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Investigation of Newly Prepared Biodegradable 32P-chromic Phosphate-polylactide-co-glycolide Seeds and Their Therapeutic Response Evaluation for Glioma Brachytherapy

32P high-dose rate brachytherapy allows high-dose radiation delivery to target lesions with less damage to adjacent tissues. The early evaluation of its therapeutic effect on tumours is vital for the optimization of treatment regimes. The most commonly used 32P-CP colloid tends to leak with blind therapeutic area after intratumour injection. We prepared 32P-chromic phosphate-polylactide-co-glycolide (32P-CP-PLGA) seeds with biodegradable PLGA as a framework and investigated their characteristics in vitro and in vivo. We also evaluated the therapeutic effect of 32P-CP-PLGA brachytherapy for glioma with the integrin αvβ3-targeted radiotracer 68Ga-3PRGD2. 32P-CP-PLGA seeds (seed group, SG, 185 MBq) and 32P-CP colloid (colloid group, CG, 18.5 MBq) were implanted or injected into human glioma xenografts in nude mice. Scanning electron microscopy (SEM) of the seeds, micro-SPECT imaging, and biodistribution studies were performed at different time points. The tumour volume was measured using a caliper, and 68Ga-3PRGD2 micro-PET-CT imaging was performed to evaluate the therapeutic effect after 32P intratumour administration. The delayed release of 32P-CP was observed with biodegradation of vehicle PLGA. Intratumoural effective half-life of 32P-CP in the SG (13.3 ± 0.3) d was longer than that in the CG (10.4 ± 0.3) d (P < 0.05), with liver appearance in the CG on SPECT. A radioactivity gradient developed inside the tumour in the SG, as confirmed by micro-SPECT and SEM. Tumour uptake of 68Ga-3PRGD2 displayed a significant increase on day 0.5 in the SG and decreased earlier (on day 2) than the volume reduction (on day 8). Thus, 32P-CP-PLGA seeds, controlling the release of entrapped 32P-CP particles, are promising for glioma brachytherapy, and 68Ga-3PRGD2 imaging shows potential for early response evaluation of 32P-CP-PLGA seeds brachytherapy.


Introduction
Glioblastoma multiforme (GBM) accounts for approximately 60% to 70% of malignant gliomas. The prognosis of patients with GBM remains dismal, with a median survival time of less than one year. This poor prognosis primarily reflects the high proliferative, infiltrative, and invasive properties of GBM [1]. Systemic administration of most potentially active drugs is not effective because of failing to cross the blood-brain barrier. Residual cells at the margins of the resection or GBM stem cells, a subpopulation of stem-like cells, frequently lead to tumour recurrence or metastasis.
Compared with external-beam radiation, brachytherapy has the advantage of delivering high doses of radiation to target sites without subjecting normal tissues to undue radiation and damage [2]. 32 P is ideal because of its relatively long halflife of 14.28 day(s), maximum energy of 1.711 MeV, mean range of 3-4 mm in soft tissues, and high relative biological effect.

