Calcium is an important mineral which the body uses to maintain strong bones and carry out many important bodily functions. According to the Dutch nutrition center people in the Netherlands consume 80-90% of their calcium through milk and other milk products. Often times people will further ’process’ milk for other food products. Examples of these include boiling milk to make hot chocolate or freezing it for ice-cream. These temperature changes can have great influence on the bio-availability of the Calcium. The goal of this thesis was to find the effect of temperature on the bio-availability of Calcium in milk. This was done through 2 experiments. Dialysing skim bovine milk at temperatures between 4-90°C and heating or cooling the bovine milk to temperatures between -18°C-95°C and then dialysing at room temperature. The dialysis separates the soluble and insoluble phase of milk allowing for the calculation of percentage soluble calcium which is a good indicator for bio-availability of calcium in milk. It was found that there is a linear relationship between the temperature of the milk and the percentage soluble Calcium, the higher the temperature of the milk the lower the amount of bio-available calcium in the milk. Furthermore when looking at the heated milk it was found that heating to 60°C and above all showed a similar drop in percentage soluble calcium indicating some permanent loss of bio-available calcium. This permanent loss is however lower than what can be observed to be lost in the milk whilst hot indicating that a portion of the calcium that becomes insoluble upon heating returns to the soluble phase once returned to its original temperature.

Breast cancer is one of the most dangerous forms of cancer worldwide. Continuous research is therefore essential in order to find realistic treatment options. 3D tumor spheroids have been proven to imitate the structural and physiological TME of in vivo tumors relatively more accurate than the more widely used 2D cell cultures. During this study, the aim was to find the optimal conditions for single spheroid formation, employing breast cancer cells and utilizing GrowDex. Different concentrations and cell densities were used, as well as differing methods (GrowDex addition prior or (days) after cell cluster formation). The liquid overlay technique was used to grow the spheroids, which were observed for a maximum of 14-16 days. Finally, an ATP viability assay was performed to determine whether the spheroids contained a proliferation zone, which corresponds to active cells. Single spheroids were obtained for all cell densities. The relatively high GrowDex concentrations responded well to a cell density of 2000 cells, while the relatively low GrowDex concentrations responded well to cell densities of 5000 and 10000 cells. Through the ATP viability assay the samples illustrated the presence of a proliferation zone, and thus active cells.

In the human body calcium is the most abundant mineral and is involved in a lot of vital processes. Calcium deficiency occurs when there is not enough calcium present in the bloodstream. In the Netherlands one in three women will face this problem in their lifetime. A major factor contributing to this problem is that the intake of calcium on average is below the recommended amount which is around 950 mg/day for adults in the Netherlands. Calcium deficiency when left unattended can lead to osteoporosis. Therefore it is important to consume enough calcium rich products or take calcium supplements. Milk is a good source of calcium and is considered a staple food. This makes bovine milk a good candidate for calcium fortification. Milk can be fortified by using calcium salts to increase the bioavailability the calcium present. In this thesis in vitro methods were used to study the effects of calcium salt fortification on the phase distribution of calcium in bovine skimmed milk under different pH conditions and by using the radioactive isotope 45Ca to track the exchange between the phase casein micelles, serum proteins and the soluble phase when fortifying the milk. For unfortified milk it was found that 36.0±0.9% was present in the soluble phase, 3.7±2.9% was present in the serum proteins phase and 60.3±3.0% in the casein micelles. Acidifying the milk to simulate the gastric environment resulted in an increase of the soluble phase from 55.1±2.7% to 97.0±3.3% which corresponds to a decrease in pH from pH 6 to pH 2. For the fortification of the milk, five calcium salts were selected for synthesis. The salts were calcium carbonate, tricalcium di-citrate, calcium gluconate, calcium lactate and tri-calcium phosphate. The fortification was performed with all of the synthesized salts except calcium lactate and tri-calcium phosphate. The starting material for the syntheses, calcium chloride, was also used for fortification. It was found that for the fortification of CaCO3 under gastric conditions a bioavailability of 100% could be achieved. However the acidic environment also resulted in coagulation of the milk and therefore the bioavailability could not be accurately assessed. Calcium gluconate had the only increase in bioavailability under normal conditions. In conclusion a better understanding of fortification with calcium salts was achieved and the influence of pH on the bioavailability of calcium in milk and the effects on the salt fortification.

