【作者】 王文博；

【导师】 于德新；

【作者基本信息】 山东大学 ， 影像医学与核医学， 2025， 硕士

【摘要】 背景全球范围内恶性肿瘤如乳腺癌等的发病率和死亡率居高不下,约80%的患者在治疗中都会使用化疗,因此对化疗疗效的精准评估至关重要。在治疗早期,在肿瘤体积和形态尚未发生显著变化时,如何有效识别肿瘤细胞死亡和生理活性改变等成为早期疗效评估的关键。但目前现有的影像学技术方法有限、检测敏感度不高,与临床需求存在很大差距。而作为金标准的活检病理学技术则因有创取材、样本有限和重复性差等原因其实际临床应用受到很大的限制。近年来随着量子检测技术的发展,基于无自旋交换弛豫磁力计(spin-exchange relaxation-free,SERF)技术的“零磁医学”检测方法,即在零磁环境下实现机体极弱磁场的检测,因其本身具有的敏感、无创、简便等特征,在心磁和脑磁疾病的极弱磁检测方面受到愈来愈多的关注。但是在检测肿瘤的磁信号方面,因肿瘤自身的磁信号极其微弱、背景噪声大等因素的干扰,其检测敏感度依然面临着巨大的挑战。因此,如何提高肿瘤的磁信号强度成为制约肿瘤零磁检测的关键问题。现有的铁磁性材料经过磁化后虽能增强肿瘤磁信号,但同时也会产生明显的背景噪声;超顺磁性材料无背景噪声干扰,但在磁化后又难以实现稳定的信号增强。因此,如何在持续增强肿瘤信号的同时最大限度地降低纳米粒子的背景噪声,是目前零磁医学肿瘤检测的瓶颈问题。基于此,制备长磁性的金属纳米体系、实现恶性肿瘤的敏感和精准零磁检测,为化疗后肿瘤内部的早期改变提供重要的检测和评估工具,具有重要的临床价值和转化潜力。目的(1)设计合成兼具铁磁性与超顺磁性且具有长磁性的新型氧化铁纳米颗粒(ferromagnetic-superparamagnetic iron oxide nanoparticles,F-SPION),使其在进入细胞并磁化后能够产生稳定的肿瘤增强磁信号,同时具备低背景噪声的特点;(2)基于F-SPION磁化后在肿瘤细胞中维持长磁性,结合SERF磁力计的高灵敏度,实现在零磁环境下肿瘤细胞磁信号的检测,验证其与细胞数量及摄取能力之间的联系;(3)分析肿瘤化疗的不同时间点,肿瘤细胞摄取F-SPION后的磁信号改变,探讨其在早期疗效评估中的价值。方法1.F-SPION纳米颗粒合成与表征将FeCl2和FeCl3溶于去离子水中,加入氨水,在氩气保护下于80℃反应1h,合成了新型氧化铁纳米颗粒。并对纳米颗粒的形态、磁学特性等方面进行了表征。2.极弱磁环境下的磁信号测定本研究采用自研的细胞零磁检测箱(配备五层磁屏蔽结构和主动磁补偿系统)构建零磁环境。在零磁环境下,使用QuSpin SERF磁力仪测量F-SPION、4T1细胞以及小鼠肿瘤组织和伤口组织的磁信号,正常组织作为对照。为了增强信号,利用铷磁铁(尺寸:10×5×0.5 cm,磁场强度:0.15 T)对F-SPION及摄取F-SPION的4T1细胞和肿瘤等组织进行磁化。极弱磁信号测量时,SERF探头对准样本的中心区域,样品与磁强计间距为1～2 mm。样品以1.7 Hz频率旋转,磁信号以200 Hz频率采集,测量时间为30 s。数据经过带通滤波和快速傅里叶变换处理,提取周期性峰值,用于评估磁信号变化。3.肿瘤细胞数量与F-SPION摄取能力对磁信号的影响评估4T1细胞(3.0×106个)在含F-SPION(100 μg/mL)的RPMI-1640培养基中分别孵育 0h、2h、4h、6h。同时,5种不同数量的 4T1 细胞(1×105、2×105、3×105、4×105、5×105个)在含F-SPION(100 μg/mL)的RPMI-1640培养基中孵育6h。为模拟药物治疗对细胞摄取能力的影响,4T1细胞(3.0×106个)与多柔比星(0.028 μg/mL)共同孵育0h、24 h、48 h后,将不同孵育时间的细胞数目调整为一致后(2.0×106个),再与含F-SPION(100μg/mL)的RPMI-1640培养基共孵育6h。所有细胞样本孵育后,直接在原培养瓶中利用上述铷磁铁进行磁化处理,磁化持续30 s,再进行磁信号检测。使用线性回归方法分析肿瘤细胞数量和信号强度之间的关系。普鲁士蓝染色用于验证前述不同孵育时间及多柔比星不同孵育时间对4T1细胞F-SPION摄取能力的影响。4.F-SPION在肿瘤组织中的磁信号对比增强及持续时间评估为评估F-SPION在增强肿瘤组织与正常组织间磁信号的差异,建立4T1细胞皮下单侧荷瘤小鼠模型。麻醉后经尾静脉注射F-SPION(2.5 mg/kg),30 min后,测量小鼠肿瘤组织及对侧正常组织在磁化前的磁信号强度。随后,利用磁铁磁化肿瘤30 s,然后测量肿瘤组织的磁信号强度;对侧正常组织采用相同的磁化方式进行处理与测量以作为对照。此外,在磁化后30 min和60 min分别再次测量两种组织的磁信号强度,以评估F-SPION介导的信号增强效应的持续性。5.评估磁化对F-SPION在肿瘤组织中分布的影响为明确磁化处理是否影响F-SPION在肿瘤组织中的分布,利用4T1细胞双侧皮下荷瘤小鼠模型,尾静脉注射F-SPION(2.5 mg/kg),30 min后随机选择一侧肿瘤组织磁化30 s。在磁化前后分别进行T2WI成像,比较两侧肿瘤各自在磁化前后的T2WI信号变化;然后处死小鼠,常规取材,行普鲁士蓝染色进行验证,观察F-SPION在肿瘤组织内的分布差异。同时,通过染色强度评估F-SPION在肿瘤组织中的积聚量,验证T2信号变化与摄取F-SPION之间的关系。6.评估血流状态对组织中F-SPION分布及磁信号的影响为明确局部血流状态是否影响F-SPION在组织中的分布及磁信号强度,在小鼠脊柱正中线上方约1 cm处建立直径10 mm的标准圆形伤口,以构建小鼠伤口模型,并随机分为对照组(不处理伤口)、充血组(滴加75%乙醇诱导血管扩张)和炎症组(腹腔注射脂多糖激活炎症反应),每组n=5。小鼠经尾静脉注射F-SPION(2.5 mg/kg),30 min后,测量三组伤口组织磁化前的磁信号强度。然后,使用铷磁铁磁化伤口组织30 s。分别在磁化后5 min、30 min和60 min,利用SERF探头测量三组伤口组织的磁信号强度,测量结束后处死小鼠,收集伤口组织并进行普鲁士蓝染色验证,评估F-SPION在三组不同血流状态组织中的分布差异。7.评估肿瘤细胞摄取F-SPION后磁化信号在疗效监测中的价值为验证肿瘤细胞摄取F-SPION并磁化后所产生的磁信号是否可用于早期肿瘤化疗反应的疗效监测,将单侧荷瘤小鼠模型随机分为对照组与治疗组(每组n=5)。治疗组每4天尾静脉注射多柔比星(2 mg/kg),共注射6次;对照组在相同时间点注射等量生理盐水。分别在第1、7、14与21天,小鼠经尾静脉注射F-SPION(2.5 mg/kg),30 min后,使用铷磁铁磁化肿瘤组织30 s,并分别在磁化后5 min、30 min与60 min测量两组小鼠肿瘤组织的磁信号强度的差异。