led to advancedtreatment delivery techniques, such as intensity-modulated radiotherapy (IMRT) andvolumetric-modulated arc therapy (VMAT), that enable the delivery of highly conformaldose distributions, minimizing the dose delivered to normal tissues [3,4]. However, anothertherapeutic avenue that is currently being pursued to increase the efficacy of radiotherapyis the incorporation of radiosensitizers to radiotherapy treatment. By supplementing tumorsites with radiosensitizers, the radiation response of tumors can be enhanced, increasingthe efficacy of the modality.In general, radiosensitizers are agents that augment the sensitivity of cells to radiation.Their integration to increase the susceptibility of tumor cells to radiation-induced damagecan help to mitigate challenges inherent to radiotherapy treatment, such as normal tissuetoxicity and tumor radiation resistance [5]. For example, the tumor microenvironmentcan become hypoxic, increasing the resistance of tumors to radiotherapy treatment [6].As a result, specific molecular agents have been developed that can elevate the oxygenlevels within the tumor microenvironment, offsetting hypoxia and thereby increasingthe sensitivity of tumors to radiation [7]. Furthermore, other radiosensitizing agents aimto target and inhibit certain cellular mechanisms that render cancer cells more prone toradiation damage, such as DNA repair pathways [8]. One of the primary concerns withradiosensitizers is the lack of specificity of some agents, which can inadvertently increasethe toxicity in healthy tissues, creating a demand for novel radiosensitizers that can offergreater specificity, and in recent years, nanoparticles have emerged as a promising solution.Due to the rapid advancements and developments in nanotechnology, the applicationof nanoparticles in cancer therapeutics has garnered much interest, offering innovativeapproaches to improve different aspects of cancer treatment. Nanoparticles (NPs), charac-terized by their size (typically ranging from 1 to 100 nanometres), possess unique physicaland chemical properties at the nanoscale that allow them to be engineered and tailored forspecific applications [9]. One of these applications is their use as radiosensitizers. Varioushigh-atomic-number (Z) NPs have shown promising radiosensitizing effects and can befunctionalized in ways such that they preferentially target tumor cells in comparison withnormal tissue [10]. Through various mechanisms of radiosensitization, introducing high-ZNPs into the tumor environment can locally enhance the therapeutic effects of radiotherapy(Figure 1) without introducing additional normal toxicity. Furthermore, high-Z NPs canserve as a targeted drug delivery system for drug-based radiosensitizing agents, serving asa bi-functional radiosensitizing NP. Currently, the spectrum of high-Z NPs that are beinginvestigated for this purpose are, but not limited to, gold (Z = 79), silver (Z = 47), bismuth(Z = 83), gadolinium (Z = 64), and hafnium (Z = 72).This article will encompass the therapeutic benefits of implementing high-Z NPsinto radiotherapy, such as their targeting capabilities and mechanisms of sensitization.The preclinical clinical data supporting their use and the current clinical status of theirapplication in radiotherapy treatment will also be discussed.Molecules 2024, 29, 2438 3 of 23Molecules 2024, 29, x FOR PEER REVIEW 3 of 23 Figure 1. Idea for using radiosensitizing high-Z NPs in radiotherapy treatment regimens. Prior to radiotherapy, high-Z NPs can be administered to the patient and allowed to accumulate in tumor tissue. This can increase the tumor’s sensitivity to radiotherapy, leading to enhanced tumor control for patients. (Created using Biorender.com). This article will encompass the therapeutic benefits of implementing high-Z NPs into radiotherapy, such as their targeting capabilities and mechanisms of sensitization. The preclinical clinical data supporting their use and the current clinical status of their appli-cation in radiotherapy treatment will also be discussed. 2. Tumor-Targeting Capabilities of Nanoparticles The tumor-targeting capabilities of NPs are instrumental in their role as promising radiosensitizers. When exploring new strategies to improve radiotherapy, enhancing dose delivery to tumor sites is critical to increase the treatment’s therapeutic index. NPs possess this ability through their proficient tumor targeting capabilities, thereby increasing the local dose deposition in tumors relative to normal tissue. The ability to modify the size and surface characteristics of NPs has enabled the abil-ity to tailor them to preferentially accumulate within tumor tissue over normal tissues, leveraging both passive and active targeting mechanisms. In passive targeting, due to their small size, NPs can preferentially accumulate in tumor tissue via the enhanced per-meability and retention (EPR) effect [11]. Conversely, active targeting uses the precision of molecular recognition, where the strategic functionalization of NPs with specific tar-geting for ligands or molecules allows them to selectively bind to cancer cell-specific markers [12]. By utilizing these two targeting strategies, NPs can be optimized to increase their therapeutic impact as radiosensitizers. 2.1. Passive Targeting The premise of the passive targeting of NPs within tumor tissue is due to the EPR effect. The EPR effect is a phenomenon in which small molecules and particles tend to preferentially accumulate within tumor sites compared with normal tissue. This selective accumulation stems from the chaotic nature of the vasculature system present within the tumor microenvironment that forms as a result of angiogenesis, a hallmark of cancer. As tumors begin to rapidly proliferate, it necessitates the formation of new blood vessels to support the growth of the tumor. However, the neovasculature system often has structural abnormalities, consisting of improper endothelial junctions that permit the seepage of small molecules into tumors [13]. Additionally, the poor lymphatic drainage and slow ve-nous return characterized in tumors impede the removal of these small molecules, pro-moting their retention [14,15]. This facilitates an increase in the accumulation of small Figure 1. Idea for using radiosensitizing high-Z NPs in radiotherapy treatment regimens. Prior toradiotherapy, high-Z Ps can be ad inistered to the patient and allo ed to accu ulate in tu orti sue. This can increase the tumor’s sensitivity to radiotherapy, leading to enhanced tumor controlfor patients. (Created using Biorender.co ).2. Tumor-Targeting Capabilities of NanoparticlesThe tumor-targeting capabil ties of NPs are instrumental in their role as pr misingradiosensitizers. When ex loring new strat gies to imp ove radiotherapy, enhancing dosedelivery to tumor sites is critical to ncrease the treatment’s therapeutic index. NPs possessthis ability through their proficient tumor targeting capabilities, thereby increasing the localdose deposition in tumors relative to norm l tissue.The ability to modify the size and surface characteristics of NPs has enabled theability to tailor them to preferentially accumulate within tumor tissue over normal tissues,leveraging both passive and active targeting mechanisms. In passive targeting, due to theirsmall size, NPs can preferentially accumulate in tumor tissue via the enhanced permeabilityand retention (EPR) effect [11]. Conversely, active targeting uses the precision of molecularrecognition, where the strategic functionalization of NPs with specific targeting for ligandsor molecules allows them to selectively bind to cancer cell-specific markers [12]. By utilizingthese two targeting strategies, NPs can be optimized to increase their therapeutic impactas radiosensitizers.2.1. Passive TargetingThe premise of the passive targeting of NPs within tumor tissue is due to the EPReffect. The EPR effect is a phenomenon in which small molecules and particles tend topreferentially accumulate within tumor sites compared with normal tissue. This selectiveaccumulation stems from the chaotic nature of the vasculature system present within thetumor microenvironment that forms as a result of angiogenesis, a hallmark of cancer. Astumors begin to rapidly proliferate, it necessitates the formation of new blood vessels tosupport the growth of the tumor. However, the neovasculature system often has structuralabnormalities, consisting of improper endothelial junctions that permit the seepage of smallmolecules into tu ors [13]. Additionally, the poor lymphatic drainage and slow venousret rn characterized in tumors impede the removal of these small olecules, promotingtheir retention [14,15]. This f cilitates n increase in the accumulation of small molecules intu or tissue c mpared with normal tissue, in which the normalized vasculatur ystemdoes no exhibit ese same characteristics (Figure 2). Therefore, the dimi utive nature ofNPs akes them ideal candidates to exploit is phenomenon to enhance their d livery totumor sit s.Molecules 2024, 29, 2438 4 of 23Molecules 2024, 29, x FOR PEER REVIEW 4 of 23 molecules in tumor tissue compared with normal tissue, in which the normalized vascu-lature system does not exhibit these same characteristics (Figure 2). Therefore, the dimin-utive nature of NPs makes them ideal candidates to exploit this phenomenon to enhance their delivery to tumor sites. Figure 2. Passive targeting of NPs. (A) The poor structural integrity and lymphatic drainage of the tumor vasculature system result in the passive preferential accumulation of NPs in tumors com-pared with normal tissue. This is referred to as the EPR effect. (Created using Biorender.com). (B) Hyperspectral imaging demonstrating blood vessels facilitating the delivery of GNPs to the sur-rounding mice tumor tissue. Reproduced with permission from open access Creative Common li-cense [16]. In order to take full advantage of the EPR effect, prolonging the blood circulation time of NPs to allow them adequate time to reach tumors is a critical aspect that needs to be considered. This is commonly achieved through the addition of ‘stealth’ molecules, like polyethylene glycol (PEG), to the surface of NPs, which enables them to evade clearance from the reticuloendothelial system, preventing opsonization and subsequent phagocyto-sis [17,18]. As NPs enter the bloodstream, opsonin molecules can adsorb onto the surface of the NP, effectively tagging it for clearance via phagocytosis. The PEGylation of NPs offers a protective hydrating layer that minimizes protein corona adsorption, thus reduc-ing their recognition by phagocytes and effectively extending their presence within the bloodstream. Unfortunately, this has also been shown to alter the NP uptake efficiency of cancer cells. Studies have reported that the addition of PEG to gold NPs (AuNPs/GNPs) resulted in a significant decrease in cellular uptake, which is attributed to the PEG moiety interfering with NPs from successfully interacting with and binding to tumor cell recep-tors [19]. To remediate this, further surface modification of NPs is necessary to promote tumor targeting, which can be achieved through active targeting mechanisms. 