Development of radiolabeled 111In-albumin liposomes for long-term imaging of tumors

Development of radiolabeled 111In-albumin liposomes for long-term imaging of tumors | 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 Development of radiolabeled 111In-albumin liposomes for long-term imaging of tumors Mohammad Ahrari, Zahra Saberi, Azam Abbasi, Jafar Rahnama Yazdi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6766595/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : The current study aims to develop a clinically applicable radiopharmaceutical agent for long-term imaging in the diagnosis and management of oncologic patients in the field of nuclear medicine. Liposomes, as pharmaceutical nanocarriers, have been extensively studied in pharmaceutical industry and depending on their structural characteristics could be disposed in various pathological sites in the body. PEGylated liposomes have smaller volume of distribution and decreased clearance, consequently, due to their more prolonged presence in the bloodstream and their stability during this time, could be used for tumor imaging. In this work, liposomal formulations encapsulating albumin were synthesized by solvent evaporation method and extrusion and were labeled by 111 In-oxine similar to leukocytes labeling method. Their biodistribution in C26-colon carcinoma tumor-bearing mice by injection dose/gram and gamma scintigraphy were studied. Results : The result of our study displayed that 111 In -radiolabeled liposomes having a size of about 130 nanometers, were capable of accumulating in tumor sites based on enhanced penetration and retention (EPR) phenomenon. These liposomes also have high stability for maintaining encapsulated albumin for a long time up to 96 hours and probably so on. In the study of biodistribution of our formulation in tumor-bearing mice, they accumulated more in the kidney, liver, spleen and tumor sites, so that even after clearance of formulation in the bloodstream, they existed in significantly high levels in the mentioned organs and furthermore tumor site up to 96 hours. In gamma scintigraphy, organs with high activity accumulation from early time from administration to 96 hours, were visible in the form of hot spots demonstrating stability in the tumor. Conclusions : Our in vitro and in vivo studies demonstrated that this PEGylated radiolabeled liposomal formulation have considerable stability and efficiency for long-term tumor imaging which merit further studies for its transformation into clinical application. 111In-albumin nanoliposome biodistribution tumor imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Gamma ray imaging or scintigraphy is a valuable tool in the optimal design of the liposomal formulation of radiopharmaceutical agents, and allows non-invasive tracking of the distribution of liposomes in the body by gamma ray (Goins and Phillips 2001 ). While radiological imaging techniques such as MRI and CT give anatomical information, centigram images display the distribution of radiopharmaceuticals in the body environment and determine the changes in physiological processes. It is well known that the diagnosis of injuries, tumors or infection centers can be determined by traditional Gama ray imaging before the appearance of anatomical abnormalities. Furthermore, the prompt diagnosis of suspected area of above abnormalities is of great importance in terms of clinical application (Boerman, Laverman et al. 2000 ). Liposomes have been labeled with gamma irradiating radiopharmaceuticals such as 67 Ga, 111 In and 99m Tc for the purpose of tumor image. Liposomes have been exploited for imaging of tumor, infection, and blood pool, and there are large amount of preclinical evidence showing radiolabeled liposomes can image tumors sites and inflammatory lesions with high specificity and sensitivity. Moreover, according to the biodistribution profiles obtained in the studies on rabbits, it has been determined that PEGylated liposomes can maintain their shielding properties more than other types of liposomes. Immediately removal of liposome by MPS phagocytizing cells in the liver and spleen greatly limited their use inside the human body. Subsequent progress in the field of long-circulating liposomes (LCLS) has created nanoliposomes that are less likely to be taken up by the cells of the mononuclear phagocyte system (MPS). The development of these LCLs by enhancing their circulating half-life has increased our ability to detect pathological processes by in vivo scintigraphy procedures (Boerman, Laverman et al. 2000 , Awasthi, Garcia et al. 2003 , Low, Yang et al. 2023 ). The researches of several decades provide a depth understanding of the effects of liposomal characteristics such as size, type and concentration of lipids, surface charge and hydrophilicity, on their pharmacokinetic after intravenous administration. In particular, modifying the surface characteristics of liposomes and the use of nanoliposomes with approximately 100nm size, containing more cholesterol and phospholipids with saturated chains enhances the circulation time of liposomes in the blood and reduces their removal by the reticuloendothelial system (Chow, Lin et al. 2009 , Man, Gawne et al. 2019 ). Today, 99m Tc radiopharmaceutical is used in nuclear medicine departments, which has a short half-life. For evaluating some diseases, especially tumors, we need a radiopharmaceutical agent with long shelf life. 111 In is a radionuclides that is obtained as 111 InCl 3 with a specific activity of 15mci/ml in 0.01 normal HCl solution in the cyclotron section of the Atomic Energy Organizations. It does not have beta irradiation and emits two gamma photons with energy of 173 Kev and 247 Kev, which are used in clinical imaging. 111 In have been used for cell radiolabeling by binding to cellular proteins. Furthermore, the binding of 111 In with proteins such as albumin is 10 12 folds stronger than its binding with oxine (Awasthi, Goins et al. 1997 , Dillehay, Henkin et al. 2006 , Phillips, Goins et al. 2009 , Drozdovitch, Brill et al. 2015 , Lamichhane, Dewkar et al. 2017 , De Silva, Fu et al. 2019 ). In this study, we used this phenomenon and prepared albumin-loaded liposomes and treated these liposomes as simulated cells. Subsequently albumin-loaded liposomes were radiolabeled with 111 In-oxine. Unlike HMPAO, which is trapped inside the liposome, oxine releases from liposomes through the lipid bilayer and transfers into the aqueous phase, after while separating from 111 In and binding of 111 In with albumin. This approach made radiolabeled liposomes to be used for tumor imaging. In imaging with radiolabeled liposomes, the metastases inside the liver and spleen are identified as cold spots (Jaggi, Khar et al. 1991 ). For this purpose, almost all traditional liposomal formulations could be used. The development of liposomal radiopharmaceuticals to determine the location of tumors was noticed since it was shown that specific liposomal formulations accumulate at the tumor site. The development of liposomes with longer half-life in the circulation has increased the hope of using these liposomal radiopharmaceuticals in tumor imaging. The ability of liposomal formulations labeled with 99m TC, 67 Ga and 111 In in identifying tumors has been proven in different studies (Presant, Proffitt et al. 1988 , Ogihara-Umeda, Sasaki et al. 1992 , Ogihara-Umeda, Sasaki et al. 1996 ). Various studies have shown that liposomes with longer half-life and higher stability in the circulation show improved tumors uptake (Boerman, Storm et al. 1995 , Ishida, Maruyama et al. 1999 , Phillips 1999 , Dams, Oyen et al. 2000 , Lee, Choi et al. 2005 ). In regarding these considerations, the present work aims to combine these approaches for long-term tumor imaging. 2. Methods 2.1. Materials: Human Albumin 20% solution was purchased from Biotest, Germany. The phospholipids, hydrogenated soya phosphatidylcholine (HSPC), methoxypolyethelene glycol (MW 2000)-distearylphosphatidylethanolamine (mPEG 2000 -DSPE), were obtained from Lipoid (Ludwigshafen, Germany). Cholesterol and Sephadex G-50 were purchased from Sigma–Aldrich (St. Louis, MO). Iodine-125 ( 125 I) was supplied from Amersham (Uppsala, Sweden). All other used chemicals were of analytical grade. 2.2. Preparation of albumin-loaded liposomes: Liposomes were prepared by thin film hydration method plus extrusion (Nikpoor, Tavakkol-Afshari et al. 2015 ). The total lipid concentration of all the liposomal formulations was adjusted 48mM. In brief, HSPC, cholesterol and mPEG2000-DSPE, that all of them were dissolved in chloroform, were mixed at the molar ratios presented in Table 1 . Then, evaporation of organic solvent was performed by a rotary evaporator in three hours, followed by three hours’ freeze drying to prepare thin film layer. The dried lipid film was hydrated with albumin solution by rotating in a water bath at 60°C and gentle mixing on a vortex mixer until thin film dissolved thoroughly. Afterward, liposomes in solution were downsized by extrusion through polycarbonate membrane filters 400nm, 200nm and 100nm (11 times each) at 60°C to reach the optimized size. The resulting liposomal products were purified by dialysis. In the dialysis method, free albumin molecules which weren’t entrapped in liposomes, removed from liposomal formulations in 300 KDa molecular weight cut-off dialysis membrane (Spectrum) by dialyzing against HEPES 10 mM and sucrose 7.5% buffer (pH ~ 7.4). The final liposomal solutions were stored at 4°C until later use. Blank liposomes (without albumin) were prepared following the same procedures mentioned before, with hydration solution excluding albumin. 2.3. Characterization of Albumin-loaded liposomes: 2.3.1. Particle size, PDI and Zeta potential measurement: Particles size, PDI (polydispersity index) and zeta potential of liposomes were measured by the dynamic light scattering technique (DLS) in a Zetasizer Nano ZS instrument (Malvern Instruments, CO., UK). The measurements were performed at 25°C after the dilution of formulation with HEPES 10 mM and sucrose 7.5% buffer (pH ~ 7.4). Zeta potential of liposomes was measured using electrophoretic light scattering by the same instrument. The results of particle size and zeta potential were expressed as mean ± S.D. (n = 3). 2.3.2. Encapsulation efficiency measurement: Encapsulation efficiency of albumin in liposomes was determined by separating free albumin from albumin-loaded liposomes using ultrafiltration method. Predialyzed samples were placed in the upper chamber of an Ultra-4 centrifugal filter unites with a molecular weight cut-off of 300 KDa (Amicon® Ultra-0.5, MWCO 300,000 Da; Millipore, Boston, USA) and centrifuged at 4000g for 20 min at 8°C. The free albumin was separated from liposomes and collected in the bottom of the tube. The concentration of albumin in filtrate was determined using BCA protein assay and encapsulation efficiency was calculated (Nikpoor, Tavakkol-Afshari et al. 2017 ). In BCA protein assay method for encapsulated insulin in liposomes, the interfering substances were removed via sample pretreatment by sodium deoxycolate and trichloroacetic acid. Encapsulation efficiency of albumin in liposomal formulations was determined by this equation: % Encapsulation: ([albumin content after purification (by dialysis)] ÷ [albumin content before purification]) × 100. 2.3.3. Phosphate assay of liposomal formulation: In order to determine phosphate content of albumin-loaded liposomes and its alteration in formulation process, Bartlett phosphate assay protocol for the liposomal formulations was performed (Shmeeda, Even-Chen et al. 2003 ). The test was performed using this procedure and then concentration of phospholipids was calculated against standards. 