通过追踪化疗过程中肿瘤组织磁信号的变化,评估肿瘤细胞摄取F-SPION并磁化后所产生的磁信号,作为化疗疗效监测指标的可行性。8.统计分析数据以均值(mean)±标准差(SD)呈现,使用GraphPad Prism 10进行统计分析,所有实验至少独立重复3次。首先使用Shapiro-Wilk检验判断数据的正态性。对于正态分布的两组数据,采用配对t检验分析组内差异。比较内容包括:F-SPION注射后,肿瘤组织与正常组织在磁化前以及磁化后不同时间点(5 min、30 min、60 min)的磁信号强度差异;对照组和治疗组小鼠肿瘤组织在第1天与第7天磁化后5 min的信号强度差异;以及在第1、7、14和21天,治疗组与对照组小鼠肿瘤组织在磁化后5 min的磁信号强度差异。对于正态分布的多组数据,采用单因素方差分析,并根据需要进行Tukey事后检验。比较内容包括:不同F-SPION孵育时间(0h、2 h、4 h、6 h)条件下4T1细胞磁化后的磁信号强度差异;不同多柔比星处理时间(0h、24 h、48 h)下,统一细胞数量(2.0×106)的4T1细胞磁化后的磁信号强度差异;不同血流状态(对照、充血、炎症)伤口组织在磁化前及磁化后不同时间(5 min、30 min、60 min)的磁信号强度差异。P<0.05视为具有统计学意义。结果1.纳米颗粒的表征测量F-SPION的尺寸为12 nm,粒径分布均一。振动样品磁强计测量结果显示,在±20,000 G磁场范围内,其饱和磁化强度为62.5 emu/g,几乎无矫顽力和剩磁,符合超顺磁性特征;在±600 G磁场范围内,磁滞回线显示轻微磁滞,说明其还具有微弱铁磁性。上述结果表明,F-SPION同时具有超顺磁性和微弱铁磁性。2.肿瘤细胞数量减少和摄取能力降低导致肿瘤细胞磁信号减弱4T1细胞与F-SPION孵育0 h、2 h、4 h、6 h后的磁信号强度分别为36.01±2.87 pT、210.42±16.50pT、224.39±5.99pT、346.64±11.46pT,组间差异具有统计学意义(P<0.001)。Tukey事后检验进一步表明,磁信号强度随孵育时间延长显著上升。磁信号强度与4T1细胞数量之间呈现高度线性相关性(R2=0.974)。4T1细胞与多柔比星孵育0h、24 h和48 h后,将细胞数统一调整为2.0×106,再分别与F-SPION共孵育6 h,随后进行磁信号测定,结果分别为267.88±5.97pT、206.02±2.23 pT和122.74±2.11 pT。组间差异具有统计学意义(P<0.001),Tukey事后检验显示磁信号强度随多柔比星孵育时间延长显著降低。普鲁士蓝染色结果显示,F-SPION孵育时间延长可增加细胞对F-SPION的摄取,随着多柔比星孵育时间延长,细胞摄取F-SPION的数量逐渐减少。上述结果提示,多柔比星处理可抑制肿瘤细胞对F-SPION的摄取能力,进而导致磁信号减弱。3.F-SPION磁化增强肿瘤组织磁信号并在肿瘤与正常组织间形成持久对比在磁化前,单侧荷瘤小鼠肿瘤组织的磁信号强度为108.47±5.29 pT,正常组织为72.60±2.88 pT,具有统计学差异(P<0.001),但整体对比度较低。磁化后5 min,肿瘤组织的磁信号强度(802.70±60.81 pT)显著高于正常组织(149.25±16.23 pT,P<0.001),该差异在磁化后30 min(肿瘤:237.34±8.70pT;正常:45.23±2.15pT)和60min(肿瘤:163.56±3.20pT;正常:42.75±1.49pT)时仍然显著(均为 P<0.001),提示 F-SPION磁化后在肿瘤组织中可维持磁信号,具有长磁性。4.磁化促进肿瘤组织中F-SPION的聚集。与磁化前相比,磁化侧肿瘤组织在T2WI成像中信号明显减弱,呈现T2低信号区域,而非磁化侧在两次成像中信号无明显变化。普鲁士蓝染色结果进一步验证了上述变化,磁化侧肿瘤组织中可见大量深蓝染色,提示F-SPION分布密集;非磁化侧仅见少量散在染色,说明磁化处理有助于F-SPION在肿瘤组织内的聚集。5.组织磁信号增强源于细胞对F-SPION的吞噬与磁激活磁化前,对照组、充血组和炎症组小鼠伤口组织的磁信号强度无显著差异(分别为36.06±1.94pT、38.10±12.25 pT、38.20±1.38 pT,P>0.05)。磁化后 5 min、30 min、60 min,炎症组伤口组织的磁信号(269.48±2.62 pT、265.88±2.20 pT、242.80±3.24pT)始终显著高于对照组(245.20±2.72 pT、166.18±1.62 pT、109.56±1.04pT)和充血组(245.00±3.00 pT、166.05±1.63 pT、110.22±2.29 pT)(P<0.001),而对照组与充血组的磁信号强度差异无统计学意义(P>0.05)。普鲁士蓝染色结果验证上述磁信号测定,显示对照组几乎无F-SPION沉积,充血组主要沉积于间质,而炎症组中F-SPION显著聚集于细胞内,提示磁信号增强源于细胞对F-SPION的吞噬与磁激活。6.肿瘤组织磁信号的变化反映肿瘤对化疗的早期响应在第7天观察到肿瘤对化疗药物的反应,治疗组小鼠肿瘤组织在第1天和第7天磁化后5min的磁信号强度分别为426.98±16.51 pT和425.41±14.37pT,差异无统计学意义(P>0.05)。而对照组小鼠肿瘤组织在相同时间段内磁化后5min的磁信号强度由481.41±3.82pT显著上升至830.72±5.90pT(P<0.001)。第14天,对照组小鼠肿瘤组织磁化后5 min磁信号强度升至1978.70±14.36pT,治疗组小鼠肿瘤组织磁化后5 min磁信号强度为346.62±5.15pT,两组差异具有统计学意义(P<0.001);第21天,对照组小鼠肿瘤组织磁化后5min磁信号强度进一步增高至4562.61±196.84pT,治疗组小鼠肿瘤组织磁化后5 min磁信号强度则降至279.04±3.84pT,差异同样显著(P<0.001)。这表明肿瘤细胞摄取F-SPION并磁化后所产生的磁信号可反映化疗的早期响应。研究结论1.肿瘤细胞数量减少和对F-SPION的摄取减弱会降低肿瘤细胞的磁信号。2.磁化作用促进F-SPION在肿瘤组织内的聚集同时增强肿瘤组织的磁信号。3.组织磁信号增强的关键是在细胞摄取F-SPION后进行磁化。4.基于F-SPION磁化后在肿瘤细胞内至少持续1小时的长磁性,结合SERF磁力计在零磁场下测量肿瘤磁信号,通过监测化疗过程中的信号降低,可以实现对肿瘤治疗效果的早期评估。更多还原