2.2. Active Targeting The active targeting capability of NPs is a key feature that has propelled the applica-tion of NPs as radiosensitizers. Unlike passive targeting, which relies on the pathophysi-ological properties of tumor tissue, active targeting aims to strategically functionalize NPs with specific targeting moieties that are selectively recognized by tumor cells. This strat-egy aims to exploit the overexpression of certain surface receptors that are exhibited by cancer cells, where the overexpressed surface receptors can be targeted by NPs conjugated with corresponding ligands that results in the preferential attachment and subsequent cel-lular internalization via receptor-mediated endocytosis (Figure 3). Examples of such tar-geting ligands that are being investigated include peptides, aptamers, antibodies, and sugar molecules. The following section will provide examples of targeting strategies that are currently being used. Figure 2. Passive targeting of NPs. (A) The poor structural integrity and lymphatic drainage ofthe tumor vasculature system result in the passive preferential accumulation of NPs in tumorscompared with normal tissue. This is referred to as the EPR effect. (Created using Biorender.com).(B) Hyperspectral imaging demonstrating blood vessels facilitating the delivery of GNPs to thesurrounding mice tumor tissue. Reproduced with permission from open access Creative Commonlicense [16].In order to take full advantage of the EPR effect, prolonging the blood circulation timeof NPs to allow them adequate time to reach tumors is a critical aspect that needs to beconsidered. This is commonly achieved through the addition of ‘stealth’ molecules, likepolyethylene glycol (PEG), to the surface of NPs, which enables them to evade clearancefr m the r ticuloendothelial system, preventing opsonizat on a d subsequent phagocyto-sis [17,18]. As NPs e ter th blood tream, opso in molecules can adsorb onto the surface ofthe NP, effectively tagging it for clearance via phagocytosis. The PEGylation of NPs offersa protective hydrating layer that minimizes protein corona adsorption, thus reducing theirrecognition by phagocytes and effectively extending their presence within the bloodstream.Unfortunately, this has also been shown to alter the NP uptake efficiency of cancer cells.Studies have reported that the addition of PEG to gold NPs (AuNPs/GNPs) resulted in asignificant decrease in cellular uptake, which is attributed to the PEG moiety interferingwith NPs from successfully interacting with and binding to tumor cell receptors [19]. Tomediate this, further surface modification of NPs is necessary to promote tumor targeting,which ca be ac ieved through a tive targeting mechanisms.2.2. Active TargetingThe active targeting capability of NPs is a key feature that has propelled the applicationof NPs as radiosensitizers. Unlike passive targeting, which relies on the pathophysiologicalproperties of tumor tissue, active targeting aims to strategically functionalize NPs withspecific targeting moieties that are selectively recognized by tumor cells. This strategyaims to exploit the overexpression of certain surface receptors that are exhibited by cancercells, where th overexpressed surface receptors can be targeted by NPs onjugated withcorresponding ligands that results in the preferential attachment and subsequent cellularinternalization via receptor-mediated endocytosis (Figure 3). Examples of such targetingligands that are being investigated include peptides, aptamers, antibodies, and sugarmolecules. The following section will provide examples of targeting strategies that arecurrently being used.Molecules 2024, 29, 2438 5 of 23Molecules 2024, 29, x FOR PEER REVIEW 5 of 23 Figure 3. Active targeting of NPs. (A) Many cancer cells overexpress various surface receptors in comparison with normal cells. The surface of NPs can be strategically modified with corresponding ligands to specifically bind to these receptors, increasing their uptake in cancer cells. (Created using Biorender.com). (B) Accumulation of cancer targeting GNPs in cancer cells versus normal fibro-blasts. Cancer cells demonstrate greater accumulation of GNPs compared with normal fibroblasts due to their overexpression of targeted receptors. Top panel: hyperspectral imaging of GNPs. Bot-tom panel: confocal imaging of GNPs (red) and microtubules (green). Reproduced with permission from open access Creative Common license [20]. 2.2.1. Antibody-Coated NPs Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine ki-nase receptor that regulates cell growth and differentiation, with many breast cancer pa-tients (20–30%) presenting an amplified expression of the protein [21]. It has also been identified to be a suitable marker for poor prognosis, where the inhibition of HER2 has been shown to increase the sensitivity of radioresistant breast cancer cells to radiotherapy [22]. HER2 has been extensively studied as a therapeutic target, and an FDA-approved monoclonal antibody, Trastuzumab, has been shown to be effective in treating patients with HER2-overexpressing breast cancer [23]. Trastuzumab selectively binds to HER2 re-ceptors, preventing them from receiving growth signals and thereby reducing cell prolif-eration. This selectivity of Trastuzumab has led researchers to conjugate the antibody to NPs to enhance their targeting effects towards cancer cells overexpressing HER2. Using trastuzumab as a targeting antibody, Cai et al. engineered a AuNP platform to enhance the targeted delivery of 111In, a radionuclide emitting low-energy (<25 keV) Auger elec-trons, in addition to its γ-emissions (Eγ = 171 keV (90%) and 245 keV (94%)) [24]. Their results showed that Trastuzumab-functionalized AuNPs were more efficiently internal-ized by HER2-positive cell lines, SK-BR-3 and MDA-MB-361, compared with their unfunc-tionalized counterparts. Furthermore, this targeted therapeutic strategy was proven to be effective in vivo, where the absorbed dose deposited in tumor tissue was 60.5 Gy com-pared with normal organs that received <0.9 Gy. More recently, affibodies that target HER2 have been developed and tested as targeting molecules. Affibody molecules are small proteins that can selectively bind to target proteins, essentially mimicking monoclo-nal antibodies. However, affibodies are much smaller, have a lower cost of production compared with monoclonal antibodies, and have high affinity and high tolerance to chem-icals, higher temperatures, and extreme pH values [25]. A study conducted by Pour-shohod et al. demonstrated their effectiveness, where AuNPs functionalized with an af-fibody that recognizes HER2 (ZHER2) increased the sensitivity of HER2-positive cell lines to radiation compared with non-functionalized AuNPs [26]. They further corroborated these findings in another study that demonstrated enhanced sensitization due to silver NPs (AgNPs) functionalized with the ZHER2 affibody [27]. Figure 3. Active targeting of NPs. (A) Many cancer cells overexpress various surface receptors incomparison with normal cells. The surface of NPs can be strategically modified with correspondingligands to specifically bind to these receptors, increasing their uptake in cancer cells. (Created usingBiorender.com). (B) Accumulation of cancer targeting GNPs in cancer cells versus normal fibroblasts.Cancer cells demonstrate greater accumulation of GNPs compared with normal fibroblasts due totheir overexpression of targeted receptors. Top panel: hyperspectral imaging of GNPs. Bottom panel:confocal imaging of GNPs (red) and microtubules (green). Reproduced with permission from openaccess Creative Common license [20].2.2.1. Antibody-Coated NPsHuman epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinasereceptor that regulates cell growth and differentiation, with many breast cancer patients(20–30%) presenting an amplified expression of the protein [21]. It has also been identi-fied to be a suitable marker for poor prognosis, where the inhibition of HER2 has beenshown to increase the sensitivity of radioresistant breast cancer cells to radiotherapy [22].HER2 has en extensively studied as a therapeutic target, and an FDA-approved mon-oclonal antibody, Trastuzumab, has been shown to be effective in treating patients withHER2-overexpressing breast cancer [23]. Trastuzumab selectively binds to HER2 receptors,preventing them from receiving growth signals and thereby reducing cell proliferation. Thisselectivity of Trastuzumab has led researchers to conjugate the antibody to NPs to enhancetheir targeting effects towards cancer cells overexpressing HER2. Using trastuzumab as atargeting antibody, Cai et al. engine red a AuNP