Abstract

   Rationale: ^212Pb, a promising in vivo alpha-particle generator of
   ^212Bi, has aroused much interest as a therapeutic radionuclide. For
   the development of targeted alpha therapy (TAT), it is important to
   determine the contribution of targeted effects in irradiated cells, and
   also of non-targeted effects in non-irradiated bystander cells.
   Currently, the critical roles of mitochondrial transfer in cellular
   crosstalk have garnered significant attention. However, the specific
   involvement of damaged mitochondrial transfer in orchestrating this
   alpha-particle radiation-induced bystander effect (RIBE) needs to be
   further explored.

   Methods: A novel alpha-emitting radiopharmaceutical, ^212Pb-hydrogel
   nanoparticles (HNPs), was synthesized and subsequently evaluated its
   theranostics effects. The impact of irradiated cell-conditioned media
   (ICCM), collected at different times post-^212Bi irradiation, on
   bystander cancer cells regarding cell viability was also investigated.
   Additionally, damaged mitochondria were isolated and cultured with
   non-irradiated bystander cells to assess their role.

   Results: ^212Pb-HNPs exhibited efficient therapeutic antitumor effects
   in vitro, including increased GSH depletion, ROS accumulation, and
   mitochondrial damage in irradiated tumor cells. In vivo studies
   demonstrated its imaging potential through SPECT/CT, and RNA sequencing
   results indicated activation of oxidative stress-related pathways in
   irradiated tumors. Additionally, ICCM influenced the viability of
   non-irradiated bystander cells, suggesting a radiation-induced
   bystander effect by the alpha-particle ^212Bi. Interestingly, damaged
   mitochondria isolated from ICCM were observed to enter co-cultured
   non-irradiated bystander cells. Further experiments confirmed that the
   transfer of damaged mitochondria results in the death of non-irradiated
   bystander cells.

   Conclusion: The present study highlights the theranostic potential of
   the alpha-particle generator ^212Pb and, more importantly, elucidates
   the role of damaged mitochondrial transfer in alpha-particle RIBE.
   These findings provide a novel theoretical mechanism for the antitumor
   effects of alpha-particles and expand the clinical application
   prospects of TAT.

   Keywords: targeted alpha therapy, lead-212, bismuth-212,
   radiation-induced bystander effect, mitochondrial transfer

Introduction

   Malignant tumors pose one of the most significant threats to human
   health, necessitating the development of new precision therapeutic
   strategies [43]^1. Notably, in recent years, radiopharmaceuticals have
   emerged as a revolutionary approach for precision oncology due to their
   superior "efficacy and safety". Historically, the use of beta-particles
   has been more prevalent both domestically and internationally. However,
   alpha-particles, with their unique clinical advantages, represent a
   novel and promising avenue in nuclear medicine for anti-tumor therapy
   [44]^2^, [45]^3. Nevertheless, the biological effects of alpha-particle
   radiation are complex, and research on the mechanisms of targeted
   alpha-therapy (TAT) has progressed more slowly than that for
   beta-particles worldwide. This slow progress has become a critical
   bottleneck in the clinical translation and application of
   alpha-particles. Therefore, elucidating the therapeutic mechanisms of
   alpha-particles is urgently needed to meet the demands of societal
   development and public health.

   One increasingly popular radionuclide is the in vivo alpha-particle
   generator ^212Pb, which emits 1 (net) alpha-particle and 2
   beta-particles within its decay chain. ^212Pb has a half-life of 10.6
   h, extending the time to deliver and target tumors with its short-lived
   ^212Bi daughter (T[½] 60.6 min) [46]^4. This results in a greater
   therapeutic impact and reduces the dose required for an effective
   therapeutic benefit. At the same time toxicity to normal tissues is
   reduced. ^212Pb possesses several advantages, including independence
   from reactors and accelerators, high radionuclide purity, well-defined
   quality control methods, and low long-term bulk supply costs. These
   attributes make ^212Pb the most scalable and competitively priced
   radionuclide for TAT. Additionally, ^212Pb emits characteristic gamma
   rays at 238 keV (43.6%), which possess potential for imaging
   applications [47]^5. Studies using^ 212Pb showed the feasibility of
   this isotope in TAT for the treatment of disseminated intraperitoneal
   disease.

   Cellular death is a crucial endpoint that has been extensively studied
   in cells exposed to radiation. It is essential to assess the
   contributions of both targeted effects in irradiated cells and
   non-targeted effects in non-irradiated bystander cells. The
   transmission of signals between irradiated and non-irradiated cells
   occurs via gap junctions or soluble signaling mediators. The resultant
   damage in adjacent non-targeted cells, collectively termed
   radiation-induced bystander effect (RIBE), includes cell death,
   chromosomal damage, mutations, and alterations in gene expression and
   protein levels [48]^6^-[49]^8. The RIBE was first identified as the
   phenomenon involving the induction of sister chromatid exchanges by
   extremely low doses of alpha-particles in non-irradiated cells [50]^9.
   However, the mechanisms by which alpha particles induce RIBE remain
   unclear, necessitating further development and refinement of the
   relevant theoretical framework.