2.3.4. Morphology of albumin-loaded liposomes by Transmission Electron Microscopy and Atomic Force Microscopy: The morphology of PEGylated albumin-loaded liposomes were determined using atomic force microscopy (AFM). This method of analysis determines surface structure of particles such as liposomes and provides three dimensional images. For AFM analysis, the formulation was diluted with dialyzed buffer (diluted 1:500) and about 30 µL of each sample were placed on a mika surface. After drying in room temperature, samples were analyzed by AFM microscope (NanoWizard®II, JPK model, Germany). Over approaching cantilever tip on samples, imaging in 2, 5 and 10 µm scales was performed; therefore 2- dimensional and 3-dimensional images of liposomes were obtained. 2.4. Radiolabeling of Albumin- loaded liposomes: 2.4.1. Measurement of the radioactivity of 111 In-oxine: First, radioactivity of 111 In-oxine complex was assessed. Briefly, by mixing 111 In-oxine with chloroform and normal saline and then incubating them in room temperature two phases were formed. Radioactivity of chloroform and normal saline were measured. Free 111 In enters saline phase due to its hydrophilicity and 111 In-oxine enters chloroform phase due to its lipophilicity. 2.4.2. Radiolabeling of Albumin- loaded liposomes with 111 In-oxine: Albumin- loaded liposomes was labeled with 111 In-oxine based on higher affinity of 111 In to albumin that was a novel labeling approach. In brief, 1 mL of albumin-loaded liposomes mixed with 5 millicurie activity of 111 In-oxine and then incubated in 55°C for 20 minutes in a rotatory state. To separate labeled albumin-loaded liposomes with 111 In-oxine from free 111 In-oxine, sephadex G-25 size exclusion column chromatography was used. Phosphate buffered saline (PBS) was used for washing. The purified labeled albumin-loaded liposomes with 111 In-oxine were collected in initial tubes and free 111 In-oxine was separated in terminal tubes. Radioactivity of each tubes was measured by dose calibrator instrument. 2.5. Sterilization of liposomal formulations: For intravenous injection, the product must be sterile. Radiolabeled liposomal formulations were sterilized by 220 nm porous filtered syringe under laminar flow hood and then collected in sterile tubes. 2.6. Stability study of radiolabeled liposomal formulation in serum: Stability of these liposomes was measured in serum medium by thin layer chromatography (TLC) method. Briefly, in the lower portion of silica gel strip, 50 microliters of radioactive liposomes is loaded and then placed in serum medium. At 0.5, 1, 4 and 24 hour, silica gel strip is cut from lower third and radioactivity of lower third and upper two third is measured. Released 111 In from liposomal formulations in the incubation period moved along silica gel strip and reached to the upper portion. Therefore, upper portion of silica gel contains released 111 In and the lower portion contains 111 In-albumin encapsulated in liposomal formulations. 2.7. In vivo studies: Mice, used in this work, were BALB-c with 6 to 8 weeks age and 18 to 20 grams, purchased from Pasteur institute of Iran (Tehran, Iran). Animal, were kept in the animal house of Pharmaceutical research center, Buo Ali research institute, Mashhad University of Medical Sciences and had free access to water and animal food under appropriate environmental conditions. All animal experiments were conducted in accordance with the approval of Institutional Ethical Committee of Mashhad University of Medical Sciences 2.7.1. Induction of Balb-c mice model of C26 colon carcinoma: C26 colon carcinoma cell line was used for induction of colon carcinoma in mice. This cell line was cultured in RPMI-1640 medium supplemented with 10% FBS and incubated in 37°C incubator with 5% CO 2 . The mice were anesthetized by intraperitoneal injection of 200µL zylazin/ketamine (5:15) and then subjected to the administration of 3 10 5 × C-26 cells (in 60 µL volume) subcutaneously at their flanks. After one week, tumors were generated with the size of about 5 millimeters in each dimension at the flank of the mice. 2.7.2. Intravenous injection of radiolabeled formulation to Balb-c mice: For this step of our work, 15 mice were used, which were in 5 groups of 3. Mice in groups 1, 2, and 3 were administrated with 200µL and groups 4 and 5 with 350µL radiolabeled liposome intravenously by syringe. Their radioactivities were measured by dose calibrator instrument. Also, after injecting into the mice and emptying the syringes, the amount of activity remaining in them was measured by dose calibrator and the quantity of radioactivity of each formulation injected into each mice was calculated. 2.7.3. Biodistribution of radiolabeled liposomes in Balb-c mice: After administration of liposomal formulations, group 1 mice were sacrificed after 4 hours, group 2 after 24 hours, group 3 after 48 hours, group 4 after 72 hours, group 5 after 96 hours, and their other organs (blood, heart, lungs, Liver, spleen, stomach, intestine, kidney, muscle tumor, tail and bone) were removed and placed into gamma tubes. The organs were washed with phosphate buffered saline, and weighted and the radioactivity of each organ was determined by a gamma counter (Delshid DL-100). The percentage of radioactivity per organ was measured as the mean injected dose per gram of organ (ID/g %) (Nikpoor, Tavakkol-Afshari et al. 2015 ). 2.7.4. Gamma scintigraphy of Balb-c mice: The biodistribution of liposomal formulations was furthermore investigated by planar photography method. Gamma cameras having the ability to receive gamma rays are used for this purpose. For this step of our experiment, 200 µCi of the formulation equivalent to 4.7 MBq of radiolabeled albumin loaded liposomes were injected intravenously through the mice tail vein. At 4, 24, 48, 96 hours after administration, the mice were anesthetized by ketamine/xylazine and then planar photography of biodistribution of liposomes were prepared by a Siemens Dual head Gamma Camera device with a matrix of 300,000 and resolution of 256×256 pixel. The characteristics of imaging instrument were: Series: Static, Camera present: In111-NMG, Matrix Size: 64×64, Zoom: 2.29, Detectors: Both detectors, Ant: 5mIn, Post: 5mIn, Orientation: Head Out/SupIne. 2.8. Statistical analysis: Data were presented as mean ± standard deviation (S.D.). The outcomes were analyzed by one-way analysis of variance (ANOVA) statistical test using GraphPad InStat version 3.05 for windows (GraphPad Software, USA). A statistical difference was considered when p value was less than 0.05. 3. Results 3.1. Formulation and characterization of albumin-loaded liposomes: 3.1.1. Particle size, PDI, zeta potential and encapsulation efficiency measurements: Liposomal formulation (Table 1 ) containing albumin could be prepared reproducibly by thin film hydration method plus extrusion. Particle size, zeta potential and encapsulation efficiency of albumin-loaded liposomes were displayed in Table 1 . Mean sizes of liposomes were about 130 nm by extrusion through polycarbonate membrane filter and encapsulation efficiency of liposomes for albumin ranged from 43–50%. PDIs of formulations were less than 0.2, indicative of almost homogenous distribution of liposomal sizes. Furthermore, phosphate concentration of the formulation was measured to be about 90 mM that was higher than expected based on formulation design, indicating evaporation duration formulation preparation. Table 1 Particle size, PDI and zeta potential of liposomes encapsulating albumin. (n = 3) Albumin-loaded liposomal formulation Molar ratio (%) Zeta average Size (nm) ± SD Polydispersity ± SD Zeta potential (mV) Entrapment efficiency% HSPC/CHOl/mPEG 2000 -DSPE (48 mM) 55/40/5 130.8 ± 1.2 0.175 ± 0.014 − 15.47 ± 3.19 46.5 ± 3 3.1.2. Morphology of albumin-loaded liposomes by Transmission Electron Microscopy and Atomic Force Microscopy: Figure 1 shows morphology of our albumin-loaded PEGylated liposomal formulation by both TEM and AFM microscopies. TEM microscopy depicts the structure and morphology of liposomes and AFM demonstrates their surface topology. As demonstrated by TEM and AFM microscopy, albumin-loaded liposomes had almost spherical shapes that were in expected correlation with the sizes obtained from DLS analysis. 3.2. Radiolabeling of Albumin- loaded liposomes: 3.2.1. Measurement of the radioactivity of 111 In-oxine for quality control: As displayed in Table 2 , the percentage of 111 In-oxine radioactivity was 20% that indicates its quality control for the study. Table 2 Quality Control of 111 In-oxine used in this experiment Phase type Radioactivity (µCi) 111 In-oxine radioactivity percent Normal saline 640 (160 / (640 + 160))*100%= 20% chloroform 160 3.2.2. Radiolabeling and purification of Albumin- loaded liposomes with 111 In-oxine: Figure 2 shows radiolabeling of albumin-loaded liposomes with 111 In-oxine and purification of them from free 111 In-oxine. The first peak indicates the radiolabeled liposomes that used in our study and second peak is pertinent to free 111 In-oxine that is excluded. 3.3. Stability study of radiolabeled liposomal formulation in serum: Stability of our formulation in serum medium was evaluated up to 24 hours. Percentage of released 111 In from liposomes indicating release percentage is presented in Fig. 3 . At 0.5, 1, 4 and 24 hours, remained radioactivity of our formulation is measured. 3.4. Biodistribution study of radiolabeled liposomes in tumor bearing mice by measurement of ID/g%: The biodistribution of our albumin-loaded PEGylated liposomal formulation was determined following 4, 24, 48, 72 and 96 hours after administration in colon carcinoma bearing Balb-C mice. The Table 3 represents the biodistribution of our formulation as ID/g% in blood and other organs, including tumor site. ID/g% represents the percentage of radioactivity of each organ per gram of that organ. Figure 4 displayed comparison of ID/g% in blood, liver, kidney, tumor and other organs. According to the Table 3 , after 4 hours post injection, the accumulated radioactive dose in all organs were increased and blood had highest dose at this time. Accumulated radioactive doses in kidney, Spleen and liver were higher in comparison to other organs at 24 hours post injection and remarkably, the accumulated dose in tumor tissue was considerably high. Moreover, the formulation in the blood was persisted significantly in this period of time and therefore has shown considerable stability. After 48 hour post injection, blood concentration of formulation was decreased but accumulated dose in kidney, spleen and liver were higher than other organs and furthermore, tumor tissue had remarkable concertation of formulation in comparison to the blood (p value < 0.05) as well. At 72 hour post administration, the accumulated dose in the liver, spleen and kidney is higher than other organs, and unlike the previous times, the liver dose is higher than the spleen. However, despite the formulation being cleared from the blood, it is still stable in the tumor site. At last in 96 hour post injection, accumulated dose in the liver, kidney, spleen and tumor is still high even after 96 hours. An important finding of our study is the lowest amount of dose reduction calculated in the tumor during 96 hours after injection as demonstrated by accumulated radioactivity in the tumor in comparison to the blood. According to Fig. 5 tumor/blood ratio, as an important