Contrast Media & Molecular Imaging
Recent studies have focused on better vehicles or carriers, and many of these studies have used 32 P bandages or films for the brachytherapy of superficial cancers and intracranial and spinal tumours [3][4][5][6][7][8][9]. However, the main hurdles to these vehicles were poor target ratios in the tumour, complex preparation, and serious side effects resulting from migration and permanent stagnation, such as lung migration and gastric fistula in vivo [7][8][9].
Polymers, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymer poly(lactide-co-glycolide) (PLGA), have extensively been used in drug delivery systems because of their biodegradability, tissue compatibility, and versatile degradation kinetics [10][11][12][13]. Compared with poly(L-lactide) (PLLA), PLGA has attracted more attention, reflecting its degradation rate and relative ease in manipulating its release of encapsulating drugs by changing the degradation-determining factors, such as its molecular weight, lactide/glycolide ratio, and degree of crystallinity [14,15]. In a previous study, we completed the formulation and determined the fabricating parameters of 32 P-CP-PLGA seeds (unpublished observations). As one potential biodegradable seed for brachytherapy, 32 P-CP-PLGA is meaningful for further preclinical studies, and the therapeutic effect evaluation of 32 P brachytherapy has rarely been reported.
Integrin v 3, specifically targeted by the arginineglycine-aspartic acid (RGD) tripeptide sequence, is highly expressed in both glioma cells and neovasculature but not in quiescent blood vessels. Integrin v 3 targeted imaging had higher sensitivity in GBM than 18 F-FDG, reflecting a clear lack of RGD affinity, particularly in brain areas where gliomas are prone to occur [16]. In an orthotopic U87MG human glioma mouse model, the tumour-to-brain ratio of radiolabelled RGD is approximately 20 times that of 18 F-FDG [17]. 68 Ga, easily accessible via elution from the 68 Ge-68 Ga generator, can produce Cerenkov radiation and allow the combination of PET-Cerenkov luminescence imaging (CLI) for both tumour diagnosis and visual guidance for further surgery [17]. 68 Ga-labelled DOTA-3PRGD 2 ( 68 Ga-3PRGD 2 ) was reported to reflect the tumour response to antiangiogenic therapy much earlier and more accurately than 18 F-FDG PET imaging [18]. Integrin-targeted imaging detects early responses preceding clinical regression as early as day 3 after the initiation of abraxane treatment, while 18 F-FDG imaging demonstrated increased tumour uptake on days 3 and 7 [19]. For 32 P brachytherapy, early response is also important for treatment direction; however, few studies have been reported.
In our previous study, we found that radiolabelling RGD is excellent for monitoring v 3 expression and glioma necrosis during tumour growth [20]. The present study reports (a) the preparation, biodegradation, and ultrastructural changes of 32 P-CP-PLGA seeds and the biodistribution of 32 P-CP particles both systemically and inside the tumour and (b) the anticancer effect on glioma in an animal model after intratumour seeds implantation and therapeutic effect evaluation with integrin v 3 receptor targeting radiotracer 68 Ga-3PRGD 2 .

Preparation of 32 P-CP-PLGA Seeds and In Vitro Study.
A total of 100 mg of PLGA (0.1-1.0 m) and 2 ml of colloidal 32 P-CP (1850 MBq/ml) were mixed with 2-3 ml of dehydrated alcohol as a dispersing agent. These components were blended and dried at 60 ∘ C in a vacuum drying oven for 9-12 h. Magnesium stearate (1.0-1.5 mg) served as a surface lubricant, and the 32 P-CP-PLGA seeds were homeprepared using an automatic precise piercer. The physical characteristics of the 32 P-CP-PLGA seeds were investigated. In vitro releasing experiments were performed by incubating the 32 P-CP-PLGA seeds in 6 ml of PBS at 37 ∘ C for 90 day(s), and 0.2 ml of the sample was withdrawn every other day from the releasing solution after stirring to generate a homogenous mixture.

Radiopharmaceutical Administration and Biodistribution
Study. Nude mice bearing U87MG human gliomas (tumour long diameter of 0.6-0.8 cm) were randomized into the 32 P-CP-PLGA seed group (SG, = 36) and the 32 P-CP colloid group (CG, = 36). One 32 P-CP-PLGA seed (18.5 MBq) or 0.02 ml of colloidal 32 P-CP (18.5 MBq) was implanted or slowly injected into the tumour core. The injection sites were gently compressed for 60 s. The mice were sacrificed at 0. 5 were further divided into 3 parts according to their distances from the core of the tumour. The 3 parts of the tumour and adjacent tissues were weighed and counted for radioactivity. 32 P distribution was expressed as the percent injected dose per gram (%ID/g). The remaining seeds and tumour tissues were prepared for SEM examination. For the mice sacrificed on day 64, micro-SPECT imaging was selectively performed, and faeces and urine were collected.