   A substantial body of evidence suggests that mitochondria may play a
   pivotal role in RIBE [51]^10^-[52]^12. Currently, mitochondrial
   research has evolved from the "powerhouse" paradigm to the "information
   processor" paradigm. As the central hub of cellular signal
   transduction, mitochondria are now recognized as essential signaling
   entities in the modulation of cellular responses to ionizing radiation.
   Historically, mitochondrial functions were thought to be confined
   primarily within the boundaries of the host cell [53]^13^-[54]^15.
   However, recent studies have demonstrated that mitochondria and their
   components can be released into the extracellular space, participating
   in intercellular communication. This process, wherein mitochondria are
   transferred from a donor cell to a recipient cell, significantly
   influences the function and fate of the recipient cell[55]^16^-[56]^20.

   In this study, we aim to develop hydrogel nanoparticles (HNPs) loaded
   with the alpha-particle generator ^212Pb to explore its theranostic
   potential. Additionally, we would like to elucidate the critical role
   of damaged mitochondrial transfer in the alpha-particle
   radiation-induced bystander effect (Figure [57]1).

Figure 1.

   [58]Figure 1
   [59]Open in a new tab

   Graphical abstract. Theranostic efficacy of alpha-particle generator
   ^212Pb and role of damaged mitochondrial transfer in alpha-particle
   radiation-induced bystander effect.

Materials and Methods

Materials

   Phosphate buffered saline (PBS), fetal bovine serum (FBS), dulbecco's
   modified eagle medium (DMEM), and a cell membrane protein extraction
   kit were purchased from Biosharp (Beijing, China). Calcein/Propidium
   Iodide (PI) cell viability/cytotoxicity assay kit, reduced glutathione
   (GSH) and oxidized glutathione disulfide (GSSG) assay kits,
   mitochondrial membrane potential assay kit with JC-1, and the Annexin
   V-Fluorescein isothiocyanate (FITC) apoptosis assay kit were obtained
   from Beyotime Biotechnology Co., Ltd. (Shanghai, China). MitoTracker™
   Red FM were purchased from Thermo Fisher (MA, USA). Rotenone was
   purchased from Sigma-Aldrich (St. Louis, USA).

Instrument

   The hydrodynamic diameter and Zeta potential of the nanoparticles were
   analyzed by the Malvern Zetasizer Nano ZS. Transmission electron
   microscopy (TEM) images were taken by FEI-Tecnai12. X-ray spectroscopy
   (EDS) mapping elemental analysis was performed at JEM-F200 (URP).
   Fluorescence imaging was performed by Carl Zeiss LSM900 confocal
   microscope and Nikon inverted fluorescence microscope. Absorbance and
   chemiluminescence were measured using the SpectraMax iD5 Multi-Mode
   Microplate Readers (Molecular Devices, USA). Flow cytometry was
   performed on a BD Fortessa flow cytometer. Imaging of ^212Pb was
   performed using single-photon emission-computed tomography (SPECT/CT)
   ((InliView-3000B, Novel Medical)).

Separation of ^212Pb and ^212Bi

   ^212Pb and^ 212Bi were obtained from a^ 224Ra-^212Pb/^212Bi generator
   prepared in our laboratory. After isolating ^224Ra from^ 228Th, it was
   absorbed on a AG50W×8 cation resin column. ^212Pb and ^212Bi was
   separated from ^224Ra by eluting the column with 2 [M] HCl and 0.5 [M]
   HCl respectively.^ 212Pb was further purified by anion exchange resin
   AGMP-1M and eluted by 0.1 [M] HNO[3] and it is possible that^ 212Pb
   exists in the form of Pb^2+. Before labelling, the pH value was
   adjusted by NH[4]OH to 6.5-7.5. ^212Bi was purified by a DGA resin
   cartridge. After loading ^212Bi onto a DGA resin column, it was finally
   eluted by 50 m[M] ammonium citrate solution with a pH value of 7.0±0.5
   and it is reasonable that ^212Bi is in the chemical form of bismuth
   citrate in solution. The activity of ^212Bi and ^212Pb was determined
   by liquid scintillation counter (LSC) after the equilibrium has been
   achieved between ^212Bi and ^212Pb [60]^21. The purity of^ 212Pb and^
   212Bi was confirmed by measuring the decay half-life using LSC.
   Radionuclides with half-lives not exceeding 5% of the reference value
   were applied in all the experiment. For ^212Pb, the purity was further
   determined by measuring the activity of^ 224Ra after 12-15 half-lives
   of ^212Pb and the results indicated that the breakthrough of ^224Ra is
   less than 0.03%.

Synthesis of HNPs and Pb/ ^212Pb-HNPs

   The material was synthesized following the procedure previously
   reported [61]^22 Typically, Span 80 (300 mg) and Tween 80 (100 mg) were
   dissolved in 15 mL of cyclohexane. Subsequently, a mixed solution
   containing 250 μL of PEGDA and 550 μL of water was added. The mixture
   was transformed into an emulsion by ultrasonication for 2 min. Next,
   100 μL of the photoinitiator 2-hydroxy-2-methylpropiophenone was added
   to the emulsion. The reaction was carried out under magnetic stirring
   at 800 rpm using a 365 nm longwave UV lamp for 4 h. The products were
   then collected by centrifugation at 12,000 rpm and washed three times
   with ethanol and water. Finally, Pb/^212Pb and water were added to the
   HNPs, ensuring that the total volume of the system was controlled at 3
   mL. The mixture was vigorously stirred for 1 h, followed by
   centrifugation and washed three times to collect the precipitate.

