Ovarian cancer (OC) is the foremost cause of mortality from gynecological malignancies. Typically diagnosed at advanced stages with extensive peritoneal metastasis due to insidious onset and atypical early symptoms, OC exhibits a 5-year overall survival (OS) of approximately 30% [1, 2]. Although cytoreductive surgery and platinum-based combination chemotherapy remain the first-line treatment option, the high risk of lymphatic metastasis and recurrence persists [3, 4]. Adjuvant therapies like intraperitoneal infusion chemotherapy and hyperthermic intraperitoneal chemotherapy offer improvements [5, 6, 7] but pose risks of drug resistance and serious complications [8]. Therefore, exploring novel treatment approaches is imperative for improving the prognosis of OC.

In view of the radiosensitivity of OC [9], molecular targeting technology offers a means of delivering precise internal irradiation to tumors and metastatic lesions after intravenous injection of radionuclides [10]. Radionuclide imaging aids in visualizing OC pathogenesis and progression [11, 12], showing diagnostic and therapeutic potential. Shabani et al. [13] synthesized Ru template gold nanoparticles and assessed the therapeutic effect of these nanoparticles on breast cancer cells. The results indicated the selective and effective anticancer function of the nanoparticles, with low cytotoxicity and superior biocompatibility [13]. Furthermore, intraperitoneal injection of radioactive agents for internal irradiation radiotherapy can generate a high local drug concentration similar to intraperitoneal chemotherapy, thereby reducing recurrence and improving survival [14, 15, 16]. Therefore, efforts focus on enhancing irradiation delivery via developing new and effective carriers for the irradiation agents, aiming to mitigate adverse events and enhance outcomes for OC.

The number and activity of folate receptor (FR), as well as the affinity of folic acid (FA) conjugates (Kd: 10${}^{-9}$–10${}^{-10}$ M) have been reported to be much higher on the surface of 90% of OC tumor cells than those in normal cells [17]. Therefore, FR-targeted therapies have attracted great attention in recent years for treating OC [18, 19, 20]. However, in vivo studies on FR-targeted carriers labeled with radionuclides (${}^{99\text{m}}$Tc, ${}^{188}$Re, ${}^{68}$Ga, ${}^{64}$Cu) [21, 22, 23, 24] revealed limited targeting effectiveness for FA conjugated carriers such as liposomes and nanoparticles [21, 22]. Herein, we used FR as the targeting molecule, the biodegradable material polyethyleneglycol-polylactic acid-co-glycolic acid (PEG-PLGA) [25] as the carrier matrix, and diethylene triamine pentaacetic acid (DOTA) as the metal chelating agent to prepare ${}^{177}$lutetium labeled nanoparticles, named ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles. In addition, both labeling yield and radiochemical purity of the nanoparticles are essential for clinical applications, they were therefore evaluated in this study. Moreover, the efficacy and safety of nanoparticles in the two treatment modes including tail vein injection and intraperitoneal infusion were evaluated utilizing mouse models bearing subcutaneously transplanted SKOV3 OC tumors or intraperitoneal metastatic SKOV3 OC tumors, respectively. The ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles prepared in this study possess targeting, degradability and nuclide internal irradiation therapeutic properties. Notably, intraperitoneal infusion of the ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles for treating OC metastatic lesions represents a novel approach. This method holds promise for collaboration with comprehensive OC treatment, potentially improving efficacy and reducing drug resistance.

FR is a glycosylphosphatidylinositol-linked protein comprising four subtypes ($\alpha$, $\beta$, $\gamma$ and $\delta$). Previous study has reported significantly higher numbers and activity of FR$\alpha$, along with enhanced affinity of folate conjugates on the surface of 90% of OC tumor cells compared to normal cells [17]. It has emerged as a promising candidate for imaging and targeted therapy of OC due to its marked expression in OC cells [26]. However, the small molecular weight of FR results in a short blood circulation time and rapid clearance from the bloodstream, leading to reduced tumor uptake.