【Abstract】 BackgroundThe global incidence and mortality rates of malignant tumors,such as breast cancer,remain high.Approximately 80%of patients undergo chemotherapy during treatment,making accurate evaluation of chemotherapy efficacy critically important.In the early stages of treatment,when there are no significant changes in tumor size or morphology,existing imaging techniques lack the sensitivity needed to effectively detect tumor cell death and changes in physiological activity,such as alterations in metabolism,apoptosis,or other vital functions.These shortcomings fall short of clinical demands.Although pathological biopsy is considered the gold standard,its invasive nature,limited sampling,and poor repeatability greatly restrict its practical clinical application.In recent years,with the development of quantum sensing technologies,zero-magnetic field detection methods based on spin-exchange relaxation-free magnetometers have attracted increasing attention in the diagnosis of cardiac and brain-related magnetic conditions,owing to their sensitivity,noninvasiveness,and simplicity.However,their application in tumor magnetic signal detection still faces significant challenges due to the inherently weak magnetic signals of tumors and high background noise.Enhancing magnetic signal strength in tumor zero-field detection has thus become a key issue.While magnetized ferromagnetic materials can amplify tumor signals,they also generate substantial background noise.Superparamagnetic materials,on the other hand,produce minimal background noise but fail to maintain stable signal enhancement after magnetization.Therefore,achieving sustained tumor signal enhancement while minimizing nanoparticle-related background noise remains a major bottleneck in zero-field tumor detection.Based on this,constructing metal nanoparticle systems with long-lasting magnetism may offer a sensitive and accurate tool for detecting malignant tumors and identifying early internal changes following chemotherapy,with important clinical value and translational potential.Objectives(1)To design and synthesize a novel type of iron oxide nanoparticles(ferromagnetic-superparamagnetic iron oxide nanoparticles,F-SPION)that combine both ferromagnetic and superparamagnetic properties and exhibit long-lasting magnetism,enabling the generation of stable tumor-enhanced magnetic signals with low background noise after entering cells and undergoing magnetization;(2)To achieve the detection of tumor cell magnetic signals in a zero-magnetic environment by leveraging the long-lasting magnetism of F-SPION after magnetization and the high sensitivity of SERF magnetometers,and to verify the correlation between magnetic signal intensity and both cell quantity and nanoparticle uptake capacity;(3)To analyze changes in magnetic signals of tumor cells at different time points during chemotherapy after F-SPION uptake,and to explore the value of these signal changes in early evaluation