Characterization of HNPs and Pb-HNPs

   The size distribution and surface zeta potential of HNPs and Pb-HNPs
   were evaluated by dynamic light scattering (DLS). The morphology of
   HNPs and Pb-HNPs was characterized by TEM. the mapping images and EDS
   showed a uniform distribution of C, N, O and Pb elements over the
   entire architecture of the Pb-HNPs.

Radiolabeling and labeling product stability assays

   The purified ^212Pb-HNPs was co-incubated with PBS and FBS (100 μL for
   each) at 37 ℃ for 1 h, 6 h, 12 h and 24 h. The labeling rate and
   stability at each time point were determined by LSC.

Cellular uptake of ^212Pb-HNPs

   LLC cells were incubated in 24-well plates at a density of
   1×10^5/mL/well for 24 h. For cell uptake experiment at different time
   points, LLC cells were treated with 0.6 kBq/mL ^212Pb-HNPs or ^212Pb,
   and incubated for 0.5 h, 1 h and 2 h. The culture medium was removed
   post incubation and cells were washed twice with PBS. Cells were lysed
   with 1[ M] sodium hydroxide solution and the cell components were
   collected. Finally, the radioactivity of cells and culture medium were
   counted by LSC.

In vitro cytotoxicity analysis

   Cell viability was measured by the CCK-8 method. LLC cells were seeded
   into 96-well plates at a density of 5×10^3 cells per well and cultured
   in DMEM containing 10% FBS (100 μL) for 24 h. The old medium was
   removed, and the cells were treated with serum-free DMEM containing
   different concentrations of ^212Pb, ^212Pb-HNPs, ^212Bi, ICCM,
   mitochondria or others. Besides, LLC cells were treated with 2 μ[M]
   rotenone, an inhibitor of mitochondrial respiratory chain, for 1 h
   prior to irradiation, then immediately washed with PBS before replacing
   with fresh media. After 24 h or other specified time points, the medium
   was sucked out, replaced with fresh DMEM, and 10 μL CCK-8 solution was
   added to each well. Then the absorbance was measured at 450 nm by a
   microplate reader, and the cell survival rate was calculated.

Detection of intracellular GSH and GSSG levels

   The ability of ^212Pb-HNPs to deplete GSH was assessed using GSH and
   GSSG assay kits. LLC cells were seeded in 6-well plates at a density of
   5×10^5 cells per well and cultured overnight to allow adherence. The
   cells were then co-incubated with HNPs (0.35 mg/mL), ^212Pb (0.7
   kBq/mL), and ^212Pb-HNPs (0.7 kBq/mL) for 24 hours. Following
   incubation, the cells were washed once with PBS buffer and collected by
   centrifugation. According to the assay kit instructions, a protein
   removal reagent was added to the collected cells, which were then
   thoroughly vortexed and subjected to two rapid freeze-thaw cycles. The
   supernatant was collected after centrifugation for 10 minutes at 4°C.
   One portion of the supernatant was used to determine total GSH, while
   the other portion was used to determine GSSG content after removing
   GSH. Absorbance at 412 nm was measured using a microplate reader, and
   total GSH and GSSG were quantified by plotting a standard curve.
   Reduced GSH was calculated by subtracting twice the amount of GSSG from
   total GSH.

Analysis of intracellular ROS generation

   The ability of drugs to induce intracellular ROS production was
   assessed by measuring the fluorescence changes resulting from the
   oxidation of the 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA)
   probe. LLC cells were cultured in 6-well plates at a density of 5×10^5
   cells per well for 24 hours. Subsequently, the cells were treated with
   HNPs (0.35 mg/mL), ^212Pb (0.7 kBq/mL) and ^212Pb-HNPs (0.7 kBq/mL) for
   an additional 24 hours. After treatment, the cells were collected and
   resuspended in diluted DCFH-DA solution, then incubated at 37°C for
   staining. The cells were washed three times with serum-free DMEM to
   thoroughly remove any DCFH-DA that had not entered the cells. Finally,
   the fluorescence intensity was analyzed by flow cytometry to quantify
   the intracellular ROS levels, and the data were processed using FlowJo
   software.

Isolation of mitochondria

   When the cell density reaches 70%-80%, irradiate with ^212Bi for 24 h.
   The cell culture medium was collected and centrifuged at 600 g for 10
   min at 4°C. The supernatant was transferred to a new centrifuge tube,
   discarding the pellet. Subsequently, the supernatant was centrifuged at
   1200 g for 10 min at 4°C. Discard the pellet, and transfer the
   supernatant to another new centrifuge tube. Finally, centrifuge at 8000
   g for 15-30 min at 4°C. Carefully remove the supernatant, and the
   resulting pellet contains the extracellular mitochondria
   [62]^23^-[63]^25.