factors in drug delivery to tumors, has increased significantly over the time period from 4 hour to 96 hour. Table 3 Biodistribution of 111 In-albumin-loaded liposomal formulation in mice at 4, 24, 48, 72 and 96 hours after administration, represented as Injection Dose/gram (ID/g). n = 3 at each time point. ID/g % for each organ, at times (h) after administration organs 4 h 24 h 48 h 72 h 96 h Blood 11.97 ± 3.07 3.89 ± 0.48 1.73 ± 0.32 1.03 ± 0.17 0.46 ± 0.02 Heart 4.56 ± 1.85 2.68 ± 0.30 3.47 ± 0.84 3.74 ± 0.90 3.21 ± 0.18 Lungs 9.28 ± 2.84 8.65 ± 2.28 7.69 ± 3.23 4.91 ± 0.93 6.71 ± 1.87 Liver 7.92 ± 2.03 12.39 ± 2.28 14.63 ± 1.11 28.36 ± 3.32 24.52 ± 2.42 Spleen 6.31 ± 2.77 20.67 ± 2.06 17.97 ± 2.57 19.25 ± 3.84 13.62 ± 1.37 Stomach 5.06 ± 4.38 1.38 ± 0.48 1.02 ± 0.51 2.43 ± 0.73 1.44 ± 0.64 Intestine 8.26 ± 6.08 6.31 ± 0.70 4.58 ± 0.82 4.67 ± 0.95 2.22 ± 0.57 Kidney 10.86 ± 3.67 34.73 ± 5.54 23.43 ± 8.06 23.31 ± 2.69 17.26 ± 2.77 Tumor 5.06 ± 1.34 8.73 ± 1.10 7.34 ± 0.35 6.77 ± 0.86 5.71 ± 1.28 Muscle 1.54 ± 0.53 1.65 ± 0.20 1.69 ± 0.40 3.08 ± 1.97 1.75 ± 0.96 Bone 3.44 ± 1.03 7.31 ± 1.65 6.54 ± 1.11 3.95 ± 0.53 5.56 ± 2.87 Tail 7.93 ± 1.54 2.80 ± 1.66 3.23 ± 2.43 3.24 ± 2.28 1.38 ± 0.40 Tumor/Blood ratio 0.42 2.24 4.24 6.57 12.41 Tumor/Muscle ratio 3.28 5.29 4.34 2.19 3.26 3.5. Gamma scintigraphy of radiolabeled liposomal formulation administered tumor bearing Balb-c mice: Gamma scintigraphy of these mice has demonstrated visual depiction of biodistribution. Figure 6 displayes static scintigraphy images of the mice subjected to formulation at times points of 4, 24, 48 and 96 hours after administration. Figure 7 represents dynamic scintigraphy image at 4 h. As demonstrated in Fig. 6 , tumor site has shown aggregation of radiolabeled liposomes and the resolution of image has enhanced over this time period in all scintigraphy images. Area with high aggregations pertains to kidneys and reticulosystem of spleen and liver, that in them entrapment of nanoliposomes is occurred. These organs are still visible as hot spots in imaging from the initial hours to 96 hours after injection of the formulation. Remarkably, according to the detected radioactivity, it seems that even at times after 96 hour, imaging can be continued and the tumor can be evaluated more clearly. 4. Discussion Various approaches have been explored to make efficient imaging of tumors and inflammatory conditions (Del Vecchio, Zannetti et al. 2007 , Gomes Marin, Nunes et al. 2020 , Weber, Czernin et al. 2020 , Bai, Qiu et al. 2023 ). Liposomes as pharmaceutical nanocarrier have been exploited in the field of drug delivery, and also tumor imaging (Man, Gawne et al. 2019 ). Characteristics of liposomes including their size and PEGylation influence their stability in vivo and their removal by phagocytes after administration (Filipczak, Pan et al. 2020 ). Radiolabeled liposomes can be particularly useful in imaging of position of infection and tumors in the body (Boerman, Storm et al. 1995 , Tzror-Azankot, Anaki et al. 2024 ). 111 In is a radionuclides emitting two gamma photons with energy of 173kev and 247kev and used in clinical imaging. In comparison to 99m TC (half-life 6 hours), the half-life of 111 In is longer and is about 67.2 hours. Furthermore 111 In has higher accumulation activity than 99m Tc (Awasthi, Goins et al. 1997 , Munekane, Kosugi et al. 2022 ). The binding of 111 In with proteins such as albumin is 10 12 folds stronger than its binding with oxine (Dillehay, Henkin et al. 2006 ). 111 In-oxine, which is a lipophilic compound, enters the liposome and interacts with encapsulated albumin in the liposome and because of this high affinity of 111 In with albumin, 111 In-albumin conjugation is formed and oxine is released and leaves the liposome (Dillehay, Henkin et al. 2006 , Owen, Thomas et al. 2020 ). In this study with using this approach for the first, we radiolabeled albumin-loaded liposomes with 111 In-oxine. In this study, we formulated PEGylated liposomes containing 111 In-albumin and investigated the characteristics of these liposomes, in vitro release of them in serum medium, biodistribution of 111 In-albumin containing liposomes in tumor-bearing mice, and their scintigraphy images. Mean size of liposomes was 130 nm, which is a proper particle size for escapement from the reticuloendothelial system, and consequently more entrapment in tumor area by EPR phenomenon. EPR mechanism is a critical consideration of drug-containing nanocarriers for permeation into tumors (Torchilin 2011 , Subhan, Parveen et al. 2023 ). PEGylation of liposomes is another factor for enhancement of EPR phenomenon (Harris and Chess 2003 , Shen and Yuan 2023 , Khajeei, Masoomzadeh et al. 2024 ). Our liposomes have zeta potential of about − 13.5 mv because of the presence of mPEG that imparts negative charge to the surface of liposomes. PDI of these nanoliposomes was less 0.1, indicating homogeneity of the size of the liposomal formulation. AFM and TEM microscopy showed almost spherical structures which also in the respect to their size were in good correlation with dynamic light scattering results. Encapsulation percentage of albumin in liposomes was approximately 45% which is similar to studies conducted on protein-encapsulating liposomes that their percentages in those studies range from about 30 to 47% (Nikpoor, Tavakkol-Afshari et al. 2015 , Yazdi, Tafaghodi et al. 2020 , Jash, Ubeyitogullari et al. 2021 ). The stability of these liposomes was first evaluated in the serum medium as in vitro study. Our results indicated that high stability of our formulation, due to its low release rate up to 24 hours. Similar to Doxil formulation, our albumin-containing liposomes have a very low release rate (maximum 10%) in the serum medium, and consequently is expected higher entrapment into the tumor area and thereafter, the phospholipases of the interstitial fluid cause degradation of the lipid bilayer of liposomes (Patel 1996 , Barenholz 2012 ). We conducted biodistribution study in mice bearing C-26 colon carcinoma. First obvious result is accumulation of liposomal formulation in reticuloendothelial system, such as the liver and spleen that is expected from their tissue structure. The important finding of our biodistribution study is increased accumulation of our formulation in tumor area through EPR phenomenon, in part because of the presence of the PEG molecule on these liposomes. Liposomes containing PEG have more stability in blood circulation compared to liposomes without PEG (Harris and Chess 2003 ). PEG molecules usually enter the liposomal bilayer and form a hydrated shell on the surface of liposomes, which protects the liposomes from being destroyed by plasma proteins. Increased stability of PEGylated liposomes derives from the PEG molecules that resulting in decreased uptake of these liposomes by the reticuloendothelial system, which, therefore, reduces their clearance from the blood, and enhances their half-life in the blood circulation. Furthermore, negative surface charge of PEGylated liposomes is lower compared to non-PEGylated liposomal formulations, which this charge induced by PEG reduces their absorption by reticuloendothelial system (Lee, Choi et al. 2005 , Jain and Stylianopoulos 2010 , Raina, Singh et al. 2021 ). In a study by Lee and his colleagues (Lee, Choi et al. 2005 ) using rats to investigate the biodistribution of radiolabeled liposomes, they concluded that liposomes not containing PEG are cleared from the blood 4 hours after injection and enter the liver and spleen. However, with adding of 5% PEG in the lipid composition of liposomal formulation, the accumulation of liposomes in the liver decreased and highest stability of liposomes in the blood was pertained to the liposomal formulation with 9.6% PEG concentration. Furthermore, another their conclusion was accumulation of liposomes in the liver is directly related to their size, so increasing in size increases their accumulation in the liver. The accumulation in the spleen, in addition to the size of the liposomes, depends on the PEG percentage of the formulation as well. The study of Awasthi and his colleagues (Awasthi, Garcia et al. 2003 ) further confirmed the finding that accumulation of liposomal formulation in the liver, spleen and blood is more than other organs after in vivo administration. The high accumulation in the RES system, such as the liver and spleen, is due to the special type of their vascular structure having pores and fenestrations, therefore liposomal particles are collected in these area. In addition, macrophages of liver and spleen break down liposomes, and their released 111 In is excreted by the kidneys. In our study, the highest accumulation was in the liver at 72 hours and in the spleen at 24 hours. The higher absorption of the spleen compared to the liver in 24 to 48 hours, and the increased absorption of the liver compared to the spleen in 72 to 96 hours are also significant. This is because of absorption of the particles larger than 160 nm in the spleen and absorption of smaller particles in the range of 30–160 nm in the liver. In the earlier hours of filtration, larger particles are filtered by the spleen, and after 48 hours, the smaller particles are removed by the liver. The increased accumulation of formulation in the kidneys is pertained to the degradation of liposomes and the clearance of released 111 In by the kidneys. One of our unexpected results was the high absorption in the kidneys, despite of the proper stability of liposomes in the first 24 hours after administration. This phenomenon could be related to the low QC of purchased 111 In-oxine radiopharmaceutical, leading to poor liposomal radiolabeling about 20%. The existence of 80% free 111 In (80%) in the formulation caused non-specifically binding to the outer surface of liposomes and after injection, free 111 In, not bonded to the albumin, separates from the liposomes over time and high excretion of this non-specific free 111 In from the kidneys was taken place. Lung, bone, stomach, muscle and intestine have similar vascular tissue, and the absorption of our radiolabeled liposomes is similar with a reflection to the blood level. The most important finding of our biodistribution study in comparison to other studies investigating biodistritribution of free radiolabeled-antibody and liposomal- 111 In not including albumin, was the relatively high and significant accumulation of our albumin- 111 In loaded liposomal formulation in tumor site after 24 hours of intravenous injection which was prolonged in the tumor up to 96 hours, therefore made it possible to evaluate the tumor up to 96 hours and probably so on after administration. The increase in Tumor/blood ratio between 4 and 96 hours was considerably significant and confirms that our formulation has greatly improved the absorption rate of the tumor compared to the blood level. Absorption of radiopharmaceuticals in tumors as well as abscesses and centers of infection and inflammation is due to the EPR phenomenon. Several studies have shown that liposomes are probably entrapped in these areas due to increased capillary penetration or damage of the layers of vascular endothelial cells (Sun, Xiang et al. 2022 , Kim, Cho et al. 2023 ). As mentioned earlier, PEGylated liposomes better demonstrates this phenomenon. One of the biggest challenges facing the use of the free form of proteins in the diagnosis and scintigraphy of tumors is their lack of sufficient accumulation in the tumor area following intravenous administration and non-specific accumulation in non-target organs. On the other