Pulsed Intra-Arterial Infusion with Synchronously Controlled Blood Flow: A Novel Strategy for Optimizing Intra-Arterial Chemotherapy

Pulsed Intra-Arterial Infusion with Synchronously Controlled Blood Flow: A Novel Strategy for Optimizing Intra-Arterial Chemotherapy | 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 Pulsed Intra-Arterial Infusion with Synchronously Controlled Blood Flow: A Novel Strategy for Optimizing Intra-Arterial Chemotherapy Yuqin Zhang, Yongqian Ge This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9331878/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 Objective: To explore a simple and convenient strategy for optimizing intra-arterial chemotherapy and fully exploit the advantages of intra-arterial chemotherapy. Materials and Methods: Pharmacokinetic study: Twelve healthy male New Zealand rabbits were randomly and equally divided into two groups: intravenous group and intra-arterial group. For the intra-arterial group, surgical puncture of the femoral artery was performed, and 3 mg/kg doxorubicin was administered via pulsed intra-arterial infusion with synchronously controlled femoral arterial blood flow (PBC-IA). The pulse frequency was 3 times per minute; the femoral artery was kept open for about 0.6 seconds during drug injection and occluded during the injection interval, with a total infusion duration of 6 hours. For the intravenous (IV) group, 3 mg/kg doxorubicin was injected via the ear vein within 2 minutes. In both groups, approximately 200 mg of gastrocnemius muscle specimens (target region) were collected at 1, 3, and 6 hours after the start of administration. At the end of the experiment, the animals were sacrificed, and approximately 200 mg of heart and lung tissue specimens were harvested. Drug concentrations in the specimens were determined by high-performance liquid chromatography (HPLC). Pharmacodynamic study: Twenty-two male rabbits bearing VX2 tumors implanted in the gastrocnemius muscle of the hind limb were randomly and equally divided into intravenous and intra-arterial groups, and underwent surgery and drug administration as described above. Tumor growth, recovery of the operated limb, and general activity of the rabbits were observed daily after administration. Tumor size was measured before treatment and on days 7, 14, and 21 post-administration, and the tumor regression rate was calculated. Results: The drug concentration in the target region (gastrocnemius muscle) was significantly higher in the intra-arterial group than in the intravenous group. During 3–6 hours after injection, the target-region drug concentration in the intra-arterial group was more than 20-fold higher than that in the intravenous group. At 6 hours post-administration, doxorubicin concentration in the heart was slightly lower in the intra-arterial group than in the intravenous group, with no statistically significant difference; the concentration in lung tissue was significantly lower in the intra-arterial group. The antitumor efficacy was markedly superior in the intra-arterial group, with a significantly higher tumor regression rate. Complete tumor regression was observed in 6 of 11 rabbits in the intra-arterial group, compared with only 1 rabbit in the intravenous group. In contrast, tumor volume increased more than 1-fold in 6 of 11 rabbits in the intravenous group during the observation period. No severe local adverse reactions in the operated limb were observed during the study. Conclusion: PBC-IA can optimize intra-arterial chemotherapy and significantly enhance the antitumor efficacy of drugs. The relationship Drug injection rate / Arterial blood flow = Target-region blood drug concentration is simple and controllable. Oncology Malignant tumor Chemotherapy Intra-arterial chemotherapy Pulsed infusion Arterial blood flow control Figures Figure 1 Figure 2 Introduction In recent years, remarkable progress has been made in the treatment of malignant tumors, with an endless emergence of therapeutic drugs and strategies. Small-molecule targeted drugs, monoclonal antibodies, and various immunotherapies have greatly prolonged the survival of cancer patients, and immunotherapy has even achieved cure in some patients. However, most small-molecule targeted drugs and some monoclonal antibodies are only effective against tumors with specific gene mutations. The response rate of immune checkpoint inhibitors, the most commonly used immunotherapeutic agents, remains low, and drug resistance is also prevalent. Tumor patients responsive to either small-molecule targeted drugs or immune checkpoint inhibitors invariably develop resistance after a period of treatment, and thus have to revert to conventional therapies such as chemotherapy. Even in current oncology practice, conventional treatments including surgery, chemotherapy, and radiotherapy account for a far larger proportion than novel therapies and remain the mainstay modalities, while new treatments often serve as adjuvants to conventional ones. Therefore, how to improve the efficacy of conventional treatments remains a worthy research topic. Chemotherapy is one of the main conventional approaches for malignant tumors. In chemotherapy, the concentration and duration of chemotherapeutic agents in tumor lesions are critical for exerting antitumor effects. A twofold increase in intratumoral drug concentration leads to an exponential increase in the number of tumor cells killed. Nevertheless, due to the toxicity of chemotherapeutic drugs, the achievable drug concentration in systemic chemotherapy is limited, which has led to the development of intra-arterial chemotherapy. In clinical practice and previous experiments, it has been observed that with most intra-arterial infusion methods, the duration of high drug concentration in tumors is limited, as agents are rapidly washed away by blood flow [ 1 – 3 ]. In contrast, transarterial chemoembolization results in an excessively small volume of drug distribution, failing to cover potential locally metastatic tumor foci [ 4 ]. To address these issues, we attempted to establish a novel strategy — pulsed intra-arterial infusion with synchronously controlled blood flow (PBC-IA) , which enables drugs to achieve ideal concentration and duration in tumor lesions while covering a certain region that includes potential local metastatic pathways of the tumor. Materials and Methods Drugs and Reagents Doxorubicin injection was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. Doxorubicin and daunorubicin reference standards were gifts from the same company. Heparin sodium injection was obtained from Changzhou Pharmaceutical Factory. Sodium phenobarbital was purchased from Shanghai DYNAMAX Biotech Co., Ltd. Other chemical reagents were of HPLC grade or analytical purity. Instruments An Agilent 1100 HPLC system was used for detection. The intra-arterial infusion and blood flow control device was a modified Model BCDB electric syringe pump manufactured by Shanghai Bochuang Medical Device Co., Ltd. Experimental Animals New Zealand rabbits were provided by the Laboratory Animal Science Department of Fudan University (License No.: SYXK [Hu]-2009-0082). Pharmacokinetic Study Surgery Twelve healthy male New Zealand rabbits, weighing 1.8–2.5 kg, were randomly and equally divided into two groups: intravenous injection group (IV) and intra-arterial injection group (PBC-IA). The ipsilateral hind limb calf was selected as the target region. Rabbits were anesthetized via ear vein injection of sodium phenobarbital at 30 mg/kg. Hair on the medial thigh and calf of one hind limb was shaved, and the skin was disinfected with 75% ethanol. For the intra-arterial group, an incision was made on the medial thigh skin, muscles were separated to expose the femoral artery and popliteal artery. An indwelling needle was inserted