Mitochondrial membrane potential measurement

   To monitor mitochondrial health, JC-1 dye was used to assess
   mitochondrial membrane potential. LLC cells subjected to different
   treatments were incubated with JC-1 (5 μ[M]) for 30 min at 37°C. JC-1
   dye accumulates in mitochondria in a potential-dependent manner,
   indicated by a fluorescence emission shift from green (Ex 485 nm/Em 516
   nm) to red (Ex 579 nm/Em 599 nm). Mitochondrial membrane potential was
   determined using flow cytometry or confocal microscopy.

Measurement of mitochondrial morphology

   The transmission electron microscopy was used to evaluate the
   capability of ^212Pb-HNPs to damage mitochondria and to demonstrate the
   presence of damaged mitochondria in ICCM. Samples were initially fixed
   in 2.5% glutaraldehyde and subsequently post-fixed in buffered 1%
   osmium tetroxide. They were then dehydrated through a graded series of
   acetone and embedded in Epon resin. Ninety-nanometer-thin sections were
   cut using a Leica EM UC6 ultramicrotome. The sections were stained with
   a saturated solution of uranyl acetate and lead citrate. Images were
   captured using a Hitachi TEM system. Mitochondrial morphology was also
   evaluated by MitoTracker™ Red FM, following the manufacturer's
   instructions. Cells were cultured on different substrates for 24 h and
   then incubated with MitoTracker™ Red dye (200 n[M]) for 20 min at 37
   °C. Images of labeled mitochondria were captured using confocal
   microscopy.

Animal model construction

   The C57BL/6 mice (female, 6 weeks old) used in the experiment were
   provided by the animal center of the China Institute for Radiation
   Protection and were raised in a specific pathogen-free (SPF)
   environment. All animal experiments were reviewed and approved by the
   Experimental Animal Welfare Ethics Committee of Shanghai Tenth People's
   Hospital. To construct a tumor-bearing mouse model, LLC cells in a good
   logarithmic growth state were used. A cell suspension containing 5×10^6
   cells in PBS buffer (100 μL) was prepared and injected subcutaneously
   into the back of each mouse.

SPECT/CT imaging

   When the tumor volume of tumor-bearing mice reached about 500 mm^3, the
   mice were anesthetized using 2% isoflurane for imaging. SPECT/CT images
   with ^212Pb or ^212Pb-HNPs (74 kBq per mouse, intratumor injection)
   were obtained on a SPECT/CT device equipped with a high-energy
   collimator detecting the ^212Pb characteristic γ-rays (238 keV, 43.6%).
   Specifically, a window width of 20% was utilized to ensure accurate
   capture of the relevant gamma emissions. The image reconstruction was
   conducted using the Ordered Subset Expectation Maximization (OSEM)
   algorithm, which is known for its efficiency in handling emission
   tomography data. Post-reconstruction, median filtering was applied to
   enhance image quality by reducing noise while preserving edges. The
   reconstructed images were configured into a 60×60 matrix with a pixel
   size of 0.7 mm, ensuring adequate resolution for detailed analysis. The
   slice thickness was also set at 0.7 mm to provide uniformity across
   slices. The iterative reconstruction process involved 40 iterations,
   which were crucial for achieving optimal convergence and image clarity.
   The scan covered a length of 54 mm, segmented into multiple
   acquisitions with a total bed step count of 88 steps. Each step
   incorporated an angular increment of 3.0 degrees to facilitate
   comprehensive volumetric data collection. Imaging sessions were
   conducted at 30 minutes of each scan at different time points (30
   minutes and 2 hours post-intratumor injection).

In vivo RNA‑seq analysis

   When the tumor grew to 50 mm^3, the tumor-bearing mice were randomly
   divided into 2 groups (n = 3 per group). it's worth mentioning that we
   could only perform injections in one mouse each for the control and
   experimental groups on the same day, followed by three repeated
   experiments. The day of grouping was designated as Day 1, marking the
   commencement of drug administration. The control group received 100 μL
   of PBS buffer, while the treatment group was administered ^212Pb-HNPs
   148 kBq) via intratumor injection. To investigate the microenvironment
   of residual tumors post-^212Pb irradiation, tumor tissues were
   harvested from mice 7 days after resection for RNA sequencing. RNA
   extraction, quality control, library construction, and sequencing were
   performed according to the company's standard procedures. The
   featureCounts (v1.5.0-p3) and DESeq2 (v1.16.1) software were used to
   quantify gene expression and perform differential expression analysis
   between the two comparison groups, respectively. Gene Ontology (GO)
   enrichment analysis and the statistical enrichment of differentially
   expressed genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathways were conducted using clusterProfiler (v3.4.4).

Live/dead cell staining assay

   The in vitro efficacy of ICCM, mitochondria and others were further
   evaluated by live/dead staining. LLC cells were seeded into 24-well
   plates (1×10^5 cells per well) and incubated overnight. The cells were
   then treated with appropriate doses of ICCM, mitochondria, and other
   substances, with an untreated group serving as the control. After 24
   hours, the cells were stained with Calcein-AM and PI. Following a
   1-hour incubation at 37°C, the staining effects were observed using a
   fluorescence microscope.

Statistical approach

   All data were expressed as mean values ± standard deviation (SD).
   Statistical analysis was implemented by using the Student's t-test
   between two groups and analysis of variance (ANOVA) when more than two
   groups. p < 0.05 is considered statistically significant.

