Comparison of cytotoxicity, radiosensitizing properties and cellular uptake of palladium nanoparticles stabilized with commercial chitosan and chitosan isolated from honey bees

Comparison of cytotoxicity, radiosensitizing properties and cellular uptake of palladium nanoparticles stabilized with commercial chitosan and chitosan isolated from honey bees | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of cytotoxicity, radiosensitizing properties and cellular uptake of palladium nanoparticles stabilized with commercial chitosan and chitosan isolated from honey bees Bartosz Klebowski, Radosław Piech, Kamil Sobczak, Marianna Gniadek, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7643260/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 22 You are reading this latest preprint version Abstract Recently, there has been increasing interest in the use of biocompatible natural polymers, especially polysaccharides, in the synthesis of nanoparticles (NPs). The undoubted advantages of such carbohydrate polymers as components of NPs include their well-defined chemical structure, biodegradability and widespread availability. This study reported the development of an effective method for the synthesis of biocompatible chitosan-stabilized palladium nanoparticles (Pd NPs). For this purpose, both commercial chitosan and chitosan isolated from honey bee corpses were used. Spherical ⁓ 20 nm Pd NPs I and ⁓ 40 nm flower-like Pd NPs II were obtained when commercial chitosan and chitosan isolated from bees (green synthesis method) were used, respectively. In vitro studies on selected glioblastoma cell lines (LN229 and U118) indicated that both types of Pd NPs have similar cytotoxicity, however Pd NPs I are characterized by improved radiosensitizing properties compared to Pd NPs II. Furthermore, real-time holotomographic observations of cells interactions with Pd NPs showed that (for the same concentrations) Pd NPs II generate more visible changes in cell morphology, including their flattening, which is particularly observed for LN229 cells. In summary, Pd NPs I seem to be more promising nanosystems for biomedical applications as radiosensitizers than Pd NPs II. chitosan palladium nanoparticles green synthesis honey bees glioblastoma radiosensitizers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Natural polymers are increasingly becoming the object of interest of scientist, especially in the context of their potential application in biomedicine. The unquestionable advantage of such natural polymers over synthetic polymers is their biodegradability, common occurrence in nature, mucoadhesive properties, as well as environmental friendliness. 1 – 3 Among natural polymers, carbohydrate polymers are particularly popular in medical-related applications. 4 One of the most popular carbohydrate polymers is chitosan. Chitosan – a product of chitin deacetylation – is a linear cationic polysaccharide composed of randomly distributed β-(1,4)-D-glucosamine and N-acetyl-D-glucosamine. 5 The presence of the –NH 3 amino group in the structure of chitosan determines its solubility in slightly acidic solutions, such as dilute acetic or formic acid. 6 Moreover, the reactive amino and hydroxyl groups in chitosan determine the possibility of relatively simple modification of this biopolymer, which may ultimately contribute to obtaining structures with improved solubility or thermal and mechanical properties. 7 , 8 The presence of these functional groups also provide an opportunity to attach drugs, which enables the use of chitosan-based nanomaterials as drug carriers. 9 – 11 Currently, chitosan is obtained from marine food industry waste, especially shrimps, crabs, krill or crayfish shells. 12 , 13 At the same time, the search for alternative sources of chitosan is ongoing, and honey bees ( Apis mellifera ) may be one of the solutions. 14 – 16 The average lifespan of a worker bee in the summer season is 2 months, while in the case of workers staying in the hive for the winter, the lifespan can be up to half a year. 17 After this time, there is a rapid replacement of workers in the hive, which is called spring die-off. It should also be noted that with environmental changes, there are more and more diseases and threats that cause significant losses in apiaries. 18 For this reason, dead bees, due to the lack of other use, may prove to be an interesting alternative to traditional sources of chitosan. The process of isolating chitosan from bees may include both chemical and enzymatic methods using chitin deacetylases that convert chitin to chitosan. 19 , 20 Chitosan can be successfully used in biomedical application due to its anti-microbial, 21 anti-oxidant (Li et al., 2019) 22 and anti-inflammatory 23 properties. However, significant attention is currently focused on the use of chitosan-based nanomaterials for applications in cancer treatment, e.g. as drug (chemotherapeutic agents) delivery systems, 24,25 radiosensitizers 26 , 27 or photosensitizers 28 , 29 in radiation/photo-based therapies. Importantly, chitosan can be either the core of the nanomaterials (e.g. nanoparticles, NPs) or only a stabilizer of other nanomaterials, e.g. metallic ones. Although there are numerous reports on the applications of chitosan-based NPs in anticancer therapies, the effect of the chitosan source on the morphology of chitosan-stabilized metallic NPs, as well as its effect on the biological activity of such NPs, is unknown. This is an aspect worth attention because it is widely known that the biological activity of nanosystems is influenced by many factors, such as their size, 30 shape, 31 charge, 32 crystal structure, 33 and even the method of synthesis. 34 Taking the above issues into account, in this work a hypothesis was put forward that the use of chitosans from different sources (commercially available chitosan vs chitosan isolated from honey bees) would result in obtaining NPs with different morphology, and moreover, these NPs would be characterized by diverse activity as radiosensitizers in vitro . Herein, it was planned to investigated whether, depending on the source of chitosan, there would be a difference in the morphology and crystal structure of NPs in the synthesis of which chitosan acted as both a reducing agent and a stabilizer. Palladium nanoparticles (Pd NPs) were selected for this purpose due to their promising potential in biomedical applications and relatively low cost of production (compared to the more popular gold or platinum-based NPs). 35 In this paper, the cytotoxicity and radiosensitizing properties of chitosan-stabilized Pd NPs in simulated proton radiotherapy (PRT) were investigated. LN229 and U118 cell line, derived from a grade IV glioblastoma, were used as an in vitro model. Finally, novel three-dimensional holotomographic imaging was also used to observe the real-time interaction of glioblastoma cells with Pd NPs, which enabled the assessment of morphology changes induced by these Pd NPs, as well as locating the preferential site of accumulation of chitosan-stabilized Pd NPs in the cells. 2. Materials and methods 2.1. General remarks Commercial chitosan (2.6∙10 6 g/mol – based on viscosity, deacetylation degree 62%), isolated chitosan (9.8∙10 5 g/mol – based on viscosity, deacetylation degree 67%), palladium (II) chloride, ascorbic acid, high-glucose DMEM (Dulbecco’s Modified Eagle Medium) were purchased from Sigma Aldrich (Burlington, MA, USA); LN229 and U118 glioblastoma cells – from ATCC (American Type Culture Collection, Manassas, VA, USA); 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test kit (Cell Titer96® Aqueous One Solution Cell Proliferation Assay) – from Promega (Madison, WI, USA). 2.2. Isolation of chitosan from honey bees 2.2.1. Samples collection The corpses of honeybees ( Apis mellifera carnica , Nyska line) were used as a source of chitosan. The bees came from a home apiary, and the reason for their death was difficult weather conditions in the winter of 2023/2024. Large temperature fluctuations in short periods of time, combined with high humidity, caused losses of 60% national population. Before initiating the isolation procedure, preliminary purification was carried out by removing visible fragments of beeswax and other solid elements. The pre-purified honeybee corpses were stored in a cool and dry place until subsequent experiments. 2.2.2. Isolation procedure The chitosan isolation process was prepared based on previous literature reports 36 , 37 with some modifications. The general procedure for isolating this polysaccharide is illustrated in Fig. 1 . Stage I – Pre-purified bee corpses were crushed in a laboratory mortar, and then 10 g of biological material were placed in a laboratory oven for 10 h (55 o C). The isolate obtained in this way was weighed and the yield of subsequent stages of chitosan isolation was calculated in relation to the determined weight ( isolate A ). Stage II – The dried bees were transferred to a beaker and then poured with 250 ml of 2 M hydrochloric acid (HCl). The Hackman demineralization reaction was carried out for 2 h at room temperature with intensive stirring. After this time, the mixture was filtered using fluted filter paper and then rinsed with water until the remaining precipitate was neutralized. Finally, the precipitate was placed in a laboratory oven (55 o C) and dried overnight ( isolate B ). Stage III – The dry isolate from the previous stage was deproteinized. For this purpose, the isolate was placed in a round-bottom flask. Then 1 M sodium hydroxide (NaOH) was added (15 ml/1 g of isolate) and the reaction mixture was placed on a heating mantle. The deproteinization process was continued for 2 h at 80 o C with gentle stirring. After this time, the mixture was filtered and dried (as before). This time, the filtered precipitate (after neutralization) was additionally rinsed with ethanol to remove lipids ( isolate C ). Stage IV – Obtained isolate was discolored using hydrogen peroxide (H 2 O 2 ). For this purpose, isolate was resuspended in water (15 ml/1 g of isolate) in a round-bottom flask placed on the heating mantle. The reaction mixture was heated to 70 o C (with gentle stirring), then 30% H 2 O 2 was gradually added until its final concentration in the flask was adjusted to 1%. The filtration and drying process was performed as before ( isolate D ). Stage V – To deactylate chitin, the isolate was placed back in the round-bottom flask on a heating mantle, followed by treated with 50% NaOH (200 ml/1 g of isolate) for 3 h at 90 o C with intensive stirring. The mixture was purified by filtration and then dried as before ( isolate E ). All isolates were stored in a dry and cool place until subsequent experiments (FTIR analyses and use for chitosan-mediated Pd NPs synthesis). 2.3. Synthesis of chitosan-stabilized Pd NPs Both commercial chitosan (Pd NPs I) and chitosan isolated from bees (Pd NPs II) were used to synthesize Pd NPs (Fig. 2 .). Due to the fact that chitosan is insoluble in water, the reaction was carried out in a slightly acidic environment, which allows the protonation of the amino groups of chitosan, making it soluble. 38 The synthesis was performed according to the procedure described in the previous paper 39 with some modification. For this purpose, 15 ml of distilled water, 50 mg of ascorbic acid and finally 10 mg of chitosan were placed in a beaker at room temperature. After that, 10 ml of aqueous 0.01 M chloropalladic acid (H 2 PdCl 4 ) solution (prepared by converting PdCl 2 with the HCl equivalent) were added. A gradual change in color from yellowish to light brown to almost black was observed within a minute. The reaction was left without stirring at room temperature for 2 h, after which the Pd NPs were collected by centrifugation (18 000 rpm, 10 minutes), washed in distilled water and centrifuged again. Pd NPs solutions were stored at room temperature until further studies. 2.4. Physicochemical characterization Fourier-transform infrared spectroscopy (FTIR) spectra of isolates from individual stages of chitosan isolation were obtained using Nicolet™ iS50 FTIR (Thermofisher, Waltham, MA, USA) enabling measurements with the attenuated total reflectance (ATR) method. Dried powder isolates were placed on an ATR crytal and then pressed with calcium fluoride (CaF 2 ) window to reduce potential noise in the spectra. Spectra were collected in the wavenumber range 4000 cm − 1 – 400 cm − 1 at a spectral resolution of 4 cm − 1 using 32 scans for each sample. The deacetylation degree (DD) of chitosan (both commercial and isolated) was also estimated by FTIR. For this purpose, the method described previously in the literature 40 was used. Knowing that: where DA is degree of acetylation (DA) calculated from the following equation: $$\:\frac{{\text{A}}_{1320}}{{\text{A}}_{1420}}\:=\:0.3822+0.0313\:DA$$ where A 1320 and A 1420 are the absorption bands at 1320 cm − 1 and 1420 cm − 1 , respectively, DD was determined. Viscometry, combined with the Mark-Houwink equation, was used to determine the average molecular weight of both types of chitosans. The viscosity parameters were measured at room temperature using rotational viscometer HAAKE Viscotester D (Thermofisher, Waltham, MA, USA). For this purpose, 0.1 g of both commercial or isolated chitosan were dispersed in 75 ml of 0.5 M aqueous solution of acetic acid. Solutions were stirred before viscosity measurements for 2 h. The determined dynamic viscosity was calculated to intrinsic viscosity (assuming the dynamic viscosity of pure solvent is 1 mPa∙s) and then the molecular weight of chitosan was estimated using the Mark-Houwink equation: $$\:\left[\text{ƞ}\right]=\:\text{K}\bullet\:\:{M}^{\alpha\:}$$ where [ƞ] is the intrisinic viscosity, M is the molecular weight, K and α are parameters characteristic of a specific polymer, solvent and temperature. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analysis enabled the evaluation of the morphology (size and shape) of the Pd NPs. For STEM analyses, purified and sonicated Pd NPs samples were dropped on a Cu carbon film coated TEM grid. Then, the grid was rinsed with fresh ethanol and dried at room temperature. Just before placing the samples into the STEM holder, they were cleaned in a plasma cleaner for 3 second. Based on the STEM images (⁓ 60 NPs), the size distribution of Pd NPs was estimated. For this purpose, FEI Talos TEM (Waltham, MA, USA) operating at 200 kV equipped with a field emission gun (FEG) cathode was used. Selected area electron diffraction (SAED) patterns were also taken in the STEM mode to determine the crystal structure of the Pd NPs. 2.5. Cell culture Two human glioblastoma cell lines (LN229 and U118) were used as an in vitro model. Both cell lines were continuously cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic mixture (penicillin, streptomycin, neomycin). Cells were cultured at 37 o C in a humid atmosphere of 5% CO 2 . Both glioblastoma cell lines were tested for Mycoplasma sp . contamination with negative results. 