through the popliteal artery to slightly above the femoral artery bifurcation. Heparin sodium was used to prevent coagulation within the needle. The calf skin was disinfected and incised to expose the gastrocnemius muscle. A blood flow control module of the modified electric infusion pump was installed above the tip of the femoral artery indwelling needle to achieve pulsed occlusion and reperfusion of the femoral artery by tightening and loosening. The infusion pump was simultaneously used for pulsed injection of chemotherapeutic agents via the indwelling needle, with injection synchronized to femoral artery opening. For the intravenous group, only a calf skin incision was made to expose the gastrocnemius muscle. After surgery, each rabbit received 500 IU/kg heparin sodium via ear vein. Drug Administration Intra-arterial group (PBC-IA) Doxorubicin at a dose of 3 mg/kg was dissolved in 60 mL normal saline and infused into the femoral artery via the indwelling needle using the syringe pump in a pulsed mode synchronized with femoral artery opening. The pulse frequency was 3 times per minute, with femoral artery open for 0.6 seconds during injection and occluded during the injection interval. The total intra-arterial infusion duration was 6 hours. Intravenous group The same dose of doxorubicin was dissolved in normal saline to a concentration of 2 mg/mL and injected via the ear vein over 2 minutes. Tissue Sample Collection and Drug Concentration Determination Approximately 200 mg of gastrocnemius muscle specimens were collected from both groups at 1, 3, and 6 hours after the start of injection. At the end of the experiment, animals were sacrificed, and approximately 200 mg of heart and lung tissue specimens were harvested. All specimens were immediately weighed and stored at − 20°C until analysis. Tissue drug concentrations were determined by high-performance liquid chromatography (HPLC) with slight modifications according to the method reported by Kümmerle A et al. [ 5 ]. The chromatographic conditions were briefly as follows: Agilent 1100 chromatography system, including a quaternary pump (G1311A), a diode array detector (G1315A), and a manual injector; Hypersil ODS C18 column (5 µm, 4.0 × 250 mm); Mobile phase: solvent A (10 mmol/L ammonium dihydrogen phosphate aqueous solution containing 3 mL glacial acetic acid and 0.8 mL triethanolamine, pH 4.5) and solvent B (acetonitrile) Gradient elution: 0–3 min, A:B = 70:30 (v/v); 3–15 min, A:B linearly changed from 70:30 to 40:60; Flow rate: 1 mL/min; detection wavelength: 254 nm. After thawing, tissue samples were minced and spiked with 50 µL of 5 µg/mL daunorubicin methanol solution as internal standard, followed by addition of 1 mL acetone and homogenization. The mixture was centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected. The precipitate was washed twice with 0.5 mL acetone, and supernatants were combined after each centrifugation. The combined supernatant was evaporated to dryness under nitrogen at 50°C. The residue was reconstituted in 50 µL methanol, and 20 µL was injected for analysis. The retention times of doxorubicin and daunorubicin were 7.6 minutes and 11.2 minutes, respectively. The detection limit was 0.1 µg/g tissue. The inter-day and intra-day variations were both < 10%. Pharmacodynamic Study VX2 Tumor Implantation One rabbit bearing VX2 tumor in the hind limb was sacrificed by air embolism via ear vein. The tumor was excised, peripheral connective tissue was removed, and fresh viable portions were retained and placed in RPMI 1640 solution. Tumor tissue was minced into approximately 1 mm³ fragments. Two fragments were implanted into the gastrocnemius muscle of the rabbit hind limb using a 17-gauge needle. Tumors were allowed to grow for 21 days before pharmacodynamic experiments. Twenty-two male New Zealand rabbits bearing VX2 tumors were used 21 days after tumor implantation and randomly and equally divided into intravenous and intra-arterial groups. Surgery was performed on the tumor-bearing thigh using the same procedures as in the pharmacokinetic study; no calf surgery was needed. Doxorubicin was administered in the same manner as in the pharmacokinetic study. Wounds were sutured after completion of drug infusion. After treatment, tumor growth, recovery of the operated limb, and general activity of rabbits were observed daily. Tumor size (calculated as length × width) was measured before treatment and on days 7, 14, and 21 post-administration, and the tumor regression rate was calculated. All animals were raised until sacrificed at 60 days post-surgery (or until death during observation). Autopsies were performed on deceased rabbits to examine tumor status and determine causes of death. Statistical Analysis Student’s t -test was used for statistical analysis of drug concentrations, AUC values, and tumor regression rates. Fisher’s exact test was applied for tumor response rates. Ethics Statement All animal care and experimental procedures were performed in accordance with the Guideline for Ethical Review of Laboratory Animals (GB/T 35892 − 2018) of the People’s Republic of China and the ARRIVE 2.0 guidelines. The experimental protocol was approved by the Animal Ethics Committee of Fudan University (approval number to be added). All operations were performed under anesthesia, and all efforts were made to minimize animal suffering. Results Pharmacokinetics Figure 1 shows the difference in doxorubicin concentration in the target region (gastrocnemius muscle) between intra-arterial and intravenous administration. In the intravenous group, the doxorubicin concentration in the gastrocnemius muscle was approximately 0.5 µg/g at 1 hour and remained at this level until 6 hours. In the intra-arterial group, the doxorubicin concentration in the gastrocnemius muscle reached 3 µg/g at 1 hour after the start of infusion, more than 5-fold higher than that in the intravenous group. From 3 to 6 hours, the difference increased to more than 20-fold. At all selected time points, the drug concentration in the gastrocnemius muscle was significantly higher in the intra-arterial group than in the intravenous group (p < 0.01). The AUC from 1 to 6 hours was 136.66 ± 58.85 in the intra-arterial group and 16.33 ± 7.84 in the intravenous group, with a statistically significant difference (p < 0.01). In heart tissue samples at 6 hours, the drug concentration was 2.93 ± 0.71 in the intra-arterial group and 3.67 ± 0.64 in the intravenous group. The concentration was slightly lower in the intra-arterial group, but the difference was not statistically significant. In lung tissue samples, the concentration was3.07 ± 0.54 in the intra-arterial group and 3.93 ± 0.63 in the intravenous group, showing a statistically significant difference (p < 0.05). Pharmacodynamics All 22 tumor-bearing rabbits completed treatment. On day 7 after treatment, 2 rabbits in the intra-arterial group and 1 rabbit in the intravenous group died accidentally. Another rabbit in the intravenous group died accidentally on day 31. None of the deaths were caused by tumor progression. Of the 2 deceased rabbits in the intra-arterial group, one showed complete tumor regression and the other showed 80% tumor shrinkage at death. In the intravenous group, the rabbit that died on day 7 showed a 16% increase in tumor size, and the one that died on day 31 showed more than a 1-fold increase to 5×5 cm. After treatment, most rabbits in the intra-arterial group showed varying degrees of tumor regression, whereas most in the intravenous group showed tumor growth. By day 21, the mean tumor volume in the intravenous group had increased approximately 1-fold (Table 1 ). Table 1 shows the tumor regression rates in both groups on days 7, 14, and 21 after treatment, with highly significant differences between groups (p < 0.01). Table 1 Regression rate of VX2 tumor in rabbits after PBC-IA and IV treatment PBC-IA (%) 7 day 14 day 21 day 70.94 ± 26.85 (n = 11) 65.60 ± 31.68 (n = 9) 59.36 ± 39.20 (n = 9) IV (%) -11.93 ± 31.37 (n = 11) -65.67 ± 82.51 (n = 10) -107.9 ± 119.7 (n = 10) P < 0.001 0.001 0.002 Table 2 summarizes the final tumor response rates in both groups. One week after treatment, 3 rabbits in the intra-arterial group showed complete tumor disappearance, and 6 showed tumor regression exceeding 50%. In the following two weeks, another 3 rabbits in the intra-arterial group achieved complete tumor regression, while 1 rabbit in the intravenous group also showed complete tumor disappearance. Three weeks after treatment, 6 rabbits in the intravenous group showed more than 1-fold tumor growth, and 1 rabbit in the intra-arterial group also showed more than 1-fold growth. For ethical reasons, these 7 rabbits were sacrificed by air embolism via the ear vein. Autopsy of sacrificed rabbits revealed all increased tumors with central necrosis, extensive old hemorrhage, and only a small amount of viable tumor tissue at the margin. All sacrificed rabbits showed tumor metastasis to the popliteal lymph nodes. One rabbit in the intravenous group had metastatic nodules on the lung surface. The rabbit in the intra-arterial group had metastatic nodules on the surface of the lungs, liver, and diaphragm. Eight rabbits in the intra-arterial group survived to day 60 after treatment: 5 showed complete tumor disappearance, and 3 had residual non-growing tumors. Four rabbits in the intravenous group survived to day 60: 1 showed complete tumor disappearance, and the other 3 had residual but non-growing tumors. Autopsy of surviving rabbits showed no evidence of tumor metastasis. Residual tumors were firm, filled with necrotic tissue, covered by a thin capsule, and no viable tumor tissue was visible macroscopically. Table 2 Response to treatment in PBC-IA and IV rabbits (n = 11) PBC-IA CR PR PD 6 4 1 IV 1 0 10 P < 0.01 – – Toxicity and Adverse Reactions Within one week after treatment, rabbits in the intra-arterial group showed edema and induration at the surgical site, with significantly limited limb movement. Edema gradually subsided and limb function recovered after one week, with full recovery by two weeks. Apart from alopecia on the dorsum of the foot in one rabbit, no other obvious local toxicities, such as necrosis, ulceration, or thrombosis in the operated limb. Approximately one week after treatment, some rabbits in both groups showed anorexia, which resolved within 1–2 days. The two accidentally deceased rabbits in the intra-arterial group mainly presented with diarrhea and anorexia; no gross abnormalities of the heart or lungs were found at autopsy. One accidentally deceased rabbit in the intravenous group presented with tachypnea, and autopsy revealed massive pleural effusion. Another showed diarrhea, with severe pulmonary and hepatic congestion at autopsy. Discussion Chemotherapy is currently one of the main therapeutic modalities for malignant tumors and will continue to play a crucial role in cancer treatment in the foreseeable future. However, due to the toxic side effects of chemotherapeutic agents, the dosage of chemotherapy drugs is limited, which in turn restricts the amount of drugs delivered to tumor lesions. The concentration of chemotherapeutic agents in tumor lesions and their duration of action are key determinants of therapeutic efficacy. To increase the amount of chemotherapeutic drugs reaching tumor lesions, numerous strategies have been explored clinically, among which intra-arterial chemotherapy is one. In addition to enhancing drug delivery and duration of action, the coverage area (target region) of chemotherapeutic agents should also be considered. This region should include potential pathways of tumor metastasis. Therefore, we believe that the vascular supply territory selected for intra-arterial chemotherapy should encompass potential routes of local tumor spread. In intra-arterial chemotherapy, arterial blood flow is the major factor determining drug concentration and duration of action. Although blood flow delivers drugs to the target region, it also rapidly washes out retained drugs from the area, so the advantage of intra-arterial injection cannot be maintained for long. Approaches to overcome this limitation include reducing flow velocity in the infusion artery; transarterial chemoembolization and drug-eluting microspheres can be regarded as technologies developed for this purpose [ 3 , 4 , 6 ]. However, these techniques either provide only short-term effects or require superselective catheterization of tumor-feeding vessels due to prolonged blood flow occlusion, resulting in limited coverage [ 7 – 11 ]. PBC-IA does not require permanent occlusion of arterial blood flow, allowing a larger coverage area and facilitating repeated treatment and administration of multiple sensitive chemotherapeutic agents. As shown in the experimental results, PBC-IA can increase target-region drug concentration by more than 20-fold, and the high concentration can be sustained for a relatively long period. Since blood flow is not completely occluded, lymphatic drainage is preserved. As the drug concentration in the target region increases, the concentration in draining lymph and small veins also rises accordingly, enhancing the killing effect on tumor cells in vascular tumor thrombi and lymph node micrometastases. Because the selected artery can cover a relatively large territory, including blood supply to lymph nodes with potential local tumor metastasis, comparable efficacy to the primary lesion can be achieved even in the presence of bulky metastatic lymph nodes. With PBC-IA, the target-region drug concentration can be adjusted to achieve ideal therapeutic effects by configuring drug concentration, injection frequency, and infusion duration. Under ideal conditions, if collateral arterial supply from other regions to the target area is blocked, the target-region blood drug concentration can be expressed by a simple mathematical formula: Cb = Vin / Vb . where Cb = blood drug concentration, Vin = drug infusion rate, and Vb = arterial blood flow. Since the infusion rate is controllable, the desired blood drug concentration in the target region can be achieved simply by measuring arterial blood flow, and the process can be readily automated. The heart and lungs are organs prone to anthracycline accumulation, and doxorubicin concentrations in cardiac and pulmonary tissues reached relatively high levels in both groups. Doxorubicin concentration in the heart was lower in the intra-arterial group than in the intravenous group, but the difference was not statistically significant. Doxorubicin concentration in lung tissue was significantly lower in the intra-arterial group (p < 0.05). This phenomenon may be attributed to the fact that at the time of tissue sampling, a portion of the drug remained locally sequestered in the target region in the intra-arterial group and had not been fully released into the systemic circulation. Preliminary antitumor efficacy experiments confirmed that PBC-IA is superior to intravenous administration. This advantage clearly arises from the high intratumoral chemotherapeutic concentration and prolonged drug exposure time. Pharmacodynamic results showed that VX2 tumors grow rapidly with a volume-doubling time of less than 21 days if treatment is ineffective. Therefore, tumor disappearance maintained for more than 60 days after treatment indicates that the VX2 tumor has been eradicated. During the experiment, edema was observed in the target region and injection site after intra-arterial infusion, which may be attributed to local inflammation induced by high-concentration doxorubicin. Worrisome complications