of therapeutic efficacy.Methods1.Synthesis and characterization of F-SPIONNovel iron oxide nanoparticles were synthesized by reacting FeCl2 and FeCl3 with ammonia in deionized water under argon protection at 80℃ for 1 hour.The resulting nanoparticles were then characterized in terms of their morphology and magnetic properties.2.Magnetic signal measurement under an ultra-weak magnetic environmentIn this study,a self-developed cell zero-magnetic detection chamber,which is equipped with a five-layer magnetic shielding structure and an active magnetic compensation system,was used to create an ultra-weak magnetic environment.In this environment,a QuSpin SERF magnetometer was used to measure the magnetic signals of F-SPION,4T1 cells,as well as mouse tumor tissues,wound tissues,and normal tissues,with normal tissues serving as the control.To enhance the signal,F-SPION,4T1 cells,and tumor tissues were magnetized using a rubidium magnet(size:10×5×0.5 cm,magnetic field strength:0.15 T).During the magnetic signal measurement,the SERF probe was aligned with the center of the sample,and the gap between the sample and the magnetometer was maintained at 1～2 mm.The sample was rotated at a frequency of 1.7 Hz,while magnetic signals were collected at a frequency of 200 Hz for 30 seconds.The data were then processed using band-pass filtering and fast Fourier transform,and periodic peaks corresponding to the sample rotation were extracted to evaluate changes in the magnetic signal.3.Evaluation of the impact of tumor cell quantity and F-SPION uptake capacity on magnetic signal4T1 cells(3.0×106)were incubated with F-SPION(100 μg/mL)in RPMI-1640 medium for 0,2,4,and 6 hours.Additionally,five different quantities of 4T1 cells(1×105,2×105,3×105,4×105,and 5×105 cells)were incubated with F-SPION(100 μg/mL)in RPMI-1640 medium for 6 hours.To simulate the effect of drug treatment on cell uptake capacity,4T1 cells(3.0×106 cells)were co-incubated with doxorubicin(0.028 μg/mL)for 0,24,and 48 hours.Afterward,the cell numbers from different incubation times were adjusted to 2.0×106 cells and co-incubated with F-SPION(100 μg/mL)in RPMI-1640 medium for 6 hours.After incubation,all cell samples were directly magnetized in the original culture flask using the aforementioned rubidium magnet for 30 seconds,followed by magnetic signal detection.The relationship between tumor cell quantity and magnetic signal intensity was analyzed using linear regression.Prussian blue staining was used to verify the effect of different incubation times and doxorubicin treatments on the F-SPION uptake capacity of 4T1 cells.4.Evaluation of F-SPION-induced magnetic signal enhancement and duration in tumor tissuesTo evaluate the magnetic signal enhancement between tumor and normal tissues,a subcutaneous unilateral 4T1 tumor-bearing mouse model was established.After anesthesia,F-SPION(2.5 mg/kg)was injected via