2.6. Proton irradiation protocol Proton irradiation was conducted using Proteus C-235 isochronus cyclotron, located in the Cyclotron Centre Bronowice. Irradiation was carried out at room temperature with dose 2 Gy (the standard dose used in radiotherapy of glioblastoma) for MTS test and additionally 1, 5 and 10 Gy for clonogenic assay. For irradiation of cells monoenergetic field with an energy of 225 MeV and dimension 20 cm x 20 nm was selected. Irradiations were performed at 1.1 cm water equivalent depth, which consist of a 1 cm RW3 phantom plate. Proton irradiation was preceded by dosimetric measurements with Markus type ionization chamber calibrated in terms of absorbed dose to water. For proton irradiation, both Pd NPs were added to the glioblastoma cells 3 h before irradiation and were incubated for another 21 h before the MTS viability test was carried out. 2.7. MTS cytotoxicity assay MTS assay was selected to determine the cellular metabolic activity. For this purpose, LN229 and U118 cells were seeded into 96-well plates (⁓8 ∙ 10 3 cells in 100 µl high-glucose DMEM medium supplemented with 10% FBS) and then incubated for 24 h. After that time, the medium was removed and 100 µl of new medium containing Pd NPs with concentration of 10, 25, 50, 75 and 100 µg/ml were added. This experiment was carried out in triplicate for each trial. Cells cultured in medium without Pd NPs served as a control. The cells were incubated for 3 and 24 h. Next, the medium was re-changed and additionally 20 µl of MTS reagent was added to each well and incubated for the following 3 h at 37 o C. Then, cell viability was determined by measuring the absorbance at 490 nm using 96-well plate reader (Spark® Tecan, Mannedorf, Switzerland). Cell survival (CS) was estimated as: $$\:\text{C}\text{S}\:=\:\frac{{ABS}_{X}}{{ABS}_{Ctrl}}\:\times\:100\%$$ when ABS x – absorbance of cells exposed to Pd NPs; ABS ctrl – absorbance of control cells (without NPs). To assess the radiosensitizing properties of Pd NPs using the MTS test, a critical concentration of Pd NPs (i.e. not causing a decrease in survival by more than 20%) was selected – 25 µg/ml for both Pd NPs. 2.8. Clonogenic assay The clonogenic assay was used to assess the ability of cells exposed to proton radiation and/or Pd NPs to form treatment-resistant colonies. For this purpose, cells were trypsynized and plated in Petri dishes at different densities depending on the stringency of the proton irradiation (from 500 to 3000 cells/dishes). The cells were left overnight and then the medium was replaced with a new one containing Pd NPs at a concentration of 25 µg/ml (the control did not containg Pd NPs). 3 h later, the cells were irradiated with various doses of radiation (1, 2, 5 and 10 Gy). After 10 days of incubation, the cells were fixed with 5% formaldehyde for 15 minutes, then rinsed with distilled water and treated with 1% crystal violet. 10 minutes later, the cells were washed again with distilled water and viable cell colonies, i.e. those consisting of at least 50 cells, were counted. The cell survival fraction (SF) was calculated as follow: $$\:\text{S}\text{F}=\:\frac{{PE}_{X}}{{PE}_{Ctrl}}\:\times\:100\%$$ when PE x – plating efficiency of cells exposed to Pd NPs/irradiation; PE ctrl – plating efficiency of control cells. The platting efficiency is defined as the ratio of the number of viable colonies to the number of seeded cells (we assume that the calculated PE for the control corresponds to SF = 1). 2.9. Holotomographic microscopy The holotomographic (Nanolive) imaging were performed using 3D Cell Explorer-Fluo (Tolochenaz, Switzerland) microscope. For experiments, ⁓8 ∙ 10 3 glioblastoma cells were seeded into a 96-well plate (special for fluorescence imaging, with glass coverslip bottom, minimal background noise and crosstalk between wells) in high-glucose DMEM medium supplemented with 10% FBS. After 24 h of incubation, the medium was changed to transparent DMEM medium without phenol red and the plate with cells was transferred into a mini-incubator coupled to a holotomographic microscope. Solutions of Pd NPs in the medium were added to individual wells in such a way as to obtain their final concentration in the medium of 25 µg/ml. Holotomographic images were collected after the injection of Pd NPs solution at different time intervals (10 min, 3 h, 6 h and 24 h). Additionally, films were recorded based on photos from a holotomographic microscope, which were taken every 5 minutes for 5 hours. During the experiment, the refractive index (RI) was measured in three dimensions using class 1 laser low power (λ = 520 nm, sample exposure 0.2 mW/mm 2 ) with a high-numerical aperture air objective. Based on the differences in intensity, RI can be digitally stained to visualize individual cells, cell components (e.g. cell membrane or cytoplasm) and Pd NPs. Moreover, to determine if the Pd NPs were accumulated outside or inside the cells, Z-axis images were also reconstructed based on the RI value. The rendering of the holotomographic images to obtain 3D reconstruction were performed using STEVE Software. 2.10. Statistical analysis The MTS test results were shown as the means ± SEM (standard error of mean). The data were analyzed using one-way analysis of variance (ANOVA) followed by post hoc Tukey test. Statistical significance was accepted when p < 0.05. The data were presented graphically using GraphPad Prism 8 and Origin Software. 3. Results and discussion 3.1. Isolation of chitosan from honeybee corpses The corpses of bees consist of proteins, chitin, melanin, as well as mineral compounds, mainly calcium, potassium, phosphorus and magnesium salts. 37 , 41 For this reason, in order to obtain chitin and, ultimately, chitosan, a multi-stage process of removing individual building blocks from bee corpses is necessary. Photographs of isolates from individual stages are provided in Fig. S1 . First, solid particles and wax were mechanically separated from the bee carcasses, and the biological material was crushed and dried. In the next stage, demineralization was performed using the Hackaman method. This stage was aimed at eliminating inorganic compounds constituting approximately 3% of dead bees. 37 2 M hydrochloric acid was used for this purpose, although – as previously shown – other acids (e.g. sulfuric acid, nitric acid or even acetic acid) can be equally effective agents used in demineralization process. 12 It is very important to select the appropriate demineralization time, because if the process is carried out too briefly, it will result in the presence of mineral residues in the isolate, and if the process is carried out for a long time, it may lead to the degradation of chitin. 42 In our case, the demineralization process lasted 2 hours at ambient temperature, which ensured over 80% efficiency of this stage (Table 1 .). As it has been shown, 43 higher temperatures can increase the effectiveness of demineralization due to easier penetration of acid into the chitin matrix, but they may negatively affect the physicochemical properties of the resulting chitin and, consequently, chitosan. In our experiments, analogous demineralization was carried out without stirring, but the efficiency of this reaction was only 44%. Table 1 Total yields and yields for individual stages of chitosan isolation. Stage yield (%) Total yield (%) Stage II 81,42 81,42 Stage III 36,89 30,03 Stage IV 48,81 14,66 Stage V 41,65 6,11 The aim of the next stage was to deproteinize biological material. This process is extremely important, especially in the context of the biomedical use of the isolation product (chitosan), because the protein component may cause allergies in humans. 12 , 44 Generally, NaOH is considered the most optimal deproteinizing compound, but KOH, 45 Ca(OH) 2 or NaHCO 3 46 are also used for this purpose. As previously shown, 47 the use of NaOH concentrations less than 1 M does not result in effective protein removal. On the other hand, too concentrated NaOH solution leads to increased chain degradation and chitin deacetylation, which will prevent the effective obtaining of chitin in its native form. Moreover, room temperature is insufficient for the effective protein removal process, hence it is advisable to use a higher temperature, which we also implemented during deproteinization. Under our reaction conditions (1 M NaOH, 2 h reaction at 80 o C), a deproteinization efficiency of approximately 37% was achieved. The isolate obtained in this way was a complex of chitin and melanin. Then, in order to decolorize (depigmentation), the obtained isolate was exposed to diluted 3% H 2 O 2 . Presumably, during alkaline hydrolysis using 1 M NaOH, as well as rinsing with ethanol some of the chitin-bound melanin had already been dissolved, but the resulting isolate was still colored (pure chitin is colorless), hence it was necessary to treat the melanin with a strong oxidant such as H 2 O 2 . By this procedure, almost debleached chitin was obtained with a yield of approximately 49% – complete discoloration occurred during deacetylation. Generally, the efficiency of this stage is influenced by the duration of the decolorization process. The highest efficiency, as previously shown, 37 is obtained with decolorization lasting 2–2.5 h. In such conditions, chitosan with a lower molecular weight (20–30 kDa) is also obtained than in the case of a shorter decolorization process (60–70 kDa). The duration of depigmentation may also depend on the source from which chitosan is isolated. For example, depigmentation in the case of chitosan isolation from Omani shrimp 36 is most optimally achieved using 30% H 2 O 2 and conducting the decolorization reaction for 3 h. Alternatively, potassium permanganate 48 or sodium hypochlorite 49 can be used for depigmentation. The final step in the chitosan isolation procedure was chitin deacetylation. The most popular way to deacetylate chitin is to use concentrated solutions of strong bases, such as NaOH, which allows obtaining water-insoluble chitosan with a deacetylation degree of 85–99%. 46 For effective deacetylation, the reaction temperature ought to be 80 o C degrees or higher. It should be borne in mind that such alkaline deacetylation may change the structural properties of chitin by e.g. rearrangement of polymer chains. Moreover, depending on whether chitin is in the form of α-chitin or β-chitin, deacetylation will result in more crystalline or amorphous chitosan, respectively. 50 In our studies, the obtained efficiency of the chitosan deacetylation step was over 40%, and the total efficiency of the chitosan isolation procedure was approximately 6%. The final yield is similar to the results of research by other authors isolating chitosan from insects. 19 , 51 The process of isolating chitosan from crustaceans 52 is usually characterized by a slightly higher efficiency (up to 15%), which is probably due to the lower content of proteins and lipids in the biomass of crustaceans compared to the biomass of insects. 53 It is noteworthy that the efficiency of chitin isolation from honey bees depends on the body part. Thus, the highest efficiency is achieved for the bee legs (13.25%) and the lowest for the thorax (6.79%). 15 The individual stages of chitosan isolation were monitored using FTIR method (Fig. 3 .). Commercial chitosan is characterized by the presence of several characteristic absorption bands in the FTIR spectrum: 3600–3000 cm − 1 (broad band from the –OH group), 3345 cm − 1 (N–H stretching vibrations), 2950–2820 cm − 1 (–C–H stretching vibrations), 1646 cm − 1 and 1550 cm − 1 (amide I band and II band), 1589 cm − 1 (–NH 2 group), 1417 cm − 1 and 1374 cm − 1 (C–H bending vibrations), 1313 cm − 1 (C–N stretching of amide III), 1150 cm − 1 , 1058 cm − 1 and 1023 cm − 1 (C–O–C stretching vibrations). 54 , 55 The FTIR spectrum for "green" chitosan is characterized by absorption bands analogous to those of commercial chitosan. However, two weak absorption bands at 2916 cm − 1 and 2849 cm − 1 are visible here (and not one, as in the case of commercial chitosan), corresponding to the symmetric and assymetric modes of –CH 2 group vibrations. 56 For the chitin spectrum, even more intense peaks in this region were visible. A more intense absorbance maximum at 1619 cm − 1 indicates the presence of hydrogen bonds between the C = O group and the hydroxyl-methyl group of the next chitin residue of the same chain. 57 Moreover, FTIR analysis showed the presence of a single peak at 1619 cm − 1 , indicating that the extracted chitin is a β-polymorph. The presence of an additional peak at 1660 cm − 1 would indicate the presence of the α-polymorph. 58 , 59 FTIR spectra of isolates from the previous stages (raw material, after demineralization and after deproteinization) are generally characterized by positions of absorbance maxima similar to those characteristic of chitin. However, changes in the intensities of some peaks are visible. At 3270 cm − 1 wavenumber, stronger absorbance was noted for isolates B and C. These peaks correspond to the –NH 2 groups characteristic of the complex of melanin and proteins, which were systematically removed at the deproteinization and depigmentation stage. A similar trend is visible for the peaks at 2916 cm − 1 and 2850 cm − 1 , the increased intensity of which is related to the presence of = CH–, –CH 2 – and –CH 3 groups in the protein-melanin complex. Finally, a peak at 1734 cm − 1 was detected, which corresponds mainly to the = C = O group of melanin, but also of proteins. For isolate D (chitin), this peak is very faint. In isolate A, no peaks characteristic of inorganic components of honey bees were observed, which is due to the fact that these mineral components constitute a small percentage (3%) compared to proteins (35–45%), melanin (30–40%) and chitin (23–32%). 47,60 In turn, the absorption band ratios A 1320 /A 1420 are 1.42 and 1.59 for chitosan isolated from bees and commercial chitosan, which translates into an estimated DD of about 67% (bee chitosan) and 62% (commercial chitosan). Finally, molecular weight of chitosans were determined. The apparent viscosity obtained directly from viscometric measurements was 3.1 mPa∙s and 2.1 mPa∙s for commercial and isolated chitosan, respectively. This resulted in an intrisinic viscosity value of 1579 ml/g (commercial chitosan) and 827 ml/g (isolated chitosan). To calculate the molecular weight of chitosan, α and K constants were assumed as 1.99∙10 − 5 dl/g and 0.59, respectively, according to Kasaai et al. 61 These constant values were chosen because they were closest to the polymer-solvent-temperature system that we dealth with in our studies. Thereby, the molecular weight of chitosans was determined as 2.6∙10 6 and 9.8∙10 5 g/mol for commercial and isolated chitosan, respectively. 