such as thrombosis, local ulceration, and necrosis of the operated limb did not occur. Besides high drug concentration, PBC-IA also causes varying degrees of ischemia and hypoxia in the target region. Therefore, further investigation is needed regarding the tolerance of different organs and tissues to high concentrations of various chemotherapeutic agents and to ischemia-hypoxia. Although the accidentally deceased rabbits exhibited diverse symptoms, the cause of death was presumed to be related to doxorubicin toxicity, suggesting that the dosage might have been relatively high. In addition, ideal drug concentrations and durations of action vary among different tumors and chemotherapeutic agents, all of which require in-depth exploration. Given the current development of arterial interventional techniques and balloon catheter technology [ 12 , 13 ], implementation of PBC-IA is technically feasible. Looking forward, with further maturation of PBC-IA, it will provide an additional therapeutic option for locally advanced inoperable malignant tumors, solitary recurrent tumors, and other clinical scenarios. References Laface C, Laforgia M, Molinari P et al (2022) Intra-Arterial Infusion Chemotherapy in Advanced Pancreatic Cancer: A Comprehensive Review. Cancers (Basel) 14(2):450 Aigner KR, Selak E, Aigner K et al (2019) Short-term intra-arterial infusion chemotherapy for head and neck cancer patients maintaining quality of life. J Cancer Res Clin Oncol 145(1):261–268 Aigner KR, ailhofer S, Selak E et al (2019) Intra-arterial infusion chemotherapy versus isolated upper abdominal perfusion for advanced pancreatic cancer: a retrospective cohort study on 454 patients. J Cancer Res Clin Oncol 145(11):2855–2862 Li QJ, He MK, Chen HW et al (2022) Hepatic Arterial Infusion of Oxaliplatin, Fluorouracil, and Leucovorin versus Transarterial Chemoembolization for Large Hepatocellular Carcinoma: A Randomized Phase III Trial. J Clin Oncol 40(2):150–160 Kümmerle A, Krueger T, Dusmet M et al (2003) A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements. J Pharm Biomed Anal 33(3):475–494 Ueda S, Hori S, Hori A et al (2022) Retrospective Study of the Efficacy and Safety of Chemoembolization with Drug-Eluting Microspheres Combined with Intra-Arterial Infusion of Bevacizumab for Unresectable Hepatocellular Carcinoma. J Hepatocell Carcinoma 9:973–985 Hsieh CY, Lein MY, Yang SN, Wang YC et al (2020) Dose-dense TPF induction chemotherapy for locally advanced head and neck cancer: a phase II study. BMC Cancer 20(1):832 Jin HY, He W, Liu Q, Wang XF et al (2016) Efficacy of intra-arterial neoadjuvant chemotherapy through the superior epigastric artery in the treatment of locally advanced triple negative breast cancer. Neoplasma 63(4):607–616 Wu CF, Chang KP, Huang CJ et al (2014) Continuous intra-arterial chemotherapy for downstaging locally advanced oral commissure carcinoma. Head Neck 36(7):1027–1033 Miyata Y, Nomata K, Ohba K et al (2015) Efficacy and safety of systemic chemotherapy and intra-arterial chemotherapy with/without radiotherapy for bladder preservation or as neo-adjuvant therapy in patients with muscle-invasive bladder cancer: a single-centre study of 163 patients. Eur J Surg Oncol 41(3):361–367 Nozato T, Koizumi T, Hayashi Y et al (2019) Thermochemoradiotherapy Using Superselective Intra-arterial Infusion for Patients with Oral Cancer with Cervical Lymph Node Metastases. Anticancer Res 39(3):1365–1373 Tanaka T, Terai Y, Fujiwara S et al (2018) Neoadjuvant intra-arterial chemotherapy using an original four-lumen double-balloon catheter for locally advanced uterine cervical cancer. Oncotarget 9(102):37766–37776 Yamamoto K, Yamamoto K, Nakai G et al (2023) Detection of the Vesical Arteries Using Three-dimensional Digital Subtraction Angiography Relevant to Intra-arterial Infusion Chemotherapy for Bladder Cancer Using Double-balloon Catheters. Interv Radiol (Higashimatsuyama) 8(2):64–69 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-9331878","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618153843,"identity":"63fbfbe4-4416-419d-853c-462d449f7b78","order_by":0,"name":"Yuqin Zhang","email":"","orcid":"","institution":"Department of Gynecologic Oncology, Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yuqin","middleName":"","lastName":"Zhang","suffix":""},{"id":618153846,"identity":"ab6a5af8-df38-45a0-b0e8-20e3f1217660","order_by":1,"name":"Yongqian Ge","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBADHgYG5gMHPlSQpoUt8eCMMyRaZHyYt4UIdfLuPYaPC35Zy/DP7vlwgLeBQZ5f7AB+LYZnzhgbz+xL55G4c3bDAckdDIYzZycQ0DIjx0yat+cwD8ON3A0HDM8wJBjcJqRl/huIFvkbOQ8OJLYRoUVegsdMmufHYR6DGzkMBw4So8WAJ63YmLchncfwRprBwYYzEoT9It9+eONjnj/W9nI3kh9//lNhI88vTciWAxwGDIxtzDC+BH7lYFsa2B8wMPxhJqBsFIyCUTAKRjQAALetSFrdmvDpAAAAAElFTkSuQmCC","orcid":"","institution":"¹Department of Pharmacy, Fudan University Shanghai Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Yongqian","middleName":"","lastName":"Ge","suffix":""}],"badges":[],"createdAt":"2026-04-06 08:57:09","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-9331878/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9331878/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106349031,"identity":"5304138a-e7cf-48c5-8412-272c0fe5d46a","added_by":"auto","created_at":"2026-04-07 16:51:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32093,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of doxorubicin in the gastrocnemius muscle of rabbits after IV and PBC‑IA administration\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9331878/v1/39f69dc1e7a4e1cd6518a5b3.png"},{"id":106349030,"identity":"152fb03a-2c30-44e4-9a86-7208e1bbe1dd","added_by":"auto","created_at":"2026-04-07 16:51:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17750,"visible":true,"origin":"","legend":"\u003cp\u003eDoxorubicin concentrations in the heart and lung tissues of rabbits at 6 hours after IV and PBC‑IA administration\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9331878/v1/bb8e77a76255f3274fd44948.png"},{"id":108490783,"identity":"c8d61001-a21f-4659-9cdb-51f286a4d282","added_by":"auto","created_at":"2026-05-05 09:48:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":237254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9331878/v1/9f80ed8d-a620-492a-b242-7cf0f6ed53bd.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003ePulsed Intra-Arterial Infusion with Synchronously Controlled Blood Flow: A Novel Strategy for Optimizing Intra-Arterial Chemotherapy\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, remarkable progress has been made in the treatment of malignant tumors, with an endless emergence of therapeutic drugs and strategies. Small-molecule targeted drugs, monoclonal antibodies, and various immunotherapies have greatly prolonged the survival of cancer patients, and immunotherapy has even achieved cure in some patients. However, most small-molecule targeted drugs and some monoclonal antibodies are only effective against tumors with specific gene mutations. The response rate of immune checkpoint inhibitors, the most commonly used immunotherapeutic agents, remains low, and drug resistance is also prevalent. Tumor patients responsive to either small-molecule targeted drugs or immune checkpoint inhibitors invariably develop resistance after a period of treatment, and thus have to revert to conventional therapies such as chemotherapy. Even in current oncology practice, conventional treatments including surgery, chemotherapy, and radiotherapy account for a far larger proportion than novel therapies and remain the mainstay modalities, while new treatments often serve as adjuvants to conventional ones. Therefore, how to improve the efficacy of conventional treatments remains a worthy research topic.