Because diagnostics have improved tremendously, nowadays breast cancer is more frequently detected at an early stage. Long-term prognosis is excellent and therefore the new treatment should focus on minimizing long-term side effects and treatment burden. This research proposes cancer treatment using nanoparticles (NPs) for a combination of heat therapy and brachytherapy. For the NP design, superparamagnetic iron-oxide nanoparticles (SPIONs) will be used. Because of their biocompatibility and good magnetic properties, they are suitable candidates for heating using an alternating magnetic field. To make the NPs applicable for brachytherapy, the NPs are doped with holmium (Ho), a lanthanide with high magnetic moment and radioisotope Ho-166. Furthermore, SPIONs and Ho change the relaxivity of water protons and therefore they can be used as contrast agents for magnetic resonance imaging. This makes it possible to image the NPs during the treatment and enables thermal planning and dose-rate calculations delivered to the cancer tissue to be performed. The incorporation of Ho inside the iron-oxide structure is expected to change the magnetic properties of the NPs. The success of using Ho as contrast agent and for brachytherapy purposes have been proven in several researches. However, the influence of Ho on the heating performance of these SPIONs is unknown. This research will characterize the magnetic properties of undoped and Ho-doped SPIONs and compare them, in order to examine the heating possibilities of Ho-doped NPs. For the synthesis of the NPs a spark discharge generator was used. Spark ablation is a synthesis method able to yield narrow size distribution of different metallic particles and alloys. However, since spark ablation is relatively new, the synthetic methodology itself had to be developed. Therefore, this research was divided into two parts: 1) the synthesis of NPs using spark ablation and 2) the characterization of Ho-doped iron NPs and the comparison with undoped SPIONs. Since the first part focused on the spark ablation synthesis, the influence of the generator settings on the primary particle size was investigated using DLS and TEM. The VS-Particle generator was able to produce 4 nm Fe-Fe and Ho-Fe NPs, a size that is smaller than expected. The generator settings did not influence the primary particle size and therefore, the settings of the generator were chosen to maximize the yield. The initial set-up had low yield and therefore, the influence of micro-bubbler, electrode configuration and bubbling column were examined and changed to improve the yield. A bronze sintered filter with pore size 0.4-20 ��, solid iron electrodes and 60 mL bubbling column volume appeared to be the most optimal. During the second part of this research, 4 nm Ho-doped iron oxide NPs and un-doped iron oxides were produced. They were characterized using TEM, DLS, XRD, ICP, SEM-EDX, Mössbauer spectroscopy, SQUID and NMR. As a result of low concentrations obtained, no good qualification of the produced particles could be obtained. All particles showed superparamagnetic behavior and influenced the relaxivity times of water protons. To what extend the relaxivity was changed upon doping remains unfortunately unclear, because the composition of the NPs is unknown. It can be concluded that the used set-up was not able to produce reproducible NPs and therefore no reliable characterization could be done on the Ho doped particles. Future research should focus on a more controllable synthesis method to be able to examine the possibilities of Ho for heating purposes.

Radionuclides are important in the diagnosis and treatment of, amongst others, cancer. A promising therapy is targeted radionuclide therapy, where the radionuclide is brought to the tumor by a targeting specific vector. Bringing the radionuclide directly to the tumor, should reduce the dose to the healthy tissue. For targeted radionuclide therapy, a radionuclide with a high specific activity is required. Some radionuclides, with promising half-lives and decay energies, are currently not produced with the required specific activity. This problem occurs mainly for radionuclides that are produced via (n,γ) reactions in nuclear reactors. For radionuclides that are produced via this reaction, it is nearly impossible to do a chemical separation between the target material and the produced radionuclide, because these are of the same element. This is why new production routes have to be investigated. In this thesis a feasibility study has been done for a new production method, which should increase the specific activity of radionuclides that are produced via a (n,γ) reaction. For this production method, the target material is labeled with a chelator. Due to the Szilard-Chalmers effect the bond with the chelator will be broken, when the target material is activated by a neutron. This enables the separation of the produced radionuclide from the target complex and therefore, the extraction of the radionuclide. The production method will be loop-based, in order to enable continuous activation of the target material and extraction of the radionuclide. Furthermore, the loop-based design should minimize the effect of radiolysis and relabeling. The loop will be placed close to the reactor core. In this thesis, the elements holmium and lutetium have been used. It has been determined which chelator is most suitable to label with holmium and lutetium. Furthermore, the stability of this complex has been investigated for: higher temperatures, time and the effect of the γ-radiation. The effect of the γ-radiation was determined because this results in radiolysis. The extraction of the radionuclide has also been investigated. These parameters have been used in the calculation of the possible achievable specific activity of 166Ho and 177Lu, when using this loop-based production method. Labeling was possible with the chelator DOTA, which resulted in a stable complex, even for higher temperatures. Labeling happened fast, which also results in fast relabeling. The effect of radiolysis, due to the γ-radiation, was determined by fitting the experimental data. The fit gave negative values for short irradiation times, which is not possible. Therefore, two possible fits were made, which did not give negative results. The first fit was shifted over the y-axis and for the second fit the negative values were assigned to be zero....