3.2. Formation process of Pd NPs and their physicochemical characterization Both Pd NPs I and Pd NPs II were synthesized using chitosan, which act as polycationic stabilizer, but also as a co-reducer. Finally, chitosan – according to previous literature reports 39 , 62 – also acts as a size-controllable agent. In general, increased chitosan concentration is associated with a decrease in the size of not only Pd NPs but also silver (Ag NPs) or gold (Au NPs) nanoparticles. 63 , 64 This is probably due to the fact that during the synthesis process of chitosan-stabilized NPs, positively charged chitosan undergoes electrostatic interaction with metal nuclei, and the strong interaction (related to the increasing chitosan concentration) blocks the binding of precursors to metal nuclei, preventing further growth of NPs. In the process of Pd NPs preparation, ascorbic avid (vitamin C) accelerates the first stage of the reaction, i.e. the conversion of Pd 2+ ions to Pd 0 , while oxidizing itself. Then, freshly produced palladium atoms preferentially grow on the pre-synthesized nuclei, and only later chitosan covers the surface of Pd NPs via interactions with the metal nuclei. As shown in our studies, it is also possible to carry out an analogous reaction using only ascorbic acid. However, in this case tha lack of chitosan covering the surface of Pd NPs will prevent the formation of a shielding barrier, which will results in further growth of Pd NPs forming irregular agglomerates precipitaing from the solution. Contrarily, the use of chitosan mass above 30 mg resulted in a Pd NPs solution that tended to form a gel. In the described studies, Pd NPs stabilized with chitosan were obtained in a single reaction. An alternative is post-synthesis modification of the obtained NPs. For this purpose, chitosan ought to be modified e.g. by thiolation, which will enable subsequent covalent binding of thiolated-chitosan to pre-synthesized Me NPs. 65 STEM and SAED analyses enabled the determination of the morphology (shape, size, and consequently size distribution) and local nanostructure of the obtained Pd NPs (Fig. 4 ). Pd NPs I ( Fig. 4a1 ) stabilized with commercial chitosan are characterized by a spherical shape and a smooth, non-wrinkled surface. The diameter of these monodisperse Pd NPs was estimated to be about 20 nm ( Fig. 4a3 ). On the other hand, Pd NPs II ( Fig. 4b1 ) prepared with chitosan isolated from bees, have a much more corrugated surface morphology, resembling nano-flowers. Moreover, Pd NPs II have about twice the diameter of Pd NPs and exhibit significantly higher polydispersity ( Fig. 4b3 ). The reason for the less regular shape of Pd NPs II is believed to be the fact that chitosan isolated from bees may contain residues of other building components of these insects, and even their small rest may influence the course of NPs synthesis. There are numerous studies showing that NPs obtained by green chemistry have an irregular shape and polydyspersity. 66 – 68 On the other hand, NPs synthesized using green chemistry may also be significantly smaller in size compared to those obtained using classical wet chemistry method and be characterized by a similar level of dispersion. 69 Finally, SAED patterns ( Fig. 4a2 and Fig. 4b2 ) of both Pd NPs show bright spots arranged at a distance proportional to the ratio of wavelength to interplanar spacing. Individual Pd NPs are monocrystalline, however, the SAED diffraction patterns is due to the presence of numerous randomly arranged Pd NPs – during SAED analysis, a field containing a lot of Pd NPs was selected with a shutter. The rings of SAED can be attributed to the (111), (200), (220), (311) and (400) lattice planes of face-centered cubic (fcc) Pd NPs (International Centre of Diffraction Data, card 00-046-1043). 3.3. Pd NPs cytotoxicity and radiosensitizing potential against glioblastoma cells Pre-synthesized Pd NPs were tested for their usefulness in combating glioblastoma multiforme ( in vitro model). This is particularly important because this type of cancer has an extremely poor prognosis and treatment options, based mainly on surgical interventions and chemotherapy with temozolomide, are limited. 70 First, the cytotoxicity of obtained Pd NPs was assessed against two selected glioblastoma lines: LN229 (epithelial morphology) and U118 (mixed morphology). For this purpose, Pd NPs solution with a wide concentration range were prepared and cell survival was estimated after 3 h and 24 h of incubation with these NPs (Fig. 5 .). The MTS colorimetric assay showed that for both LN229 (Fig. 5 a) and U118 (Fig. 5 b) cell lines a systematic decrease in survival was observed with increasing Pd NPs concentration, but for concentration of 10 µg/ml and 25 µg/ml this decrease is insignificant. Moreover, for higher concentration (75 µg/ml and 100 µg/ml) a clear difference in the survival of both cell lines was observed depending on the incubation time used. A similar trend was observed for copper and zinc oxide NPs, where after 3 h of incubation the cells remained slightly damaged, while after 24 h an intensive decrease in survival was observed. 71 It is worth emphasizing that NPs generate a similar cytotoxic effect against both tested glioma cell lines. The concentration of 25 µg/ml was chosen for further radiosensitization experiments because it was considered to be the maximum critical allowable concentration that did not cause any destructive effect on the glioma cells tested. Also, for the study of radiosensitizing properties of Pd NPs in simulated PRT, the optimal radiation dose of 2 Gy (standard dose used in radiotherapy for glioblastoma) was chosen ( Fig. S2 ). By selecting a non-toxic radiation dose and Pd NPs concentration, it was possible to reliably assess whether the combined effect of Pd NPs and proton beam would be observed in the simulated PRT supported by Pd NPs. The results of radiosensitization studies using the MTS assay are illustrated below (Fig. 6 ). Without a doubt, the first conclusion that can be drawn from the conducted research is that PRT supported by Pd NPs (both types) turned out to be significantly more effective than standard PRT without the use of NPs. In detail, Pd NPs I-based radiosensitizers inhibited the proliferation of approximately 64% (LN229) and 52% (U118) of glioma cells. Similarly, Pd NPs reduced the survival rates by 56% and 39% for LN229 and U118, respectively. The radiosensitizing effect of these Pd NPs can be elucidated by both physical (Compton–, photoelectric– or Auger effect) and biological-chemical (oxidative stress, DNA damage, cell cycle effect, bystander effect) mechanisms of radiosensitization. 72 The second intriguing finding is the observation of differences in the radiosensitizing activity (especially in relation to U118 cells) of both Pd NPs used. It is interesting that despite the similar cytotoxicity of both types of Pd NPs, the combined radiosensitizing effect is diverse. As mentioned in Chap. 3.2., in the process of chitosan isolation from bees, residues of other bee building components could of course remain in the final isolate. On the one hand, these residues themselves could remain neutral with respect to the obtained glioma cells; on the other hand, bee products (honey, royal jelly, propolis, bee venom) – as previously shown in the literature – can act as radioprotectors, preventing the effects of radiotherapy. 73 , 74 Moreover, as concluded in section 3.1., chitosan isolated from bees is probably characterized by a slighy larger DD than commercial chitosan. So far, this parameter has not been shown to be associated with a change in cell radiosensitization, although in relation to biological systems it has been confirmed that the DD affects the inflammatory/innate immune cells response. 75 , 76 Finally, the structure of chitosan (crystalline vs amorphous) – determined by whether chitin is in its α-polymorph or β-polymorph form – may also be important in the context of cell radiosensitization, although no confirmation of this theory has been found in the literature to date. Of course, the above theories may overlap and all described aspects may, to a greater or lesser extent, influence the final radiosensitizing effect of the tested Pd NPs. The MTS test shows the short-term effect of NPs and/or radiation. In order to obtain information whether cells subjected to a specific therapy will demonstrate the ability to create clones resistant to a given treatment after a dozen or so days, the clonogenic test – which is the gold standard for assessing the long-term effects of radiotherapy – was performed. Of course, this test can also be successfully used to evaluate the effect of Pd NPs themselves, which was also performed in the present study ( Fig. S3 ). In Fig. 7 . the survival data for LN229 and U118 glioblastoma cells of clonogenic assay are plotted against proton irradiation dose. The survival curves show that with increasing radiation dose, a reduction in the number of cell colonies resistant to simulated proton therapy is observed. In particular, for higher radiation doses, it can be clearly observed that the final effect of proton beam irradiation is dependent on the Pd NPs used, with Pd NPs I being more satisfactory radiosensitizers for both tested cell line. Thus, both tests used confirmed the same tendency for radiosensitization by Pd NPs and Pd NPs II. To complete the discussion, it should be noted that synthesized Pd NPs are characterized by both different shape and size, which makes their objective comparison in the context of cytotoxic properties or as nano-radioenhancers difficult. The final cytotoxicity, as well as the radiosensitizing effect, is the result of many factors, such as (apart from the mentioned morphology) the method of synthesis, charge, coating, solubility or chemical composition of NPs. 77 – 79 Theoretically, NPs with a more folded structure (such as Pd NPs II) would seem to be better radiosensitizers due to the possible larger surface area of ​​interaction with protons, and consequently – the generation of, for example, an increased amount of reactive oxygen species (ROS) or secondary electrons determining apoptosis. Contrarily, smaller spherical NPs are characterized by a very high surface to volume ratio, which is also positive in the context of their application as radiosensitizers. 3.4. Cellular uptake of Pd NPs by glioblastoma cells Holotomographic microscopy was used as a technique for real-time and three-dimensional imaging of cell interactions with Pd NPs. After adding Pd NPs, holotomographic images were collected every 5 min for 5 h, which allowed recording movies of the interaction of LN229 ( Movie S1 and Movie S2 ) and U118 cells ( Movie S3 and Movie S4 ) with Pd NPs I and Pd NPs II, respectively. Additionally, holotomographic images were taken after 6 h and 24 h of incubation of Pd NPs with cells. Data obtained from holotomographic images allowed the assessment of changes in cell morphology, the preferred site of Pd NPs accumulation, as well as the estimation of the volume occupied by them in cells as a function of time. The exemplary results (3D holotomographic images, as well as Z-axis reconstruction) for LN229 and U118 cells incubated with Pd NPs I are depicted below (Fig. 8 and Fig. 9 ). Analogous analyses for Pd NPs II are provided in Supplementary Materials ( Fig. S4 and Fig. S5 ). LN229 (Fig. 8 ), as well as U118 (Fig. 9 ) cells exposed to Pd NPs I did not undergo significant morphological changes within 6 h of incubation. Quite the contrary, further growth and division of these cells was observed. Taking into account the fact that cytoplasm and cell membrane have different RI value compared to Pd NPs, it was possible to assess the preferred localization of these NPs at given time points. The assessment of the Pd NPs site of accumulation was facilitated by Z-axis reconstruction (Fig. 8 a and Fig. 9 a) and imaging of cell cross-sections in different layers (Fig. 8 z1, z2, z3 and Fig. 9 z1, z2, z3 ). To clearly visualize the procedure for determining the place of Pd NPs accumulation, helpful videos have been recorded ( Movie S5 ). For all tested combinations, it was observed that after 10 minutes Pd NPs penetrated into the glioblastoma cells. It was not noted that NPs had any preferred locations in the cells – they were rather evenly distributed inside the cells (sections z2 in Fig. 8 and Fig. 9 are more rich in red marked Pd NPs than outer z1 and z3 sections), although initially (which is visible for U118 + Pd NPs II) Pd NPs were also located on the cell periphery (see Fig. 8 , z 1 slice, 10 min). Despite the initial growth and effective cell divisions, after 24 h a change in cell morphology was observed – a significant part of the cells was detached from the substrate and took on a spherical-like shape. In some combinations – which is particularly visible for LN229 + Pd NPs II – significant cell thinning was observed in the field of view. It should be borne in mind that during measurements with a holotomographic microscope, we only analyze a narrow area imposed by the microscope and are not able to image what is happening outside of it. The cells, under the influence of the Pd NPs solution, presumably detached and transferred to another place in the well, where they (at least the fraction most resistant to Pd NPs) could adhere again and continue to grow. The fact that most glioma cells overcome the initial stress associated with NPs exposure is evidenced by the results of the clonogenic assay determining the fraction of surviving cells after 10 days. Finally, the cell volumes occupied by NPs were also determined as a function of time (Table 2 ). Moreover, by analyzing holotomographic images using STEVE Software, it was also possible to estimate the volume of the cytoplasm or cell membrane ( Fig. S6 ). For both cell lines used, increased cellular uptake of Pd NPs II was observed compared to Pd NPs I. This is consistent with previous literature reports, which found that NPs of 30–50 nm in size are more readily absorbed by cells than their smaller/larger counterparts. 80 – 82 However, after 24 h of incubation, a clear decrease in the volume occupied by Pd NPs II was observed, which results – as previously mentioned – from the fact that a significant part of the cells were detached from the substrate and floated out of the field of view of the microscope. These experiments show that holotomography measurements of cellular uptake are effective for short incubation times of NPs with cells or for the use of such types or concentrations of NPs that will not cause dramatic changes in the behavior of cells resulting in their detachment. Table 2 Values of RI volume (µm 3 ) corresponding to the both types of Pd NPs incubated for different times with LN229 and U118 glioblastoma cells. 