Translating Research for the Radiotheranostics of Nanotargeted 188Re-Liposome

Nanoliposomes are one of the leading potential nano drug delivery systems capable of targeting chemotherapeutics to tumor sites because of their passive nano-targeting capability through the enhanced permeability and retention (EPR) effect for cancer patients. Recent advances in nano-delivery systems have inspired the development of a wide range of nanotargeted materials and strategies for applications in preclinical and clinical usage in the cancer field. Nanotargeted 188Re-liposome is a unique internal passive radiotheranostic agent for nuclear imaging and radiotherapeutic applications in various types of cancer. This article reviews and summarizes our multi-institute, multidiscipline, and multi-functional studied results and achievements in the research and development of nanotargeted 188Re-liposome from preclinical cells and animal models to translational clinical investigations, including radionuclide nanoliposome formulation, targeted nuclear imaging, biodistribution, pharmacokinetics, radiation dosimetry, radiation tumor killing effects in animal models, nanotargeted radionuclide and radio/chemo-combination therapeutic effects, and acute toxicity in various tumor animal models. The systemic preclinical and clinical studied results suggest 188Re-liposome is feasible and promising for in vivo passive nanotargeted radionuclide theranostics in future cancer care applications.


Introduction
Over the past few decades, theranostics has emerged as a field in which diagnosis and targeted therapy are combined to achieve a personalized treatment approach to the patient [1]. The progress in molecular biology and the understanding of malignant transformation and tumorigenesis have revealed recognition of the widespread applicability of the hallmarks of cancer concepts. These novel concepts increasingly affect the development of new means to treat human cancer in personalized and translational medicine. Conventionally, anticancer drugs are used to systemically destroy any residual or metastatic tumor cells, but they cannot discriminate between neoplastic and non-neoplastic cells [2]. Besides, poor solubility and distribution, unfavorable pharmacokinetics, and high-tissue damage or toxicity are also noted in various therapeutic agents. Therefore, an effective therapeutic strategy targeting specific mechanisms involved in tumorigenesis is needed [3]. The biggest challenges in cancer diagnostics and therapeutic radionuclide agents in clinical applications include low drug bioavailability within cancer cells and the high toxicities to normal organs [4]. An ideal delivery system can carry radionuclides into the cancer targeting area and releases the desired cytotoxic concentration at the right time. The importance of nanotechnology to radionuclide delivery systems in molecular imaging and targeted radiotherapy has been recognized this decade [5][6][7]. Several innovative radionuclides and drug delivery systems focused on cancer nanotechnology have been developed in recent significantly reactivated the p53 pathway, suppressing the epithelial-to-mesenchymal transition (EMT) process [23,24] and the reversal of glycolysis in an ovarian tumor model [23]. 188 Re-liposome also effectively suppressed the expression of stemness-relevant markers of cancer-stem cells and may be a novel treatment for ovarian cancer when delivered intraperitoneally [23]. In addition, 188 Re-liposome could induce the autophagy/mitophagy of cancer stem cells, leading to tumor regression in the ovarian tumor animal model [25].
MicroRNA, an abundant family of short (19-25 nucleotides) noncoding RNAs, can modulate gene expression upon binding to target mRNAs. The aberrant expression of miRNAs can regulate diverse cellular processes, including the apoptosis of tumor cells [26]. Microarray array analysis revealed the tumor suppressor microRNA let-7 could be induced by 188 Re-liposome to regulate downstream genes in human head and neck squamous cell carcinoma (HNSCC) [27]. Most 188 Re-liposome that up-regulate microRNAs, including miR-206-3p, were categorized as tumor suppressors, while down-regulate microRNAs, including miR-142-5P, were oncogenic [28]. Studies on the regulation of microRNAs correlate to the therapeutic efficacy of 188 Re-liposome in HNSCC tumor [27,28]. The evidence indicated 188 Re-liposome enhances various anti-tumor mechanisms in tumor therapy. Figure 1 overviews the possible mechanisms in the anticancer effects of 188 Re-liposome. the molecular mechanism is still an unmet need. We demonstrated the colon tumors from treated mice had a 26-fold increase in the numbers of apoptotic cells compared with those from the normal saline control mice at 8 h after treatment with 188 Re-liposome [22]. 188 Re-liposome significantly reactivated the p53 pathway, suppressing the epithelial-to-mesenchymal transition (EMT) process [23,24] and the reversal of glycolysis in an ovarian tumor model [23]. 188 Re-liposome also effectively suppressed the expression of stemness-relevant markers of cancer-stem cells and may be a novel treatment for ovarian cancer when delivered intraperitoneally [23]. In addition, 188 Re-liposome could induce the autophagy/mitophagy of cancer stem cells, leading to tumor regression in the ovarian tumor animal model [25]. MicroRNA, an abundant family of short (19-25 nucleotides) noncoding RNAs, can modulate gene expression upon binding to target mRNAs. The aberrant expression of miRNAs can regulate diverse cellular processes, including the apoptosis of tumor cells [26]. Microarray array analysis revealed the tumor suppressor microRNA let-7 could be induced by 188 Re-liposome to regulate downstream genes in human head and neck squamous cell carcinoma (HNSCC) [27]. Most 188 Re-liposome that up-regulate mi-croRNAs, including miR-206-3p, were categorized as tumor suppressors, while down-regulate microRNAs, including miR-142-5P, were oncogenic [28]. Studies on the regulation of microRNAs correlate to the therapeutic efficacy of 188 Re-liposome in HNSCC tumor [27,28]. The evidence indicated 188 Re-liposome enhances various anti-tumor mechanisms in tumor therapy. Figure 1 overviews the possible mechanisms in the anticancer effects of 188 Re-liposome. Our preclinical cell and animal model investigations demonstrated the efficacy and safety of nanotargeted radiotheranostic 188 Re-liposome or radio/chemotheranostic 188 Redoxorubicin (DXR)-liposome, which includes tumor site targeting and nuclear imaging, pharmacokinetics, biodistribution, dosimetry, the radiation killing effect, and synergistic radio/chemo-combination effects. We also completed a phase 0 clinical trial for 12 pa- Our preclinical cell and animal model investigations demonstrated the efficacy and safety of nanotargeted radiotheranostic 188 Re-liposome or radio/chemotheranostic 188 Redoxorubicin (DXR)-liposome, which includes tumor site targeting and nuclear imaging, pharmacokinetics, biodistribution, dosimetry, the radiation killing effect, and synergistic radio/chemo-combination effects. We also completed a phase 0 clinical trial for 12 patients, which showed the potential of 188 Re-liposome as a new nanotargeted anti-cancer radiotherapeutic [29]. The current review and summaries focus on the systemic overview and comparisons of our preclinical nanotargeted 188 Re-liposome pharmacology, therapeutic effects, and safety studies in animal models, leading to translational clinical investigations' achievements (Table 1) and promoting the advancement and utilization of nanotargeted 188 Re-liposome in future cancer clinical research and practical applications.