Micro-SPECT and
Micro-SPECT/CT Imaging. Anesthesia of mice (SG and CG) was induced using isoflurane. Micro-SPECT images were obtained on days 1, 4, 16, and 64 using a micro-SPECT scanner equipped with a 0.6 mm multipinhole collimator. The specified 32 P-CP-PLGA seeds and 32 P-CP colloids, same as those used in the SG and CG, were served as markers and imaged, respectively. Regions of interest (ROIs) of the targeted tumour (T) and the marker (T mk ) were drawn to estimate the T/T mk ratio and intratumoural effective half-life of 32 P-CP. After SPECT acquisition (75 projections over 30 min per frame, 2 frames), SPECT reconstruction was performed using a POSEM (pixelated ordered subsets by expectation maximization) algorithm with 6 iterations and 16 subsets. Micro-CT imaging was added on day 1 using the "normal" acquisition settings at 45 kV and 500 A. CT data were reconstructed using a cone-beam filtered back-projection algorithm (NRecon v1.6.3, Skyscan). After reconstruction, the SPECT and CT data were automatically coregistered according to the movement of the robotic stage and then resampled to equivalent voxel sizes. Coregistered images were further rendered and visualized using PMOD software (PMOD Technologies, Zurich, Switzerland).
2.6. 68 Ga-3PRGD 2 Micro-PET-CT Imaging. We prepared 68 Ga-3PRGD 2 as previously described [18]. In brief, 68 GaCl 3 was eluted from 68 Ge/ 68 Ga-generator with 0.05 M HCl. The conjugation of 3PRGD 2 with DOTA-OSu was performed for the synthesis of DOTA-3PRGD 2 . The DOTA-3PRGD 2 and 68 GaCl 3 were mixed and heated in a water bath at 90 ∘ C for 15 min. The radiochemical purity was then determined with radio-HPLC. 0.1 mL of 68 Ga-3PRGD 2 (74 MBq/mL) was injected via tail vein under isoflurane anesthesia. Ten-minute static PET scans were acquired and images were reconstructed by an OSEM 3D (Three-Dimensional Ordered Subsets Expectation Maximum) algorithm followed by MAP (Maximization/Maximum A Posteriori). The 3D ROIs were drawn over the tumour guided by CT images and tracer uptake was measured using the software of Inveon Research Workplace (IRW) 3.0. Mean standardized uptake values (SUV) were determined by dividing the relevant ROI concentration by the ratio of the injected activity to the body weight.

Seeds Ultrastructure Changes and Glioma Integrin v 3
Expression. SEM examination was performed to investigate the ultrastructure changes of 32 P-CP-PLGA seeds. Frozen glioma tissues were cut into 5 m slices and blocked with 10% goat serum for 30 min and then incubated with the hamster anti-integrin 3 antibody and rat anti-CD31 antibody for 1 h at room temperature. After incubating with the Cy3-conjugated goat anti-hamster and fluorescein-(FITC-) conjugated goat anti-rat secondary antibodies and washing with PBS, the fluorescence was visualized using a fluorescence microscope.

Statistical
Analysis. Data are shown as mean ± SD. Differences in tissues uptake between SG and CG were statistically analyzed at each time point with SPSS 11.5 software. Student's t-test was used for statistical analysis. values less than 0.05 were considered statistically significant.

Basic Characteristics and Biodegradation of 32 P-CP-PLGA
Seeds. 32 P-CP-PLGA seeds are cylindrical with a diameter of (0.9 ± 0.02) mm, length of (1.9 ± 0.2) mm, weight of (1.2 ± 0.15) mg each, hardness of (5.7 ± 1.4) N, and apparent radioactivity of (18.5±3.7) MBq. The release of 32 P-CP-PLGA seeds can be divided into the rapid release phase (0∼8 d, phase I), the stable release phase (8∼60 d, phase II), and the disintegration release phase (60 d ∼, phase III) according to time-release curve slope (Figure 1(a)). Figure 1(b) displays the SEM results of 32 P-CP-PLGA seeds before and 8, 16, 32, and 64 days after intratumour implantation. The seed was uniform and dense in texture, showing a mosaic of 32 P-CP particles prior to implantation. An increasing number of micropores, tunnels, cellular structures, cavities, and fragments were observed with increasing seed brittleness and inability to maintain shape during release. Figure 1(c) displays the ultrastructure of 32 P-CP particles before the preparation of 32 P-CP-PLGA seeds and those released from the 32 P-CP-PLGA seeds.  Figure 3 demonstrated the distribution of the released 32 P-CP particles in tumour and adjacent normal tissues in the CG or SG after eradication and radioactivity counting of the remaining 32 P-CP-PLGA seeds. Most of released 32 P-CP particles in the SG were stagnant in the tumour core with a decreasing gradient towards the periphery in the SG (Figure 3(b)). This finding was confirmed by using SEM (Figure 3(c)). The gradient existed for approximately 2 weeks and became insignificant on day 14. The %ID/g tumour uptake of 32 P-CP significantly increased on day 16, primarily reflecting tumour shrinkage. Conversely, the intratumour distribution of 32 P-CP in the CG was irregular and a slight increase of %ID/g tumour uptake was observed with tumour shrinkage. The regional peak uptake in tumour tissues (t2) on day 16 was higher than day 8 (t1) (Figure 3(a)).