hand, the EPR mechanism is a main mechanism for the entry of nanocarrier such as liposomes smaller than 200 nm into the tumor area. As a result, based on our biodistribution results, 111 In-albumin encapsulated in PEGylated liposomes is efficient approach for radio imaging because of high stability in bloodstream and more accumulation in the tumor area. In addition, the high half-life of 111 In and its high binding strength with albumin and stability of this formulation confirmed by imaging up to 96 hours, have led to the possibility of imaging the tumor in a longer period of time for clinical application. In a study, Boerman and his colleagues (Boerman, Storm et al. 1995 ) investigated the biodistribution of PEGylated liposomes labeled with 111 In compared to 111 In bound to IgG antibody in rats with focal S.areus infections. In that study, they found that the removal of radioactive labeled material in abscesses with In-containing liposomes is 2 times the removal of In-bound IgG. Comparison of the biodistribution of our 111 In-albumin liposomal formulation to the biodistribution of In-IgG and In-liposome in Boerman's study shows that formulation stability in the blood and Tumor/blood ratio as well as tumor accumulation have been significantly improved. Especially, with our formulation, it is possible to examine the tumor for up to 96 hours. In this research, scintigraphy imaging in mice was also performed, and the information obtained from the scintigraphy was consistent with the results obtained from biodistribution study. In the period of time, the contrast between the tumor and the background was improved and the high uptake in the tumor as well as in the liver, spleen and kidneys forms a distinct localization in scintigraphy image. 5. Conclusions Carefully designed radiopharmaceutical agents play a core role in nuclear medicine. In this study for the first time, radiolabeled 111 In-albumin encapsulated liposomes were prepared by using high binding affinity of 111 In to the albumin molecules entrapped into the liposome. Our biodistribution study and scintigraphy images demonstrated that 111 In-albumin encapsulated liposomes have high stability and efficiency that could be exploited in long-term tumor imaging. Further studies including randomized clinical trial is needed for its clinical application. Declarations Authorship contributions: M.A: Investigation, Formal analysis, Writing - original draft. Z.A, A.A and J.RY: Investigation, Visualization, Software, Writing - original draft. MR.J: Project administration, Formal analysis, Conceptualization, Writing - review and editing, Resources. K.S: Project administration, Formal analysis, Conceptualization, Writing - review and editing, Resources. All authors read and approved this final manuscript. Funding: This study was a part of Mohammad Ahrari M.Sc. dissertation with master thesis code 11130512942015. This study was supported by Vice Chancellor for Research and Technology, Mashhad University of Medical Sciences. Availability of data and material: The data produced during this research study are available from the corresponding authors upon request. Ethics approval and consent to participate: All animal experiments were conducted in accordance with the approval of Institutional Ethical Committee of Mashhad University of Medical Sciences in accordance with the principles of the Declaration of Helsinki. Consent to participate was not applicable as this study did not involve human subjects. (Master thesis code: 11130512942015) Consent to publish: Not applicable as t his study did not include human subjects. Acknowledgements: The support of the Nanotechnology Research Center, Mashhad University of Medical Sciences and Department of nuclear medicine, Ghaem Hospital, Mashhad University of Medical Sciences is gratefully acknowledged. We would like to thank Dr Amin Reza Nikpoor for his excellent assistance. Competing interests: The authors declare no competing financial or nonfinancial interests or personal relationships that could have appeared to influence the work reported in this article. References Awasthi, V., D. Garcia, B. Goins and W. Phillips (2003). "Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits." International journal of pharmaceutics 253 (1-2): 121-132. Awasthi, V., B. Goins, R. Klipper, R. Loredo, D. Korvick and W. Phillips (1997). Comparison of dual radiolabeled liposomes (DRL), Tc-99m-DMP and GA-67 citrate for imaging osteomyelitis in rabbit model. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6766595","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":469065443,"identity":"be1b4278-f5b3-4f1c-b579-3891649b5838","order_by":0,"name":"Mohammad 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1","display":"","copyAsset":false,"role":"figure","size":698463,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of albumin-loaded PEGylated liposomes by (A) Transmission Electron Microscopy and (B) Atomic Force Microscopy\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6766595/v1/af2e52560165040f711e0f42.png"},{"id":84545130,"identity":"b21b7383-66ff-4927-9f98-c1ab3887d312","added_by":"auto","created_at":"2025-06-13 09:12:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43160,"visible":true,"origin":"","legend":"\u003cp\u003eGraphic representation of \u003csup\u003e111\u003c/sup\u003eIn radiolabeled albumin-loaded liposomes in peak 1 vs free \u003csup\u003e111\u003c/sup\u003eIn in peak 2.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6766595/v1/a70707ee74cdb9d1bc5a9eb7.png"},{"id":84547927,"identity":"65654ac0-ab1d-4ff2-85bc-b3afef39cc95","added_by":"auto","created_at":"2025-06-13 09:36:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":23155,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of remaining radioactivity in albumin-\u003csup\u003e111\u003c/sup\u003eIn-loaded liposomes in serum medium. n=3\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6766595/v1/911d5a01fb966ee806df89e5.png"},{"id":84545134,"identity":"cc51b97b-494d-423b-8ff1-4a7d62b20102","added_by":"auto","created_at":"2025-06-13 09:12:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97504,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of biodistribution of \u003csup\u003e111\u003c/sup\u003eIn-albumin-loaded liposomal formulation in mice at 4, 24, 48, 72 and 96 hours after administration in specified organs, represented as Injection Dose/gram (ID/g). n=3 at each time point.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6766595/v1/11f2007b3f62c1686904d46b.png"},{"id":84546023,"identity":"f855d749-33ca-46ad-ac20-0a91dc6e5f60","added_by":"auto","created_at":"2025-06-13 09:20:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":52497,"visible":true,"origin":"","legend":"\u003cp\u003eTumor/Blood ratio of \u003csup\u003e111\u003c/sup\u003eIn-albumin-loaded liposomal formulation in mice at 4, 24, 48, 72 and 96 hours after administration\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6766595/v1/47f3a4be6270c19e18755fc6.png"},{"id":84545140,"identity":"5f5cec3f-939e-4285-8a18-724021f14d6a","added_by":"auto","created_at":"2025-06-13 09:12:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":329929,"visible":true,"origin":"","legend":"\u003cp\u003ePlanar scintigraphy images of mice administrated \u003csup\u003e111\u003c/sup\u003eIn-albumin-loaded liposomal formulation at (A) 4, (B) 24, (C) 48, and (D) 96 hours post injection. Ant: Anterior view, Post: Posterior view.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6766595/v1/1c0ce3a2c2308906afdf4e74.png"},{"id":84545142,"identity":"ef9fd1f3-ac01-48f5-8d5e-a0861cabb229","added_by":"auto","created_at":"2025-06-13 09:12:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":407404,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic scintigraphy image at 4 h. Ant: Anterior view, Post: Posterior view.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6766595/v1/5adf3950beff6b56608990f2.png"},{"id":85456187,"identity":"36965e63-5585-410b-8c52-18e8e6824e5a","added_by":"auto","created_at":"2025-06-26 06:30:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3508348,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6766595/v1/fb563744-a634-406b-af14-2fc6fecd29d3.pdf"}],"financialInterests":"","formattedTitle":"Development of radiolabeled 111In-albumin liposomes for long-term imaging of tumors","fulltext":[{"header":"1.\tIntroduction","content":"\u003cp\u003eGamma ray imaging or scintigraphy is a valuable tool in the optimal design of the liposomal formulation of radiopharmaceutical agents, and allows non-invasive tracking of the distribution of liposomes in the body by gamma ray (Goins and Phillips \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). While radiological imaging techniques such as MRI and CT give anatomical information, centigram images display the distribution of radiopharmaceuticals in the body environment and determine the changes in physiological processes. It is well known that the diagnosis of injuries, tumors or infection centers can be determined by traditional Gama ray imaging before the appearance of anatomical abnormalities. Furthermore, the prompt diagnosis of suspected area of above abnormalities is of great importance in terms of clinical application (Boerman, Laverman et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Liposomes have been labeled with gamma irradiating radiopharmaceuticals such as \u003csup\u003e67\u003c/sup\u003eGa, \u003csup\u003e111\u003c/sup\u003eIn and \u003csup\u003e99m\u003c/sup\u003eTc for the purpose of tumor image. Liposomes have been exploited for imaging of tumor, infection, and blood pool, and there are large amount of preclinical evidence showing radiolabeled liposomes can image tumors sites and inflammatory lesions with high specificity and sensitivity. Moreover, according to the biodistribution profiles obtained in the studies on rabbits, it has been determined that PEGylated liposomes can maintain their shielding properties more than other types of liposomes. Immediately removal of liposome by MPS phagocytizing cells in the liver and spleen greatly limited their use inside the human body. Subsequent progress in the field of long-circulating liposomes (LCLS) has created nanoliposomes that are less likely to be taken up by the cells of the mononuclear phagocyte system (MPS). The development of these LCLs by enhancing their circulating half-life has increased our ability to detect pathological processes by in vivo scintigraphy procedures (Boerman, Laverman et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Awasthi, Garcia et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Low, Yang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The researches of several decades provide a depth understanding of the effects of liposomal characteristics such as size, type and concentration of lipids, surface charge and hydrophilicity, on their pharmacokinetic after intravenous administration. In particular, modifying the surface characteristics of liposomes and the use of nanoliposomes with approximately 100nm size, containing more cholesterol and phospholipids with saturated chains enhances the circulation time of liposomes in the blood and reduces their removal by the reticuloendothelial system (Chow, Lin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Man, Gawne et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Today, \u003csup\u003e99m\u003c/sup\u003eTc radiopharmaceutical is used in nuclear medicine departments, which has a short half-life. For evaluating some diseases, especially tumors, we need a radiopharmaceutical agent with long shelf