Anaemia affects a third of women and almost 50% of infants and toddlers worldwide. Iron deficiency is the most common cause of anaemia, and the most common nutritional disorder in the world. To increase dietary iron intake, iron fortification of staple foods commonly consumed by the general public is an effective approach to prevent and control iron deficiency anaemia. It is important to maximise the iron bioavailability (absorption by the human body) in these fortified products, since iron overload is also known to cause Since the diet of infants and toddlers revolves around milk, milk is a good food carrier to address iron deficiency anaemia in children. The aim of this project is to assess the influence of five factors on the bioavailability of iron fortified skim milk: (1) iron concentration, (2) molecular form of the added iron, (3) storage time after fortification, (4) the addition of an iron enhancer and (5) the addition of an iron inhibitor. Skim milk was fortified with FeC l2 and FeC l3 to 5 and 20 mmol Fe L−1 and stored for 24 hours or one week. Part of the milk was further left untreated and to another part ascorbic acid (vitamin C) or tannic acid were added as respectively iron enhancer and iron inhibitor. The bioavailability of the iron in these milk samples was assessed on phase distribution and oxidation state, since it is believed that iron in the soluble phase of the milk and in the oxidation state Fe2+ is more bioavailable. Analysis showed that the difference in iron concentration induced a pH decrease, which was greater for the higher concentrations of iron and the molecular form FeC l3. The pH difference caused the percentage of iron present in the casein fraction to increase, in the protein fraction to decrease and did not influence the iron in the soluble phase. It was also found that the 20 mmol L −1 FeC l2 showed more iron in the soluble phase and more Fe2+, whereas no statistical difference was observed for the 5 mmol L −1 , which might also be due to the pH difference. The increase in storage time to one week was reflected by an increase in iron bound to the caseins. This shift was not reflected in a change of oxidation state. The addition of the iron enhancer (ascorbic acid) increased the iron present in the protein fraction and soluble phase, which was strengthened by the further decrease in pH. The chelation of iron by ascorbic acid did not necessarily reduce Fe3+ tot Fe2+; the Fe2+ decreased for the 20 mmol L −1 FeC l2, whereas it increased for the 20 mmol L −1 FeC l3. Lastly the addition of the iron inhibitor resulted in a significant decrease of Fe2+. Most iron was bound to the casein or precipitated as iron-tannic acid complex. Overall, this research confirmed that the bioavailability of milk is not easily evaluated, since the behaviour of iron in milk depends on many different factors that can not be studied isolated. Only by evaluating many possible combinations of concentration, molecular forms, additives and storage conditions, the bigger puzzle can be solved. This project attributed to a part of the solution and is therefore a valuable contribution in the development of bioavailable milk products, that will reduce iron deficiency anaemia in children.

A radioisotope used in radionuclide therapy is Holmium-166 (Ho-166). The treatment effectiveness of Ho-166 could be improved by the use of a so called in vivo Dysprosium-166(Dy-166)/Ho-166 generator. The application of this generator is hindered by an effect called internal conversion (IC). This affect can arise after the decay of Dy-166 to Ho-166, which can cause separation of Ho-166 from its carrier. Polymeric micelles might form a solution in the application of the Dy-166/Ho-166 generator in radionuclide therapy. The main goal of this thesis was to investigate and understand the loading mechanism of metallic species and polymeric micelles with a focus on the loading of Dy/Dy-166 and Ho-166. It was found that it was not effective to load metallic species (Dy/Dy-166) as free ions or as solid precipitates. Loading metallic species as aqueous hydroxides showed to be crucial for achieving a good loading and high stability. The second goal was to study if polymeric micelles were able to retain Ho-166 inside their core under the effects of internal conversion. No additional losses of Ho-166 were found when Dy/Dy-166 and Ho-166 were loaded into the micelles. It was concluded that the PCL-PEO micelles prevented the loss of Ho-166 under internal conversion effects.