Radioactivity Gradient Developed in Tumour and Surrounding Tissues.
Micro-SPECT/CT Imaging. 32 P bremsstrahlung facilitates SPECT-CT imaging to monitor the intratumour administration of 32 P-CP colloid (Figure 4(a)) and 32 P-CP-PLGA seeds (Figure 4(b)). As shown in Figure 4(b), 32 P-CP-PLGA seeds showed dot-like focal radioactivity accumulation on days 1 and 4. The released 32 P-CP showed a vague appearance of tumour on days 16 and 64 in the SG. 32 P-CP colloids displayed irregular shapes in and/or close to the tumour location, with abnormal distribution in the liver in the CG on day 16, and this effect was even more apparent on day 64. The T/T mk decreased to 89.3% ± 2.4% (SG) and 65.8% ± 3.1% (CG) on day 64 based on micro-SPECT/CT images.

68 Ga-3PRGD 2 Micro-PET/CT Imaging and Tumour
Growth Monitoring. As shown in Figure 5, the tumour uptake heterogeneity of 68 Ga-3PRGD 2 was observed on days 2 and 4 with internal absence on day 8. Scar formation was observed on day 16 in the SG. In the P-CP-PLGA seed group, the distribution of 68 Ga-3PRGD 2 was uniform for 8 day(s), and Contrast Media & Molecular Imaging 5 Table 1: Biodistribution of 32 P-CP particle in nude mice bearing U87MG glioma after intratumor implantation of 32 P-CP-PLGA seed ( = 5).

Tissue
Radioactivity distribution (%ID/g) 0. 5     internal heterogeneity was observed on day 8, reflecting natural necrosis. The tumour uptake (%ID/cm 3 ) of 68 Ga-3PRGD 2 in the SG (8.50 ± 1.32) was higher than that in the P-CP-PLGA group (7.15 ± 1.49, < 0.05) on day 0.5, while the tumour uptake in the SG (5.13 ± 1.06) was lower than that in the P-CP-PLGA group (8.16±1.26, < 0.01) on day 2. The tumour time-growth curve displayed no significant changes during 10 days in the SG or CG after 32 P administration, and 40% (2/5) of the mice demonstrated tumour growth near the scar on day 20.

Immunohistochemistry Examination.
Immunofluorescence staining was performed to examine the v 3 and CD31 expression levels in tumour tissues from the xenografted gliomas in the SG and P-CP-PLGA group on days 0.5, 2, and 4; as shown in Figure 6, compared with the P-CP-PLGA seed group, the quantitation of the glioma anti-v 3 immunofluorescence levels in the SG revealed a 1.37-fold (±0.15) mean increase (±SD) on day 0.5 and 0.74-fold (±0.22) and 0.15-fold (±0.05) decreases on days 2 and 4, respectively. IHC examination was not performed on days 8 and 16 due to increased areas of necrosis and scar formation.