platform to enhance the targ ted deliveryof 111In, a radionuclide emitting low-energy (<25 keV) Auger electro s, in addition toits γ-emissions (Eγ = 171 keV (90%) and 245 keV (94%)) [24]. Their results showed thatTrastuzumab-functionalized AuNPs were more efficiently internalized by HER2-positivecell lines, SK-BR-3 and MDA-MB-361, compared with their unfunctionalized counterparts.Furthermore, this targeted therapeutic strategy was proven to be effective in vivo, wherethe absorbed dose deposited in tumor tissue was 60.5 Gy compared with normal organsthat receive <0.9 Gy. More recently, affibodies that target HER2 have been developedand tested as targeting molecules. Affibody molecules are small proteins that can selec-tively bind to target proteins, essentially mimicking monoclonal antibodies. However,affibodies are much smaller, have a lower cost of production compared with monoclonalantibodies, and have high affinity and high tolerance to chemicals, higher temperatures,and extreme pH values [25]. A study conducted by Pourshohod et al. demonstratedtheir effectiveness, where AuNPs functionalized with an affibody that recognizes HER2(ZHER2) creased the s nsi ity of HER2-positive cell lines to radiation compared withnon-functionalized AuNPs [26]. They further corroborated these findings in another studyMolecules 2024, 29, 2438 6 of 23that demonstrated enhanced sensitization due to silver NPs (AgNPs) functionalized withthe ZHER2 affibody [27].Similar to HER2, CXC chemokine receptor 4 (CXCR4) is commonly overexpressed bymost breast cancers, making it an attractive targeting option for the treatment of breastcancers. One of the benefits of targeting CXCR4 is that this receptor is still expressedby triple-negative breast cancer, characterized by the absence of estrogen receptors (ER),progesterone receptors (PR), and HER2. Bhattarai et al. highlighted the prospect of targetingCXCR4 to enhance the efficacy of GNPs as radiosensitizers [28]. In their study, PEGylatedGNPs were targeted towards this receptor via conjugation to an anti-CXR4 antibody.Dark-field images confirmed that the functionalized anti-CXCR4 GNPs (cGNP) resultedin enhanced uptake by cells compared with the PEGylated GNPs (pGNP) (Figure 4A).Furthermore, when combined with radiotherapy, it was found that the efficacy of thetreatment strategy was dependent on the CXCR4 expression of various cell lines, displayingthe dependence of receptor expression for targeting and treatment efficacy.2.2.2. Folate-Coated NPsFolic acid or folate is an essential molecule for various crucial cellular processes, suchas DNA synthesis and repair, RNA synthesis, amino acid metabolism, and phospholipidbiosynthesis, making it an effective targeting ligand for the application of targeted cancertherapeutics. Due to the rapid division and proliferation of cancer cells, they often havean increased demand for compounds required for DNA synthesis. This results in theupregulation of folate receptors on the surface of cancer cells in attempts to receive anincreased supply of folate from the surrounding environment to meet the high demandnecessary to support the heightened growth rate of tumors. Therefore, folic acid is aprevalent targeting molecule to enhance tumor selectivity, and it has been used for NPtargeted applications, facilitated by its favorable conjugation chemistry to the surface ofNPs [29]. A study conducted by Daniele et al. investigated the targeting efficiency offolic acid, comparing the cellular internalization of PEGylated AuNPs functionalized withfolic acid (FA AuNPs) versus non-functionalized PEGylated AuNPs [30]. Their resultsdisplayed that the accumulation of FA AuNPs was significantly increased in folate receptor-overexpressing KB cells compared with their unfunctionalized counterparts, with theincrease being dependent on the AuNP surface density of folate (Figure 4B). However, theinternalization of AuNPs was significantly less in MCF-7 cells that exhibit low expressionof folate receptors, highlighting the selective targeting of folate-functionalized NPs. Thesetargeting capabilities of folic acid were further demonstrated by Kefayat et al. using a C6glioma tumor model [31]. Folic acid and BSA-decorated gold nanoclusters (FA-AuNCs)were shown to have 2.5 times higher cellular accumulation in C6 cancer cells compared withL929 normal cells, effectively displaying the preferential selectivity towards cancer cells.This was shown to translate in vivo with a higher concentration of FA-AuNCs localizedin brain tumors compared with the surrounding normal tissue. When combined with aradiation dose of 6 Gy, the median survival of tumor-bearing mice increased from 18 days(radiotherapy alone) to 24.5 days (radiotherapy + FA-AuNPs), indicating the applicabilityof folic acid-coated NPs as promising radiosensitizers.Molecules 2024, 29, 2438 7 of 23Molecules 2024, 29, x FOR PEER REVIEW 7 of 23 Figure 4. Enhanced cellular uptake of nanoparticles via targeting moieties. (A) Dark-field imaging of gold nanoparticles (yellow color) in MDA-MB-231 cells incubated with PBS (control), anti-CXCR4 functionalized GNPs (cGNP), or PEGylated GNPs (pGNP). Cell nuclei are stained in blue. (B) Cell uptake of rhodium-labeled folate functionalized AuNPs (Rho-FAx-AuNPs) by KB (overexpression of folate receptors) and MCF-7 (low expression of folate receptors) cells. AuNPs were functionalized with various surface concentrations of folate. Statistical significance was calculated versus non-tar-geted particles (Rho-Fa0 -AuNPs): **** p < 0.0001, ns nonsignificant. (C) Effects of PEG and RGD on the cellular accumulation of GNPs. Reproduced with permission from open access Creative Com-mon license [16,28,30]. 2.2.3. Peptide-Capped NPs Commonly used targeting ligands are peptides containing the arginine–glycine–as-partic acid (RGD) sequence. RGD exhibits a high affinity for the integrin receptor ανβ3, an integrin receptor that is highly overexpressed by many solid tumors. The ability of RGD to effectively target various forms of cancer has made it a versatile tool in oncological ap-plications, which has extended to its application in NP-mediated therapeutics. Highlight-ing this, research by Cruje et al. demonstrated that PEGylated GNPs functionalized with RGD displayed enhanced GNP uptake by MDA-MB-231 breast cancer cells compared with unfunctionalized PEGylated GNPs [32]. Interestingly, this effect was only realized for GNPs with a diameter of 14 nm, but not for larger GNPs with a diameter of 74 nm. This was attributed to the smaller NPs having an increased surface curvature that aided the RGD peptide in accessing the integrin receptors on the surface of the cells, suggesting that parameters such as NP size must be taken into consideration when functionalizing NPs for targeting purposes. Additionally, Yang et al. found similar results in MIA-PaCa-2 cells, with RGD significantly enhancing the cellular uptake of GNPs, where an increase in RGD density resulted in a greater accumulation of GNPs (Figure 4C) [16]. This correla-tion between RGD density and cell accumulation also extended to in vivo environments, with a greater density yielding enhanced tumor accumulation and retention, demonstrat-ing the tumor-targeting abilities of RGD-functionalized NPs. Figure 4. Enhanced cellular uptake of nanoparticles via t rgeting moiet s. (A) Dark-field imagin ofgold nanoparticles (yellow color) in MDA-MB-231 cells incubated with PBS (control), anti-CXCR4functionalized GNPs (cGNP), or PEGylated GNPs (pGNP). Cell nuclei are stained in blue. (B) Celluptake of rhodium-labeled folate functionalized AuNPs (Rho-FAx-AuNPs) by KB (overexpression offolate receptors) and MCF-7 (low expression of folate receptors) cells. AuNPs were functionalizedwith various surface concentrations of folate. Statistical significance was calculated versus non-targeted particles (Rho-Fa0 -AuNPs): **** p < 0.0001, ns nonsignificant. (C) Effects of PEG and RGDon the cellular accumulation of GNPs. Reproduced with permission from open access CreativeCommon license [16,28,30].2.2.3. Peptide-Capped NPsCommonly u d targ ting ligands are peptides containing the arginine–glycine–aspartic acid (RGD) sequence. RGD exhibit a high affinity for the integrin receptor ανβ3,an integrin receptor that is highly overexpressed by many solid tumors. The ability ofRGD to effectively target various forms of cancer has made it a versatile tool in oncologicalapplications, which has extended to its application in NP-mediated therapeutics. High-lighting this, research by Cruje et al. demonstrated that PEGylated GNPs functionalizedwith RGD displayed enhanced GNP uptake by MDA-MB-231 breast cancer cells comparedwith unfunctionalized PEGylated GNPs [32]. Interestingly, this effect was only realizedfor GNPs with a diameter of 14 nm, but not for larger GNPs with a diameter of 74 nm.This was attributed to the smaller NPs having an increased surface curvature that aidedthe RGD peptide in accessing the integrin receptors on the surface of the cells, suggestingthat parameters such as NP size must be taken into consideration when functionalizingNPs for targeting purposes. Additionally, Yang et al. found similar results in MIA-PaCa-2cells, with RGD significantly enhancing the cellular uptake of GNPs, where an increase inRGD density resulted in a greater accumulation of GNPs (Figure 4C) [16]. This correlationbetween RGD density and cell accumulation also extended to in vivo environments, with aMolecules 2024, 29, 2438 8 of 23greater density yielding enhanced tumor accumulation and retention, demonstrating thetumor-targeting abilities of RGD-functionalized NPs.3. Mechanisms of Radiosensitization3.1. Physical Mechanism of NP-Based RadiosensitizationThe deposition of a dose of ionizing radiation is facilitated through the transfer of itsenergy to absorbing materials, and in the case of ionizing photons, this transfer of energyprimarily occurs via ionizing interactions. The