\u003c/p\u003e \u003cp\u003eChemotherapy is one of the main conventional approaches for malignant tumors. In chemotherapy, the concentration and duration of chemotherapeutic agents in tumor lesions are critical for exerting antitumor effects. A twofold increase in intratumoral drug concentration leads to an exponential increase in the number of tumor cells killed. Nevertheless, due to the toxicity of chemotherapeutic drugs, the achievable drug concentration in systemic chemotherapy is limited, which has led to the development of intra-arterial chemotherapy.\u003c/p\u003e \u003cp\u003eIn clinical practice and previous experiments, it has been observed that with most intra-arterial infusion methods, the duration of high drug concentration in tumors is limited, as agents are rapidly washed away by blood flow [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In contrast, transarterial chemoembolization results in an excessively small volume of drug distribution, failing to cover potential locally metastatic tumor foci [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. To address these issues, we attempted to establish a novel strategy \u0026mdash; \u003cb\u003epulsed intra-arterial infusion with synchronously controlled blood flow (PBC-IA)\u003c/b\u003e, which enables drugs to achieve ideal concentration and duration in tumor lesions while covering a certain region that includes potential local metastatic pathways of the tumor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eDrugs and Reagents\u003c/p\u003e \u003cp\u003eDoxorubicin injection was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. Doxorubicin and daunorubicin reference standards were gifts from the same company. Heparin sodium injection was obtained from Changzhou Pharmaceutical Factory. Sodium phenobarbital was purchased from Shanghai DYNAMAX Biotech Co., Ltd. Other chemical reagents were of HPLC grade or analytical purity.\u003c/p\u003e \u003cp\u003eInstruments\u003c/p\u003e \u003cp\u003eAn Agilent 1100 HPLC system was used for detection. The intra-arterial infusion and blood flow control device was a modified Model BCDB electric syringe pump manufactured by Shanghai Bochuang Medical Device Co., Ltd.\u003c/p\u003e \u003cp\u003eExperimental Animals\u003c/p\u003e \u003cp\u003eNew Zealand rabbits were provided by the Laboratory Animal Science Department of Fudan University (License No.: SYXK [Hu]-2009-0082).\u003c/p\u003e \u003cp\u003ePharmacokinetic Study\u003c/p\u003e \u003cp\u003eSurgery\u003c/p\u003e \u003cp\u003eTwelve healthy male New Zealand rabbits, weighing 1.8\u0026ndash;2.5 kg, were randomly and equally divided into two groups: intravenous injection group (IV) and intra-arterial injection group (PBC-IA). The ipsilateral hind limb calf was selected as the target region.\u003c/p\u003e \u003cp\u003eRabbits were anesthetized via ear vein injection of sodium phenobarbital at 30 mg/kg. Hair on the medial thigh and calf of one hind limb was shaved, and the skin was disinfected with 75% ethanol.\u003c/p\u003e \u003cp\u003eFor the intra-arterial group, an incision was made on the medial thigh skin, muscles were separated to expose the femoral artery and popliteal artery. An indwelling needle was inserted through the popliteal artery to slightly above the femoral artery bifurcation. Heparin sodium was used to prevent coagulation within the needle. The calf skin was disinfected and incised to expose the gastrocnemius muscle. A blood flow control module of the modified electric infusion pump was installed above the tip of the femoral artery indwelling needle to achieve pulsed occlusion and reperfusion of the femoral artery by tightening and loosening. The infusion pump was simultaneously used for pulsed injection of chemotherapeutic agents via the indwelling needle, with injection synchronized to femoral artery opening.\u003c/p\u003e \u003cp\u003eFor the intravenous group, only a calf skin incision was made to expose the gastrocnemius muscle. After surgery, each rabbit received 500 IU/kg heparin sodium via ear vein.\u003c/p\u003e \u003cp\u003eDrug Administration\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIntra-arterial group (PBC-IA)\u003c/strong\u003e \u003cp\u003eDoxorubicin at a dose of 3 mg/kg was dissolved in 60 mL normal saline and infused into the femoral artery via the indwelling needle using the syringe pump in a pulsed mode synchronized with femoral artery opening. The pulse frequency was 3 times per minute, with femoral artery open for 0.6 seconds during injection and occluded during the injection interval. The total intra-arterial infusion duration was 6 hours.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIntravenous group\u003c/strong\u003e \u003cp\u003eThe same dose of doxorubicin was dissolved in normal saline to a concentration of 2 mg/mL and injected via the ear vein over 2 minutes.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eTissue Sample Collection and Drug Concentration Determination\u003c/p\u003e \u003cp\u003eApproximately 200 mg of gastrocnemius muscle specimens were collected from both groups at 1, 3, and 6 hours after the start of injection. At the end of the experiment, animals were sacrificed, and approximately 200 mg of heart and lung tissue specimens were harvested. All specimens were immediately weighed and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eTissue drug concentrations were determined by high-performance liquid chromatography (HPLC) with slight modifications according to the method reported by K\u0026uuml;mmerle A et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The chromatographic conditions were briefly as follows:\u003c/p\u003e \u003cp\u003eAgilent 1100 chromatography system, including a quaternary pump (G1311A), a diode array detector (G1315A), and a manual injector; Hypersil ODS C18 column (5 \u0026micro;m, 4.0 \u0026times; 250 mm); Mobile phase: solvent A (10 mmol/L ammonium dihydrogen phosphate aqueous solution containing 3 mL glacial acetic acid and 0.8 mL triethanolamine, pH 4.5) and solvent B (acetonitrile) Gradient elution: 0\u0026ndash;3 min, A:B\u0026thinsp;=\u0026thinsp;70:30 (v/v); 3\u0026ndash;15 min, A:B linearly changed from 70:30 to 40:60; Flow rate: 1 mL/min; detection wavelength: 254 nm.\u003c/p\u003e \u003cp\u003eAfter thawing, tissue samples were minced and spiked with 50 \u0026micro;L of 5 \u0026micro;g/mL daunorubicin methanol solution as internal standard, followed by addition of 1 mL acetone and homogenization. The mixture was centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected. The precipitate was washed twice with 0.5 mL acetone, and supernatants were combined after each centrifugation. The combined supernatant was evaporated to dryness under nitrogen at 50\u0026deg;C. The residue was reconstituted in 50 \u0026micro;L methanol, and 20 \u0026micro;L was injected for analysis.\u003c/p\u003e \u003cp\u003eThe retention times of doxorubicin and daunorubicin were 7.6 minutes and 11.2 minutes, respectively. The detection limit was 0.1 \u0026micro;g/g tissue. The inter-day and intra-day variations were both \u0026lt;\u0026thinsp;10%.\u003c/p\u003e \u003cp\u003ePharmacodynamic Study\u003c/p\u003e \u003cp\u003eVX2 Tumor Implantation\u003c/p\u003e \u003cp\u003eOne rabbit bearing VX2 tumor in the hind limb was sacrificed by air embolism via ear vein. The tumor was excised, peripheral connective tissue was removed, and fresh viable portions were retained and placed in RPMI 1640 solution. Tumor tissue was minced into approximately 1 mm\u0026sup3; fragments. Two fragments were implanted into the gastrocnemius muscle of the rabbit hind limb using a 17-gauge needle. Tumors were allowed to grow for 21 days before pharmacodynamic experiments.