the tail vein.Thirty minutes later,magnetic signals of the tumor and contralateral normal tissues were measured before magnetization.The tumor was then magnetized for 30 seconds using a magnet,followed by signal measurement.The contralateral normal tissue underwent the same magnetization and measurement procedure as a control.Magnetic signals of both tissues were also measured at 30 and 60 minutes post-magnetization to assess the persistence of F-SPION-mediated enhancement.5.Evaluation of the impact of magnetization on F-SPION distribution in tumor tissuesTo determine whether magnetization affects the distribution of F-SPION in tumor tissues,a bilateral subcutaneous 4T1 tumor-bearing mouse model was used.After intravenous injection of F-SPION(2.5 mg/kg),tumor tissues were magnetized for 30 seconds on one side 30 minutes later.T2-weighted imaging was performed before and after magnetization,and the T2WI signal changes in both sides of the tumor were compared.The mice were then euthanized,and the tumor tissues were collected for routine processing and Prussian blue staining to verify the results.The distribution of F-SPION in tumor tissues was observed,and the accumulation of F-SPION in the tissues was evaluated based on staining intensity.The relationship between T2 signal changes and F-SPION uptake was also validated.6.Evaluation of the impact of blood flow status on F-SPION distribution and magnetic signal in tissuesTo investigate whether local blood flow status affects F-SPION distribution and magnetic signal intensity,a standard circular wound(10 mm in diameter)was created approximately 1 cm above the spinal midline in mice.The animals were randomly divided into three groups:control(untreated wound),congestion(75%ethanol applied to induce vascular dilation),and inflammation(lipopolysaccharide injection to activate inflammation),with 5 mice in each group.F-SPION(2.5 mg/kg)was intravenously injected via the tail vein.Thirty minutes later,magnetic signal intensity of wound tissues in all groups was measured before magnetization.The wounds were then magnetized for 30 seconds using a rubidium magnet.Magnetic signal intensity was measured again at 5,30,and 60 minutes post-magnetization using a SERF probe.After measurements,the mice were euthanized,and wound tissues were collected for Prussian blue staining to assess F-SPION distribution across the three groups.7.Evaluation of the magnetic signal of tumor cell uptake of F-SPION in therapeutic monitoringTo verify whether the magnetic signals produced by tumor cells after the uptake of F-SPION and magnetization can be used for early monitoring of tumor chemotherapy response,a unilateral tumor-bearing mouse model was randomly divided into two groups:a control group and a treatment group(n=5 in each group).The treatment group received intravenous injections of doxorubicin(2 mg/kg)every 4 days for a total of 6 