primary ionizing interactions throughwhich incident photons in the keV to low MeV range lose energy are the Compton andphotoelectric effects. In the Compton effect, incident photons are scattered due to collisionswith weakly bound electrons, transferring a portion of their energy that results in theelectron being ejected from the atom. These photons, which retain most of their energy, havelong ranges, leading to a sparse distribution of ionizing events. In the case of photoelectricinteractions, electrons from the inner atomic orbitals absorb the incident photons, causingthe ejection of these inner-shell electrons. The resultant inner shell vacancy is subsequentlyfilled by an outer shell electron, thereby releasing energy that causes the emission of low-energy fluorescent photons and a cascade of secondary electrons known as Auger electrons.These Auger electrons are low energy, depositing their energy in close proximity to theabsorbing material with an ionizing track ranging up to a few micrometers, resulting inhighly localized ionizing events and enhancing local dose deposition [33].The physical mechanism of NP-mediated radiosensitization primarily hinges on thedifference in energy absorption properties between soft tissue (Z~7.3) and high-Z NPs,arising from the difference in atomic number between the two absorbing materials. Whilethe X-ray cross-section (probability of interaction) for the Compton effect is largely inde-pendent of the atomic number of the absorbing material, the X-ray cross-section for thephotoelectric effect is approximately proportional to Z3–Z4. Consequently, this leads to anincrease in ionizing interactions, particularly the photoelectric effect, for high-Z-absorbingmaterials. By incorporating high-Z NPs, such as gold (Z = 79), silver (Z = 47), bismuth(Z = 83), and gadolinium (Z = 64) into tumor sites, they can provide a significantly greateramount of energy per unit mass due to their high energy absorption compared with that ofsoft tissue, thus enhancing the local deposition of dose in tumors (Figure 5).However, the extent to which this physical dose enhancement contributes to the ob-served radiosensitization of cells remains unclear. The increased photoelectric interactionsby high-Z NPs are more pronounced for incident photons produced by kilovoltage (kV)X-ray sources, where the strong photoelectric absorption at lower keV energies leads toa dramatic increase in the absorbed dose [34,35]. In contrast, for photons produced bymegavoltage (MV) X-ray sources, which is prevalent in most clinical scenarios, the primaryinteraction is the Compton effect, where the X-ray cross-section is essentially independentof atomic number. Thus, it was thought that the differential in dose deposition due to theincorporation of high-Z NPs would be insignificant when using MV X-ray sources [36,37].Interestingly, in vitro and in vivo experimental evidence indicates radiosensitization effectsthat often surpass the expected physical dose enhancement. For instance, a study con-ducted by Chithrani et al. demonstrated that HeLa cells exposed to GNPs with a diameterof 50 nm exhibited a radiosensitization-enhancement factor (REF) varying with the energyof the X-ray sources (1.66 with 105 kVp, 1.43 with 220 kVp, and 1.17 with 6 MV) [38].Although their study effectively displayed the energy dependence of the radiosensitizationdue to GNPs, it suggested that GNPs were effective in increasing the radiosensitization ofcells when treated with MV X-rays. These results have also been further corroborated byvarious studies that effectively display the sensitizing effects of high-Z NPs when irradiatedby MV sources, indicating that other mechanisms could be responsible for the observedradiosensitization [20,39–42].Molecules 2024, 29, 2438 9 of 23Molecules 2024, 29, x FOR PEER REVIEW 9 of 23 Figure 5. Mechanisms of high-Z NP radiosensitization. NPs can increase the sensitivity to radiation of cells through physical, chemical, and biological mechanisms. NPs interact with ionizing radia-tion, producing a shower of secondary electrons that can exert damage upon the cell. The electron-ically active surface NPs also catalyze the production of DNA-damaging ROS and free radicals, damaging critical cellular components. NPs can also increase the sensitivity of cells to radiation through oxidative stress and cell cycle effects. (Created using Biorender.com). 3.2. Production of Reactive Oxygen Species (ROS) and Oxidative Stress One of the potential mechanisms of NP-based radiosensitization is attributed to the production of reactive oxygen species (ROS) (Figure 5). The production of ROS and the subsequent oxidative stress is an important pathway that is responsible for cellular dam-age during radiotherapy. When exposed to radiation, water molecules within the cell un-dergo radiolysis that subsequently leads to the production of ROS, such as superoxide anion radicals (O2−), hydroxyl radicals (OH−), and hydrogen peroxide (H2O2). These reac-tive species can directly damage DNA, triggering various cellular responses, including cell death. In addition, the excessive production of ROS can exert oxidative stress within the cell as a result of ROS levels surpassing the reducing capacity of the cell. This can impair various cellular functions due to the oxidation of proteins and lipids, and damage induced to the mitochondria, which can ultimately lead to apoptosis. Numerous studies have shown that NPs can enhance ROS production following ra-diotherapy treatment. A study conducted by Misawa et al. demonstrated this concept, where the addition of AuNPs significantly enhanced the production of ROS in a water environment, with their results displaying 1.46- and 7.68-fold increases in the production of OH− and O2− radicals, respectively, under 100 kVp irradiation [43]. They attributed the rise in O2− radicals to the electron transfer from the photo- and auger electrons, whereas the increased production of OH- radicals was attributed to the secondary radiolysis of wa-ter by fluorescent X-rays, suggesting that high-Z NPs facilitate a significant production in ROS under irradiation. Furthermore, this enhancement in ROS has been demonstrated to translate in cell cultures for various NPs [44–48]. For instance, Bemidinezhad et al. found that glucose-coated AuNPs significantly increased the ROS levels in cell cultures post-ir-radiation, resulting in an upregulation in caspase 3/7 activity, crucial enzymes in the Figure 5. Mechanisms of high-Z NP radiosensitization. NPs can increase the sensitivity to radiationof cells through physical, chemical, and biological mechanisms. NPs interact with ionizing radiation,producing a shower of s condary electrons that can exert damage upon the cell. Th electroni allyactive surfac NPs also catalyze the production f DNA-damaging ROS and free radicals, damagingcritical cellular components. NPs can also increase the sensitivity of cells to radiation throughoxidative stress and cell cycle effects. (Created using Biorender.com).3.2. Production of Reactive Oxygen Species (ROS) and Oxidative StressOne of the potential mechanisms of NP-based radiosensitization is attributed to theproduction of reactive oxygen species (ROS) (Figure 5). The production of ROS and thesubsequent oxidative stress is an important pathway that is responsible for cellular d mageduring radiotherapy. When exposed to radiation, water molecules within the cell undergoradiolysis that subsequently leads to the production of ROS, such as superoxide anionradicals (O2−), hydroxyl radicals (OH−), and hydrogen peroxide (H2O2). These reactivespecies can directly damage DNA, triggering various cellular responses, including celldeath. In addition, the excessive production of ROS can exert oxidative stress within thecell as a result of ROS levels surpassing the reducing capacity of the cell. This can impairvarious cellular functions due to the oxidation of proteins and lipids, and damage inducedto the mitochondria, which can ultimately lead to apoptosis.Numerous studies have shown that NPs can enhance ROS production followingradiotherapy treatment. A study conducted by Misawa et al. demonstrated this concept,where the addition of AuNPs significantly enhanced the production of ROS in a waterenvironment, with their results displaying 1.46- and 7.68-fold increases in the productionof OH− and O2− radicals, respectively, under 100 kVp irradiation [43]. They attributed therise in O2− radicals to the electron transfer from the photo- and auger electrons, whereasthe increased production of OH− radicals was attributed to the secondary radiolysis ofwater by fluorescent X-rays, suggesting that high-Z NPs facilitate a significant productionin ROS under irradiation. Furthermore, this enhancement in ROS has been demonstratedto translate in cell cultures for various NPs [44–48]. For instance, Bemidinezhad et al.found that glucose-coated AuNPs significantly increased the ROS levels in cell culturespost-irradiation, resulting in an upregulation in caspase 3/7 activity, crucial enzymes in theMolecules 2024, 29, 2438 10 of 23apoptosis pathway, which was correlated with an increase in the therapeutic response toradiotherapy treatment [49].Interestingly, NPs have also been shown to amplify ROS production in the absenceof radiation [50–53]. It is thought that NPs with catalytic surfaces can catalyze chemi-cal reactions and increase the production of ROS, where radiation further enhances thiseffect due to the highly reactive environment. For example, while it was once widelyaccepted that AuNPs are chemically inert, evidence suggests that the surface of AuNPsis electronically active and can facilitate the surface-mediated transfer of electrons fromelectron donors, interacting with molecular oxygen to generate ROS such as superoxideradicals [54]. However, contrary evidence from a recent study has suggested that, althoughAuNPs catalyze the reduction of O2, the production