Discussion
32 P-CP-PLGA seed is a biodegradable dosage form of 32 P, utilizing PLGA as the controlled release carrier for drug delivery. In addition to its easy preparation in large amounts, the main advantage of 32 P-CP-PLGA seeds is their delivery of high-dose radiation to the tumour and long intratumoural radioactive effective half-life with minimal distribution and damage to systemic tissues. A radioactivity gradient formed inside the tumour with potential effect to enhance the intratumour fire effect, reduce damage to adjacent tissues, and avoid the implantation of low radioactivity seeds in the peripheral part of the tumours. No significant difference was observed in tumour uptake of 32 P-CP particles in the SG and CG immediately after The regional peak uptake in tumour tissues (t2) on day 16 was higher than day 8 (t1) (# means < 0.05) [t1, t2, and t3 indicate tumour tissues at different distance from the seed (SG) or the core of the tumour (CG), near to far, and "s" indicates tissues surrounding the tumour]. (b) 32 P-CP distribution in tumour and adjacent normal tissues in the SG (significant difference between ti and t2, t2 and t3, and t3 and s, < 0.05) (c). SEM results of tumour and adjacent normal tissues (t1, t2, t3, and s). The 32 P-CP particles are indicated with a black arrow. Irregularity of radioactivity distribution can be observed in the tumour and adjacent tissues in the CG, while a radioactivity gradient formed in the SG. The results were confirmed by SEM.
administration, except for the occurrence of leakage of colloidal 32 P-CP in the CG. However, the dynamic radioactivity distribution indicated more retention and longer effective half-life of 32 P-CP in the tumour and less or even undetectable distribution in other tissues in the SG compared with that in the CG. The principal mechanism likely involved the arrest of 32 P-CP particles inside the seeds using the framework of PLGA, and their release and slow drainage was determined by the biodegradation of PLGA. A radioactivity concentration gradient, formed by the 32 P-CP particles released from the 32 P-CP-PLGA seeds, was observed inside the tumour. The tumour uptake (%ID/g) was extremely high, primarily because more than 95% (%ID) of the implanted 32 P remained in the small tumour tissues (approximately 0.2 cm 3 ) during the first few days. The increased uptake primarily reflected tumour shrinkage on day 16 and scar formation. The released 32 P-CP particles increase the killing range and enhance the therapeutic effect without noticeable damage to adjacent tissues. The gradient gap was predominant in the first few days and narrowed with increasing release of 32 P-CP particles and tumour shrinkage. However, the irregular and uneven intratumour distribution of 32 P-CP colloids in the CG led to a therapeutic blind area, tumour relapse, and leakage from the injection site, resulting in damage to the adjacent tissues. The 32 P-CP particles drained from the tumour were primarily distributed throughout reticuloendothelial systems, such as the liver in the CG, and were less than reported [9]. This effect potentially reflects the use of gelatine sponge to prevent or reduce the leakage of 32 P-CP colloids. The uptake of 32 P-CP particles in normal tissues peaked at 16 days and likely involved the following three mechanisms. First, 32 P-CP particles at the surface or inlaying the outer 1/3 of the seeds tended to be released, while those inlaid near the core stagnated inside, although there was gradual formation of tunnels and cavities. Second, the decrease or lack of regional circulation around the seeds resulting from tumour tissue necrosis and scar formation may inhibit the drainage of released 32 P-CP particles. Third, the experimental period used in the present study may not be long enough.
Pathological changes of important organs were absent in the SG. 32 P bremsstrahlung SPECT imaging facilitates the positioning, effective for half-life evaluation under physical conditions [21]. Any immigration or abnormal uptake can easily be detected, and SPECT-CT fusion techniques with a certain postprocessing attenuation correction algorithm may contribute to precise localization, dosiology studies, and future establishment of brachytherapy in clinical settings [6]. 18 F-FDG PET-CT imaging was widely used to monitor therapeutic effect of tumour treatment. However, two potential disadvantages limit its use in gliomas. One disadvantage is the high distribution of glioma in normal brain tissues, particularly in the cerebral cortex, basal ganglia, and thalamus, where glioma is prone to occur. The other disadvantage is the increased accumulation of tumour tissue when inflammatory cells, such as macrophages, are present, particularly at the early stages of treatment [19]. 18 F-FPPRGD 2 is superior to 18 F-FDG because the uptake of 18 F-FPPRGD 2 is not influenced by tumour-associated macrophages after chemotherapy, and the remarkable distribution of 68 Ga-3PRGD 2 in the U87MG xenografts is observed with low background in the normal brain tissues compared with 18 F-FDG [17,19]. 68 Ga is easily accessible from a 68 Ge-68 Ga generator and showed similar labelling characteristics with therapeutic isotopes, such as 177 Lu, with DOTA and NOTA as linkers. In the present study, the initial increased uptake of 68 Ga-3PRGD 2 in glioma was observed on day 0.5 after 32 P-CP-PLGA seed implantation. The possible mechanism involves the rapid upregulation of integrin expression induced by a low-tointermediate dose of radiation, as previously reported [22]. Thus, the increased glioma uptake of 68 Ga-3PRGD 2 on day 0.5 in the SG is possibly due to radiation, while the decrease on day 2 is due to cell damage resulting from accumulated 32 P radiation and therapeutic effects [23]. This effect occurred much earlier than the tumour volume reduction and was confirmed using IHC. 68 Ga-3PRGD 2 imaging has potential to serve as an important tool for the therapeutic evaluation of 32 P brachytherapy of glioma as early as day 2. Thus, the precise relationship between the − radiation dose and integrin expression upregulation needs further study.