Calcium is an important nutrient for both young and elderly people which can, among others, stimulate healthy bone growth and prevent osteoporosis. Therefore, nutrition with sufficient calcium is of great importance. The highest calcium nutrient density for all foods can be found in bovine milk, which contains on average 1.2 g Ca / L. To increase the amount of bioavailable calcium, milk can be fortified by the addition of different calcium salts. In this research the bioavailability and exchangeability of calcium in unfortified and fortified skimmed milk was studied by in vitro methods using equilibrium dialysis and ultracentrifugation to separate different fractions in milk. Radioactive 45Ca was used to follow the exchange behaviour of calcium between the three different fractions in milk: casein micelles, serum proteins, and soluble calcium. For unfortified milk it was found that 34.9 ± 2.2% of the calcium was present in the soluble phase, 9.1 ± 0.6% was bound to serum proteins, and 56 ± 1.6% was present in the casein micelles, which confirmed the results in literature. In addition, with the investigation of the exchange behaviour of 45Ca, it was confirmed that ~40% of calcium in casein micelles is hardly exchanged.To investigate the influence of calcium fortification on the calcium equilibrium in milk, six different calcium salts were selected to be synthesized: calcium chloride, calcium carbonate, calciumgluconate, tri-calcium di-citrate, calcium lactate, and tri-calcium phosphate. Of these, calcium chloride, calcium carbonate, calcium gluconate, and tri-calcium di-citrate were successfully synthesized. Further experiments were performed with calcium chloride and calcium carbonate, intrinsically labelled with 45Ca. From fractionation with ultracentrifugation, it was concluded that addition of calcium chloride led to an increase of calcium in the soluble phase, which might indicate an increase in bioavailability. Fortification of milk with 45Ca labelled calciumcarbonate did not lead to an exchange of 45Ca with the calcium present in milk, indicating that calcium carbonate is unlikely to make calcium in milk more bioavailable. In conclusion, a better understanding of the solubility and exchangeability of Ca in milk and the influence of milk fortification with calcium chloride and calcium carbonate is obtained by using 45Ca as a tracer.

Background: Cancer is one of the leading causes of death worldwide. Targeted radionuclide therapy has become a important treatment, this is when a carrier molecule is attached to a radionuclide to deliver cytotoxic radiation levels to diseased cells. β − emitters are frequently used in RNT because they have a long penetration depth, such as the dysprosium-166/holmium-166 (166Dy/166Ho) in vivo generator. The 166Dy/166Ho in vivo generator shows great potential for large tumors because of the long half-life time of the mother nuclide 166Dy and the emission of high energy β− from the daughter nuclide 166Ho. Previous research shows the release of 72 % of the 166Ho when 166Ho is bound to conventional chelators due to internal conversion after the β− decay. The aim of this thesis is to synthesize an iron oxide nanoparticle as carrier for the 166Dy/166Ho in vivo generator. This iron oxide nanoparticles (SPIONs) should prevent internal conversion and thus the loss of the daughter nuclide 166Ho. Results: The dysprosium doped iron oxide nanoparticles were synthesized successfully with an average diameter of 6.1 ± 1.5 nm, measured with TEM. The DLS and TEM results showed that the SPIONs were aggregating. The labeling efficiency represents how much Dy is retained in the Dy doped SPIONs, for the non-radioactive Dy this was measured with the ICP/OES. The labelling efficiency was 3.21 ± 0.8 %. The 166Dy doped iron oxide nanoparticles were also synthesized successfully with a radiolabelling efficiency of 3.12 ± 2.2 %, measured with a 2480 Wizard 2 Gamma counter. The retention of 166Dy + 166Ho was 79.2 ± 1.8 % after 96 h. Conclusion: The retention of 166Dy + 166Ho in iron oxide nanoparticles was 79.2±1.8 % after 96 hours. This is much higher than the retention of 28% by conventional chelators. This means that the iron oxide nanoparticls is a safer carrier for the 166Dy/166Ho in vivo generator than the conventional chelator.