of ROS is merely transient surfaceproducts produced as intermediate products in the reduction process, not contributing toROS reactivity [55]. Rather, the oxidative stress observed in cells could be due to AuNPscatalyzing redox reactions, subsequently oxidizing antioxidants and reducing the redoxcapacity of cells, eventually leading to oxidative stress.This reduction in the redox capacity of cells mediated by the introduction of NPs tothe cellular environment is a mechanism that has been previously explored. One of the keymechanisms underlying this effect is through the inhibition of endogenous reducing agents,such as glutathione (GSH), an essential molecule in maintaining the redox homeostasis ofcells. NPs can interact with reducing agents, leading to their depletion or inactivation. Forexample, NPs that have a high affinity for thiol groups can bind to antioxidants containingthiol groups, such as GSH, effectively inhibiting the reducing ability of the molecule andthereby decreasing the reduction capacity of the cell [56]. A study conducted by Jawaid et al.demonstrated this, where 2 nm AuNPs were shown to decrease the intracellular levelsof GSH, which led to an increase in intracellular ROS [57]. Similarly, Abudayyak et al.reported that Bi2O3 NPs also facilitated a decrease in the intracellular levels of GSH forvarious cell lines [58]. Regarding GSH, another reducing agent that has also been shown tobe affected by NPs is thioredoxin reductase 1 (TrxR1). Similar to GSH, TrxR1 is importantin maintaining redox equilibrium, regulating cellular redox processes, with its activityhaving been shown to be inhibited in the presence of Au clusters, leading to an increasein apoptosis [59]. Collectively, these studies suggest that NPs have the capacity to inhibitcertain redox mechanisms and elevate ROS production, particularly in conjunction withradiotherapy. However, the full extent and implication of these interactions in which theycontribute to the radiosensitization of cells remains to be elucidated.3.3. Cell Cycle EffectsThe cell cycle is a key determinant in the radiosensitivity of cells, with distinct phasesdemonstrating varying degrees of sensitivity. In general, cells in the late G2 and mitosis(G2/M) phase of the cycle exhibit enhanced sensitivity to radiation, whereas cells in thelate S phase are markedly more radioresistant. Furthermore, the cell cycle is also integral tothe DNA damage response following exposure to ionizing radiation, where the activationof cell cycle checkpoints is the second major effector pathway in the DNA damage responseof cells [2]. These checkpoints either slow or halt the progression of cells through the cellcycle, maintaining genomic integrity by repairing radiation-induced damage or initiatingapoptosis, thus preventing the replication of damaged DNA.NPs have been shown to alter the cell cycle, with some studies demonstrating anenhancement in the population of the cells in the G2/M phase, causing a potential increasein the radiosensitivity of cells (Figure 5). Roa et al. observed that glucose-coated AuNPsincreased the G2/M phase population 24 h after exposure to AuNPs [60]. This effect waslinked to the activation of cyclin-dependent kinases, causing the acceleration of DU-145cells through the G0/G1 phase towards G2/M, thereby augmenting their radiosensitivity.Similarly, Wang et al. observed that glucose-capped AuNPs arrested MDA-MB-231 cells inthe G2/M phase, resulting in a sensitization enhancement ratio of 1.86 [61]. However, notall studies agree on the impact that NPs have on the cell cycle, with some research revealingMolecules 2024, 29, 2438 11 of 23negligible effects [62–64], which could be due to the effects of NPs varying for differentcell types, as well as the different distinct physicochemical properties of NPs. Homila et al.studied the effects of AgNPs on a panel of lung cell lines, and their results found thatAgNPs enhanced the accumulation of cells in the G2/M phase in both A549 and Calu-1cell lines, while inducing S-phase accumulation in BEAS-2B cells and showing no effecton NCI-H358 cells [65]. Regarding physicochemical properties, the functionalization ofNPs has also been shown to influence cell cycle distribution, where bovine serum albumin(BSA)-coated AuNPs induced G2/M arrest in RAW264.7 cells, contrasting with the G0/G1arrest caused by non-functionalized AuNPs [66]. Together, these studies suggest that thealteration of the cell cycle induced by NPs could be dependent on both cell type and theproperties of NPs, leading to the different results observed by various studies.However, given the diversity in the physicochemical properties of NPs and the broadrange of cell lines that have been studied, it is difficult to draw general conclusions on theeffect that NPs have on the cell cycle and its contribution to the radiosensitization of cells;thus, further research is required to elicit definitive answers to understand how NPs andtheir properties can cause alterations. Nevertheless, existing studies indicate that NPs canmodify the cell cycle distribution to enhance the sensitivity of cells to radiotherapy, wherefurther research could elucidate ways to exploit this mechanism for therapeutic purposes.4. Application of Nanoparticles as Radiosensitizers4.1. High-Z Nanoparticles as RadiosensitizersThe use of high-Z nanoparticles as radiosensitizers has emerged as a novel tool inradiotherapy treatment. Initially, high-Z materials were proposed as radiosensitizers dueto their potential to enhance dose deposition, a concept based on the increased X-rayabsorption correlating with the atomic number of the absorbing material. This was firstdemonstrated using iodine (Z = 53) as a radiosensitizer, where Matsudaira et al. observedthat iodine augmented the effects of X-rays in cell cultures over 40 years ago [67]. Followingthat study, it was shown that injecting iodine directly into tumors prior to RT suppressedtumor growth by 80% [68]. Together, these studies effectively demonstrated the feasibilityof augmenting the effects of radiotherapy through the introduction of high-Z absorbers intotarget sites. Since then, advancements in nanotechnology have propelled the applicationof high-Z NPs as radiosensitizers. These NPs also aim to exploit the X-ray absorptionphenomenon to augment the effects of radiotherapy, but they have also been shownto increase ROS production and alter the cell cycle dynamics, thereby enhancing theradiosensitivity of cells. Moreover, their physicochemical properties can be tailored forbetter biocompatibility, prolonged circulation time, and tumor targeting, making themsuitable candidates as radiosensitizers.Among other nanoparticles, AuNPs have been the most widely investigated in the fieldof high-Z NPs. A pioneering study conducted by Hainfeld et al. revealed their significantpotential as radiosensitizers, where it was shown that injecting 1.9 nm AuNPs into micebearing subcutaneous EMT-6 mammary carcinomas prior to 250 kVp X-ray irradiationsignificantly improved the one-year survival from 20% with radiotherapy alone to 86%with the combined treatment [69]. These promising results were also replicated in furtherstudies using head and neck squamous carcinoma (SCCVII) and malignant glioma (Tu-2449)tumor models, providing compelling evidence about the potential applicability of AuNPsin radiotherapy treatment [70,71]. In these studies, mice were injected with AuNPs at aconcentration ranging from ~2 to 4 g Au/kg body weight of mice. It was initially thoughtthat such concentrations were required to achieve significant radiosensitization, whichraised questions about the clinical translation of the strategy due to economic and safetyconcerns. Subsequent research, however, has indicated that substantial radiosensitizingeffects can be realized at much lower AuNP concentrations, making the approach moreviable for clinical use.A study by Liu et al. demonstrated the effectiveness of AuNPs at significantly reducedconcentrations using bovine serum albumin (BSA)-functionalized AuNPs [72]. For theirMolecules 2024, 29, 2438 12 of 23study, mice bearing H22 hepatoma tumors received injections of 4 mg Au/kg body weight,a thousand-fold reduction compared with the aforementioned studies. Their findingsrevealed that both 8 and 50 nm BSA-AuNPs suppressed tumor growth when used inconjunction with radiotherapy (5 Gy, 6 MV), achieving enhancement factors of 1.93 and2.02, respectively. Similar results were also found by Bhattari et al., where they showedsignificant reductions in tumor growth in MDA-MB-231 xenograft models after a combinedtreatment of anti-CXCR4-functionalized GNPs (cGNP) and radiotherapy using a concen-tration of 2 mg Au/kg body weight [28]. This treatment led to a significant reduction intumor growth, where tumor size reduced to baseline levels and regrowth did not occur forover 50 days post-treatment (Figure 6A). However, these radiosensitization effects werenot realized with non-targeted PEGylated GNPs (pGNP), underscoring the importance oftumor-targeted NPs for radiosensitizing purposes.Beyond conventional X-ray irradiation, the effectiveness of AuNPs can also extend toother radiation types. Electron radiotherapy, which is generally used for the treatment ofsuperficial tumors, has been shown to benefit from AuNPs. Mehrnia et al. demonstratedthis, where AuNPs under 4 MeV electron irradiation significantly decreased cell viabilityin both MCF-7 and MDA-MB-231 cells, with sensitizer enhancement ratios of 1.43 and 1.62,respectively [73]. These radiosensitizing effects were also observed in B16F10 melanomatumor-bearing mice, where AuNPs significantly enhanced apoptotic cell death whenirradiated with a 6 MeV electron beam, inducing the retardation of tumor growth [49].Additionally, while not a common form of treatment, AuNPs have been shown to augmentproton therapy. In a proof of principle study by Cunningham et al., AuNPs were found toincrease cell death