\u003c/p\u003e \u003cp\u003eTwenty-two male New Zealand rabbits bearing VX2 tumors were used 21 days after tumor implantation and randomly and equally divided into intravenous and intra-arterial groups. Surgery was performed on the tumor-bearing thigh using the same procedures as in the pharmacokinetic study; no calf surgery was needed. Doxorubicin was administered in the same manner as in the pharmacokinetic study. Wounds were sutured after completion of drug infusion.\u003c/p\u003e \u003cp\u003eAfter treatment, tumor growth, recovery of the operated limb, and general activity of rabbits were observed daily. Tumor size (calculated as length \u0026times; width) was measured before treatment and on days 7, 14, and 21 post-administration, and the tumor regression rate was calculated. All animals were raised until sacrificed at 60 days post-surgery (or until death during observation). Autopsies were performed on deceased rabbits to examine tumor status and determine causes of death.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStudent\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used for statistical analysis of drug concentrations, AUC values, and tumor regression rates. Fisher\u0026rsquo;s exact test was applied for tumor response rates.\u003c/p\u003e \u003cp\u003eEthics Statement\u003c/p\u003e \u003cp\u003eAll animal care and experimental procedures were performed in accordance with the \u003cb\u003eGuideline for Ethical Review of Laboratory Animals (GB/T 35892\u0026thinsp;\u0026minus;\u0026thinsp;2018)\u003c/b\u003e of the People\u0026rsquo;s Republic of China and the ARRIVE 2.0 guidelines. The experimental protocol was approved by the Animal Ethics Committee of Fudan University (approval number to be added). All operations were performed under anesthesia, and all efforts were made to minimize animal suffering.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003ePharmacokinetics\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the difference in doxorubicin concentration in the target region (gastrocnemius muscle) between intra-arterial and intravenous administration. In the intravenous group, the doxorubicin concentration in the gastrocnemius muscle was approximately 0.5 \u0026micro;g/g at 1 hour and remained at this level until 6 hours. In the intra-arterial group, the doxorubicin concentration in the gastrocnemius muscle reached 3 \u0026micro;g/g at 1 hour after the start of infusion, more than 5-fold higher than that in the intravenous group. From 3 to 6 hours, the difference increased to more than 20-fold.\u003c/p\u003e \u003cp\u003eAt all selected time points, the drug concentration in the gastrocnemius muscle was significantly higher in the intra-arterial group than in the intravenous group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eThe AUC from 1 to 6 hours was 136.66\u0026thinsp;\u0026plusmn;\u0026thinsp;58.85 in the intra-arterial group and 16.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.84 in the intravenous group, with a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eIn heart tissue samples at 6 hours, the drug concentration was 2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 in the intra-arterial group and 3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 in the intravenous group. The concentration was slightly lower in the intra-arterial group, but the difference was not statistically significant.\u003c/p\u003e \u003cp\u003eIn lung tissue samples, the concentration was3.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 in the intra-arterial group and 3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 in the intravenous group, showing a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePharmacodynamics\u003c/p\u003e \u003cp\u003eAll 22 tumor-bearing rabbits completed treatment.\u003c/p\u003e \u003cp\u003eOn day 7 after treatment, 2 rabbits in the intra-arterial group and 1 rabbit in the intravenous group died accidentally. Another rabbit in the intravenous group died accidentally on day 31. None of the deaths were caused by tumor progression. Of the 2 deceased rabbits in the intra-arterial group, one showed complete tumor regression and the other showed 80% tumor shrinkage at death. In the intravenous group, the rabbit that died on day 7 showed a 16% increase in tumor size, and the one that died on day 31 showed more than a 1-fold increase to 5\u0026times;5 cm.\u003c/p\u003e \u003cp\u003eAfter treatment, most rabbits in the intra-arterial group showed varying degrees of tumor regression, whereas most in the intravenous group showed tumor growth. By day 21, the mean tumor volume in the intravenous group had increased approximately 1-fold (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the tumor regression rates in both groups on days 7, 14, and 21 after treatment, with highly significant differences between groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRegression rate of VX2 tumor in rabbits after PBC-IA and IV treatment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePBC-IA (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21 day\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.94\u0026thinsp;\u0026plusmn;\u0026thinsp;26.85 (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.60\u0026thinsp;\u0026plusmn;\u0026thinsp;31.68\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.36\u0026thinsp;\u0026plusmn;\u0026thinsp;39.20\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-11.93\u0026thinsp;\u0026plusmn;\u0026thinsp;31.37 (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-65.67\u0026thinsp;\u0026plusmn;\u0026thinsp;82.51\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-107.9\u0026thinsp;\u0026plusmn;\u0026thinsp;119.7\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the final tumor response rates in both groups. One week after treatment, 3 rabbits in the intra-arterial group showed complete tumor disappearance, and 6 showed tumor regression exceeding 50%.\u003c/p\u003e \u003cp\u003eIn the following two weeks, another 3 rabbits in the intra-arterial group achieved complete tumor regression, while 1 rabbit in the intravenous group also showed complete tumor disappearance.\u003c/p\u003e \u003cp\u003eThree weeks after treatment, 6 rabbits in the intravenous group showed more than 1-fold tumor growth, and 1 rabbit in the intra-arterial group also showed more than 1-fold growth. For ethical reasons, these 7 rabbits were sacrificed by air embolism via the ear vein. Autopsy of sacrificed rabbits revealed all increased tumors with central necrosis, extensive old hemorrhage, and only a small amount of viable tumor tissue at the margin. All sacrificed rabbits showed tumor metastasis to the popliteal lymph nodes. One rabbit in the intravenous group had metastatic nodules on the lung surface. The rabbit in the intra-arterial group had metastatic nodules on the surface of the lungs, liver, and diaphragm.\u003c/p\u003e \u003cp\u003eEight rabbits in the intra-arterial group survived to day 60 after treatment: 5 showed complete tumor disappearance, and 3 had residual non-growing tumors.\u003c/p\u003e \u003cp\u003eFour rabbits in the intravenous group survived to day 60: 1 showed complete tumor disappearance, and the other 3 had residual but non-growing tumors. Autopsy of surviving rabbits showed no evidence of tumor metastasis. Residual tumors were firm, filled with necrotic tissue, covered by a thin capsule, and no viable tumor tissue was visible macroscopically.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResponse to treatment in PBC-IA and IV rabbits (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePBC-IA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCR\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePR\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePD\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003eToxicity and Adverse Reactions\u003c/p\u003e \u003cp\u003eWithin one week after treatment, rabbits in the intra-arterial group showed edema and induration at the surgical site, with significantly limited limb movement. Edema gradually subsided and limb function recovered after one week, with full recovery by two weeks.