10 min 3 h 6 h 24 h LN229 + Pd NPs I 1 240 2 882 4 502 3 205 LN229 + Pd NPs II 2 686 3 512 4 882 376 U118 + Pd NPs I 2 740 3 227 4 021 4 668 U118 + Pd NPs II 3 142 3 741 2 956 1 887 4. Conclusions In summary, we demonstrated that – depending on the source of chitosan, which is a stabilizer and reducing agents – Pd NPs with different morphology (size and shape) were prepared. In this study the pre-synthesized Pd NPs were examined for their possible application in biomedicine as nano-radiosensitizers in simulated PRT of glioblastoma. It was shown that Pd NPs I stabilized with commercial chitosan are characterized by increased biocompatibility than Pd NPs II, as revealed by the MTS survival test. Analyzes of changes in cell morphology observed in real time using holotomographic microscopy correlate with the results of the MTS test, as more intense cell destruction by Pd NPs II was demonstrated. Moreover, using the same concentration of both Pd NPs, a joined effect of NPs-assisted PRT was noticed, and this effect was stronger for Pd NPs I (⁓ 15% lower cell survival). It has therefore been shown that, without a shadow of a doubt, the source of the carbohydrate-based polymer determines not only the morphology of the NPs obtained from it, but also the activity towards biological systems, which should be taken into account when designing such nanosystems, especially in medical-related applications. Declarations CRediT authorship contribution statement Bartosz Klębowski : Conceptualization, investigation, visualization, writing – original draft. Radosław Piech : Investigation, writing – original draft. Kamil Sobczak : Investigation. Marianna Gniadek : Investigation. Joanna Depciuch : Writing – review and editing, supervision. Declaration of competing interest We have nothing to declare. Data availability The data supporting this article have been included as part of the Supplementary Information. Other raw data will be available upon request from corresponding author Dr Bartosz Klebowski ( [email protected] ). Acknowledgements This research was funded by a grant from the National Science Centre (UMO-2020/37/N/ST5/02414). The authors also thank the Institute of Engineering Materials and Biomaterials of the Silesian University of Technology for the use of the Titan FEI TEM instrument and Department of Chemistry, University of Warsaw for the access to the Talos F200 FEI TEM instrument. The help of Prof. Magdalena Parlińska-Wojtan with STEM analysis is highly acknowledged. Appendix A. Supplementary material Supplementary material related to this article can be found, in the online version, at doi: … References Satchanska, G.; Davidowa, S.; Petrov, P. 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1","display":"","copyAsset":false,"role":"figure","size":155930,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral scheme of chitosan isolation.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/145e755c0889ea795b77ff40.png"},{"id":92752715,"identity":"21e59b08-2264-4de0-970f-7254b9f7d32e","added_by":"auto","created_at":"2025-10-04 00:20:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61108,"visible":true,"origin":"","legend":"\u003cp\u003eSimplified scheme for the synthesis of chitosan-stabilized Pd NPs.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/bc1b5c27c6015642120c5400.png"},{"id":92752969,"identity":"1d86067b-11e6-43b5-bdf8-c70327afc262","added_by":"auto","created_at":"2025-10-04 00:28:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210411,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra for commercial chitosan and isolates from individual stages of chitosan isolation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/b2899ad67dfb0b1e91420359.png"},{"id":92752630,"identity":"487d9145-3b47-422c-adc4-a7d38cef1572","added_by":"auto","created_at":"2025-10-04 00:12:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":143580,"visible":true,"origin":"","legend":"\u003cp\u003eSTEM HAADF images (scale bar 20 nm) of Pd NPs I (a1) and Pd NPs II (b1) with corresponding SAED patterns (a2, b2), as well as size distribution based on STEM analysis (a3, b3).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/8f3a5deb7dcd958e89db0fdc.png"},{"id":92752646,"identity":"d9749db2-b080-4d84-92f5-b82ee0b46c20","added_by":"auto","created_at":"2025-10-04 00:12:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":297199,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the MTS survival test for LN229 (a) and U118 (b) glioblastoma cells cultured with Pd NPs I / Pd NP II of various concentration ranges. Data were considered significant when *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e control.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/0ad0d461937f1330d16235ba.png"},{"id":92752722,"identity":"b14abf08-c87b-4633-9851-e524b5984b81","added_by":"auto","created_at":"2025-10-04 00:20:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169221,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the MTS survival test for LN229 and U118 glioblastoma cells exposed to Pd NPs–assisted proton irradiation (total dose 2 Gy). Data were considered significant when *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e control; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 – differences between both tested cell lines; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 – differences between both tested Pd NPs.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/b6339640d1387e852951764c.png"},{"id":92752637,"identity":"1ecc872f-55e5-41e8-95da-1766faaf26b9","added_by":"auto","created_at":"2025-10-04 00:12:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":82945,"visible":true,"origin":"","legend":"\u003cp\u003eClonogenic survival of protons-irradiated LN229 (a) and U118 (b) glioblastoma cells.\u003c/p\u003e","description":"","filename":"floatimage71.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/e8272f9fdea6946308d07c73.png"},{"id":92752727,"identity":"bfff78ac-b160-4e9d-b025-9786ae393513","added_by":"auto","created_at":"2025-10-04 00:20:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":566535,"visible":true,"origin":"","legend":"\u003cp\u003e3D holotomographic images (a) of LN229 cells cultured with Pd NPs I reconstructed based on the RI of: Pd NPs I (red color), nuclei and cytoplasms (green color) and cell membrane (blue color); Z-axis reconstruction of holotomographic images (b) with slice thicknesses marked at ≈ 15 μm (z1), ≈ 7 μm (z2) and ≈ 0 μm (z3); holotomographic images of the respective slices (z1, z2, z3). Scale bar 20 μm.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/a5c0be7d4e63c28c60401aef.png"},{"id":92752720,"identity":"56229300-de57-48a9-bf55-a1f24b63d49e","added_by":"auto","created_at":"2025-10-04 00:20:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":596333,"visible":true,"origin":"","legend":"\u003cp\u003e3D holotomographic images (a) of U118 cells cultured with Pd NPs I reconstructed based on the RI of: Pd NPs I (red color), nuclei and cytoplasms (green color) and cell membrane (blue color); Z-axis reconstruction of holotomographic images (b) with slice thicknesses marked at ≈ 15 μm (z1), ≈ 7 μm (z2) and ≈ 0 μm (z3); holotomographic images of the respective slices (z1, z2, z3). Scale bar 20 μm.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/cac1e547eb2f22050072bd2f.png"},{"id":92753272,"identity":"c3e22847-0a4a-4e47-9c7e-f3daae01bd59","added_by":"auto","created_at":"2025-10-04 00:44:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3756586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/f8b369eb-27bc-40ea-8291-67d81b64e4af.pdf"},{"id":92752718,"identity":"75422354-27ad-41f9-aeeb-b1c25ca540f2","added_by":"auto","created_at":"2025-10-04 00:20:01","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2842636,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS1LN229PdNPsI.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/c0ed69231bf8fe5255417800.mp4"},{"id":92752640,"identity":"3e2446d0-8728-407d-b6b2-43ad0445a548","added_by":"auto","created_at":"2025-10-04 00:12:01","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3035898,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS2LN229PdNPsII.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/605fcbe48e8b474f4d89875a.mp4"},{"id":92752633,"identity":"b42c646f-dc24-4c15-96e4-0bf1a6647b4a","added_by":"auto","created_at":"2025-10-04 00:12:01","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3694143,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS3U118PdNPsI.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/03e0a7e1f18bce7fa931a3fa.mp4"},{"id":92752724,"identity":"896903ad-869b-4bd3-895a-0c59824f7d96","added_by":"auto","created_at":"2025-10-04 00:20:02","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2559613,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS4U118PdNPsII.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/34a47046bca89fc3709bc6d5.mp4"},{"id":92752670,"identity":"28377cd8-97fc-4542-ab79-9b57e7c30731","added_by":"auto","created_at":"2025-10-04 00:12:14","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":193257472,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS5.avi","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/d12a1728f95e6227b5206c98.avi"},{"id":92752649,"identity":"dbd37769-e6dc-4936-9922-b9803ff2de51","added_by":"auto","created_at":"2025-10-04 00:12:02","extension":"doc","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":6930432,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.doc","url":"https://assets-eu.researchsquare.com/files/rs-7643260/v1/7e3101d1dc53868ccf41fe34.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of cytotoxicity, radiosensitizing properties and cellular uptake of palladium nanoparticles stabilized with commercial chitosan and chitosan isolated from honey bees","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNatural polymers are increasingly becoming the object of interest of scientist, especially in the context of their potential application in biomedicine. The unquestionable advantage of such natural polymers over synthetic polymers is their biodegradability, common occurrence in nature, mucoadhesive properties, as well as environmental friendliness.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Among natural polymers, carbohydrate polymers are particularly popular in medical-related applications.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e One of the most popular carbohydrate polymers is chitosan.\u003c/p\u003e\u003cp\u003eChitosan \u0026ndash; a product of chitin deacetylation \u0026ndash; is a linear cationic polysaccharide composed of randomly distributed β-(1,4)-D-glucosamine and N-acetyl-D-glucosamine.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e The presence of the \u0026ndash;NH\u003csub\u003e3\u003c/sub\u003e amino group in the structure of chitosan determines its solubility in slightly acidic solutions, such as dilute acetic or formic acid.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Moreover, the reactive amino and hydroxyl groups in chitosan determine the possibility of relatively simple modification of this biopolymer, which may ultimately contribute to obtaining structures with improved solubility or thermal and mechanical properties.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The presence of these functional groups also provide an opportunity to attach drugs, which enables the use of chitosan-based nanomaterials as drug carriers.\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Currently, chitosan is obtained from marine food industry waste, especially shrimps, crabs, krill or crayfish shells.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e At the same time, the search for alternative sources of chitosan is ongoing, and honey bees (\u003cem\u003eApis mellifera\u003c/em\u003e) may be one of the solutions.\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e The average lifespan of a worker bee in the summer season is 2 months, while in the case of workers staying in the hive for the winter, the lifespan can be up to half a year.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e After this time, there is a rapid replacement of workers in the hive, which is called spring die-off. It should also be noted that with environmental changes, there are more and more diseases and threats that cause significant losses in apiaries.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e For this reason, dead bees, due to the lack of other use, may prove to be an interesting alternative to traditional sources of chitosan. The process of isolating chitosan from bees may include both chemical and enzymatic methods using chitin deacetylases that convert chitin to chitosan.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eChitosan can be successfully used in biomedical application due to its anti-microbial,\u003csup\u003e21\u003c/sup\u003e anti-oxidant (Li et al., 2019)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and anti-inflammatory\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e properties. However, significant attention is currently focused on the use of chitosan-based nanomaterials for applications in cancer treatment, e.g. as drug (chemotherapeutic agents) delivery systems,\u003csup\u003e24,25\u003c/sup\u003e radiosensitizers\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e or photosensitizers\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e in radiation/photo-based therapies. Importantly, chitosan can be either the core of the nanomaterials (e.g. nanoparticles, NPs) or only a stabilizer of other nanomaterials, e.g. metallic ones. Although there are numerous reports on the applications of chitosan-based NPs in anticancer therapies, the effect of the chitosan source on the morphology of chitosan-stabilized metallic NPs, as well as its effect on the biological activity of such NPs, is unknown. This is an aspect worth attention because it is widely known that the biological activity of nanosystems is influenced by many factors, such as their size,\u003csup\u003e30\u003c/sup\u003e shape,\u003csup\u003e31\u003c/sup\u003e charge,\u003csup\u003e32\u003c/sup\u003e crystal structure,\u003csup\u003e33\u003c/sup\u003e and even the method of synthesis.