Combining different treatment modalities has proven to improve patient outcome compared to applying single treatments. Consequently, mild hyperthermia (HT) and radiotherapy (RT) are combined to radiosensitize tumour cells by inhibiting DNA damage repair mechanisms. In this study, it was investigated if combining both treatment modalities shows an improved therapeutic effect on 3D tumour models made of FaDu cells. To this end, the spheroid size and cell viability were measured over time after treatment. It was also attempted to obtain further qualitative information of the effect on the spheroids using confocal microscopy. During experiments spheroids were formed of FaDu tumour cells, cells of a head-and-neck squamous cell carcinoma, using Matrigel®. These spheroids grew fast within the first 10 days of seeding up to a size of 550-600 𝜇m reliably. These spheroids were then exposed to both single HT and RT as well as combined HT and RT. Both single treatment modalities showed a decrease in cell growth and cell viability with increasing treatment dose. The thermal doses of 30 and 120 CEM43 and radiation doses of 2 and 6 Gy were chosen to be used in the combined treatment experiments. Combined treatment showed an improved effect on the cell growth and cell viability for most treatment groups, both for HT followed by RT and the other way around, with the determining factor seemingly being the thermal dose. The thermal dose of 120 CEM43 combined with 6 Gy of radiation dose showed the highest cell killing potential, with 9±2% of the cell viability remaining when using radiotherapy before hyperthermia and 18±3% when starting with HT before using RT. The most significant difference in effect due to the order in which RT and HT were administered was seen in the group treated with 2 Gy of radiation dose and 120 CEM43 of thermal dose. In this group RT preceding HT resulted in a cell viability of 13±2% on day 7 after treatment, whilst HT preceding RT resulted in a cell viability of 48±8%. Visual inspection of the spheroids showed an increased effect in the form of less growth, as well as flaking around the edges of the spheroids. Obtaining qualitative information on the effect that combined treatment had on spheroids using confocal microscopy was unsuccessful. Both using a live and dead cell staining kit, as well as PI staining proved ineffective in accurately visualizing the dead cells in spheroids.

In radionuclide therapy, radioisotopes are used to irradiate tumours from within the body. Usually beta-emitters coupled to tumour-targeting molecules are used, which specifically accumulate at the tumour site. Instead of using beta-emitters, it is also possible to use radionuclides which emit an alpha particle upon decay. Alpha particles have a shorter range and are much more effective in destroying tumour cells. Alpha radionuclide therapy is steadily gaining interest, although currently in most studies radionuclides with relatively short half-life are used. Long lived radionuclides like the 225Ac employed in this thesis are ideal for the treatment of tumours which take a longer time to reach. The long halflife of 225Ac combined with four alpha particles in its decay chain ensure long irradiation of the targeted tissue. However, upon alpha-decay the daughter nuclide receives a recoil energy decoupling it from any targeting agent, allowing it to diffuse throughout the body to irradiate healthy tissue. The main goal of this thesis is to develop polymeric nanocarriers, so-called polymersomes, which retain the recoiling daughter atoms of 225Ac in order to limit healthy tissue toxicity in alpha radionuclide therapy.@en

Cancer is one of the leading causes of death worldwide and the number of cases is expected to keep increasing in the next years. Even though nowadays most employed cancer treatments in clinical practice (surgery, chemo-, and radiotherapy) are effective, they are still associated with multiple limitations and side effects. The main pitfall stays in their non-specificity to tumour cells, which leads to affecting healthy tissues. Therefore, alternative treatments able to overcome the oncologic challenges of the current treatment regimens by specifically treating only the cancer cells, be minimally invasive, and limit short and long-term side effects are highly needed. As the number of patients diagnosed with cancer in incipient stages is constantly increasing, such alternative treatments are currently even more attractive. Due to the advances in nanotechnology, cancer nanomedicine is a fast-advancing field, employing nanoparticles to both diagnose and deliver therapy of cancer, namely, nanotheranostics. Nanobrachytherapy is the brachytherapy treatment delivered via injection of radioactive nanoparticles into the tumour. The great advantage is that nanobrachytherapy retains the characteristics of brachytherapy, such as precise and targeted dose delivery, while allowing a less invasive administration and a more uniform dose distribution in the tumour. However, the radio-resistance exhibited by the tumour cells can hinder the success of nanobrachytherapy, but the synergetic combination of cell damaging agents, as well as radioactivity and heating, is wellknown. Thermal treatments, such as hyperthermia, offer a hyperthermic radiosensitization making the tumour more susceptible to irradiation, while thermal ablation can serve as surgery replacement. Furthermore, thermal treatments can be delivered by injection of colloidal suspensions of magnetic nanoparticles (MNPs) in tumours and heating them via exposure to an externally applied alternating magnetic field. An additional advantage of such magnetic nanoparticles is their ability to ensure visualization via magnetic resonance imaging (MRI), a non-invasive technique, helpful in monitoring the treatment effects. This thesis aims to develop a nanotheranostic agent, able to deliver therapeutic effects via radiation and heating, with additional imaging via magnetic resonance imaging. We envision the nanotheranostic as a core-shell hybrid nanoparticle in the form of 103Pd:Pd/Fe-oxide. The palladium core is radiolabelled with 103Pd radioisotope, responsible for the required radiation dose, whereas the iron oxide coating ensures hyperthermia/thermal ablation and imaging…@en