in CHO-K1 cells under 200 MeV proton irradiation by 27% and 44% atdoses of 2 Gy and 6 Gy, respectively (Figure 6B) [74]. Collectively, these studies outline thebroad application of AuNPs in radiotherapy, showcasing their potential to augment theeffects of various types of radiation therapies.Molecules 2024, 29, x FOR PEER REVIEW 12 of 23 A study by Liu et al. demonstrated the effectiveness of AuNPs at significantly re-duced concentrations using bovine serum albumin (BSA)-functionalized AuNPs [72]. For their study, mice bearing H22 hepatoma tumors received injections of 4 mg Au/kg body weight, a thousand-fold reduction compared with the aforementioned studies. Their find-ings revealed that both 8 and 50 nm BSA-AuNPs suppressed tumor growth when used in conjunction with radiotherapy (5 Gy, 6 MV), achieving enhancement factors of 1.93 and 2.02, respectively. Similar results were also found by Bhattari et al., where they showed significant reductions in tumor growth in MDA-MB-231 xenograft models after a com-bined treatment of anti-CXCR4-functionalized GNPs (cGNP) and radiotherapy using a concentration of 2 mg Au/kg body weight [28]. This tre tment led to a significant reduc-tion in tumor growth, where tumor size reduced to baseline level nd regrowth did not occur for over 50 days post-treatment (Figure 6A). However, these radiosensitization ef-fects were not realized with non-targeted PEGylated GNPs (pGNP), underscoring the im-portance of tumor-targeted NPs for radiosensitizing purposes. Beyond conventional X-ray irradiation, the effectiveness of AuNPs can also extend to other radiation types. Electron radiotherapy, which is generally used for the treatment of superficial tumors, has been shown to benefit from AuNPs. Mehrnia et al. demonstrated this, where AuNPs und r 4 MeV electron irradiation significantly decreased cell viability in both MCF-7 and MDA-MB-231 cells, with sensitizer en anc ment ratio of 1.43 and 1.62, respectively [73]. These radiosensitizing effects were also observed in B16F10 mela-noma tumor-bearing mice, where AuNPs significantly enhanced apoptotic cell death when irradiated with a 6 MeV electron beam, inducing the retardation of tumor growth [49]. Additionally, while not a common form of treatment, AuNPs have been shown to augment proton therapy. In a proof of principle study by Cunningham et al., AuNPs were found to increase cell death in CHO-K1 cells under 200 MeV proton irradiation by 27% and 44% at doses of 2 Gy and 6 Gy, respectively (Figure 6B) [74]. Collectively, these studies outline the broad application of AuNPs in radiotherapy, showcasing their potential to augment the effects of various types of radiation therapi s. Figure 6. Radiosensitization due to high-Z nanoparticles. (A) Normalized tumor volumes of MDA-MB-231 xenograft tumor-bearing mice post treatment with cGNP alone, radiation (10 Gy single dose) alone, pGNP + radiation (RT), or cGNP + RT (B) Survival fraction curves of CHO-K1 cells Figure 6. Ra itization due to high-Z na oparticles. (A) Normalized tumor volumes of MDA-MB-231 raft tumor-bearing mice post treatment with cGNP alone, radiation (10 Gy single dose)Molecules 2024, 29, 2438 13 of 23alone, pGNP + radiation (RT), or cGNP + RT (B) Survival fraction curves of CHO-K1 cells treatedwith AuNPs and irradiated at various doses with a 200 MeV proton beam. (C) Kaplan–Meier survivalcurves for U251 tumor-bearing mice treated with AuNPs or AgNPs with or without ionizing radiation(IR). Mice were irradiated with a single dose of 8 Gy with a 6 MV X-ray beam. Mice were treated withAgNPs at the same molar concentration (mol-AgNPs) or mass concentration (mas-AgNPs) as AuNPs.(D) Apoptosis of U251 cells after treatment with AgNPs or AuNPs combined with a radiation dose of6 Gy. * p < 0.05, *** p < 0.001 compared with the control group. ## p < 0.01, ### p < 0.001 comparedwith the corresponding AuNPs-treated group (E) Tumor volume growth curves after treatment withHA-Gd2O3 NPs and radiation (R + HA-Gd). Mice were administered 3 fractions of 3 Gy with a 6 MeVbeam over a 7-day period for a total of 9 Gy. Reproduced with permission from open access CreativeCommon license [28,74–76].Similar to gold, other high-Z nanoparticles, such as AgNPs, GdNPs, and BiNPs, havebeen explored as radiosensitizers. For instance, Liu et al. investigated the impact of Ag-NPs and radiotherapy in C6 glioma-bearing rats [77]. With the combined treatment, theyobserved anti-proliferative and pro-apoptotic effects, markedly improving survival timescompared with the other treatment groups. Building on this research, they found similar re-sults using a U251 glioblastoma mouse model [75]. Interestingly, their study displayed thatAgNPs outperformed AuNPs in radiosensitization at equivalent concentrations (Figure 6C).It was reported that AgNPs significantly increased autophagy levels and pro-apoptoticactivity (Figure 6D) when paired with radiation in comparison to AuNPs, which wasattributed to the enhancement in the therapeutic results. Additionally, Gadolinium NPs(GdNPs) have been shown to be effective radiosensitizers, but they are also effective MRIcontrast agents, enabling them to be a valuable tool for theranostics, which has led to theircurrent clinical investigation. In a study by Wu et al., they found that hyaluronic acid-functionalized gadolinium oxide NPs (HA-Gd2O3 NPs) were effective in both roles [76].The radiotherapeutic results displayed that HA-Gd2O3 NPs showed efficient suppressionof tumor growth when combined with RT, with a 62% reduction in tumor volume 10 dayspost treatment (Figure 6E). Furthermore, studies have also demonstrated the potentialradiosensitizing effects of BiNPs. A study by Deng et al. indicated that Folate-modifiedBiNPs enhanced the radiotherapeutic effects in 4T1 tumor-bearing mice, reducing the tumorgrowth and increasing the survival time compared with control groups without inducingany apparent toxicity, indicating the safety and efficacy of the treatment strategy [78].4.2. Targeted Delivery of Radiosensitizing Chemotherapeutics via High-Z NanoparticlesWhile radiotherapy predominantly aims to enhance local tumor control, chemotherapyis generally used to target metastasis, with the combination of these treatments becomingincreasingly more common in the management of patients with locally advanced disease.In addition, various chemotherapeutic agents are known to augment the effects of ra-diotherapy, resulting in synergistic therapeutic benefits that can be exploited to improvepatient outcomes. This concomitant use of radiotherapy and chemotherapy is a strategythat is applied in certain cancer treatment regimens, aiming to capitalize on the comple-mentary mechanisms of both treatments to achieve better tumor control and curative rates,where studies have demonstrated increased loco-regional control and overall survival ofpatients [79–82]. However, issues that are innate to chemotherapeutics limit the overallefficacy of the treatment. Often, these drugs have poor solubility and a short circulationtime, resulting in suboptimal concentrations reaching tumor sites. Furthermore, the lackof specificity causes systemic toxicity, inducing adverse effects that restrict the amount ofdose that can be administered to patients.In recent years, the advent of nano drug delivery systems (DDSs) has presented asolution to mitigate these challenges. Through encapsulation or the adsorption of drugsonto their surface, NPs can improve the pharmacokinetics, pharmacodynamics, and thetargeted delivery of chemotherapeutics, ensuring that higher concentrations are deliveredto tumors while simultaneously lowering normal tissue toxicity. The implementation ofMolecules 2024, 29, 2438 14 of 23nano DDSs has already been proven to be effective, with nano DDSs such as Doxil (lipo-somal doxorubicin) and Abraxane (nab-paclitaxel) currently used in clinical settings [83].Extending from this, the coupling of radiosensitizing chemotherapeutic agents with high-Znanoparticles has been proposed to produce nanomedicines with dual-radiosensitizingeffects, streamlining the therapeutic process.Utilizing high-Z nanoparticles as DDSs for radiosensitizing agents presents a uniqueopportunity to introduce a bi-functional nanoplatform to improve cancer therapeutics. Aspreviously discussed, high-Z nanoparticles inherently augment the effects of radiotherapy,and their combination with drug-based radiosensitizers can offer a dual sensitizationeffect that can be used to increase tumor responses to radiotherapy treatment. Their highsurface area-to-volume ratio and adaptable surface chemistry facilitate the straightforwardconjugation of moieties to their surface, including chemotherapeutics, and by leveragingtheir intrinsic therapeutic benefits, such as their tumor targeting and extended circulationtime, drug delivery to tumors can be improved, leading to favorable therapeutic effects.One chemotherapy agent that has been delivered using high-Z NPs is docetaxel (DTX).DTX is a member of the taxane family that is used to effectively treat various forms of cancer,including head and neck, breast, and prostate. Its mechanism of action involves stabilizingmicrotubules within cells, thereby preventing the depolymerization required to progressthrough the cell cycle and undergo cell division, causing cells to be arrested in the G2/Mphase. This phase is notably susceptible to radiation-induced damage, positioning DTXas an ideal radiosensitizer, with studies demonstrating synergistic effects between DTXand radiotherapy [84,85]. Additionally, DTX has been shown to increase NP accumulationin cells, both in vitro and in vivo, suggesting enhanced radiosensitization potential whenused in combination with high-Z NPs [86–89]. A study by Guo et al. demonstrated thetherapeutic prospect of synthesizing a AuNP-based DDS for DTX to enhance the effects ofradiotherapy [90]. In their study, DUPA-modified AuNPs were loaded with DTX (Au@DTX-DUPA) to