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Taking the above issues into account, in this work a hypothesis was put forward that the use of chitosans from different sources (commercially available chitosan \u003cem\u003evs\u003c/em\u003e chitosan isolated from honey bees) would result in obtaining NPs with different morphology, and moreover, these NPs would be characterized by diverse activity as radiosensitizers \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eHerein, it was planned to investigated whether, depending on the source of chitosan, there would be a difference in the morphology and crystal structure of NPs in the synthesis of which chitosan acted as both a reducing agent and a stabilizer. Palladium nanoparticles (Pd NPs) were selected for this purpose due to their promising potential in biomedical applications and relatively low cost of production (compared to the more popular gold or platinum-based NPs).\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e In this paper, the cytotoxicity and radiosensitizing properties of chitosan-stabilized Pd NPs in simulated proton radiotherapy (PRT) were investigated. LN229 and U118 cell line, derived from a grade IV glioblastoma, were used as an \u003cem\u003ein vitro\u003c/em\u003e model. Finally, novel three-dimensional holotomographic imaging was also used to observe the real-time interaction of glioblastoma cells with Pd NPs, which enabled the assessment of morphology changes induced by these Pd NPs, as well as locating the preferential site of accumulation of chitosan-stabilized Pd NPs in the cells.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. General remarks\u003c/h2\u003e\u003cp\u003eCommercial chitosan (2.6∙10\u003csup\u003e6\u003c/sup\u003e g/mol \u0026ndash; based on viscosity, deacetylation degree 62%), isolated chitosan (9.8∙10\u003csup\u003e5\u003c/sup\u003e g/mol \u0026ndash; based on viscosity, deacetylation degree 67%), palladium (II) chloride, ascorbic acid, high-glucose DMEM (Dulbecco\u0026rsquo;s Modified Eagle Medium) were purchased from Sigma Aldrich (Burlington, MA, USA); LN229 and U118 glioblastoma cells \u0026ndash; from ATCC (American Type Culture Collection, Manassas, VA, USA); 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test kit (Cell Titer96\u0026reg; Aqueous One Solution Cell Proliferation Assay) \u0026ndash; from Promega (Madison, WI, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Isolation of chitosan from honey bees\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Samples collection\u003c/h2\u003e\u003cp\u003eThe corpses of honeybees (\u003cem\u003eApis mellifera carnica\u003c/em\u003e, Nyska line) were used as a source of chitosan. The bees came from a home apiary, and the reason for their death was difficult weather conditions in the winter of 2023/2024. Large temperature fluctuations in short periods of time, combined with high humidity, caused losses of 60% national population. Before initiating the isolation procedure, preliminary purification was carried out by removing visible fragments of beeswax and other solid elements. The pre-purified honeybee corpses were stored in a cool and dry place until subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Isolation procedure\u003c/h2\u003e\u003cp\u003eThe chitosan isolation process was prepared based on previous literature reports\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e with some modifications. The general procedure for isolating this polysaccharide is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eStage I\u003c/b\u003e \u0026ndash; Pre-purified bee corpses were crushed in a laboratory mortar, and then 10 g of biological material were placed in a laboratory oven for 10 h (55\u003csup\u003eo\u003c/sup\u003eC). The isolate obtained in this way was weighed and the yield of subsequent stages of chitosan isolation was calculated in relation to the determined weight (\u003cb\u003eisolate A\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStage II\u003c/b\u003e \u0026ndash; The dried bees were transferred to a beaker and then poured with 250 ml of 2 M hydrochloric acid (HCl). The Hackman demineralization reaction was carried out for 2 h at room temperature with intensive stirring. After this time, the mixture was filtered using fluted filter paper and then rinsed with water until the remaining precipitate was neutralized. Finally, the precipitate was placed in a laboratory oven (55\u003csup\u003eo\u003c/sup\u003eC) and dried overnight (\u003cb\u003eisolate B\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStage III\u003c/b\u003e \u0026ndash; The dry isolate from the previous stage was deproteinized. For this purpose, the isolate was placed in a round-bottom flask. Then 1 M sodium hydroxide (NaOH) was added (15 ml/1 g of isolate) and the reaction mixture was placed on a heating mantle. The deproteinization process was continued for 2 h at 80\u003csup\u003eo\u003c/sup\u003eC with gentle stirring. After this time, the mixture was filtered and dried (as before). This time, the filtered precipitate (after neutralization) was additionally rinsed with ethanol to remove lipids (\u003cb\u003eisolate C\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStage IV\u003c/b\u003e \u0026ndash; Obtained isolate was discolored using hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). For this purpose, isolate was resuspended in water (15 ml/1 g of isolate) in a round-bottom flask placed on the heating mantle. The reaction mixture was heated to 70\u003csup\u003eo\u003c/sup\u003eC (with gentle stirring), then 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was gradually added until its final concentration in the flask was adjusted to 1%. The filtration and drying process was performed as before (\u003cb\u003eisolate D\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStage V\u003c/b\u003e \u0026ndash; To deactylate chitin, the isolate was placed back in the round-bottom flask on a heating mantle, followed by treated with 50% NaOH (200 ml/1 g of isolate) for 3 h at 90\u003csup\u003eo\u003c/sup\u003eC with intensive stirring. The mixture was purified by filtration and then dried as before (\u003cb\u003eisolate E\u003c/b\u003e). All isolates were stored in a dry and cool place until subsequent experiments (FTIR analyses and use for chitosan-mediated Pd NPs synthesis).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Synthesis of chitosan-stabilized Pd NPs\u003c/h2\u003e\u003cp\u003eBoth commercial chitosan (Pd NPs I) and chitosan isolated from bees (Pd NPs II) were used to synthesize Pd NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.). Due to the fact that chitosan is insoluble in water, the reaction was carried out in a slightly acidic environment, which allows the protonation of the amino groups of chitosan, making it soluble.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e The synthesis was performed according to the procedure described in the previous paper\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e with some modification. For this purpose, 15 ml of distilled water, 50 mg of ascorbic acid and finally 10 mg of chitosan were placed in a beaker at room temperature. After that, 10 ml of aqueous 0.01 M chloropalladic acid (H\u003csub\u003e2\u003c/sub\u003ePdCl\u003csub\u003e4\u003c/sub\u003e) solution (prepared by converting PdCl\u003csub\u003e2\u003c/sub\u003e with the HCl equivalent) were added. A gradual change in color from yellowish to light brown to almost black was observed within a minute. The reaction was left without stirring at room temperature for 2 h, after which the Pd NPs were collected by centrifugation (18 000 rpm, 10 minutes), washed in distilled water and centrifuged again. Pd NPs solutions were stored at room temperature until further studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Physicochemical characterization\u003c/h2\u003e\u003cp\u003eFourier-transform infrared spectroscopy (FTIR) spectra of isolates from individual stages of chitosan isolation were obtained using Nicolet\u0026trade; iS50 FTIR (Thermofisher, Waltham, MA, USA) enabling measurements with the attenuated total reflectance (ATR) method. Dried powder isolates were placed on an ATR crytal and then pressed with calcium fluoride (CaF\u003csub\u003e2\u003c/sub\u003e) window to reduce potential noise in the spectra. Spectra were collected in the wavenumber range 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026ndash; 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a spectral resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using 32 scans for each sample. The deacetylation degree (DD) of chitosan (both commercial and isolated) was also estimated by FTIR. For this purpose, the method described previously in the literature\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e was used. Knowing that:\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere DA is degree of acetylation (DA) calculated from the following equation:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\frac{{\\text{A}}_{1320}}{{\\text{A}}_{1420}}\\:=\\:0.3822+0.0313\\:DA$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere A\u003csub\u003e1320\u003c/sub\u003e and A\u003csub\u003e1420\u003c/sub\u003e are the absorption bands at 1320 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1420 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, DD was determined.\u003c/p\u003e\u003cp\u003eViscometry, combined with the Mark-Houwink equation, was used to determine the average molecular weight of both types of chitosans. The viscosity parameters were measured at room temperature using rotational viscometer HAAKE Viscotester D (Thermofisher, Waltham, MA, USA). For this purpose, 0.1 g of both commercial or isolated chitosan were dispersed in 75 ml of 0.5 M aqueous solution of acetic acid. Solutions were stirred before viscosity measurements for 2 h. The determined dynamic viscosity was calculated to intrinsic viscosity (assuming the dynamic viscosity of pure solvent is 1 mPa∙s) and then the molecular weight of chitosan was estimated using the Mark-Houwink equation:\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:\\left[\\text{ƞ}\\right]=\\:\\text{K}\\bullet\\:\\:{M}^{\\alpha\\:}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere [ƞ] is the intrisinic viscosity, M is the molecular weight, K and α are parameters characteristic of a specific polymer, solvent and temperature.\u003c/p\u003e\u003cp\u003eHigh-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analysis enabled the evaluation of the morphology (size and shape) of the Pd NPs. For STEM analyses, purified and sonicated Pd NPs samples were dropped on a Cu carbon film coated TEM grid. Then, the grid was rinsed with fresh ethanol and dried at room temperature. Just before placing the samples into the STEM holder, they were cleaned in a plasma cleaner for 3 second. Based on the STEM images (⁓ 60 NPs), the size distribution of Pd NPs was estimated. For this purpose, FEI Talos TEM (Waltham, MA, USA) operating at 200 kV equipped with a field emission gun (FEG) cathode was used. Selected area electron diffraction (SAED) patterns were also taken in the STEM mode to determine the crystal structure of the Pd NPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Cell culture\u003c/h2\u003e\u003cp\u003eTwo human glioblastoma cell lines (LN229 and U118) were used as an \u003cem\u003ein vitro\u003c/em\u003e model. Both cell lines were continuously cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic mixture (penicillin, streptomycin, neomycin). Cells were cultured at 37\u003csup\u003eo\u003c/sup\u003eC in a humid atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. Both glioblastoma cell lines were tested for \u003cem\u003eMycoplasma sp\u003c/em\u003e. contamination with negative results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Proton irradiation protocol\u003c/h2\u003e\u003cp\u003eProton irradiation was conducted using Proteus C-235 isochronus cyclotron, located in the Cyclotron Centre Bronowice. Irradiation was carried out at room temperature with dose 2 Gy (the standard dose used in radiotherapy of glioblastoma) for MTS test and additionally 1, 5 and 10 Gy for clonogenic assay. For irradiation of cells monoenergetic field with an energy of 225 MeV and dimension 20 cm x 20 nm was selected. Irradiations were performed at 1.1 cm water equivalent depth, which consist of a 1 cm RW3 phantom plate. Proton irradiation was preceded by dosimetric measurements with Markus type ionization chamber calibrated in terms of absorbed dose to water. For proton irradiation, both Pd NPs were added to the glioblastoma cells 3 h before irradiation and were incubated for another 21 h before the MTS viability test was carried out.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.7. MTS cytotoxicity assay\u003c/h2\u003e\u003cp\u003eMTS assay was selected to determine the cellular metabolic activity. For this purpose, LN229 and U118 cells were seeded into 96-well plates (⁓8 ∙ 10\u003csup\u003e3\u003c/sup\u003e cells in 100 \u0026micro;l high-glucose DMEM medium supplemented with 10% FBS) and then incubated for 24 h. After that time, the medium was removed and 100 \u0026micro;l of new medium containing Pd NPs with concentration of 10, 25, 50, 75 and 100 \u0026micro;g/ml were added. This experiment was carried out in triplicate for each trial. Cells cultured in medium without Pd NPs served as a control. The cells were incubated for 3 and 24 h. Next, the medium was re-changed and additionally 20 \u0026micro;l of MTS reagent was added to each well and incubated for the following 3 h at 37\u003csup\u003eo\u003c/sup\u003eC. Then, cell viability was determined by measuring the absorbance at 490 nm using 96-well plate reader (Spark\u0026reg; Tecan, Mannedorf, Switzerland). Cell survival (CS) was estimated as:\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:\\text{C}\\text{S}\\:=\\:\\frac{{ABS}_{X}}{{ABS}_{Ctrl}}\\:\\times\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhen ABS\u003csub\u003ex\u003c/sub\u003e \u0026ndash; absorbance of cells exposed to Pd NPs; ABS\u003csub\u003ectrl\u003c/sub\u003e \u0026ndash; absorbance of control cells (without NPs).\u003c/p\u003e\u003cp\u003eTo assess the radiosensitizing properties of Pd NPs using the MTS test, a critical concentration of Pd NPs (i.e. not causing a decrease in survival by more than 20%) was selected \u0026ndash; 25 \u0026micro;g/ml for both Pd NPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Clonogenic assay\u003c/h2\u003e\u003cp\u003eThe clonogenic assay was used to assess the ability of cells exposed to proton radiation and/or Pd NPs to form treatment-resistant colonies. For this purpose, cells were trypsynized and plated in Petri dishes at different densities depending on the stringency of the proton irradiation (from 500 to 3000 cells/dishes). The cells were left overnight and then the medium was replaced with a new one containing Pd NPs at a concentration of 25 \u0026micro;g/ml (the control did not containg Pd NPs). 