life. \u003csup\u003e111\u003c/sup\u003eIn is a radionuclides that is obtained as \u003csup\u003e111\u003c/sup\u003eInCl\u003csub\u003e3\u003c/sub\u003e with a specific activity of 15mci/ml in 0.01 normal HCl solution in the cyclotron section of the Atomic Energy Organizations. It does not have beta irradiation and emits two gamma photons with energy of 173 Kev and 247 Kev, which are used in clinical imaging. \u003csup\u003e111\u003c/sup\u003eIn have been used for cell radiolabeling by binding to cellular proteins. Furthermore, the binding of \u003csup\u003e111\u003c/sup\u003eIn with proteins such as albumin is 10\u003csup\u003e12\u003c/sup\u003e folds stronger than its binding with oxine (Awasthi, Goins et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Dillehay, Henkin et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Phillips, Goins et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Drozdovitch, Brill et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Lamichhane, Dewkar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, De Silva, Fu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, we used this phenomenon and prepared albumin-loaded liposomes and treated these liposomes as simulated cells. Subsequently albumin-loaded liposomes were radiolabeled with \u003csup\u003e111\u003c/sup\u003eIn-oxine. Unlike HMPAO, which is trapped inside the liposome, oxine releases from liposomes through the lipid bilayer and transfers into the aqueous phase, after while separating from \u003csup\u003e111\u003c/sup\u003eIn and binding of \u003csup\u003e111\u003c/sup\u003eIn with albumin. This approach made radiolabeled liposomes to be used for tumor imaging. In imaging with radiolabeled liposomes, the metastases inside the liver and spleen are identified as cold spots (Jaggi, Khar et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). For this purpose, almost all traditional liposomal formulations could be used. The development of liposomal radiopharmaceuticals to determine the location of tumors was noticed since it was shown that specific liposomal formulations accumulate at the tumor site. The development of liposomes with longer half-life in the circulation has increased the hope of using these liposomal radiopharmaceuticals in tumor imaging. The ability of liposomal formulations labeled with \u003csup\u003e99m\u003c/sup\u003eTC, \u003csup\u003e67\u003c/sup\u003eGa and \u003csup\u003e111\u003c/sup\u003eIn in identifying tumors has been proven in different studies (Presant, Proffitt et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1988\u003c/span\u003e, Ogihara-Umeda, Sasaki et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Ogihara-Umeda, Sasaki et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Various studies have shown that liposomes with longer half-life and higher stability in the circulation show improved tumors uptake (Boerman, Storm et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, Ishida, Maruyama et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Phillips \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Dams, Oyen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Lee, Choi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In regarding these considerations, the present work aims to combine these approaches for long-term tumor imaging.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials:\u003c/h2\u003e \u003cp\u003eHuman Albumin 20% solution was purchased from Biotest, Germany. The phospholipids, hydrogenated soya phosphatidylcholine (HSPC), methoxypolyethelene glycol (MW 2000)-distearylphosphatidylethanolamine (mPEG\u003csub\u003e2000\u003c/sub\u003e-DSPE), were obtained from Lipoid (Ludwigshafen, Germany). Cholesterol and Sephadex G-50 were purchased from Sigma\u0026ndash;Aldrich (St. Louis, MO). Iodine-125 (\u003csup\u003e125\u003c/sup\u003eI) was supplied from Amersham (Uppsala, Sweden). All other used chemicals were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of albumin-loaded liposomes:\u003c/h2\u003e \u003cp\u003eLiposomes were prepared by thin film hydration method plus extrusion (Nikpoor, Tavakkol-Afshari et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The total lipid concentration of all the liposomal formulations was adjusted 48mM. In brief, HSPC, cholesterol and mPEG2000-DSPE, that all of them were dissolved in chloroform, were mixed at the molar ratios presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Then, evaporation of organic solvent was performed by a rotary evaporator in three hours, followed by three hours\u0026rsquo; freeze drying to prepare thin film layer. The dried lipid film was hydrated with albumin solution by rotating in a water bath at 60\u0026deg;C and gentle mixing on a vortex mixer until thin film dissolved thoroughly. Afterward, liposomes in solution were downsized by extrusion through polycarbonate membrane filters 400nm, 200nm and 100nm (11 times each) at 60\u0026deg;C to reach the optimized size. The resulting liposomal products were purified by dialysis. In the dialysis method, free albumin molecules which weren\u0026rsquo;t entrapped in liposomes, removed from liposomal formulations in 300 KDa molecular weight cut-off dialysis membrane (Spectrum) by dialyzing against HEPES 10 mM and sucrose 7.5% buffer (pH\u0026thinsp;~\u0026thinsp;7.4). The final liposomal solutions were stored at 4\u0026deg;C until later use. Blank liposomes (without albumin) were prepared following the same procedures mentioned before, with hydration solution excluding albumin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization of Albumin-loaded liposomes:\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Particle size, PDI and Zeta potential measurement:\u003c/h2\u003e \u003cp\u003eParticles size, PDI (polydispersity index) and zeta potential of liposomes were measured by the dynamic light scattering technique (DLS) in a Zetasizer Nano ZS instrument (Malvern Instruments, CO., UK). The measurements were performed at 25\u0026deg;C after the dilution of formulation with HEPES 10 mM and sucrose 7.5% buffer (pH\u0026thinsp;~\u0026thinsp;7.4). Zeta potential of liposomes was measured using electrophoretic light scattering by the same instrument. The results of particle size and zeta potential were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Encapsulation efficiency measurement:\u003c/h2\u003e \u003cp\u003eEncapsulation efficiency of albumin in liposomes was determined by separating free albumin from albumin-loaded liposomes using ultrafiltration method. Predialyzed samples were placed in the upper chamber of an Ultra-4 centrifugal filter unites with a molecular weight cut-off of 300 KDa (Amicon\u0026reg; Ultra-0.5, MWCO 300,000 Da; Millipore, Boston, USA) and centrifuged at 4000g for 20 min at 8\u0026deg;C. The free albumin was separated from liposomes and collected in the bottom of the tube. The concentration of albumin in filtrate was determined using BCA protein assay and encapsulation efficiency was calculated (Nikpoor, Tavakkol-Afshari et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In BCA protein assay method for encapsulated insulin in liposomes, the interfering substances were removed via sample pretreatment by sodium deoxycolate and trichloroacetic acid. Encapsulation efficiency of albumin in liposomal formulations was determined by this equation: % Encapsulation: ([albumin content after purification (by dialysis)] \u0026divide; [albumin content before purification]) \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Phosphate assay of liposomal formulation:\u003c/h2\u003e \u003cp\u003eIn order to determine phosphate content of albumin-loaded liposomes and its alteration in formulation process, Bartlett phosphate assay protocol for the liposomal formulations was performed (Shmeeda, Even-Chen et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The test was performed using this procedure and then concentration of phospholipids was calculated against standards.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. Morphology of albumin-loaded liposomes by Transmission Electron Microscopy and Atomic Force Microscopy:\u003c/h2\u003e \u003cp\u003eThe morphology of PEGylated albumin-loaded liposomes were determined using atomic force microscopy (AFM). This method of analysis determines surface structure of particles such as liposomes and provides three dimensional images. For AFM analysis, the formulation was diluted with dialyzed buffer (diluted 1:500) and about 30 \u0026micro;L of each sample were placed on a mika surface. After drying in room temperature, samples were analyzed by AFM microscope (NanoWizard\u0026reg;II, JPK model, Germany). Over approaching cantilever tip on samples, imaging in 2, 5 and 10 \u0026micro;m scales was performed; therefore 2- dimensional and 3-dimensional images of liposomes were obtained.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Radiolabeling of Albumin- loaded liposomes:\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Measurement of the radioactivity of \u003csup\u003e111\u003c/sup\u003eIn-oxine:\u003c/h2\u003e \u003cp\u003eFirst, radioactivity of \u003csup\u003e111\u003c/sup\u003eIn-oxine complex was assessed. Briefly, by mixing \u003csup\u003e111\u003c/sup\u003eIn-oxine with chloroform and normal saline and then incubating them in room temperature two phases were formed. Radioactivity of chloroform and normal saline were measured. Free \u003csup\u003e111\u003c/sup\u003eIn enters saline phase due to its hydrophilicity and \u003csup\u003e111\u003c/sup\u003eIn-oxine enters chloroform phase due to its lipophilicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Radiolabeling of Albumin- loaded liposomes with \u003csup\u003e111\u003c/sup\u003eIn-oxine:\u003c/h2\u003e \u003cp\u003eAlbumin- loaded liposomes was labeled with \u003csup\u003e111\u003c/sup\u003eIn-oxine based on higher affinity of \u003csup\u003e111\u003c/sup\u003eIn to albumin that was a novel labeling approach. In brief, 1 mL of albumin-loaded liposomes mixed with 5 millicurie activity of \u003csup\u003e111\u003c/sup\u003eIn-oxine and then incubated in 55\u0026deg;C for 20 minutes in a rotatory state. To separate labeled albumin-loaded liposomes with \u003csup\u003e111\u003c/sup\u003eIn-oxine from free \u003csup\u003e111\u003c/sup\u003eIn-oxine, sephadex G-25 size exclusion column chromatography was used. Phosphate buffered saline (PBS) was used for washing. The purified labeled albumin-loaded liposomes with \u003csup\u003e111\u003c/sup\u003eIn-oxine were collected in initial tubes and free \u003csup\u003e111\u003c/sup\u003eIn-oxine was separated in terminal tubes. Radioactivity of each tubes was measured by dose calibrator instrument.