Conclusions
32 P-CP-PLGA seeds, controlling the release of entrapped 32 P-CP particles, are promising for glioma brachytherapy. 68 Ga-3PRGD 2 imaging shows potential for early response evaluation of 32 P-CP-PLGA seeds brachytherapy.  Figure 5: 68 Ga-3PRGD 2 micro-PET-CT imaging during 32 P brachytherapy and tumour volume measurement using a caliper. (a) The 68 Ga-3PRGD 2 micro-PET/CT transverse images of nude mice bearing glioma on days 0.5, 2, 4, 8, and 16 after intratumoural implantation of radioactive 32 P-CP-PLGA seeds (A) and nonradioactive P-CP-PLGA seeds (B). (b) Tumour volume was measured using a caliper every other day after intratumoural administration of 32 P-CP-PLGA seeds, 32 P-CP colloid, and nonradioactive P-CP-PLGA seed. (c) Tumour uptake of 68 Ga-3PRGD 2 was quantified based on micro-PET/CT imaging and compared between radioactive and nonradioactive seeds group. Tumour uptake (%ID/g) of 68 Ga-3PRGD 2 was significantly different at the same points. In 32 P-CP-PLGA seeds group, distribution heterogeneity of 68 Ga-3PRGD 2 was observed on day 2 and was even significant and toroidal on day 4, with slight accumulation of radiotracer on day 8 and being almost negative on day 16. The distribution of 68 Ga-3PRGD 2 was uniform at 8 days after nonradioactive seed implantation. Internal absence was observed on day 16, reflecting ulcer formation. # means tumour uptake of 68 Ga-3PRGD 2 in P-CP-PLGA seeds group was significantly lower than 32 P-CP-PLGA seeds group ( < 0.05) while * means tumour uptake of 68 Ga-3PRGD 2 in P-CP-PLGA seeds group was significantly higher than 32 P-CP-PLGA seeds group ( < 0.05). The white arrows refer to tumors.

Disclosure
Guoqiang Shao, Yuebing Wang, and Xianzhong Liu are cofirst authors. An earlier version of this work was presented as an abstract at MTAI: Educational Exhibits Posters 2017.

CD31
Overlay 3 (f) Figure 6: Representative immunohistochemical images for frozen glioma slices from xenografted glioma tumours at different time points (day 0.5: (a, b); day 2: (c, d); day 4: (e, f)) after intratumoural implantation of radioactive 32 P-CP-PLGA seeds (a, c, e) and nonradioactive P-CP-PLGA seeds (b, d, f). The CD31 was used to label the tumour endothelial cells on blood vessels. The integrin 3 was visualized with Cy3 (red), and CD31 was visualized with fluorescein isothiocyanate (green) under an Olympus fluorescence system, scale bar: 50 m.