Several strategies have been explored to overcome these limitations. Some studies have utilized small molecule albumin conjugates to noncovalently couple antibody fragments and folate conjugates with plasma proteins, effectively increasing the blood circulation time and drug concentration in tumors. However, this approach often led to high radioactivity uptake in the kidneys [27, 28]. Additionally, pre-administration of antifolates has been employed to reduce the reabsorption of ${}^{111}$In-DTPA-FA in the proximal renal tubules, but the underlying mechanism remains unclear, and results are often poorly reproducible [27]. Based on the property of PEG to covalently bind to folate ligands and their analogues [29], Bao et al. [30] prepared FA-DOTA-PEG-PLGA nanocarriers. These nanoparticles were designed to modify the nanoparticle surface using the hydrophilic polymer material PEG, creating long-circulating nanoparticles, also known as stealth nanoparticles (SNP) [30]. This modification aimed to reduce recognition and phagocytosis by the liver and spleen reticuloendothelial system, enhancing the blood circulation time of the nanoparticles. To address the concern about nanoparticle retention in the kidneys, we utilized PLGA as the nanocarrier, known to significantly reduce renal drug distribution. Additionally, we controlled the size range of nanoparticles to be greater than 20 nm, a parameter shown to reduce renal drug excretion [31]. Histological distribution analysis in this study revealed partial radioactivity uptake in blood circulation 72 h after tail vein injection of nanoparticles, with elimination of radioactivity uptake in the blood at 168 h. This suggested a prolonged retention time of ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles in the blood circulation. Furthermore, the peak value of radioactivity uptake in the kidneys was only 1.646 %ID/g, significantly lower than that observed in previous study on FR radionuclide drug [32].

Micro-SPECT/CT imaging revealed a notable increase in radioactivity uptake in subcutaneous tumors of the mice at 4 h after the tail vein injection of nanoparticles, with peak uptake observed at 24 h. These findings suggested an active targeting effect and rapid tumor entry of the nanoparticles. While there was no significant difference in tumor volume between the nanoparticle group and the control group, nanoparticle-treated tumors displayed a discernible trend of suppression. It is noteworthy that the tumor-suppressing effect of the nanoparticles weakened from day 9 to day 14, potentially attributed to the diminished antitumor efficacy of the ${}^{177}$Lu after multiple decays. This implied that the optimal therapeutic time of a single administration of the nanoparticles spans approximately one week. An escalation in the radiopharmaceutical dose may enhance the antitumor efficacy of the nanoparticles.

Intraperitoneal injection of radiopharmaceuticals has been under development for nearly 50 years as a treatment for peritoneal metastasis and ascites of OC. Early studies utilized ${}^{32}$P colloid, which could be attached to the inner wall of the body cavity or the surface of organs for radiotherapy. However, the lack of a targeting effect limited its maximum dose [14, 16]. Subsequent studies attempted to use radionuclide-labeled antibody agents for targeted therapy, but their transient residence and action time in the abdominal cavity resulted in limited advantages for local treatment [33, 34]. Moreover, ${}^{32}$P colloid demonstrated uneven distribution in the body after administration, leading to potential intestinal toxicity and adverse events [15]. On this basis, we performed further in vivo experiments to explore the therapeutic efficacy of intraperitoneal injection of nanoparticles in treating mice with peritoneal metastasis.

In this study, the histological distribution results revealed persistent radioactivity uptake in the blood within 72 h after intraperitoneal injection of nanoparticles, indicating a continuous influx of nanoparticles into the bloodstream from the abdominal cavity during this period. The peritoneum, characterized by abundant capillaries and lymphatic vessels, serves as a bidirectional semipermeable membrane, permitting the passage of water, electrolytes, and some small molecular substances. The ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles, being macromolecules with a size of 20–80 nm, have the capability to traverse the abdominal cavity but not the peritoneum. With the gradual dissolution and decomposition of the matrix PLGA [31], the nanoparticles underwent subsequent breakdown into small molecular fragments, which were then absorbed by peritoneal capillaries and lymphatic vessels. Some of these fragments entered tumors under the influence of targeting effect, while the remaining fragments were excreted in urine. This slow degradation process of nanoparticles ensured prolonged residence time of radiopharmaceuticals in the abdominal cavity. The absorption into the bloodstream post-degradation mitigated the impact of uneven distribution of radiopharmaceuticals, especially in some patients with intestinal adhesions [35], where encapsulated fluid may cause uneven drug dispersion, resulting in residual lesions and suboptimal therapeutic effects.