\u003c/p\u003e \u003cp\u003eApart from alopecia on the dorsum of the foot in one rabbit, no other obvious local toxicities, such as necrosis, ulceration, or thrombosis in the operated limb.\u003c/p\u003e \u003cp\u003eApproximately one week after treatment, some rabbits in both groups showed anorexia, which resolved within 1\u0026ndash;2 days.\u003c/p\u003e \u003cp\u003eThe two accidentally deceased rabbits in the intra-arterial group mainly presented with diarrhea and anorexia; no gross abnormalities of the heart or lungs were found at autopsy.\u003c/p\u003e \u003cp\u003eOne accidentally deceased rabbit in the intravenous group presented with tachypnea, and autopsy revealed massive pleural effusion. Another showed diarrhea, with severe pulmonary and hepatic congestion at autopsy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eChemotherapy is currently one of the main therapeutic modalities for malignant tumors and will continue to play a crucial role in cancer treatment in the foreseeable future. However, due to the toxic side effects of chemotherapeutic agents, the dosage of chemotherapy drugs is limited, which in turn restricts the amount of drugs delivered to tumor lesions. The concentration of chemotherapeutic agents in tumor lesions and their duration of action are key determinants of therapeutic efficacy. To increase the amount of chemotherapeutic drugs reaching tumor lesions, numerous strategies have been explored clinically, among which intra-arterial chemotherapy is one. In addition to enhancing drug delivery and duration of action, the coverage area (target region) of chemotherapeutic agents should also be considered. This region should include potential pathways of tumor metastasis. Therefore, we believe that the vascular supply territory selected for intra-arterial chemotherapy should encompass potential routes of local tumor spread.\u003c/p\u003e \u003cp\u003eIn intra-arterial chemotherapy, arterial blood flow is the major factor determining drug concentration and duration of action. Although blood flow delivers drugs to the target region, it also rapidly washes out retained drugs from the area, so the advantage of intra-arterial injection cannot be maintained for long. Approaches to overcome this limitation include reducing flow velocity in the infusion artery; transarterial chemoembolization and drug-eluting microspheres can be regarded as technologies developed for this purpose [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, these techniques either provide only short-term effects or require superselective catheterization of tumor-feeding vessels due to prolonged blood flow occlusion, resulting in limited coverage [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. PBC-IA does not require permanent occlusion of arterial blood flow, allowing a larger coverage area and facilitating repeated treatment and administration of multiple sensitive chemotherapeutic agents.\u003c/p\u003e \u003cp\u003eAs shown in the experimental results, PBC-IA can increase target-region drug concentration by more than 20-fold, and the high concentration can be sustained for a relatively long period. Since blood flow is not completely occluded, lymphatic drainage is preserved. As the drug concentration in the target region increases, the concentration in draining lymph and small veins also rises accordingly, enhancing the killing effect on tumor cells in vascular tumor thrombi and lymph node micrometastases. Because the selected artery can cover a relatively large territory, including blood supply to lymph nodes with potential local tumor metastasis, comparable efficacy to the primary lesion can be achieved even in the presence of bulky metastatic lymph nodes.\u003c/p\u003e \u003cp\u003eWith PBC-IA, the target-region drug concentration can be adjusted to achieve ideal therapeutic effects by configuring drug concentration, injection frequency, and infusion duration. Under ideal conditions, if collateral arterial supply from other regions to the target area is blocked, the target-region blood drug concentration can be expressed by a simple mathematical formula:\u003c/p\u003e \u003cp\u003e \u003cb\u003eCb\u0026thinsp;=\u0026thinsp;Vin / Vb\u003c/b\u003e. where \u003cb\u003eCb\u003c/b\u003e\u0026thinsp;=\u0026thinsp;blood drug concentration, \u003cb\u003eVin\u003c/b\u003e\u0026thinsp;=\u0026thinsp;drug infusion rate, and \u003cb\u003eVb\u003c/b\u003e\u0026thinsp;=\u0026thinsp;arterial blood flow. Since the infusion rate is controllable, the desired blood drug concentration in the target region can be achieved simply by measuring arterial blood flow, and the process can be readily automated.\u003c/p\u003e \u003cp\u003eThe heart and lungs are organs prone to anthracycline accumulation, and doxorubicin concentrations in cardiac and pulmonary tissues reached relatively high levels in both groups. Doxorubicin concentration in the heart was lower in the intra-arterial group than in the intravenous group, but the difference was not statistically significant. Doxorubicin concentration in lung tissue was significantly lower in the intra-arterial group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This phenomenon may be attributed to the fact that at the time of tissue sampling, a portion of the drug remained locally sequestered in the target region in the intra-arterial group and had not been fully released into the systemic circulation.\u003c/p\u003e \u003cp\u003ePreliminary antitumor efficacy experiments confirmed that PBC-IA is superior to intravenous administration. This advantage clearly arises from the high intratumoral chemotherapeutic concentration and prolonged drug exposure time. Pharmacodynamic results showed that VX2 tumors grow rapidly with a volume-doubling time of less than 21 days if treatment is ineffective. Therefore, tumor disappearance maintained for more than 60 days after treatment indicates that the VX2 tumor has been eradicated.\u003c/p\u003e \u003cp\u003eDuring the experiment, edema was observed in the target region and injection site after intra-arterial infusion, which may be attributed to local inflammation induced by high-concentration doxorubicin. Worrisome complications such as thrombosis, local ulceration, and necrosis of the operated limb did not occur. Besides high drug concentration, PBC-IA also causes varying degrees of ischemia and hypoxia in the target region. Therefore, further investigation is needed regarding the tolerance of different organs and tissues to high concentrations of various chemotherapeutic agents and to ischemia-hypoxia.\u003c/p\u003e \u003cp\u003eAlthough the accidentally deceased rabbits exhibited diverse symptoms, the cause of death was presumed to be related to doxorubicin toxicity, suggesting that the dosage might have been relatively high. In addition, ideal drug concentrations and durations of action vary among different tumors and chemotherapeutic agents, all of which require in-depth exploration.\u003c/p\u003e \u003cp\u003eGiven the current development of arterial interventional techniques and balloon catheter technology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], implementation of PBC-IA is technically feasible. Looking forward, with further maturation of PBC-IA, it will provide an additional therapeutic option for locally advanced inoperable malignant tumors, solitary recurrent tumors, and other clinical scenarios.