Results

Synthesis and characterization of HNPs and Pb-HNPs

   The synthesis process of HNPs and ^212Pb-HNPs was illustrated in Figure
   [64]2A. DLS data indicated a slight reduction in both particle size and
   zeta potential of Pb-HNPs compared to HNPs (Figure [65]2B-D). The
   morphology of the synthesized HNPs and Pb-HNPs was characterized using
   TEM (Figure [66]2E, F). The images revealed that the size of HNPs
   ranged from 100 to 200 nm, exhibiting a spherical structure. Besides,
   upon doping with lead, the morphology remained unchanged. The mapping
   images and EDS showed a uniform distribution of C, N, O and Pb elements
   over the entire architecture of the Pb-HNPs (Figure [67]2G).

Figure 2.

   [68]Figure 2
   [69]Open in a new tab

   Synthesis and Characterization of HNPs and Pb-HNPs. A) Schematic
   illustration of the formation of HNPs and ^212Pb-HNPs. B and C) DLS of
   HNPs and Pb-HNPs. D) Zeta potential of HNPs and Pb-HNPs. E and F) TEM
   image of HNPs and Pb-HNPs. G) The EDS mapping of C, N, O and Pb of
   prepared Pb-HNPs.

Characterization and antitumor properties of ^212Pb-HNPs in vitro

   The radiolabeled ^212Pb-HNPs exhibited a radiochemical purity of up to
   95% and showed prolonged radiolabeling stability under the conditions
   of PBS and FBS at 37℃. The results demonstrated that the residual
   radiolabeling stability could nearly reach 80% after 24 hours, which is
   sufficient for killing tumors (Figure [70]3A). First, the cellular
   uptake efficiency of ^212Pb-HNPs was validated. The results indicated
   that ^212Pb-HNPs exhibited a higher cellular uptake rate compared to
   free ^212Pb (Figure [71]3B). This enhanced uptake is likely due to the
   material-bound radionuclides precipitating around the cells, whereas
   free radionuclides may remain suspended in the cell culture medium. We
   employed the CCK-8 assay to assess the impact of ^212Pb-HNPs on the
   proliferation of LLC cells (Figure [72]3C). Due to the short range of
   alpha-particles, which is only 5-10 cell diameters, cells treated with
   the radionuclide-loaded nanomaterials are more susceptible to
   radiation-induced damage compared to those exposed to free
   radionuclides. As demonstrated in Figures [73]3D and [74]3E,
   ^212Pb-HNPs exhibited the most pronounced GSH depletion ability, while
   other groups also showed a certain degree of GSH depletion. To further
   validate the ROS generation capability of ^212Pb-HNPs, we employed
   DCFH-DA as a probe and conducted flow cytometry analysis. Upon cellular
   internalization, DCFH-DA undergoes hydrolysis catalyzed by esteraze,
   resulting in the formation of DCFH. Subsequently, ROS oxidize the
   initially non-fluorescent DCFH, converting it into fluorescent DCF. The
   results revealed that the ROS levels generated by ^212Pb-HNPs were
   significantly higher compared to HNPs and ^212Pb alone (Figures [75]3F
   and [76]3G), indicating that this novel radiopharmaceutical exhibits
   the strongest ability to induce ROS production. Additionally, to
   investigate the effects of the alpha-particle generator ^212Pb on the
   mitochondria of irradiated cells, JC-1 staining was employed to assess
   the decline in mitochondrial membrane potential. As shown in Figure
   [77]3H and I, the extent of mitochondrial membrane potential decrease
   became more pronounced with increasing radiation doses, indicating more
   severe mitochondrial damage. Furthermore, TEM was used to observe the
   morphology of mitochondria in irradiated tumor cells, compared to the
   control group, the mitochondria in ^212Pb-irradiated tumor cells
   exhibited a morphological shift from their typical ovoid shape to a
   more rounded appearance (Figure [78]3J). These results suggest that the
   alpha-emitter generator ^212Pb induces mitochondrial damage in
   irradiated tumor cells.

Figure 3.

   [79]Figure 3
   [80]Open in a new tab

   Characterization and antitumor properties of ^212Pb-HNPs in vitro. A)
   The stability analysis of ^212Pb-HNPs incubated in PBS and FBS (37℃)
   for 1 h, 6 h, 12 h and 24 h. B) The uptake comparison of^ 212Pb and^
   212Pb-HNPs in LLC cells. C) The cytotoxic effect of ^212Pb and
   ^212Pb-HNPs with gradient concentration on LLC cells for 24 h was
   evaluated by CCK-8 assays (n=4 per group). D and E) GSSG and GSH levels
   in LLC cells after different treatments. F and G) Fluorescence images
   and the corresponding mean fluorescence intensity (MFI) of ROS in LLC
   cells stained with DCFH-DA after various treatments. H and I) Flow
   cytometry patterns of JC-1 decrease in LLC cells after different
   treatments. J) Representative pictures of mitochondrial morphology
   observed under transmission electron microscope (red triangle, left),
   with a high magnification (yellow dashed circle, right).