3 h later, the cells were irradiated with various doses of radiation (1, 2, 5 and 10 Gy). After 10 days of incubation, the cells were fixed with 5% formaldehyde for 15 minutes, then rinsed with distilled water and treated with 1% crystal violet. 10 minutes later, the cells were washed again with distilled water and viable cell colonies, i.e. those consisting of at least 50 cells, were counted. The cell survival fraction (SF) was calculated as follow:\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{F}=\\:\\frac{{PE}_{X}}{{PE}_{Ctrl}}\\:\\times\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhen PE\u003csub\u003ex\u003c/sub\u003e \u0026ndash; plating efficiency of cells exposed to Pd NPs/irradiation; PE\u003csub\u003ectrl\u003c/sub\u003e \u0026ndash; plating efficiency of control cells. The platting efficiency is defined as the ratio of the number of viable colonies to the number of seeded cells (we assume that the calculated PE for the control corresponds to SF\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Holotomographic microscopy\u003c/h2\u003e\u003cp\u003eThe holotomographic (Nanolive) imaging were performed using 3D Cell Explorer-Fluo (Tolochenaz, Switzerland) microscope. For experiments, ⁓8 ∙ 10\u003csup\u003e3\u003c/sup\u003e glioblastoma cells were seeded into a 96-well plate (special for fluorescence imaging, with glass coverslip bottom, minimal background noise and crosstalk between wells) in high-glucose DMEM medium supplemented with 10% FBS. After 24 h of incubation, the medium was changed to transparent DMEM medium without phenol red and the plate with cells was transferred into a mini-incubator coupled to a holotomographic microscope. Solutions of Pd NPs in the medium were added to individual wells in such a way as to obtain their final concentration in the medium of 25 \u0026micro;g/ml. Holotomographic images were collected after the injection of Pd NPs solution at different time intervals (10 min, 3 h, 6 h and 24 h). Additionally, films were recorded based on photos from a holotomographic microscope, which were taken every 5 minutes for 5 hours. During the experiment, the refractive index (RI) was measured in three dimensions using class 1 laser low power (λ\u0026thinsp;=\u0026thinsp;520 nm, sample exposure 0.2 mW/mm\u003csup\u003e2\u003c/sup\u003e) with a high-numerical aperture air objective. Based on the differences in intensity, RI can be digitally stained to visualize individual cells, cell components (e.g. cell membrane or cytoplasm) and Pd NPs. Moreover, to determine if the Pd NPs were accumulated outside or inside the cells, Z-axis images were also reconstructed based on the RI value. The rendering of the holotomographic images to obtain 3D reconstruction were performed using STEVE Software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe MTS test results were shown as the means\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;SEM (standard error of mean). The data were analyzed using one-way analysis of variance (ANOVA) followed by \u003cem\u003epost hoc\u003c/em\u003e Tukey test. Statistical significance was accepted when \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The data were presented graphically using GraphPad Prism 8 and Origin Software.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Isolation of chitosan from honeybee corpses\u003c/h2\u003e\u003cp\u003eThe corpses of bees consist of proteins, chitin, melanin, as well as mineral compounds, mainly calcium, potassium, phosphorus and magnesium salts.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e For this reason, in order to obtain chitin and, ultimately, chitosan, a multi-stage process of removing individual building blocks from bee corpses is necessary. Photographs of isolates from individual stages are provided in \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. First, solid particles and wax were mechanically separated from the bee carcasses, and the biological material was crushed and dried. In the next stage, demineralization was performed using the Hackaman method. This stage was aimed at eliminating inorganic compounds constituting approximately 3% of dead bees.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e 2 M hydrochloric acid was used for this purpose, although \u0026ndash; as previously shown \u0026ndash; other acids (e.g. sulfuric acid, nitric acid or even acetic acid) can be equally effective agents used in demineralization process.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e It is very important to select the appropriate demineralization time, because if the process is carried out too briefly, it will result in the presence of mineral residues in the isolate, and if the process is carried out for a long time, it may lead to the degradation of chitin.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e In our case, the demineralization process lasted 2 hours at ambient temperature, which ensured over 80% efficiency of this stage (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.). As it has been shown,\u003csup\u003e43\u003c/sup\u003e higher temperatures can increase the effectiveness of demineralization due to easier penetration of acid into the chitin matrix, but they may negatively affect the physicochemical properties of the resulting chitin and, consequently, chitosan. In our experiments, analogous demineralization was carried out without stirring, but the efficiency of this reaction was only 44%.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTotal yields and yields for individual stages of chitosan isolation.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStage yield (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal yield (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStage II\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e81,42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e81,42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStage III\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36,89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30,03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStage IV\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e48,81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14,66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStage V\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e41,65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6,11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe aim of the next stage was to deproteinize biological material. This process is extremely important, especially in the context of the biomedical use of the isolation product (chitosan), because the protein component may cause allergies in humans.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Generally, NaOH is considered the most optimal deproteinizing compound, but KOH,\u003csup\u003e45\u003c/sup\u003e Ca(OH)\u003csub\u003e2\u003c/sub\u003e or NaHCO\u003csub\u003e3\u003c/sub\u003e \u003csup\u003e46\u003c/sup\u003e are also used for this purpose. As previously shown,\u003csup\u003e47\u003c/sup\u003e the use of NaOH concentrations less than 1 M does not result in effective protein removal. On the other hand, too concentrated NaOH solution leads to increased chain degradation and chitin deacetylation, which will prevent the effective obtaining of chitin in its native form. Moreover, room temperature is insufficient for the effective protein removal process, hence it is advisable to use a higher temperature, which we also implemented during deproteinization. Under our reaction conditions (1 M NaOH, 2 h reaction at 80\u003csup\u003eo\u003c/sup\u003eC), a deproteinization efficiency of approximately 37% was achieved. The isolate obtained in this way was a complex of chitin and melanin.\u003c/p\u003e\u003cp\u003eThen, in order to decolorize (depigmentation), the obtained isolate was exposed to diluted 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Presumably, during alkaline hydrolysis using 1 M NaOH, as well as rinsing with ethanol some of the chitin-bound melanin had already been dissolved, but the resulting isolate was still colored (pure chitin is colorless), hence it was necessary to treat the melanin with a strong oxidant such as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. By this procedure, almost debleached chitin was obtained with a yield of approximately 49% \u0026ndash; complete discoloration occurred during deacetylation. Generally, the efficiency of this stage is influenced by the duration of the decolorization process. The highest efficiency, as previously shown,\u003csup\u003e37\u003c/sup\u003e is obtained with decolorization lasting 2\u0026ndash;2.5 h. In such conditions, chitosan with a lower molecular weight (20\u0026ndash;30 kDa) is also obtained than in the case of a shorter decolorization process (60\u0026ndash;70 kDa). The duration of depigmentation may also depend on the source from which chitosan is isolated. For example, depigmentation in the case of chitosan isolation from Omani shrimp\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e is most optimally achieved using 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and conducting the decolorization reaction for 3 h. Alternatively, potassium permanganate\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e or sodium hypochlorite\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e can be used for depigmentation.\u003c/p\u003e\u003cp\u003eThe final step in the chitosan isolation procedure was chitin deacetylation. The most popular way to deacetylate chitin is to use concentrated solutions of strong bases, such as NaOH, which allows obtaining water-insoluble chitosan with a deacetylation degree of 85\u0026ndash;99%.\u003csup\u003e46\u003c/sup\u003e For effective deacetylation, the reaction temperature ought to be 80\u003csup\u003eo\u003c/sup\u003eC degrees or higher. It should be borne in mind that such alkaline deacetylation may change the structural properties of chitin by e.g. rearrangement of polymer chains. Moreover, depending on whether chitin is in the form of α-chitin or β-chitin, deacetylation will result in more crystalline or amorphous chitosan, respectively.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e In our studies, the obtained efficiency of the chitosan deacetylation step was over 40%, and the total efficiency of the chitosan isolation procedure was approximately 6%. The final yield is similar to the results of research by other authors isolating chitosan from insects.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e The process of isolating chitosan from crustaceans\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e is usually characterized by a slightly higher efficiency (up to 15%), which is probably due to the lower content of proteins and lipids in the biomass of crustaceans compared to the biomass of insects.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e It is noteworthy that the efficiency of chitin isolation from honey bees depends on the body part. Thus, the highest efficiency is achieved for the bee legs (13.25%) and the lowest for the thorax (6.79%).\u003csup\u003e15\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe individual stages of chitosan isolation were monitored using FTIR method (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCommercial chitosan is characterized by the presence of several characteristic absorption bands in the FTIR spectrum: 3600\u0026ndash;3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (broad band from the \u0026ndash;OH group), 3345 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N\u0026ndash;H stretching vibrations), 2950\u0026ndash;2820 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u0026ndash;C\u0026ndash;H stretching vibrations), 1646 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1550 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide I band and II band), 1589 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (\u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e group), 1417 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1374 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026ndash;H bending vibrations), 1313 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026ndash;N stretching of amide III), 1150 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1058 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1023 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026ndash;O\u0026ndash;C stretching vibrations).\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e The FTIR spectrum for \"green\" chitosan is characterized by absorption bands analogous to those of commercial chitosan. However, two weak absorption bands at 2916 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2849 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are visible here (and not one, as in the case of commercial chitosan), corresponding to the symmetric and assymetric modes of \u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e group vibrations.\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e For the chitin spectrum, even more intense peaks in this region were visible. A more intense absorbance maximum at 1619 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicates the presence of hydrogen bonds between the C\u0026thinsp;=\u0026thinsp;O group and the hydroxyl-methyl group of the next chitin residue of the same chain.\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e Moreover, FTIR analysis showed the presence of a single peak at 1619 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating that the extracted chitin is a β-polymorph. The presence of an additional peak at 1660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e would indicate the presence of the α-polymorph.\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e FTIR spectra of isolates from the previous stages (raw material, after demineralization and after deproteinization) are generally characterized by positions of absorbance maxima similar to those characteristic of chitin. However, changes in the intensities of some peaks are visible. At 3270 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wavenumber, stronger absorbance was noted for isolates B and C. These peaks correspond to the \u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e groups characteristic of the complex of melanin and proteins, which were systematically removed at the deproteinization and depigmentation stage. A similar trend is visible for the peaks at 2916 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2850 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the increased intensity of which is related to the presence of =\u0026thinsp;CH\u0026ndash;, \u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e\u0026ndash; and \u0026ndash;CH\u003csub\u003e3\u003c/sub\u003e groups in the protein-melanin complex. Finally, a peak at 1734 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was detected, which corresponds mainly to the\u0026thinsp;=\u0026thinsp;C\u0026thinsp;=\u0026thinsp;O group of melanin, but also of proteins. For isolate D (chitin), this peak is very faint. In isolate A, no peaks characteristic of inorganic components of honey bees were observed, which is due to the fact that these mineral components constitute a small percentage (3%) compared to proteins (35\u0026ndash;45%), melanin (30\u0026ndash;40%) and chitin (23\u0026ndash;32%).