injections,while the control group received an equal volume of saline at the same time points.On days 1,7,14,and 21,F-SPION(2.5 mg/kg)was injected via the tail vein,and 30 minutes later,the tumor tissues were magnetized.The magnetic signal intensity of the tumor tissues was measured at 5,30,and 60 minutes after magnetization.The differences in magnetic signal intensity between the two groups were analyzed.By tracking the changes in tumor tissue magnetic signals during chemotherapy,the feasibility of using the magnetic signal produced by F-SPION uptake and magnetization as an indicator for early chemotherapy efficacy monitoring was evaluated.8.Statistical analysisAll data are presented as mean ± standard deviation.Statistical analyses were performed using GraphPad Prism 10.Each experiment was independently repeated at least three times.The Shapiro-Wilk test was first applied to assess the normality of the data.For normally distributed paired data,paired t-tests were used to evaluate within-group differences.These comparisons included:magnetic signal intensity of tumor versus normal tissues before and after magnetization at different time points(5,30,and 60 min)following F-SPION injection;magnetic signal differences between day 1 and day 7 at 5 min post-magnetization within each group(control and treatment);and magnetic signal intensity at 5 min post-magnetization between the two groups at four time points(day 1,7,14,and 21).For normally distributed multi-group comparisons,one-way ANOVA was performed,followed by Tukey’s post hoc test as appropriate.These comparisons included:magnetic signal differences of 4T1 cells after magnetization under different F-SPION incubation times(0,2,4,and 6 h);signal differences under different doxorubicin pretreatment durations(0,24,and 48 h)in 4T1 cells at a fixed quantity(2.0×106);and signal intensity differences in wound tissues under different blood flow states(control,congestion,inflammation)before and after magnetization at different time points(5,30,and 60 min).P<0.05 was considered statistically significant.Results1.Nanoparticle characterizationThe F-SPION nanoparticles had an average size of 12 nm with a uniform particle size distribution.Vibrating sample magnetometry revealed a high saturation magnetization of 62.5 emu/g under a large magnetic field(±20,000 G),with negligible coercivity and remanence,indicating superparamagnetic behavior.Under a small magnetic field(±600 G),a slight magnetic hysteresis was observed,suggesting that the nanoparticles also possess weak ferromagnetic properties.These results demonstrate that F-SPION exhibits both superparamagnetic and weak ferromagnetic characteristics.2.Tumor cell number reduction and decreased uptake capacity lead to weakened tumor cell magnetic signalThe magnetic signal intensities of 4T1 cells incubated with F-SPION for 0 h,2 h,4 h,and 6 h were 36.01±2.87 pT,210.42± 16.50 