Both the nanoparticle group and chemotherapy group exhibited tumor suppression and a reduction in ascitic fluid volume, especially the nanoparticle group. In addition, side effects such as intestinal perforation may be induced after intraperitoneal injection of the nanoparticles due to the direct contact between nanoparticles and organs. This concern was addressed through HE staining results, which showed the absence of side effects in the small intestine and colon tissues of the mice, indicating the safety of the ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles. Our findings indicated that, in comparison with ${}^{32}$P colloid [15], ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles exhibited reduced intra-abdominal local retention and a diminished potential for adverse reactions. Collectively, these findings provide a novel perspective for the treatment and recurrence reduction of OC.

In addition to their targeting and intraperitoneal retention properties, ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles may exhibit other therapeutic advantages. Firstly, the $\beta$-ray range of ${}^{177}$Lu extends up to 2 mm, possessing a specific range of action and penetration (about 0.2–0.3 mm in soft tissues). This characteristic makes it more likely to exert the ‘cross-effect’ of radiation in confined spaces such as the abdominal cavity, particularly in discreet sites. Conventional chemotherapy drugs, in contrast, are limited to surface perfusion on abdominal tissues and lack the local penetration capability of ionizing radiation. For instance, the capsules of liver and spleen are common sites for peritoneal metastasis in OC patients. Achieving a high drug concentration in these local areas is challenging due to their elevated position and deep narrow cavities. In addition to the therapeutic role in intraabdominal tumors, the nanoparticles absorbed by the liver and spleen can effectively target tumors in the capsules and adjacent areas. Furthermore, the PEG long chains on the nanoparticle surface can bind a significant number of water molecules. This property contributes to maintaining the small size, monodisperity, and stable state of the nanoparticles, consequently enhancing their tumor-targeted aggregation. In addition, PEG plays a crucial role in stabilizing the nanoparticles, ensuring their persistent retention in tumors [36].

This study has several limitations. We did not explore the potential benefits of combining systemic and intraperitoneal administration of the nanoparticles for treatment. Besides, the optimal dosage, long-term efficacy, and safety of the ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles have not been thoroughly investigated. Furthermore, our understanding on the controlled release dynamics of nanoparticles in the body remains limited. These aspects merit increased attention in future research endeavors.

We successfully engineered PEG surface-modified ${}^{177}$Lu-FA-DOTA-PEG-PLGA long-circulating nanoparticles with a prolonged blood circulation time of 72 h and a remarkably low renal radioactivity uptake of only 1.646% ID/g. This uptake was significantly lower than that of previous FR radionuclide drugs. Notably, these nanoparticles had targeting, degradability, and nuclide internal irradiation therapeutic properties. Moreover, our findings indicated that the nanoparticles showed no damage to intestinal tissues, reducing systemic toxicity and side effects commonly associated with conventional chemotherapy. The nanoparticles demonstrated notable antitumor efficacy against subcutaneous tumors via FR targeting. Furthermore, their sustained presence in the peritoneal cavity enables effective targeting of intraperitoneal metastatic OC tumors, leading to a reduction in ascitic fluid volume. In summary, the unique properties of the ${}^{177}$Lu-FA-DOTA-PEG-PLGA nanoparticles offer significant promise for improving therapeutic outcomes of OC by addressing challenges associated with drug delivery.

The original data presented in the study are included in the article, further inquiries can be directed to the corresponding author.

ZG and YD: methodology, formal analysis, data curation, writing. JZ and SS: investigation, software, writing. JW: conceptualization, supervision, writing-review & editing. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All animal care and experimental protocols were approved by the Ethical Committee of Xuzhou Medical University Experimental Animal Center (No. 202101w015).

Not applicable.

This study was support by the Jiangsu Provincial Health Committee Key scientific research projects (No. ZD2021053).

The authors declare no conflict of interest.