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Sterilization of liposomal formulations:\u003c/h2\u003e \u003cp\u003eFor intravenous injection, the product must be sterile. Radiolabeled liposomal formulations were sterilized by 220 nm porous filtered syringe under laminar flow hood and then collected in sterile tubes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Stability study of radiolabeled liposomal formulation in serum:\u003c/h2\u003e \u003cp\u003eStability of these liposomes was measured in serum medium by thin layer chromatography (TLC) method. Briefly, in the lower portion of silica gel strip, 50 microliters of radioactive liposomes is loaded and then placed in serum medium. At 0.5, 1, 4 and 24 hour, silica gel strip is cut from lower third and radioactivity of lower third and upper two third is measured. Released \u003csup\u003e111\u003c/sup\u003eIn from liposomal formulations in the incubation period moved along silica gel strip and reached to the upper portion. Therefore, upper portion of silica gel contains released \u003csup\u003e111\u003c/sup\u003eIn and the lower portion contains \u003csup\u003e111\u003c/sup\u003eIn-albumin encapsulated in liposomal formulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7. In vivo studies:\u003c/h2\u003e \u003cp\u003eMice, used in this work, were BALB-c with 6 to 8 weeks age and 18 to 20 grams, purchased from Pasteur institute of Iran (Tehran, Iran). Animal, were kept in the animal house of Pharmaceutical research center, Buo Ali research institute, Mashhad University of Medical Sciences and had free access to water and animal food under appropriate environmental conditions. All animal experiments were conducted in accordance with the approval of Institutional Ethical Committee of Mashhad University of Medical Sciences\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Induction of Balb-c mice model of C26 colon carcinoma:\u003c/h2\u003e \u003cp\u003eC26 colon carcinoma cell line was used for induction of colon carcinoma in mice. This cell line was cultured in RPMI-1640 medium supplemented with 10% FBS and incubated in 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. The mice were anesthetized by intraperitoneal injection of 200\u0026micro;L zylazin/ketamine (5:15) and then subjected to the administration of 3 10\u003csup\u003e5\u003c/sup\u003e \u0026times; C-26 cells (in 60 \u0026micro;L volume) subcutaneously at their flanks. After one week, tumors were generated with the size of about 5 millimeters in each dimension at the flank of the mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. Intravenous injection of radiolabeled formulation to Balb-c mice:\u003c/h2\u003e \u003cp\u003eFor this step of our work, 15 mice were used, which were in 5 groups of 3. Mice in groups 1, 2, and 3 were administrated with 200\u0026micro;L and groups 4 and 5 with 350\u0026micro;L radiolabeled liposome intravenously by syringe. Their radioactivities were measured by dose calibrator instrument. Also, after injecting into the mice and emptying the syringes, the amount of activity remaining in them was measured by dose calibrator and the quantity of radioactivity of each formulation injected into each mice was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3. Biodistribution of radiolabeled liposomes in Balb-c mice:\u003c/h2\u003e \u003cp\u003eAfter administration of liposomal formulations, group 1 mice were sacrificed after 4 hours, group 2 after 24 hours, group 3 after 48 hours, group 4 after 72 hours, group 5 after 96 hours, and their other organs (blood, heart, lungs, Liver, spleen, stomach, intestine, kidney, muscle tumor, tail and bone) were removed and placed into gamma tubes. The organs were washed with phosphate buffered saline, and weighted and the radioactivity of each organ was determined by a gamma counter (Delshid DL-100). The percentage of radioactivity per organ was measured as the mean injected dose per gram of organ (ID/g %) (Nikpoor, Tavakkol-Afshari et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.7.4. Gamma scintigraphy of Balb-c mice:\u003c/h2\u003e \u003cp\u003eThe biodistribution of liposomal formulations was furthermore investigated by planar photography method. Gamma cameras having the ability to receive gamma rays are used for this purpose. For this step of our experiment, 200 \u0026micro;Ci of the formulation equivalent to 4.7 MBq of radiolabeled albumin loaded liposomes were injected intravenously through the mice tail vein. At 4, 24, 48, 96 hours after administration, the mice were anesthetized by ketamine/xylazine and then planar photography of biodistribution of liposomes were prepared by a Siemens Dual head Gamma Camera device with a matrix of 300,000 and resolution of 256\u0026times;256 pixel. The characteristics of imaging instrument were: Series: Static, Camera present: In111-NMG, Matrix Size: 64\u0026times;64, Zoom: 2.29, Detectors: Both detectors, Ant: 5mIn, Post: 5mIn, Orientation: Head Out/SupIne.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis:\u003c/h2\u003e \u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (S.D.). The outcomes were analyzed by one-way analysis of variance (ANOVA) statistical test using GraphPad InStat version 3.05 for windows (GraphPad Software, USA). A statistical difference was considered when p value was less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Formulation and characterization of albumin-loaded liposomes:\u003c/h2\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1. Particle size, PDI, zeta potential and encapsulation efficiency measurements:\u003c/h2\u003e\n \u003cp\u003eLiposomal formulation (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) containing albumin could be prepared reproducibly by thin film hydration method plus extrusion. Particle size, zeta potential and encapsulation efficiency of albumin-loaded liposomes were displayed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Mean sizes of liposomes were about 130 nm by extrusion through polycarbonate membrane filter and encapsulation efficiency of liposomes for albumin ranged from 43\u0026ndash;50%. PDIs of formulations were less than 0.2, indicative of almost homogenous distribution of liposomal sizes. Furthermore, phosphate concentration of the formulation was measured to be about 90 mM that was higher than expected based on formulation design, indicating evaporation duration formulation preparation.\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eParticle size, PDI and zeta potential of liposomes encapsulating albumin. (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlbumin-loaded liposomal formulation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMolar ratio (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZeta average Size (nm)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePolydispersity\u003c/p\u003e\n \u003cp\u003e\u0026plusmn; SD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZeta potential (mV)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEntrapment efficiency%\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHSPC/CHOl/mPEG\u003csub\u003e2000\u003c/sub\u003e-DSPE (48 mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55/40/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e130.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.175\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;15.47\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2. Morphology of albumin-loaded liposomes by Transmission Electron Microscopy and Atomic Force Microscopy:\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows morphology of our albumin-loaded PEGylated liposomal formulation by both TEM and AFM microscopies. TEM microscopy depicts the structure and morphology of liposomes and AFM demonstrates their surface topology. As demonstrated by TEM and AFM microscopy, albumin-loaded liposomes had almost spherical shapes that were in expected correlation with the sizes obtained from DLS analysis.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Radiolabeling of Albumin- loaded liposomes:\u003c/h2\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1. Measurement of the radioactivity of \u003csup\u003e111\u003c/sup\u003eIn-oxine for quality control:\u003c/h2\u003e\n \u003cp\u003eAs displayed in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the percentage of \u003csup\u003e111\u003c/sup\u003eIn-oxine radioactivity was 20% that indicates its quality control for the study.\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eQuality Control of \u003csup\u003e111\u003c/sup\u003eIn-oxine used in this experiment\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhase type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRadioactivity (\u0026micro;Ci)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e111\u003c/sup\u003eIn-oxine radioactivity percent\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal saline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e(160 / (640\u0026thinsp;+\u0026thinsp;160))*100%= 20%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echloroform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e160\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2. Radiolabeling and purification of Albumin- loaded liposomes with \u003csup\u003e111\u003c/sup\u003eIn-oxine:\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows radiolabeling of albumin-loaded liposomes with \u003csup\u003e111\u003c/sup\u003eIn-oxine and purification of them from free \u003csup\u003e111\u003c/sup\u003eIn-oxine. The first peak indicates the radiolabeled liposomes that used in our study and second peak is pertinent to free \u003csup\u003e111\u003c/sup\u003eIn-oxine that is excluded.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Stability study of radiolabeled liposomal formulation in serum:\u003c/h2\u003e\n \u003cp\u003eStability of our formulation in serum medium was evaluated up to 24 hours. Percentage of released \u003csup\u003e111\u003c/sup\u003eIn from liposomes indicating release percentage is presented in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. At 0.5, 1, 4 and 24 hours, remained radioactivity of our formulation is measured.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Biodistribution study of radiolabeled liposomes in tumor bearing mice by measurement of ID/g%:\u003c/h2\u003e\n \u003cp\u003eThe biodistribution of our albumin-loaded PEGylated liposomal formulation was determined following 4, 24, 48, 72 and 96 hours after administration in colon carcinoma bearing Balb-C mice. The Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e represents the biodistribution of our formulation as ID/g% in blood and other organs, including tumor site. ID/g% represents the percentage of radioactivity of each organ per gram of that organ. Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e displayed comparison of ID/g% in blood, liver, kidney, tumor and other organs.\u003c/p\u003e\n \u003cp\u003eAccording to the Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, after 4 hours post injection, the accumulated radioactive dose in all organs were increased and blood had highest dose at this time. Accumulated radioactive doses in kidney, Spleen and liver were higher in comparison to other organs at 24 hours post injection and remarkably, the accumulated dose in tumor tissue was considerably high. Moreover, the formulation in the blood was persisted significantly in this period of time and therefore has shown considerable stability. After 48 hour post injection, blood concentration of formulation was decreased but accumulated dose in kidney, spleen and liver were higher than other organs and furthermore, tumor tissue had remarkable concertation of formulation in comparison to the blood (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) as well. At 72 hour post administration, the accumulated dose in the liver, spleen and kidney is higher than other organs, and unlike the previous times, the liver dose is higher than the spleen. However, despite the formulation being cleared from the blood, it is still stable in the tumor site. At last in 96 hour post injection, accumulated dose in the liver, kidney, spleen and tumor is still high even after 96 hours. An important finding of our study is the lowest amount of dose reduction calculated in the tumor during 96 hours after injection as demonstrated by accumulated radioactivity in the tumor in comparison to the blood. According to Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e tumor/blood ratio, as an important factors in drug delivery to tumors, has increased significantly over the time period from 4 hour to 96 hour.