pT,224.39±5.99 pT,and 346.64±11.46 pT,respectively,with statistically significant differences among groups(P<0.001).Tukey’s post-hoc test further confirmed that the magnetic signal intensity significantly increased with prolonged incubation time.A strong linear correlation was observed between magnetic signal intensity and 4T1 cell number(R2=0.974).To evaluate the effect of doxorubicin on magnetic signal intensity,4T1 cells were incubated with doxorubicin for 0 h,24 h,and 48 h.After incubation,the cell number in each group was adjusted to 2.0×106,followed by co-incubation with F-SPION in RPMI-1640 medium for 6 hours.The measured magnetic signal intensities were 267.88±5.97 pT,206.02±2.23 pT,and 122.74±2.11 pT,respectively.Statistically significant differences were found among groups(P<0.001),and Tukey’s post-hoc test indicated that magnetic signal intensity significantly decreased with prolonged doxorubicin incubation.Prussian blue staining showed that prolonged incubation with F-SPION enhanced nanoparticle uptake by 4T1 cells.In contrast,cells pretreated with doxorubicin exhibited a time-dependent reduction in staining intensity,indicating decreased nanoparticle uptake.These findings suggest that doxorubicin treatment reduces both tumor cell number and their ability to internalize F-SPION,thereby leading to a significant decline in magnetic signal intensity.3.Magnetically activated F-SPION enhances tumor tissue magnetic signal and forms sustained contrast between tumor and normal tissuesBefore magnetization,the magnetic signal intensity of tumor tissues in single-side tumor-bearing mice was 108.47±5.29 pT,and that of normal tissues was 72.60±2.88 pT,showing a statistically significant difference(P<0.001),but the overall contrast was low.At 5 minutes after magnetization,the magnetic signal intensity of tumor tissues(802.70±60.81 pT)was significantly higher than that of normal tissues(149.25±16.23 pT,P<0.001).This difference remained significant at 30 minutes after magnetization(tumor:237.34±8.70 pT;normal:45.23±2.15 pT)and 60 minutes(tumor:163.56±3.20 pT;normal:42.75±1.49 pT)(all P<0.001),suggesting that magnetized F-SPION can maintain magnetic signals in tumor tissues,exhibiting long-lasting magnetism.4.Magnetization promotes the aggregation of F-SPION in tumor tissuesCompared with pre-magnetization,the magnetized tumor tissue exhibited a markedly reduced signal in T2-weighted imaging,appearing as a low-signal region,while the non-magnetized side showed no obvious signal change in either of the two imaging sessions.Prussian blue staining further confirmed these findings:abundant dark blue staining was observed in the magnetized tumor tissue,indicating a dense distribution of F-SPION;in contrast,only a small amount of scattered staining was seen in the non-magnetized tissue,suggesting that magnetization facilitates the aggregation of F-SPION within tumor