The potential theranostic efficacy of ^212Pb-HNPs in vivo

   Based on these findings, it is suggested that ^212Pb-HNPs holds
   substantial promise for in vivo tumor therapy. To further investigate
   the theranostics potential of^ 212Pb, subcutaneous LLC xenograft tumor
   models were established in mice (Figure [81]4A). Small animal SPECT/CT
   was employed to validate the imaging capabilities of ^212Pb. Upon
   intratumoral injection of ^212Pb-HNPs, radioactivity uptake at the
   tumor site was observed even at 6 h post-injection, whereas there was
   little radioactivity in free ^212Pb (Figure [82]4B). This result
   suggests that ^212Pb-HNPs exhibit prolonged retention in the tumor
   region. Although we observed accumulation of free ^212Pb in the bladder
   of mice at 30 minutes post-injection, we did not perform
   biodistribution studies by sacrificing the mice at different time
   points following imaging. In this current study, our conclusion is
   limited to demonstrating the imaging potential of ^212Pb. Future
   studies are necessary to investigate the pharmacokinetics of ^212Pb.

Figure 4.

   [83]Figure 4
   [84]Open in a new tab

   The potential theranostic efficacy of ^212Pb-HNPs in vivo. A) Schematic
   representation of the experimental design for the subsequent study. B)
   Representative SPECT/CT imaging of LLC tumor-bearing mice
   post-intratumor injection of ^212Pb or ^212Pb-HNPs at different time
   points. C) Volcano plots showing the DEGs between the ^212Pb-HNPs and
   control groups. D) Heatmap of DEGs between the ^212Pb-HNPs and control
   groups. E) Biological process analysis. F) Cellular component analysis.
   G) Molecular function analysis. H) KEGG pathways enrichment analysis
   (TOP 20).

   Due to the limited yield of ^212Pb, we were unable to meet the demand
   for animal experiments. We could only perform injections in one mouse
   each for the control and experimental groups on the same day, followed
   by three repeated experiments. Based on the limitations of radionuclide
   yield, we divided the subjects into three groups: control group, 74 kBq
   group, and 148 kBq group. Tumor development was monitored according to
   the administration schedule. Regrettably, due to the small therapeutic
   doses administered, although partial tumor regression was observed in
   the experimental groups, the overall therapeutic efficacy was not
   satisfactory. Therefore, we did not present the data. However, RNA
   sequencing was performed on tumor samples from the mice after 7 days
   treatment (Control group: PBS; Treatment group: 148 kBq ^212Pb-HNPs
   i.t.) to analyze the related results. The findings suggested that
   ^212Pb-HNPs could induce oxidative stress in the tumor tissues (Figure
   [85]4C-H). These findings are preliminary but significant as they
   provide initial insights into the molecular mechanisms of oxidative
   stress induction by ^212Pb-HNPs. Combined with results from ROS
   measurement and GSH determination at the cellular level, these findings
   suggest the therapeutic potential of ^212Pb.

Radiation-induced bystander effect triggered by alpha-particle ^212Bi

   To further validate the radiobiological effects of ^212Pb on
   non-targeted tumor cells, the ^212Pb-induced bystander effect was
   investigated. However, due to the 10.6 h half-life of^ 212Pb, it was
   challenging to completely eliminate the radiation impact of residual
   ^212Pb in the supernatant. Although ^212Pb properties are different
   from its daughter nuclide^ 212Bi, under specific conditions, they can
   be used to simulate and validate radiation-induced cellular damage
   effectively. Therefore, we chose to use ^212Bi due to its immediate
   radiation effects which are easier to observe and record at the
   cellular level. This choice allowed us to gain preliminary insights
   into radiation-induced cellular damage mechanisms. Consequently, we
   employed ^212Bi directly for cell-level validation (Figure [86]5A).

Figure 5.

   [87]Figure 5
   [88]Open in a new tab

   Radiation-induced bystander effect triggered by alpha-particle ^212Bi.
   A) Decay scheme of alpha-particle generator ^212Pb. B) Schematic
   representation of the experimental design for the subsequent cytotoxic
   effect study. C, D, and E) The cytotoxic effect of ^212Bi with gradient
   concentration on donor cells for 6 h(C), 12h (D), 24 h (E),
   respectively (n=5). F, G, and H) The cytotoxic effect of supernatant
   from donor cells after 6 h (F), 12 h (G), and 24 h (H)
   ^212Bi-irradiation with gradient concentration on recipient cells for
   24 h (n=5). I) Flow cytometry patterns of recipient cells and donor
   cells stained with annexin Ⅴ-FITC and propidium iodide (PI) for
   determining the apoptosis level. J) The cytotoxic effect of supernatant
   after 24 h ^212Bi-irradiation with gradient concentration on
   rotenone-pretreated recipient cells for 24 h.