\u003csup\u003e47,60\u003c/sup\u003e In turn, the absorption band ratios A\u003csub\u003e1320\u003c/sub\u003e/A\u003csub\u003e1420\u003c/sub\u003e are 1.42 and 1.59 for chitosan isolated from bees and commercial chitosan, which translates into an estimated DD of about 67% (bee chitosan) and 62% (commercial chitosan). Finally, molecular weight of chitosans were determined. The apparent viscosity obtained directly from viscometric measurements was 3.1 mPa∙s and 2.1 mPa∙s for commercial and isolated chitosan, respectively. This resulted in an intrisinic viscosity value of 1579 ml/g (commercial chitosan) and 827 ml/g (isolated chitosan). To calculate the molecular weight of chitosan, α and K constants were assumed as 1.99∙10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e dl/g and 0.59, respectively, according to Kasaai et al.\u003csup\u003e61\u003c/sup\u003e These constant values were chosen because they were closest to the polymer-solvent-temperature system that we dealth with in our studies. Thereby, the molecular weight of chitosans was determined as 2.6∙10\u003csup\u003e6\u003c/sup\u003e and 9.8∙10\u003csup\u003e5\u003c/sup\u003e g/mol for commercial and isolated chitosan, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Formation process of Pd NPs and their physicochemical characterization\u003c/h2\u003e\u003cp\u003eBoth Pd NPs I and Pd NPs II were synthesized using chitosan, which act as polycationic stabilizer, but also as a co-reducer. Finally, chitosan \u0026ndash; according to previous literature reports\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e \u0026ndash; also acts as a size-controllable agent. In general, increased chitosan concentration is associated with a decrease in the size of not only Pd NPs but also silver (Ag NPs) or gold (Au NPs) nanoparticles.\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e This is probably due to the fact that during the synthesis process of chitosan-stabilized NPs, positively charged chitosan undergoes electrostatic interaction with metal nuclei, and the strong interaction (related to the increasing chitosan concentration) blocks the binding of precursors to metal nuclei, preventing further growth of NPs. In the process of Pd NPs preparation, ascorbic avid (vitamin C) accelerates the first stage of the reaction, i.e. the conversion of Pd\u003csup\u003e2+\u003c/sup\u003e ions to Pd\u003csup\u003e0\u003c/sup\u003e, while oxidizing itself. Then, freshly produced palladium atoms preferentially grow on the pre-synthesized nuclei, and only later chitosan covers the surface of Pd NPs \u003cem\u003evia\u003c/em\u003e interactions with the metal nuclei. As shown in our studies, it is also possible to carry out an analogous reaction using only ascorbic acid. However, in this case tha lack of chitosan covering the surface of Pd NPs will prevent the formation of a shielding barrier, which will results in further growth of Pd NPs forming irregular agglomerates precipitaing from the solution. Contrarily, the use of chitosan mass above 30 mg resulted in a Pd NPs solution that tended to form a gel. In the described studies, Pd NPs stabilized with chitosan were obtained in a single reaction. An alternative is post-synthesis modification of the obtained NPs. For this purpose, chitosan ought to be modified e.g. by thiolation, which will enable subsequent covalent binding of thiolated-chitosan to pre-synthesized Me NPs.\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eSTEM and SAED analyses enabled the determination of the morphology (shape, size, and consequently size distribution) and local nanostructure of the obtained Pd NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePd NPs I (\u003cb\u003eFig.\u0026nbsp;4a1\u003c/b\u003e) stabilized with commercial chitosan are characterized by a spherical shape and a smooth, non-wrinkled surface. The diameter of these monodisperse Pd NPs was estimated to be about 20 nm (\u003cb\u003eFig.\u0026nbsp;4a3\u003c/b\u003e). On the other hand, Pd NPs II (\u003cb\u003eFig.\u0026nbsp;4b1\u003c/b\u003e) prepared with chitosan isolated from bees, have a much more corrugated surface morphology, resembling nano-flowers. Moreover, Pd NPs II have about twice the diameter of Pd NPs and exhibit significantly higher polydispersity (\u003cb\u003eFig.\u0026nbsp;4b3\u003c/b\u003e). The reason for the less regular shape of Pd NPs II is believed to be the fact that chitosan isolated from bees may contain residues of other building components of these insects, and even their small rest may influence the course of NPs synthesis. There are numerous studies showing that NPs obtained by green chemistry have an irregular shape and polydyspersity.\u003csup\u003e\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e On the other hand, NPs synthesized using green chemistry may also be significantly smaller in size compared to those obtained using classical wet chemistry method and be characterized by a similar level of dispersion.\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eFinally, SAED patterns (\u003cb\u003eFig.\u0026nbsp;4a2\u003c/b\u003e and \u003cb\u003eFig.\u0026nbsp;4b2\u003c/b\u003e) of both Pd NPs show bright spots arranged at a distance proportional to the ratio of wavelength to interplanar spacing. Individual Pd NPs are monocrystalline, however, the SAED diffraction patterns is due to the presence of numerous randomly arranged Pd NPs \u0026ndash; during SAED analysis, a field containing a lot of Pd NPs was selected with a shutter. The rings of SAED can be attributed to the (111), (200), (220), (311) and (400) lattice planes of face-centered cubic (fcc) Pd NPs (International Centre of Diffraction Data, card 00-046-1043).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Pd NPs cytotoxicity and radiosensitizing potential against glioblastoma cells\u003c/h2\u003e\u003cp\u003ePre-synthesized Pd NPs were tested for their usefulness in combating glioblastoma multiforme (\u003cem\u003ein vitro\u003c/em\u003e model). This is particularly important because this type of cancer has an extremely poor prognosis and treatment options, based mainly on surgical interventions and chemotherapy with temozolomide, are limited.\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e First, the cytotoxicity of obtained Pd NPs was assessed against two selected glioblastoma lines: LN229 (epithelial morphology) and U118 (mixed morphology). For this purpose, Pd NPs solution with a wide concentration range were prepared and cell survival was estimated after 3 h and 24 h of incubation with these NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe MTS colorimetric assay showed that for both LN229 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) and U118 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) cell lines a systematic decrease in survival was observed with increasing Pd NPs concentration, but for concentration of 10 \u0026micro;g/ml and 25 \u0026micro;g/ml this decrease is insignificant. Moreover, for higher concentration (75 \u0026micro;g/ml and 100 \u0026micro;g/ml) a clear difference in the survival of both cell lines was observed depending on the incubation time used. A similar trend was observed for copper and zinc oxide NPs, where after 3 h of incubation the cells remained slightly damaged, while after 24 h an intensive decrease in survival was observed.\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e It is worth emphasizing that NPs generate a similar cytotoxic effect against both tested glioma cell lines. The concentration of 25 \u0026micro;g/ml was chosen for further radiosensitization experiments because it was considered to be the maximum critical allowable concentration that did not cause any destructive effect on the glioma cells tested. Also, for the study of radiosensitizing properties of Pd NPs in simulated PRT, the optimal radiation dose of 2 Gy (standard dose used in radiotherapy for glioblastoma) was chosen (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). By selecting a non-toxic radiation dose and Pd NPs concentration, it was possible to reliably assess whether the combined effect of Pd NPs and proton beam would be observed in the simulated PRT supported by Pd NPs.\u003c/p\u003e\u003cp\u003eThe results of radiosensitization studies using the MTS assay are illustrated below (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWithout a doubt, the first conclusion that can be drawn from the conducted research is that PRT supported by Pd NPs (both types) turned out to be significantly more effective than standard PRT without the use of NPs. In detail, Pd NPs I-based radiosensitizers inhibited the proliferation of approximately 64% (LN229) and 52% (U118) of glioma cells. Similarly, Pd NPs reduced the survival rates by 56% and 39% for LN229 and U118, respectively. The radiosensitizing effect of these Pd NPs can be elucidated by both physical (Compton\u0026ndash;, photoelectric\u0026ndash; or Auger effect) and biological-chemical (oxidative stress, DNA damage, cell cycle effect, bystander effect) mechanisms of radiosensitization.\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e The second intriguing finding is the observation of differences in the radiosensitizing activity (especially in relation to U118 cells) of both Pd NPs used. It is interesting that despite the similar cytotoxicity of both types of Pd NPs, the combined radiosensitizing effect is diverse. As mentioned in Chap.\u0026nbsp;3.2., in the process of chitosan isolation from bees, residues of other bee building components could of course remain in the final isolate. On the one hand, these residues themselves could remain neutral with respect to the obtained glioma cells; on the other hand, bee products (honey, royal jelly, propolis, bee venom) \u0026ndash; as previously shown in the literature \u0026ndash; can act as radioprotectors, preventing the effects of radiotherapy.\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e Moreover, as concluded in section 3.1., chitosan isolated from bees is probably characterized by a slighy larger DD than commercial chitosan. So far, this parameter has not been shown to be associated with a change in cell radiosensitization, although in relation to biological systems it has been confirmed that the DD affects the inflammatory/innate immune cells response.\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e Finally, the structure of chitosan (crystalline \u003cem\u003evs\u003c/em\u003e amorphous) \u0026ndash; determined by whether chitin is in its α-polymorph or β-polymorph form \u0026ndash; may also be important in the context of cell radiosensitization, although no confirmation of this theory has been found in the literature to date. Of course, the above theories may overlap and all described aspects may, to a greater or lesser extent, influence the final radiosensitizing effect of the tested Pd NPs.\u003c/p\u003e\u003cp\u003eThe MTS test shows the short-term effect of NPs and/or radiation. In order to obtain information whether cells subjected to a specific therapy will demonstrate the ability to create clones resistant to a given treatment after a dozen or so days, the clonogenic test \u0026ndash; which is the gold standard for assessing the long-term effects of radiotherapy \u0026ndash; was performed. Of course, this test can also be successfully used to evaluate the effect of Pd NPs themselves, which was also performed in the present study (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). In Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. the survival data for LN229 and U118 glioblastoma cells of clonogenic assay are plotted against proton irradiation dose. The survival curves show that with increasing radiation dose, a reduction in the number of cell colonies resistant to simulated proton therapy is observed. In particular, for higher radiation doses, it can be clearly observed that the final effect of proton beam irradiation is dependent on the Pd NPs used, with Pd NPs I being more satisfactory radiosensitizers for both tested cell line. Thus, both tests used confirmed the same tendency for radiosensitization by Pd NPs and Pd NPs II.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo complete the discussion, it should be noted that synthesized Pd NPs are characterized by both different shape and size, which makes their objective comparison in the context of cytotoxic properties or as nano-radioenhancers difficult. The final cytotoxicity, as well as the radiosensitizing effect, is the result of many factors, such as (apart from the mentioned morphology) the method of synthesis, charge, coating, solubility or chemical composition of NPs.\u003csup\u003e\u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e Theoretically, NPs with a more folded structure (such as Pd NPs II) would seem to be better radiosensitizers due to the possible larger surface area of ​​interaction with protons, and consequently \u0026ndash; the generation of, for example, an increased amount of reactive oxygen species (ROS) or secondary electrons determining apoptosis. Contrarily, smaller spherical NPs are characterized by a very high surface to volume ratio, which is also positive in the context of their application as radiosensitizers.