tissues.5.Enhancement of tissue magnetic signals due to cell uptake of F-SPION and magnetic activationBefore magnetization,there was no significant difference in the magnetic signal intensity of wound tissues among the control,congestion,and inflammation groups(36.06±1.94 pT,38.10±12.25 pT,and 38.20±1.38 pT,respectively;P>0.05).At 5,30,and 60 minutes after magnetization,the magnetic signals of wound tissues in the inflammation group(269.48±2.62 pT,265.88±2.20 pT,and 242.80±3.24 pT)were consistently and significantly higher than those in the control group(245.20±2.72 pT,166.18±1.62 pT,and 109.56±1.04 pT)and the congestion group(245.00±3.00 pT,166.05±1.63 pT,and 110.22±2.29 pT)(P<0.001),while no significant difference was observed between the control and congestion groups(P>0.05).Prussian blue staining confirmed the magnetic signal measurements,showing almost no F-SPION deposition in the control group,deposition mainly in the stroma in the congestion group,and prominent intracellular accumulation of F-SPION in the inflammation group.These findings suggest that magnetic signal enhancement results from cellular phagocytosis and magnetic activation of F-SPION.6.Changes in tumor tissue magnetic signals reflect early response to chemotherapyOn day 7,the tumor response to chemotherapy was observed.In the treatment group,the magnetic signal intensities of tumor tissues at 5 minutes after magnetization on days 1 and 7 were 426.98±16.51 pT and 425.41±14.37 pT,respectively,with no significant difference(P>0.05).In contrast,in the control group,the magnetic signal at the same time points significantly increased from 481.41±3.82 pT to 830.72±5.90 pT(P<0.001).On day 14,the tumor tissue magnetic signal in the control group increased to 1978.70±14.36 pT,while that in the treatment group was 346.62±5.15 pT,with a significant difference between groups(P<0.001).On day 21,the magnetic signal in the control group further increased to 4562.61±196.84 pT,while that in the treatment group decreased to 279.04±3.84 pT,and the difference remained significant(P<0.001).These results indicate that the magnetic signals generated by tumor cells after F-SPION uptake and magnetization can reflect the early response to chemotherapy.Conclusions1.A reduction in tumor cell quantity and weakened uptake of F-SPION lead to decreased magnetic signals in tumor cells.2.Magnetization promotes the accumulation of F-SPION within tumor tissue and enhances its magnetic signal.3.The key to signal enhancement lies in magnetizing the tissue after cellular uptake of F-SPION.4.Based on the long-lasting magnetism of F-SPION,which persists for at least one hour after magnetization within tumor cells,combined with the SERF magnetometer’s ability to measure tumor magnetic signals under zero-field conditions,monitoring signal attenuation during chemotherapy allows for early evaluation of tumor therapeutic efficacy.更多还原