   As Figure [89]5B shows, tumor cells were irradiated with ^212Bi (donor
   cells) at a gradient dose, and the cell supernatant was extracted at 6
   h, 12 h, and 24 h post-irradiation. The viability of these donor cells
   was subsequently examined (Figure C-E). This supernatant was then added
   to non-irradiated cells (bystander cells or recipient cells), and the
   activity of the bystander cells was monitored (Figure F-H). The results
   indicated varying degrees of cell death in the bystander cells, which
   exhibited a dose-dependent relationship. It is suggested that the
   presence of some intercellular communication factors in the supernatant
   affect the viability of the bystander cells. Furthermore, apoptosis
   assays were employed to further confirm the presence of alpha-particle
   RIBE (Figure [90]5I). Tumor cells were irradiated using a 6-well plate
   format. Each well received ^212Bi at a radioactivity concentration of 3
   kBq/mL. The chosen radioactivity concentration of 3 kBq/mL was
   determined based on preliminary dose-response studies to ensure an
   optimal balance between inducing measurable biological effects and
   maintaining cell viability. Given that the previous studies
   demonstrated that ^212Pb-HNPs could induce ROS generation in tumor
   cells, we hypothesized that ROS might be a critical influencing factor
   in this context. To investigate this, we pretreated the bystander cells
   with rotenone, an ROS inhibitor, prior to exposing them to the
   supernatant from donor cells. The results showed a significant increase
   in the viability of the bystander cells. This suggests that ROS is one
   of the key factors involved (Figure [91]5J).

Damaged mitochondrial transfer induced by ^212Bi

   To further investigate whether alpha-particles can induce the transfer
   of damaged mitochondria, we employed gradient density centrifugation to
   isolate mitochondria from irradiated cell-conditioned media (ICCM)
   (Figure [92]6A). As shown in Figure [93]6B, TEM confirmed that this
   isolation method successfully extracted damaged mitochondria that had
   transferred into the ICCM. Flow cytometry analysis revealed a gradual
   increase in the number of damaged mitochondria with extended
   irradiation time (Figure [94]6C and [95]Figure S1). To determine
   whether the damaged mitochondria in ICCM could enter non-irradiated
   tumor cells, we labeled the mitochondria of donor cells with
   MitoTracker™ Red FM and co-incubated the isolated mitochondria with
   recipient cells. Confocal microscopy revealed that the mitochondria
   from donor cells were marked in red and exhibited abnormal morphology,
   indicating mitochondrial damage. Interestingly, red-labeled
   mitochondria were also observed in bystander cells, suggesting that the
   mitochondria from irradiated donor cells could transfer to recipient
   cells (Figure [96]6D). These experimental results indicate that
   alpha-particle ^212Bi can drive the transfer of damaged mitochondria.

Figure 6.

   [97]Figure 6
   [98]Open in a new tab

   Damaged mitochondrial transfer induced by ^212Bi. A) Outline the
   protocol for an isolation of mitochondria from irradiated cells. B)
   Transmission electron microscope demonstrating the free mitochondria
   (red triangle, left) in culture medium of LLC cells after different
   treatments, with a high magnification (yellow dashed circle, right). C)
   The number of free mitochondria in culture medium following ^212Bi
   irradiation at various time intervals. D) Visualization of Mito-Tracker
   (red) in donor cells and recipient cells, indicating the free
   mitochondria in culture medium of donor can be transferred to
   recipient.

The transfer of damaged mitochondria results in alpha-particle
radiation-induced bystander effect

   To further investigate the role of transferred damaged mitochondria in
   alpha-particle radiation-induced bystander effects, recipient cells
   were treated with the ICCM from ^212Bi-irradiated cells, the
   mitochondria transferred into the ICCM, and other remaining substances,
   respectively, and observed the viability of the recipient cells (Figure
   [99]7A). CCK8 assay results indicated that the transferred damaged
   mitochondria significantly inhibited the viability of recipient cells,
   exhibiting a similar effect to that of ICCM (Figure [100]7B-D).
   Apoptosis assays and live/dead cell staining further confirmed the
   anti-tumor effects of transferred damaged mitochondria (Figure
   [101]7E-G). Additionally, confocal microscopy analysis of JC-1-stained
   recipient cell mitochondria revealed an increase in JC-1 monomers
   following treatment with damaged mitochondria, indicating a decrease in
   mitochondrial membrane potential and suggesting mitochondrial damage
   within the recipient cells (Figure [102]7H).

Figure 7.

   [103]Figure 7
   [104]Open in a new tab

   The transfer of damaged mitochondria results in alpha-particle
   radiation-induced bystander effect. A) Schematic representation of the
   experimental design for the subsequent study. B, C, and D) The
   cytotoxic effect of various treatments with gradient concentration on
   recipient cells for 24 h was evaluated by CCK-8 assays (n=5 per group).
   E and F) Flow cytometry patterns of recipient cells stained with
   annexin Ⅴ-FITC and PI after various treatments for determining the
   apoptosis level. G) Cytotoxicity of various treatments on recipient
   cells by using co-staining with PI and Calcein-AM. H) Visualization of
   JC-1 monomer (Green) and JC-1 aggregate (Red) in recipient cells after
   various treatments.

Discussion

   Due to the fact that alpha-particles exhibit unique clinical advantages
   such as "high energy, short range, hypoxia resistance, and ease of
   shielding." These properties highlight their tremendous potential in
   the precise treatment of tumors in nuclear medicine, suggesting they
   may drive significant shifts in therapeutic paradigms and concepts
   within the field. However, the therapeutic mechanisms of
   alpha-particles are more complex compared to beta-particles, and
   research on alpha-particles is still in its infancy globally [105]^26.
   Numerous fundamental theoretical "uncharted territories" exist, posing
   key bottlenecks that hinder their clinical translation and application.