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLaface C, Laforgia M, Molinari P et al (2022) Intra-Arterial Infusion Chemotherapy in Advanced Pancreatic Cancer: A Comprehensive Review. Cancers (Basel) 14(2):450\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAigner KR, Selak E, Aigner K et al (2019) Short-term intra-arterial infusion chemotherapy for head and neck cancer patients maintaining quality of life. J Cancer Res Clin Oncol 145(1):261\u0026ndash;268\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAigner KR, ailhofer S, Selak E et al (2019) Intra-arterial infusion chemotherapy versus isolated upper abdominal perfusion for advanced pancreatic cancer: a retrospective cohort study on 454 patients. J Cancer Res Clin Oncol 145(11):2855\u0026ndash;2862\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi QJ, He MK, Chen HW et al (2022) Hepatic Arterial Infusion of Oxaliplatin, Fluorouracil, and Leucovorin versus Transarterial Chemoembolization for Large Hepatocellular Carcinoma: A Randomized Phase III Trial. J Clin Oncol 40(2):150\u0026ndash;160\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK\u0026uuml;mmerle A, Krueger T, Dusmet M et al (2003) A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements. J Pharm Biomed Anal 33(3):475\u0026ndash;494\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUeda S, Hori S, Hori A et al (2022) Retrospective Study of the Efficacy and Safety of Chemoembolization with Drug-Eluting Microspheres Combined with Intra-Arterial Infusion of Bevacizumab for Unresectable Hepatocellular Carcinoma. J Hepatocell Carcinoma 9:973\u0026ndash;985\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsieh CY, Lein MY, Yang SN, Wang YC et al (2020) Dose-dense TPF induction chemotherapy for locally advanced head and neck cancer: a phase II study. BMC Cancer 20(1):832\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin HY, He W, Liu Q, Wang XF et al (2016) Efficacy of intra-arterial neoadjuvant chemotherapy through the superior epigastric artery in the treatment of locally advanced triple negative breast cancer. Neoplasma 63(4):607\u0026ndash;616\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu CF, Chang KP, Huang CJ et al (2014) Continuous intra-arterial chemotherapy for downstaging locally advanced oral commissure carcinoma. Head Neck 36(7):1027\u0026ndash;1033\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyata Y, Nomata K, Ohba K et al (2015) Efficacy and safety of systemic chemotherapy and intra-arterial chemotherapy with/without radiotherapy for bladder preservation or as neo-adjuvant therapy in patients with muscle-invasive bladder cancer: a single-centre study of 163 patients. Eur J Surg Oncol 41(3):361\u0026ndash;367\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNozato T, Koizumi T, Hayashi Y et al (2019) Thermochemoradiotherapy Using Superselective Intra-arterial Infusion for Patients with Oral Cancer with Cervical Lymph Node Metastases. Anticancer Res 39(3):1365\u0026ndash;1373\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka T, Terai Y, Fujiwara S et al (2018) Neoadjuvant intra-arterial chemotherapy using an original four-lumen double-balloon catheter for locally advanced uterine cervical cancer. Oncotarget 9(102):37766\u0026ndash;37776\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamamoto K, Yamamoto K, Nakai G et al (2023) Detection of the Vesical Arteries Using Three-dimensional Digital Subtraction Angiography Relevant to Intra-arterial Infusion Chemotherapy for Bladder Cancer Using Double-balloon Catheters. Interv Radiol (Higashimatsuyama) 8(2):64\u0026ndash;69\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Fudan University Shanghai Cancer Center","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":"Malignant tumor, Chemotherapy, Intra-arterial chemotherapy, Pulsed infusion, Arterial blood flow control","lastPublishedDoi":"10.21203/rs.3.rs-9331878/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9331878/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To explore a simple and convenient strategy for optimizing intra-arterial chemotherapy and fully exploit the advantages of intra-arterial chemotherapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods:\u003c/strong\u003ePharmacokinetic study: Twelve healthy male New Zealand rabbits were randomly and equally divided into two groups: intravenous group and intra-arterial group. For the intra-arterial group, surgical puncture of the femoral artery was performed, and 3 mg/kg doxorubicin was administered via pulsed intra-arterial infusion with synchronously controlled femoral arterial blood flow (PBC-IA). The pulse frequency was 3 times per minute; the femoral artery was kept open for about 0.6 seconds during drug injection and occluded during the injection interval, with a total infusion duration of 6 hours. For the intravenous (IV) group, 3 mg/kg doxorubicin was injected via the ear vein within 2 minutes. In both groups, approximately 200 mg of gastrocnemius muscle specimens (target region) were collected at 1, 3, and 6 hours after the start of administration. At the end of the experiment, the animals were sacrificed, and approximately 200 mg of heart and lung tissue specimens were harvested. Drug concentrations in the specimens were determined by high-performance liquid chromatography (HPLC).\u003c/p\u003e\n\u003cp\u003ePharmacodynamic study: Twenty-two male rabbits bearing VX2 tumors implanted in the gastrocnemius muscle of the hind limb were randomly and equally divided into intravenous and intra-arterial groups, and underwent surgery and drug administration as described above. Tumor growth, recovery of the operated limb, and general activity of the rabbits were observed daily after administration. Tumor size was measured before treatment and on days 7, 14, and 21 post-administration, and the tumor regression rate was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The drug concentration in the target region (gastrocnemius muscle) was significantly higher in the intra-arterial group than in the intravenous group. During 3–6 hours after injection, the target-region drug concentration in the intra-arterial group was more than 20-fold higher than that in the intravenous group. At 6 hours post-administration, doxorubicin concentration in the heart was slightly lower in the intra-arterial group than in the intravenous group, with no statistically significant difference; the concentration in lung tissue was significantly lower in the intra-arterial group.\u003c/p\u003e\n\u003cp\u003eThe antitumor efficacy was markedly superior in the intra-arterial group, with a significantly higher tumor regression rate. Complete tumor regression was observed in 6 of 11 rabbits in the intra-arterial group, compared with only 1 rabbit in the intravenous group. In contrast, tumor volume increased more than 1-fold in 6 of 11 rabbits in the intravenous group during the observation period. No severe local adverse reactions in the operated limb were observed during the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e PBC-IA can optimize intra-arterial chemotherapy and significantly enhance the antitumor efficacy of drugs. The relationship \u003cstrong\u003eDrug injection rate / Arterial blood flow = Target-region blood drug concentration\u003c/strong\u003e is simple and controllable.\u003c/p\u003e","manuscriptTitle":"Pulsed Intra-Arterial Infusion with Synchronously Controlled Blood Flow: A Novel Strategy for Optimizing Intra-Arterial Chemotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 16:51:36","doi":"10.21203/rs.3.rs-9331878/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":"193ac72b-cc39-4225-a9ff-2679a3f1d0c2","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65775839,"name":"Oncology"}],"tags":[],"updatedAt":"2026-04-07T16:51:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 16:51:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9331878","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9331878","identity":"rs-9331878","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}