\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBiodistribution of \u003csup\u003e111\u003c/sup\u003eIn-albumin-loaded liposomal formulation in mice at 4, 24, 48, 72 and 96 hours after administration, represented as Injection Dose/gram (ID/g). n\u0026thinsp;=\u0026thinsp;3 at each time point.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eID/g % for each organ, at times (h) after administration\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eorgans\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e24 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e48 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e96 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBlood\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLungs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.69\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.36\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStomach\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;4.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIntestine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.26\u0026thinsp;\u0026plusmn;\u0026thinsp;6.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.73\u0026thinsp;\u0026plusmn;\u0026thinsp;5.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.43\u0026thinsp;\u0026plusmn;\u0026thinsp;8.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMuscle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTail\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor/Blood ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor/Muscle ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Gamma scintigraphy of radiolabeled liposomal formulation administered tumor bearing Balb-c mice:\u003c/h2\u003e\n \u003cp\u003eGamma scintigraphy of these mice has demonstrated visual depiction of biodistribution. Figure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e displayes static scintigraphy images of the mice subjected to formulation at times points of 4, 24, 48 and 96 hours after administration. Figure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e represents dynamic scintigraphy image at 4 h. As demonstrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, tumor site has shown aggregation of radiolabeled liposomes and the resolution of image has enhanced over this time period in all scintigraphy images. Area with high aggregations pertains to kidneys and reticulosystem of spleen and liver, that in them entrapment of nanoliposomes is occurred. These organs are still visible as hot spots in imaging from the initial hours to 96 hours after injection of the formulation. Remarkably, according to the detected radioactivity, it seems that even at times after 96 hour, imaging can be continued and the tumor can be evaluated more clearly.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eVarious approaches have been explored to make efficient imaging of tumors and inflammatory conditions (Del Vecchio, Zannetti et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Gomes Marin, Nunes et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Weber, Czernin et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Bai, Qiu et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Liposomes as pharmaceutical nanocarrier have been exploited in the field of drug delivery, and also tumor imaging (Man, Gawne et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Characteristics of liposomes including their size and PEGylation influence their stability in vivo and their removal by phagocytes after administration (Filipczak, Pan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Radiolabeled liposomes can be particularly useful in imaging of position of infection and tumors in the body (Boerman, Storm et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, Tzror-Azankot, Anaki et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003csup\u003e111\u003c/sup\u003eIn is a radionuclides emitting two gamma photons with energy of 173kev and 247kev and used in clinical imaging. In comparison to \u003csup\u003e99m\u003c/sup\u003eTC (half-life 6 hours), the half-life of \u003csup\u003e111\u003c/sup\u003eIn is longer and is about 67.2 hours. Furthermore \u003csup\u003e111\u003c/sup\u003eIn has higher accumulation activity than \u003csup\u003e99m\u003c/sup\u003eTc (Awasthi, Goins et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Munekane, Kosugi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The binding of \u003csup\u003e111\u003c/sup\u003eIn with proteins such as albumin is 10\u003csup\u003e12\u003c/sup\u003e folds stronger than its binding with oxine (Dillehay, Henkin et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). \u003csup\u003e111\u003c/sup\u003eIn-oxine, which is a lipophilic compound, enters the liposome and interacts with encapsulated albumin in the liposome and because of this high affinity of \u003csup\u003e111\u003c/sup\u003eIn with albumin, \u003csup\u003e111\u003c/sup\u003eIn-albumin conjugation is formed and oxine is released and leaves the liposome (Dillehay, Henkin et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Owen, Thomas et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study with using this approach for the first, we radiolabeled albumin-loaded liposomes with \u003csup\u003e111\u003c/sup\u003eIn-oxine.\u003c/p\u003e \u003cp\u003eIn this study, we formulated PEGylated liposomes containing \u003csup\u003e111\u003c/sup\u003eIn-albumin and investigated the characteristics of these liposomes, in vitro release of them in serum medium, biodistribution of \u003csup\u003e111\u003c/sup\u003eIn-albumin containing liposomes in tumor-bearing mice, and their scintigraphy images. Mean size of liposomes was 130 nm, which is a proper particle size for escapement from the reticuloendothelial system, and consequently more entrapment in tumor area by EPR phenomenon. EPR mechanism is a critical consideration of drug-containing nanocarriers for permeation into tumors (Torchilin \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Subhan, Parveen et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). PEGylation of liposomes is another factor for enhancement of EPR phenomenon (Harris and Chess \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Shen and Yuan \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Khajeei, Masoomzadeh et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our liposomes have zeta potential of about \u0026minus;\u0026thinsp;13.5 mv because of the presence of mPEG that imparts negative charge to the surface of liposomes. PDI of these nanoliposomes was less 0.1, indicating homogeneity of the size of the liposomal formulation. AFM and TEM microscopy showed almost spherical structures which also in the respect to their size were in good correlation with dynamic light scattering results. Encapsulation percentage of albumin in liposomes was approximately 45% which is similar to studies conducted on protein-encapsulating liposomes that their percentages in those studies range from about 30 to 47% (Nikpoor, Tavakkol-Afshari et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Yazdi, Tafaghodi et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Jash, Ubeyitogullari et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The stability of these liposomes was first evaluated in the serum medium as in vitro study. Our results indicated that high stability of our formulation, due to its low release rate up to 24 hours. Similar to Doxil formulation, our albumin-containing liposomes have a very low release rate (maximum 10%) in the serum medium, and consequently is expected higher entrapment into the tumor area and thereafter, the phospholipases of the interstitial fluid cause degradation of the lipid bilayer of liposomes (Patel \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Barenholz \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe conducted biodistribution study in mice bearing C-26 colon carcinoma. First obvious result is accumulation of liposomal formulation in reticuloendothelial system, such as the liver and spleen that is expected from their tissue structure. The important finding of our biodistribution study is increased accumulation of our formulation in tumor area through EPR phenomenon, in part because of the presence of the PEG molecule on these liposomes. Liposomes containing PEG have more stability in blood circulation compared to liposomes without PEG (Harris and Chess \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). PEG molecules usually enter the liposomal bilayer and form a hydrated shell on the surface of liposomes, which protects the liposomes from being destroyed by plasma proteins. Increased stability of PEGylated liposomes derives from the PEG molecules that resulting in decreased uptake of these liposomes by the reticuloendothelial system, which, therefore, reduces their clearance from the blood, and enhances their half-life in the blood circulation. Furthermore, negative surface charge of PEGylated liposomes is lower compared to non-PEGylated liposomal formulations, which this charge induced by PEG reduces their absorption by reticuloendothelial system (Lee, Choi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Jain and Stylianopoulos \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Raina, Singh et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In a study by Lee and his colleagues (Lee, Choi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) using rats to investigate the biodistribution of radiolabeled liposomes, they concluded that liposomes not containing PEG are cleared from the blood 4 hours after injection and enter the liver and spleen. However, with adding of 5% PEG in the lipid composition of liposomal formulation, the accumulation of liposomes in the liver decreased and highest stability of liposomes in the blood was pertained to the liposomal formulation with 9.6% PEG concentration. Furthermore, another their conclusion was accumulation of liposomes in the liver is directly related to their size, so increasing in size increases their accumulation in the liver. The accumulation in the spleen, in addition to the size of the liposomes, depends on the PEG percentage of the formulation as well. The study of Awasthi and his colleagues (Awasthi, Garcia et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) further confirmed the finding that accumulation of liposomal formulation in the liver, spleen and blood is more than other organs after in vivo administration. The high accumulation in the RES system, such as the liver and spleen, is due to the special type of their vascular structure having pores and fenestrations, therefore liposomal particles are collected in these area. In addition, macrophages of liver and spleen break down liposomes, and their released \u003csup\u003e111\u003c/sup\u003eIn is excreted by the kidneys. In our study, the highest accumulation was in the liver at 72 hours and in the spleen at 24 hours. The higher absorption of the spleen compared to the liver in 24 to 48 hours, and the increased absorption of the liver compared to the spleen in 72 to 96 hours are also significant. This is because of absorption of the particles larger than 160 nm in the spleen and absorption of smaller particles in the range of 30\u0026ndash;160 nm in the liver. In the earlier hours of filtration, larger particles are filtered by the spleen, and after 48 hours, the smaller particles are removed by the liver. The increased