   ^212pb is the current cutting-edge hotspot in the field of targeted
   nuclide therapy, and its decay releases the alpha-particle ^212Bi with
   an energy of 6.05 MeV (half-life 60.6 min), which is often used as an
   in vivo alpha-particle generator. Compared with other long half-life
   alpha-particles (e.g. ^225Ac,^ 223Ra), ^212Pb (half-life 10.6 hours)
   has a simpler and more efficient decay chain, which can rapidly decay
   and release alpha-particles, and the energy generated from the decay of
   this more 'high-dose-rate' alpha-particles can be rapidly deposited
   into the tumor, with off-target toxicity and daughter nuclide recoil.
   The off-target toxicity caused by daughter nucleus recoil and the toxic
   side effects caused by daughter nuclide redistribution are much
   smaller. In addition, the ^212Pb decay process can release 238.6 keV
   γ-rays, which can be detected by SPECT/CT, and is expected to realize
   in vivo dynamic imaging, which is very promising for theranostics
   [106]^27^-[107]^30. In previous studies, ^203Pb and ^212 Pb are usually
   paired for the purpose of theranostics. Among them, ^203Pb are usually
   administered at 18.5 MBq or 7.4 MBq per mouse, and there are also
   studies that used ^212 Pb (7.4 MBq or 1.44 MBq) for in vivo imaging
   studies and 185 kBq or 100 kBq for postmortem biodistribution studies.
   All of these studies were administered at higher doses than we used.
   Due to the limitation of the technology, the maximum yield of the
   ^212Pb that we can obtain at each time is only about 370 kBq at most.
   Besides, As the drug continues to decay, the dosage of each mouse (for
   free ^212Pb or ^212Pb-HNPs imaging) can only reach 74 kBq. Although the
   dose is very low, this study still demonstrates the great imaging
   potential of ^212Pb after intratumor injection. In the future, we will
   aim to expand the yield of ^212Pb and further improve the experiments
   on the theranostics of ^212Pb. Besides, it is necessary to investigate
   the pharmacokinetics of ^212Pb in vivo.

   Currently, the therapeutic mechanisms of alpha-particles may be
   categorized into three main aspects. a) Nuclear DNA Damage Effect:
   Alpha-particles possess high linear energy transfer, which can induce
   irreparable double-strand breaks in nuclear DNA, thereby directly
   killing tumor cells. b) Immune Synergistic Effect: Alpha-particles
   could drive immunogenic cell death, with subsequently activating
   systemic anti-tumor immune responses, which can synergize with
   immunotherapies to enhance therapeutic efficacy. c) Bystander Effect:
   When only a small number of tumor cells are irradiated and damaged by
   alpha-particles, neighboring non-irradiated tumor cells can also
   exhibit damage effects [108]^31. However, among various types of
   ionizing radiation, alpha-particles exhibit a unique pattern of
   heterogeneous ionization. The significant energy difference between
   regions of high ionization density and surrounding non-irradiated areas
   results in stronger biological signals that are more readily
   transmitted to non-irradiated cells [109]^32^-[110]^35. Consequently,
   the bystander effect induced by alpha-particles warrants particular
   attention in research and clinical applications.

   In this study, we found that after treatment of tumor cells with the
   alpha-particle ^212Bi, damaged mitochondria could be transferred to the
   extracellular environment. Upon further extraction and co-incubation of
   these damaged mitochondria with non-irradiated tumor cells, it was
   observed that the non-irradiated tumor cells could uptake the damaged
   mitochondria, resulting in reduced viability of these non-irradiated
   cells. This confirms that alpha-particles can drive the transfer of
   damaged mitochondria, which in turn can induce damage in non-irradiated
   tumor cells. Therefore, alpha-particles may mediate the bystander
   effect through the transfer of damaged mitochondria. Existing research
   has provided evidence that the transfer of damaged mitochondria can
   occur through three main pathways: tunneling nanotubes, extracellular
   vesicles, and free mitochondria [111]^36. Once transferred to recipient
   cells via these mechanisms, the damaged mitochondria can significantly
   impact the function and fate of the recipient cells. Potential
   mechanisms by which transferred damaged mitochondria exert their
   effects include alterations in mitochondrial dynamics, increased
   genomic instability, and elevated oxidative stress [112]^37^-[113]^39.
   However, our study did not identify specific mechanisms related to
   alpha-particle. In future research, we intend to investigate these
   potential mechanisms more thoroughly. These mechanisms collectively
   contribute to the therapeutic potential of alpha-particles in oncology.

Conclusion

   This study validates the theranostics potential of the in vivo
   alpha-particle generator ²¹²Pb, with a particular focus on its ability
   to induce robust bystander effect. By elucidating the functional role
   of damaged mitochondrial transfer in the alpha-particle
   radiation-induced bystander effect, this research contributes to
   advancing the scientific understanding and clinical applications of
   targeted alpha therapy.

Supplementary Material

   Supplementary figure.
   [114]thnov14p6768s1.pdf^ (637.5KB, pdf)

Acknowledgments