enhance the radiosensitization of tumors. Following treatment with Au@DTX-DUPA and radiotherapy, 22RV1 tumor-bearing mice demonstrated significant therapeuticbenefits due to the sensitization effects of both DTX and AuNPs, with a significant reductionin tumor growth being observed for the combined treatment (Figure 7A). This effect couldbe attributed to the enhanced sensitization from the combination of both AuNPs andDTX. As shown in Figure 7B, there was an enhancement in AuNP accumulation in tumortissue 24 h post-injection for mice treated with Au@DTX-DUPA, the timepoint at whichmice were irradiated. Furthermore, Au@DTX-DUPA successfully induced cell arrest inthe G2/M phase of the cell cycle, indicating increased levels of sensitization (Figure 7C).This resulted in a significant decrease in Ki67 expression for the combined treatment ofAu@DTX-DUPA and radiotherapy, signaling a significant inhibition of the proliferativecapacity of tumors (Figure 7D) and highlighting the promising capabilities of high-Z NPsas a dual-sensitizing DDS.Similar to DTX, cisplatin is another chemotherapeutic agent known to enhance cell sen-sitivity to radiotherapy. The platinum-based chemotherapeutic agent functions by bindingto DNA, inducing both intrastrand and interstrand crosslinks. Like other chemotherapeu-tics, the efficacy of cisplatin is often compromised by systemic toxicity that stems from itslack of specificity. Therefore, implementing a DDS has the potential to mitigate this issue.A recent study by Chen et al. demonstrated this using AuNPs as a DDS for cisplatin [91]. Itwas noted that cisplatin in its free form caused acute systemic toxicity in mice, characterizedby a significant decrease in body weight a week after injection, where this was not realizedfor mice treated with Au-cisplatin NPs. Furthermore, Au-cisplatin NPs were shown toalso diminish nephrotoxicity and hepatotoxicity associated with cisplatin in its free form.When combined with ablative radiotherapy, Au-cisplatin NPs demonstrated enhancedradiosensitization effects, decreasing tumor growth and improving the overall survival ofmice. It was also shown to elicit immunogenic cell death, which enhanced the recruitmentof tumor-infiltrating immune cells, such as natural killer T cells and CD8+ T cells. Notably,this resulted in distant non-irradiated tumors experiencing growth suppression, showingMolecules 2024, 29, 2438 15 of 23the potential of Au-cisplatin NPs to prime the immune system to reduce the metastaticpotential and induce abscopal effects.Molecules 2024, 29, x FOR PEER REVIEW 15 of 23 Figure 7. Dual radiosensitization through docetaxel-loaded gold nanoparticles. (A) Normalized tu-mor growth curves of 22RV1 xenograft tumor-bearing mice after irradiation of 6 Gy with 6 MeV electron beam. (B) Concentration of Au in 22RV1 xenograft tumor post-injection with Au-DUPA NPs or Au@DTX-DUPA NPs. (C) Cell cycle analysis of 22RV1 cells after treatment with Au-DUPA NPs, free DTX, or Au@DTX-DUPA NPs. (D) Ki67 staining assay of tumors 15 days post treatment to in-dicate proliferative capacity. * p < 0.05, ** p < 0.01. Reproduced with permission from open access Creative Common license [90]. Similar to DTX, cisplatin is another chemotherapeutic agent known to enhance cell sensitivity to radiotherapy. The platinum-based chemotherapeutic agent functions by binding to DNA, inducing both intrastrand and interstrand crosslinks. Like other chemo-therapeutics, the efficacy of cisplatin is often compromised by systemic toxicity that stems from its lack of specificity. Therefore, implementing a DDS has the potential to mitigate this issue. A recent study by Chen et al. demonstrated this using AuNPs as a DDS for cisplatin [91]. It was noted that cisplatin in its free form caused acute systemic toxicity in mice, characterized by a significant decrease in body weight a week after injection, where this was not realized for mice treated with Au-cisplatin NPs. Furthermore, Au-cisplatin NPs were shown to also diminish nephrotoxicity and hepatotoxicity associated with cis-platin in its free form. When combined with ablative radiotherapy, Au-cisplatin NPs demonstrated enhanced radiosensitization effects, decreasing tumor growth and improv-ing the overall survival of mice. It was also shown to elicit immunogenic cell death, which enhanced the recruitment of tumor-infiltrating immune cells, such as natural killer T cells and CD8+ T cells. Notably, this resulted in distant non-irradiated tumors experiencing growth suppression, showing the potential of Au-cisplatin NPs to prime the immune sys-tem to reduce the metastatic potential and induce abscopal effects. 4.3. Challenges and Considerations in the Application of High-Z NPs as Radiosensitizers Despite the compelling evidence that promotes the application of high-Z NPs in ra-diotherapy treatment, there are still challenges that are apparent that can limit their clini-cal translation. Various factors and parameters, such as NP material, size, surface modifi-cations, and concentrations need to be considered when designing NPs to obtain optimal Figure 7. al ra i se sitization through docetaxel-loaded gold nanoparticles. (A) Normalizedtumor growth curves of 22RV1 xenograft tumor-bearing mice after irradiation of 6 Gy with 6 MeVelectron beam. (B) Concentration of Au in 22RV1 xenograft tumor post-injection with Au-DUPANPs or Au@DTX-DUPA NPs. (C) Cell cycle analysis of 22RV1 cells after treatment with Au-DUPANPs, free DTX, or Au@DTX-DUPA NPs. (D) Ki67 staining assay of tumors 15 days post treatment toindicate proliferative capacity. * p < 0.05, ** p < 0.01. Reproduced with permission from open accessCreative Common license [90].4.3. Challenges and Considerations in the Application of High-Z NPs as RadiosensitizersDespite the compelling evidence that promotes the application of high-Z NPs inradiotherapy treatment, there are still challenges that are apparent that can limit theirclinical translation. Various factors and parameters, such as NP material, size, surfacemodifications, and concentrations need to be considered when designing NPs to obtainoptimal results. However, when taking these factors into consideration, it is not abundantlyclear as to which parameters will provide the best therapeutic results.Many studies have shown a correlation between the size of NPs and their cellular up-take. An early study conducted by Chithrani et al. demonstrated a difference in the cellularuptake of GNPs with diameters ranging from 14–100 nm, with the results concluding that50 nm GNPs had the greatest uptake [92]. Similar studies have also shown that ~50 nmdiameter GNPs had a significant increase in uptake in human prostate carcinoma PC-3 cellsin comparison with their smaller and larger counterparts, suggesting that 50 nm NPs areideal to obtain optimal uptake, and thus radiosensitization [93]. Interstingly, when movinginto more complex models, such as multilayer cell structures and 3D tumor spheroids,smaller NPs appear to be more advantageous. One study compared the uptake of 20 nmand 50 nm GNPs in tissue-like multilayer cellular structures that were grown to modelpost-vascular tumor environments [94]. Although uptake was greater for 50 nm GNPs atthe monolayer level, 20 nm GNPs penetrated multilayer cellular structures more efficiently,suggesting a reversal in size dep d ncy fo ultilayer structures. This as also beensupported by uptake data from three-dimensional spheroids. GNPs of 15 m increased thedepth pene ration by 33% and 17% for CAL-27 and HeLa spheroids, respectively, comparedMolecules 2024, 29, 2438 16 of 23with 50 nm GNPs [95], which can be attributed to the extracellular matrix (ECM) and itscomposition. As NPs diffuse through the ECM, they interact with the network structurevia hydrodynamic interactions, physical interactions, as well as electrostatic interactionsthat can constitute a barrier to some NPs. One of these barriers is the inter-matrix spacingin the ECM, an important component when considering the optimal size of NPs. Collagenfibrils that reside in the ECM present a size limit for NPs where particles cannot penetratethe matrix barrier if the diameters surpass the size of the network space. Despite this,smaller NPs (<10 nm) can also present some disadvantages. These smaller NPs can enterthe nucleus of a cell, which may result in toxicity [96,97]. In addition, NPs with a size of<10 nm can also experience rapid clearance from the RES, resulting in inadequate deliveryof NPs to target sites.To achieve optimal therapeutic results, it is paramount to ensure that adequate con-centrations of NPs in tumors are met to achieve radiosensitization, as more NPs present inthe tumor will lead to a greater sensitizing effect. However, simply increasing the concen-trations administered can lead to an increased risk of toxicity. Biodistribution studies havedemonstrated the accumulation of NPs in organs such as the liver, spleen, and kidneys,raising concerns about their use [98]. As such, the safety and biocompatibility of NPsshould be of utmost importance to avoid acute and late toxicities that could hinder theirtherapeutic efficacy. As a result, many preclinical studies focus on exploring the surfacemodification of NPs to improve their biocompatibility, circulation time, and targeting. Awide variety of functionalization strategies have been explored in preclinical studies, withmost suggesting beneficial therapeutic results. However, this poses a challenge in itselfto pinpoint what array of NPs are the most beneficial, or rather most clinically feasible.Perhaps one aspect that should be prioritized when deciding this is their ability to beproduced on a large scale that is required for clinical applications. Although studies demon-strate tremendous success in synthesizing and functionalizing a wide variety of NPs, thecommercialization required when transitioning from bench to bedside necessitates largescaled-up synthesis that can