Computed Tomography (CT) imaging is an important step in treatment planning for proton therapy. The conversion from Hounsfield Unit (HU) to stopping power ratio (SPR) accounts for more than half of the 3.5% error that is considered in treatment planning. The aim of the study is to investigate the efficacy of photon-counting CT (PCCT) for predicting the SPR in proton therapy. Its performance will be compared to single-energy CT (SECT) and dual-energy CT (DECT). Fresh tissue samples—steak, ground beef, and bone marrow—were secured in 3D-printed holders to minimize air inclusion and maintain tissue stability during measurements. These samples were secured in a head and a body configuration of a phantom and CT scans were conducted using standard clinical protocols. SPR values were derived from the CT data using automated DirectSPR software for the DECT and PCCT scans, and converted from HUs to SPR using a Hounsfield look up table (HLUT) for the SECT scans. Results were validated against actual SPR measurements obtained via proton beam measurements. Two measurement series have been performed. For the validation of the first measurement series, proton beam energies of 150 and 175 MeV both resulted in an SPR value of 1.00 for the ground beef sample. For the steak sample, SPR values of 1.02 and 1.03 were found. The CT-based SPR predictions resulted in errors up to 1.5% for ground beef and 3.5% for steak. For the validation of the second measurement series, SPR values for ground beef (0.89-0.92), steak (0.96-0.98), and bone marrow (0.87-0.91) were obtained. The percentage error between the CT-based SPR predictions and the validation measurements showed no substantial differences between the CT-modalities, except for SECT providing lower SPRs for the bone marrow samples than DECT and PCCT. The validation measurements of the second series resulted in lower SPR values than found in literature and the first series, indicating possible methodological inconsistencies. Factors such as sample heterogeneities and measurement errors might have contributed to these inconsistencies, but do not account for these inconsistencies completely. These results highlight the need for further research with standardized phantoms and animal tissue samples to evaluate PCCT’s true potential in combination with DirectSPR prediction. From this study it cannot be concluded that PCCT demon- strates an advantage over SECT and DECT as CT-modality for the HU-SPR conversion in proton therapy treatment planning

Computed Tomography (CT) imaging is an important step in treatment planning for proton therapy. The conversion from Hounsfield Unit (HU) to stopping power ratio (SPR) accounts for more than half of the 3.5% error that is considered in treatment planning. The aim of the study is to investigate the efficacy of photon-counting CT (PCCT) for predicting the SPR in proton therapy. Its performance will be compared to single-energy CT (SECT) and dual-energy CT (DECT). Fresh tissue samples—steak, ground beef, and bone marrow—were secured in 3D-printed holders to minimize air inclusion and maintain tissue stability during measurements. These samples were secured in a head and a body configuration of a phantom and CT scans were conducted using standard clinical protocols. SPR values were derived from the CT data using automated DirectSPR software for the DECT and PCCT scans, and converted from HUs to SPR using a Hounsfield look up table (HLUT) for the SECT scans. Results were validated against actual SPR measurements obtained via proton beam measurements. Two measurement series have been performed. For the validation of the first measurement series, proton beam energies of 150 and 175 MeV both resulted in an SPR value of 1.00 for the ground beef sample. For the steak sample, SPR values of 1.02 and 1.03 were found. The CT-based SPR predictions resulted in errors up to 1.5% for ground beef and 3.5% for steak. For the validation of the second measurement series, SPR values for ground beef (0.89-0.92), steak (0.96-0.98), and bone marrow (0.87-0.91) were obtained. The percentage error between the CT-based SPR predictions and the validation measurements showed no substantial differences between the CT-modalities, except for SECT providing lower SPRs for the bone marrow samples than DECT and PCCT. The validation measurements of the second series resulted in lower SPR values than found in literature and the first series, indicating possible methodological inconsistencies. Factors such as sample heterogeneities and measurement errors might have contributed to these inconsistencies, but do not account for these inconsistencies completely. These results highlight the need for further research with standardized phantoms and animal tissue samples to evaluate PCCT’s true potential in combination with DirectSPR prediction. From this study it cannot be concluded that PCCT demon- strates an advantage over SECT and DECT as CT-modality for the HU-SPR conversion in proton therapy treatment planning