accumulation of formulation in the kidneys is pertained to the degradation of liposomes and the clearance of released \u003csup\u003e111\u003c/sup\u003eIn by the kidneys. One of our unexpected results was the high absorption in the kidneys, despite of the proper stability of liposomes in the first 24 hours after administration. This phenomenon could be related to the low QC of purchased \u003csup\u003e111\u003c/sup\u003eIn-oxine radiopharmaceutical, leading to poor liposomal radiolabeling about 20%. The existence of 80% free \u003csup\u003e111\u003c/sup\u003eIn (80%) in the formulation caused non-specifically binding to the outer surface of liposomes and after injection, free \u003csup\u003e111\u003c/sup\u003eIn, not bonded to the albumin, separates from the liposomes over time and high excretion of this non-specific free \u003csup\u003e111\u003c/sup\u003eIn from the kidneys was taken place. Lung, bone, stomach, muscle and intestine have similar vascular tissue, and the absorption of our radiolabeled liposomes is similar with a reflection to the blood level. The most important finding of our biodistribution study in comparison to other studies investigating biodistritribution of free radiolabeled-antibody and liposomal-\u003csup\u003e111\u003c/sup\u003eIn not including albumin, was the relatively high and significant accumulation of our albumin-\u003csup\u003e111\u003c/sup\u003eIn loaded liposomal formulation in tumor site after 24 hours of intravenous injection which was prolonged in the tumor up to 96 hours, therefore made it possible to evaluate the tumor up to 96 hours and probably so on after administration. The increase in Tumor/blood ratio between 4 and 96 hours was considerably significant and confirms that our formulation has greatly improved the absorption rate of the tumor compared to the blood level.\u003c/p\u003e \u003cp\u003eAbsorption of radiopharmaceuticals in tumors as well as abscesses and centers of infection and inflammation is due to the EPR phenomenon. Several studies have shown that liposomes are probably entrapped in these areas due to increased capillary penetration or damage of the layers of vascular endothelial cells (Sun, Xiang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Kim, Cho et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As mentioned earlier, PEGylated liposomes better demonstrates this phenomenon. One of the biggest challenges facing the use of the free form of proteins in the diagnosis and scintigraphy of tumors is their lack of sufficient accumulation in the tumor area following intravenous administration and non-specific accumulation in non-target organs. On the other hand, the EPR mechanism is a main mechanism for the entry of nanocarrier such as liposomes smaller than 200 nm into the tumor area. As a result, based on our biodistribution results, \u003csup\u003e111\u003c/sup\u003eIn-albumin encapsulated in PEGylated liposomes is efficient approach for radio imaging because of high stability in bloodstream and more accumulation in the tumor area. In addition, the high half-life of \u003csup\u003e111\u003c/sup\u003eIn and its high binding strength with albumin and stability of this formulation confirmed by imaging up to 96 hours, have led to the possibility of imaging the tumor in a longer period of time for clinical application.\u003c/p\u003e \u003cp\u003eIn a study, Boerman and his colleagues (Boerman, Storm et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) investigated the biodistribution of PEGylated liposomes labeled with \u003csup\u003e111\u003c/sup\u003eIn compared to \u003csup\u003e111\u003c/sup\u003eIn bound to IgG antibody in rats with focal S.areus infections. In that study, they found that the removal of radioactive labeled material in abscesses with In-containing liposomes is 2 times the removal of In-bound IgG. Comparison of the biodistribution of our \u003csup\u003e111\u003c/sup\u003eIn-albumin liposomal formulation to the biodistribution of In-IgG and In-liposome in Boerman's study shows that formulation stability in the blood and Tumor/blood ratio as well as tumor accumulation have been significantly improved. Especially, with our formulation, it is possible to examine the tumor for up to 96 hours. In this research, scintigraphy imaging in mice was also performed, and the information obtained from the scintigraphy was consistent with the results obtained from biodistribution study. In the period of time, the contrast between the tumor and the background was improved and the high uptake in the tumor as well as in the liver, spleen and kidneys forms a distinct localization in scintigraphy image.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eCarefully designed radiopharmaceutical agents play a core role in nuclear medicine. In this study for the first time, radiolabeled \u003csup\u003e111\u003c/sup\u003eIn-albumin encapsulated liposomes were prepared by using high binding affinity of \u003csup\u003e111\u003c/sup\u003eIn to the albumin molecules entrapped into the liposome. Our biodistribution study and scintigraphy images demonstrated that \u003csup\u003e111\u003c/sup\u003eIn-albumin encapsulated liposomes have high stability and efficiency that could be exploited in long-term tumor imaging. Further studies including randomized clinical trial is needed for its clinical application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship contributions:\u003c/strong\u003e M.A: Investigation, Formal analysis, Writing - original draft. Z.A, A.A and J.RY: Investigation, Visualization, Software, Writing - original draft. \u0026nbsp;MR.J: Project administration, Formal analysis, Conceptualization, Writing - review and editing, Resources. K.S: Project administration, Formal analysis, Conceptualization, Writing - review and editing, Resources. All authors read and approved this final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was a part of Mohammad Ahrari M.Sc. dissertation with master thesis code 11130512942015. This study was supported by Vice Chancellor for Research and Technology, Mashhad University of Medical Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e The data produced during this research study are available from the corresponding authors upon request.\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e All animal experiments were conducted in accordance with the approval of Institutional Ethical Committee of Mashhad University of Medical Sciences in accordance with the principles of the Declaration of Helsinki. Consent to participate was not applicable as this study did not involve human subjects. (Master thesis code: 11130512942015)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u0026nbsp;\u003c/strong\u003eNot applicable as\u003cstrong\u003e\u0026nbsp;t\u003c/strong\u003ehis study did not include human subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe support of the Nanotechnology Research Center, Mashhad University of Medical Sciences and Department of nuclear medicine, Ghaem Hospital, Mashhad University of Medical Sciences is gratefully acknowledged. We would like to thank Dr Amin Reza Nikpoor for his excellent assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing financial or nonfinancial interests or personal relationships that could have appeared to influence the work reported in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAwasthi, V., D. Garcia, B. Goins and W. Phillips (2003). \u0026quot;Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits.\u0026quot; International journal of pharmaceutics \u003cstrong\u003e253\u003c/strong\u003e(1-2): 121-132.\u003c/li\u003e\n \u003cli\u003eAwasthi, V., B. Goins, R. Klipper, R. Loredo, D. Korvick and W. Phillips (1997). Comparison of dual radiolabeled liposomes (DRL), Tc-99m-DMP and GA-67 citrate for imaging osteomyelitis in rabbit model. 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Graham (2020). \u0026quot;The future of nuclear medicine, molecular imaging, and theranostics.\u0026quot; Journal of Nuclear Medicine \u003cstrong\u003e61\u003c/strong\u003e(Supplement 2): 263S-272S.\u003c/li\u003e\n \u003cli\u003eYazdi, J. R., M. Tafaghodi, K. Sadri, M. Mashreghi, A. R. Nikpoor, S. Nikoofal-Sahlabadi, J. Chamani, R. Vakili, S. A. Moosavian and M. R. Jaafari (2020). \u0026quot;Folate targeted PEGylated liposomes for the oral delivery of insulin: In vitro and in vivo studies.\u0026quot; Colloids and Surfaces B: Biointerfaces \u003cstrong\u003e194\u003c/strong\u003e: 111203.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"111In-albumin, nanoliposome, biodistribution, tumor imaging","lastPublishedDoi":"10.21203/rs.3.rs-6766595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6766595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: The current study aims to develop a clinically applicable radiopharmaceutical agent for long-term imaging in the diagnosis and management of oncologic patients in the field of nuclear medicine. Liposomes, as pharmaceutical nanocarriers, have been extensively studied in pharmaceutical industry and depending on their structural characteristics could be disposed in various pathological sites in the body. PEGylated liposomes have smaller volume of distribution and decreased clearance, consequently, due to their more prolonged presence in the bloodstream and their stability during this time, could be used for tumor imaging.\u003c/p\u003e\n\u003cp\u003eIn this work, liposomal formulations encapsulating albumin were synthesized by solvent evaporation method and extrusion and were labeled by \u003csup\u003e111\u003c/sup\u003eIn-oxine similar to leukocytes labeling method. Their biodistribution in C26-colon carcinoma tumor-bearing mice by injection dose/gram and gamma scintigraphy were studied.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The result of our study displayed that \u003csup\u003e111\u003c/sup\u003eIn -radiolabeled liposomes having a size of about 130 nanometers, were capable of accumulating in tumor sites based on enhanced penetration and retention (EPR) phenomenon. These liposomes also have high stability for maintaining encapsulated albumin for a long time up to 96 hours and probably so on. In the study of biodistribution of our formulation in tumor-bearing mice, they accumulated more in the kidney, liver, spleen and tumor sites, so that even after clearance of formulation in the bloodstream, they existed in significantly high levels in the mentioned organs and furthermore tumor site up to 96 hours. In gamma scintigraphy, organs with high activity accumulation from early time from administration to 96 hours, were visible in the form of hot spots demonstrating stability in the tumor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Our in vitro and in vivo studies demonstrated that this PEGylated radiolabeled liposomal formulation have considerable stability and efficiency for long-term tumor imaging which merit further studies for its transformation into clinical application.\u003c/p\u003e","manuscriptTitle":"Development of radiolabeled 111In-albumin liposomes for long-term imaging of tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-13 09:12:22","doi":"10.21203/rs.3.rs-6766595/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86fb5d46-11d5-4463-95b9-17bccfb371cb","owner":[],"postedDate":"June 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-26T06:22:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-13 09:12:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6766595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6766595","identity":"rs-6766595","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}