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Cellular uptake of Pd NPs by glioblastoma cells\u003c/h2\u003e\u003cp\u003eHolotomographic microscopy was used as a technique for real-time and three-dimensional imaging of cell interactions with Pd NPs. After adding Pd NPs, holotomographic images were collected every 5 min for 5 h, which allowed recording movies of the interaction of LN229 (\u003cb\u003eMovie S1\u003c/b\u003e and \u003cb\u003eMovie S2\u003c/b\u003e) and U118 cells (\u003cb\u003eMovie S3\u003c/b\u003e and \u003cb\u003eMovie S4\u003c/b\u003e) with Pd NPs I and Pd NPs II, respectively. Additionally, holotomographic images were taken after 6 h and 24 h of incubation of Pd NPs with cells. Data obtained from holotomographic images allowed the assessment of changes in cell morphology, the preferred site of Pd NPs accumulation, as well as the estimation of the volume occupied by them in cells as a function of time. The exemplary results (3D holotomographic images, as well as Z-axis reconstruction) for LN229 and U118 cells incubated with Pd NPs I are depicted below (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Analogous analyses for Pd NPs II are provided in Supplementary Materials (\u003cb\u003eFig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e and \u003cb\u003eFig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLN229 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), as well as U118 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) cells exposed to Pd NPs I did not undergo significant morphological changes within 6 h of incubation. Quite the contrary, further growth and division of these cells was observed. Taking into account the fact that cytoplasm and cell membrane have different RI value compared to Pd NPs, it was possible to assess the preferred localization of these NPs at given time points. The assessment of the Pd NPs site of accumulation was facilitated by Z-axis reconstruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea) and imaging of cell cross-sections in different layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003ez1, z2, z3\u003c/b\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u003cb\u003ez1, z2, z3\u003c/b\u003e). To clearly visualize the procedure for determining the place of Pd NPs accumulation, helpful videos have been recorded (\u003cb\u003eMovie S5\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eFor all tested combinations, it was observed that after 10 minutes Pd NPs penetrated into the glioblastoma cells. It was not noted that NPs had any preferred locations in the cells \u0026ndash; they were rather evenly distributed inside the cells (sections z2 in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e are more rich in red marked Pd NPs than outer z1 and z3 sections), although initially (which is visible for U118\u0026thinsp;+\u0026thinsp;Pd NPs II) Pd NPs were also located on the cell periphery (see Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, z\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e slice, 10 min). Despite the initial growth and effective cell divisions, after 24 h a change in cell morphology was observed \u0026ndash; a significant part of the cells was detached from the substrate and took on a spherical-like shape. In some combinations \u0026ndash; which is particularly visible for LN229\u0026thinsp;+\u0026thinsp;Pd NPs II \u0026ndash; significant cell thinning was observed in the field of view. It should be borne in mind that during measurements with a holotomographic microscope, we only analyze a narrow area imposed by the microscope and are not able to image what is happening outside of it. The cells, under the influence of the Pd NPs solution, presumably detached and transferred to another place in the well, where they (at least the fraction most resistant to Pd NPs) could adhere again and continue to grow. The fact that most glioma cells overcome the initial stress associated with NPs exposure is evidenced by the results of the clonogenic assay determining the fraction of surviving cells after 10 days.\u003c/p\u003e\u003cp\u003eFinally, the cell volumes occupied by NPs were also determined as a function of time (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, by analyzing holotomographic images using STEVE Software, it was also possible to estimate the volume of the cytoplasm or cell membrane (\u003cb\u003eFig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u003c/b\u003e). For both cell lines used, increased cellular uptake of Pd NPs II was observed compared to Pd NPs I. This is consistent with previous literature reports, which found that NPs of 30\u0026ndash;50 nm in size are more readily absorbed by cells than their smaller/larger counterparts.\u003csup\u003e\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e However, after 24 h of incubation, a clear decrease in the volume occupied by Pd NPs II was observed, which results \u0026ndash; as previously mentioned \u0026ndash; from the fact that a significant part of the cells were detached from the substrate and floated out of the field of view of the microscope. These experiments show that holotomography measurements of cellular uptake are effective for short incubation times of NPs with cells or for the use of such types or concentrations of NPs that will not cause dramatic changes in the behavior of cells resulting in their detachment.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eValues of RI volume (\u0026micro;m\u003csup\u003e3\u003c/sup\u003e) corresponding to the both types of Pd NPs incubated for different times with LN229 and U118 glioblastoma cells.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6 h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24 h\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLN229\u0026thinsp;+\u0026thinsp;Pd NPs I\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026nbsp;240\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 882\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 502\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3 205\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLN229\u0026thinsp;+\u0026thinsp;Pd NPs II\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2 686\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 882\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e376\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eU118\u0026thinsp;+\u0026thinsp;Pd NPs I\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2 740\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 227\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4 668\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eU118\u0026thinsp;+\u0026thinsp;Pd NPs II\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 142\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 741\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 956\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u0026nbsp;887\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, we demonstrated that \u0026ndash; depending on the source of chitosan, which is a stabilizer and reducing agents \u0026ndash; Pd NPs with different morphology (size and shape) were prepared. In this study the pre-synthesized Pd NPs were examined for their possible application in biomedicine as nano-radiosensitizers in simulated PRT of glioblastoma. It was shown that Pd NPs I stabilized with commercial chitosan are characterized by increased biocompatibility than Pd NPs II, as revealed by the MTS survival test. Analyzes of changes in cell morphology observed in real time using holotomographic microscopy correlate with the results of the MTS test, as more intense cell destruction by Pd NPs II was demonstrated. Moreover, using the same concentration of both Pd NPs, a joined effect of NPs-assisted PRT was noticed, and this effect was stronger for Pd NPs I (⁓ 15% lower cell survival). It has therefore been shown that, without a shadow of a doubt, the source of the carbohydrate-based polymer determines not only the morphology of the NPs obtained from it, but also the activity towards biological systems, which should be taken into account when designing such nanosystems, especially in medical-related applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBartosz Klębowski\u003c/strong\u003e: Conceptualization, investigation, visualization, writing \u0026ndash; original draft. \u003cstrong\u003eRadosław Piech\u003c/strong\u003e: Investigation, writing \u0026ndash; original draft. \u003cstrong\u003eKamil Sobczak\u003c/strong\u003e: Investigation. \u0026nbsp;\u003cstrong\u003eMarianna Gniadek\u003c/strong\u003e: Investigation. \u0026nbsp;\u003cstrong\u003eJoanna Depciuch\u003c/strong\u003e: Writing \u0026ndash; review and editing, supervision. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have nothing to declare.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this article have been included as part of the Supplementary Information. Other raw data will be available upon request from corresponding author Dr Bartosz Klebowski ([email protected]). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by a grant from the National Science Centre (UMO-2020/37/N/ST5/02414). The authors also thank the Institute of Engineering Materials and Biomaterials of the Silesian University of Technology for the use of the Titan FEI TEM instrument and Department of Chemistry, University of Warsaw for the access to the Talos F200 FEI TEM instrument. The help of Prof. Magdalena Parlińska-Wojtan with STEM analysis is highly acknowledged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary material related to this article can be found, in the online version, at doi: \u0026nbsp;\u0026hellip;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSatchanska, G.; Davidowa, S.; Petrov, P. D. Natural and synthetic polymers for biomedical and environmental applications. \u003cem\u003ePolymers (Basel).\u003c/em\u003e\u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e16\u003c/em\u003e, No. 1159. \u003c/li\u003e\n\u003cli\u003eBekchanov, D.; Mukhamediev, M.; Yarmanov, S.; Lieberzeit, P.; Mujahid, A. Functionalizing natural polymers to develop green adsorbent for wasterwater treatment applications. \u003cem\u003eCarbohydr. Polym\u003c/em\u003e. \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e321\u003c/em\u003e, No. 121397.\u003c/li\u003e\n\u003cli\u003eSionkowska, A. 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Optimization of bio-nano interface using gold nanostructures as a model of nanoparticle system. \u003cem\u003eInsciences J\u003c/em\u003e. \u003cstrong\u003e2011\u003c/strong\u003e, \u003cem\u003e1\u003c/em\u003e, 115-135. \u003c/li\u003e\n\u003cli\u003eKettler, K.; Veltman, K.; van de Meent, D.; van Wezel, A.; Hendriks, J. Cellular uptake of nanoparticles as determined by particles properties, experimental conditions, and cell type. \u003cem\u003eEnviron. Toxicol. Chem\u003c/em\u003e. \u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e33\u003c/em\u003e, 481-492. \u003c/li\u003e\n\u003cli\u003eSabourian, P.; Yazdani, G.; Ashraf, S. S.; Frounchi, M.; Mashayekhan, S.; Kiani, S.; Kakkar, A. Effect of physico-chemical properties of nanoparticles on their intracellular uptake. \u003cem\u003eInt. J. Mol. Sci\u003c/em\u003e. \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e21\u003c/em\u003e, No. 8019. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Appendix A","content":"\u003cp\u003eThe file for Appendix A is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"chitosan, palladium nanoparticles, green synthesis, honey bees, glioblastoma, radiosensitizers","lastPublishedDoi":"10.21203/rs.3.rs-7643260/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7643260/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecently, there has been increasing interest in the use of biocompatible natural polymers, especially polysaccharides, in the synthesis of nanoparticles (NPs). The undoubted advantages of such carbohydrate polymers as components of NPs include their well-defined chemical structure, biodegradability and widespread availability. This study reported the development of an effective method for the synthesis of biocompatible chitosan-stabilized palladium nanoparticles (Pd NPs). For this purpose, both commercial chitosan and chitosan isolated from honey bee corpses were used. Spherical ⁓ 20 nm Pd NPs I and ⁓ 40 nm flower-like Pd NPs II were obtained when commercial chitosan and chitosan isolated from bees (green synthesis method) were used, respectively. \u003cem\u003eIn vitro\u003c/em\u003e studies on selected glioblastoma cell lines (LN229 and U118) indicated that both types of Pd NPs have similar cytotoxicity, however Pd NPs I are characterized by improved radiosensitizing properties compared to Pd NPs II. Furthermore, real-time holotomographic observations of cells interactions with Pd NPs showed that (for the same concentrations) Pd NPs II generate more visible changes in cell morphology, including their flattening, which is particularly observed for LN229 cells. In summary, Pd NPs I seem to be more promising nanosystems for biomedical applications as radiosensitizers than Pd NPs II.\u003c/p\u003e","manuscriptTitle":"Comparison of cytotoxicity, radiosensitizing properties and cellular uptake of palladium nanoparticles stabilized with commercial chitosan and chitosan isolated from honey bees","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-04 00:11:56","doi":"10.21203/rs.3.rs-7643260/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-15T16:53:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T20:34:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T09:53:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-02T23:53:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-02T08:31:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-29T19:53:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-27T11:00:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-26T04:17:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152620133498456631398581910969862198134","date":"2025-09-26T04:08:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T19:02:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139232134399344984823598276781562471958","date":"2025-09-25T12:03:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277730098335151092364470950858897639044","date":"2025-09-23T18:23:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216139517640724196042610931429818059190","date":"2025-09-23T09:54:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256899654388975436304355842569487746680","date":"2025-09-23T06:16:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208376507497150574402146217884173815920","date":"2025-09-22T19:35:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33311717554604597445117316437457868320","date":"2025-09-22T17:21:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197249114508535507713299335268576664482","date":"2025-09-22T17:04:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52336387195408668233178861769161123286","date":"2025-09-22T15:52:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-22T15:49:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-19T13:16:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-19T13:16:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Polymers and the Environment","date":"2025-09-17T19:39:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b212251-87fa-4851-955d-075ef07a2819","owner":[],"postedDate":"October 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-02T15:09:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-04 00:11:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7643260","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7643260","identity":"rs-7643260","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}