present obstacles, such as reproducibility and quality control.This begs the question of whether the design of certain NPs used as radiosensitizers is foracademic purposes or for clinical intentions. The synthesis of NPs at the lab scale allowsfor the precise synthesis conditions required to produce highly uniform and reproducibleNPs. On the other hand, scaled-up synthesis often can introduce variability, resulting invariations in the properties of the NPs. Therefore, it is important to consider the feasibilityof using cost-effective, well-controlled, and good manufacturing practices to produce largereproducible batches of NPs, and this should be considered early on in preclinical studiesto ease their clinical translation.4.4. Clinical Translation of High-Z Nanoparticles as RadiosensitizersAlthough preclinical studies have suggested that the application of high-Z nanopar-ticles in radiotherapy can provide significant benefits, their clinical translation remainslimited in comparison with more commonly studied organic NPs, such as liposomes andpolymers. High-Z NPs encounter distinct challenges in terms of their clinical adaptation,including concerns regarding their biocompatibility, toxicity, biodistribution, and the com-plexities associated with scaled-up synthesis. Despite this, two radiosensitizing high-ZNPs, NBTXR3 and AGuIX, have transitioned into clinical trials, indicating a potential shiftin the clinical application of high-Z NPs as radiosensitizers.NBTXR3, developed by Nanobiotix, is a hafnium oxide (HfO2) NP currently underclinical investigation. Similar to other high-Z nanoparticles, its radiosensitizing abilitiesarise from hafnium’s high atomic number (Z = 72), increasing dose deposition via physicalmechanisms. When present in tumors, hafnium increases the interactions with ionizingradiation, yielding a local enhancement in the generation of dose-depositing electronsand DNA-damaging free radicals, thereby amplifying DNA damage and the cytotoxicitybeyond that of conventional radiotherapy. Currently, clinical studies are investigatingNBTXR3’s radiosensitizing effects across various types of cancers, including soft tissueMolecules 2024, 29, 2438 17 of 23sarcoma, head and neck, prostate, lung, and brain metastases (Table 1). Interestingly,in contrast to many preclinical studies that examined the efficacy of high-Z NPs as ra-diosensitizers, these clinical trials are analyzing the effects of intratumoral injections ofNBTXR3 combined with radiotherapy, a strategy that might ease the clinical translation ofradiosensitizing NPs. Intratumoral injections address concerns such as systemic toxicityand biodistribution issues that are associated with intravenous injections, where NPs oftenaccumulate in organs like the kidneys, liver, and spleen, despite their functionalizationwith targeting moieties [87–89]. This raises concerns about the toxicity of the treatment,where lower concentrations would need to be administered at the cost of reducing doseenhancement. Intratumoral injections can help to mitigate this trade-off, enabling higherconcentrations in tumors without increasing the risk of toxicity. A recent phase II/IIIclinical trial (NCT02379845) has shown encouraging results, with intratumoral injections ofNBTXR3 demonstrating increased anticancer effects and tolerability in patients with locallyadvanced soft-tissue sarcoma [99]. That study found an increase in complete pathologicalresponse for patients who received NBTXR3 in conjunction with preoperative radiotherapycompared with preoperative radiotherapy alone, suggesting promising therapeutic benefitsof incorporating high-Z NPs to complement the effects of radiotherapy. Additionally,preclinical studies have suggested that NBTXR3 can enhance the efficacy of immunother-apies by bolstering the antitumor immune response, prompting clinical investigationsof the combined treatment modalities (NCT05039632, NCT04862455, NCT03589339, andNCT04892173) [100–102].Table 1. Current clinical studies investigating the effects of high-Z NPs for radiotherapy.Nanoparticle Condition Phase Status IdentifierNBTXR3Adult soft-tissue sarcoma Phase I Completed NCT01433068Pancreatic ductal adenocarcinoma Phase I Recruiting NCT04484909Lung non-small-cell carcinoma Phase I Recruiting NCT04505267Metastatic malignant solid neoplasm Phase I/II Recruiting NCT05039632Esophageal adenocarcinoma Phase I Recruiting NCT04615013Head and neck squamous cell carcinoma Phase II Recruiting NCT04862455Advanced cancers Phase I Recruiting NCT03589339Head and neck squamous Phase I Active NCT01946867Adult soft-tissue sarcoma Phase II/III Completed NCT02379845Head and neck squamous cell carcinoma Phase III Recruiting NCT04892173AGuIXGlioblastoma Phase I/II Recruiting NCT04881032Brain metastases Phase I Completed NCT02820454Brain metastases Phase II Recruiting NCT03818386Gynecological cancers Phase I Recruiting NCT03308604Brain metastases Phase II Recruiting NCT04899908Lung tumors and pancreatic cancer Phase I/II Recruiting NCT04789486Recurrent cancer Phase I Not yet recruiting NCT04784221In addition to NBTXR3, AGuIX, developed by NH TherAguix, is another nanopar-ticle currently under clinical investigation. These sub 5 nm nanoparticles are composedof gadolinium (Z = 64)-chelated polysiloxane, where the strategic choice of gadoliniumenables them to be effective radiosensitizers and MRI contrast agents. When administeredintravenously to patients, AGuIX can preferentially accumulate in tumors due to the EPR ef-fect, allowing for enhanced concentrations in tumor tissue compared with the surroundinghealthy tissue. Furthermore, their small size promotes effective renal clearance, avoidingpotential toxicity that could be induced by long-term retention [103]. Upon irradiation,the Gd chelates enhance the local dose deposition through increased interactions withionizing radiation, where preclinical studies have demonstrated favorable radiotherapeuticeffects [104–106]. Additionally, the enhanced MRI contrast provided by the accumulationof AGuIX NPs in tumor sites facilitates better imaging, improving the quality of treatmentplanning and real-time patient monitoring that can be used to improve adaptive radiothera-Molecules 2024, 29, 2438 18 of 23peutic techniques. A current clinical trial is aiming to exploit these features, coupling AGuIXwith stereotactic magnetic resonance-guided adaptive radiation therapy (SMART) for thetreatment of lung tumors and pancreatic cancer (NCT04789486). This cutting-edge deliverytechnique is designed to provide high-resolution, real-time MR imaging to ensure the pre-cise delivery of stereotactic radiation therapy, thus leveraging AGuIX’s image enhancementand dose deposition capabilities to potentially further improve patient outcomes. Alongwith this study, there are other clinical trials that are investigating the effects of AGuIX andradiotherapy for various types of cancer as shown in Table 1. Despite these studies beingin their early stages, results from a recent phase I clinical study (NCT02820454) indicatedAGuIX’s safety and tolerability, along with potential clinical benefits when combined withradiotherapy [107].5. ConclusionsThe field of nanotechnology has promoted a transformative shift in cancer therapeutics,offering innovative approaches to enhance treatment efficacy and patient outcomes. Amongthese advances, the application of nanoparticles as radiosensitizers presents a promisingfrontier to improve radiotherapy treatment. High-Z nanoparticles have emerged as anovel solution to overcome challenges inherent to radiotherapy, such as normal tissuetoxicity and radiation resistance of tumors. Their unique physico-chemical properties canbe tailored to preferentially target tumors, maximizing the therapeutic impact on tumorswithout inducing additional damage to surrounding healthy tissue. Preclinical studieshave already demonstrated the significant therapeutic benefits that high-Z nanoparticlescan provide, including their potential application as drug delivery systems to furtheraugment the effects of radiotherapy. Yet, the clinical application and efficacy of high-Z NPs remain to be fully investigated, with few clinical trials exploring the effects ofhigh-Z NPs as radiosensitizers. This could be due to several factors, such as the lack ofunderstanding behind the mechanisms of sensitization, safety concerns, and the challengesin the up-scaled synthesis of NPs required for clinical applications. However, as furtherresearch is conducted on NPs, it will elicit a better understanding on their mechanismsof sensitization, their interactions with biological systems, and their potential toxicities,enabling the production of optimized nanoparticle systems to take full advantage of theirabilities to improve radiotherapy. Furthermore, encouraging results in the current clinicaltrials will provide a rationale for high-Z NPs to improve radiotherapy, and may result in ashift towards more high-Z NPs being clinically investigated, laying the foundation requiredto transition their use into radiotherapy treatment regimens to improve the prognosis forcancer patients.Author Contributions: Writing—original draft preparation, N.J., D.C., W.B. and D.B.C.; writing—reviewand editing, N.J., D.C., W.B. and D.B.C. All authors have read and agreed to the published version ofthe manuscript.Funding: This research was funded by a Nanomedicines Innovation Network (NMIN) StrategicInitiative (SI) grant, 2021-RES-SI-05; Natural Sciences and Engineering Research Council (NSERC)Discovery Grant (DG), RGPIN-2017-04501; and Canadian Institute for Health Research (CIHR),CIHR PJT-399879.Data Availability Statement: Data sharing is not applicable.Conflicts of Interest: The authors declare no conflicts of interest.References1. 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Application of High-Z Nanoparticles to Enhance Current Radiotherapy Treatment
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