Rhenium-188 Production
The most important achievement in using 188 Re is the development of a 188 W/ 188 Regenerator. The 188 W/ 188 Re-generator is easy to manipulate and practical, especially in some remote areas where it is difficult to transport short-living radionuclides routinely. Originally, 188 W is produced by the 186 W (2n,γ) 188 W nuclear reaction [49,50]. This reaction involves two successive neutron captures, making the probability of a reaction proportional to the square of the neutron flux. Consequently, the production depends on the neutron flux. However, only very high neutron flux (10 15 /cm 2 /second) can produce sufficient 188 W. Currently, a 188 W/ 188 Re-generator design is based on 188 Re separation from 188 W using an alumina column. For instance, the parent nuclide, 188 W, is loaded on the column and a 188 Re daughter radionuclide is eluted with normal saline. The half-life of 188 W is 69 days. Therefore, the shelf life of an 188 W/ 188 Re-generator can be longer than four months, based on the activity levels of 188 Re required. The interval of 188 Re elution to achieve the maximum radioactivity is approximately three days [51]. For the preparation of 188 Re-liposome, 188 Re could be milked from a homemade 188 W/ 188 Re-generator of the Institute of Nuclear Energy Research [52] or a commercialized Good Manufacturing Practice (GMP)/Pharmaceutical-Grade 188 W/ 188 Re generator by IRE (Institut National des Radioelements, Fleurus, Belgium).

Preparation and Characterization of 188 Re-Liposome
188 Re-liposome is composed of liposome (Carrier), N,N-bis(2-mercaptoethyl)-N ,Ndiethyl-ethylenediamine (BMEDA, chelator) and 188 Re (Radionuclide) by a remote-loading method [19]. BMEDA is a nitrogen and sulfur donor (SNS) pattern ligand with a tridentate structure that has one nitrogen and two sulfur atoms ( Figure 2A). These three atoms are able to offer electrons to 186 Re, 188 Re and 99 Tc for organizing a lipophilic complex in a neutral state [19] ( Figure 2B). The SNS/S complexes have a neutral core coordinate structure which can cross the lipophilic double membrane of a liposome. Carrier-free 188 Reperrhenate (NaReO 4 ) and BMEDA were used to form 188 Re-BMEDA [9]. The labeling efficiency of 188 Re-BMEDA is usually more than 95%. Liposomes encapsulating (NH 4 ) 2 SO 4 have been chosen as the drug delivery scaffold [9]. With 188 Re-BMEDA crossing the lipophilic bilayer of liposome, the lipophilic/hydrophilic characterization can trigger a gel-like formation and anions trapping ( Figure 2C). The 188 Re-liposome was separated from free 188 Re-BMEDA using a PD-10 column eluted with normal saline. The overall yield of 188 Re-liposome is about 70%. For clinical trials, 188 Re-liposome was further filtered with a 0.22 micrometer filter in our Pharmaceutical Inspection Convention and Pharmaceutical Inspection Cooperation Scheme (PIC/S) GMP radiopharmaceutical production center. The specification for the concentration of phospholipid were 3~6 µmol/mL, a particle size of 80~100 nm, zeta potential of −3~2 mV and radiochemical purity of more than 90%. Figure 3 shows the scheme for the preparation of 188 Re-liposomes [32]. Studies have indicated 188 Re-liposome is stable in normal saline, serum and plasma [30,32,53]. The results from in vitro and in vivo studies indicated that about 92-98% of 188 Re-liposomes maintain high radiochemical purity (RCP) for up to 72 h of incubation [30][31][32]53]. The evidence indicates 188 Re-liposome has high radiochemical purity and drug encapsulation stability.

Pharmacokinetics and Biodistribution
For pharmacokinetics, whole blood samples were collected for up to 168 h (lung metastatic model up to 96 h) post administration through the tail vein. Radioactivity in whole blood samples was normalized to the percentage of injected dose per gram at various time points following the intravenous injection of 188 Re-liposome. Pharmacokinetic parameters were determined using the WinNonlin software v5.0.1 (Pharsight Corp., Mountain View, CA, USA). Non-compartmental analysis was used with the log/linear trapezoidal rule. The pharmacokinetic parameters, including time to achieve maximum concentration (Tmax), maximum concentration (Cmax), clearance (Cl), area under the tissue concentration-time curve (AUC), and mean residence time (MRT) were determined. Pharmacokinetic parameters of 188 Re-liposome from blood in various cancer-bearing mice models have been estimated [30,32,36,37]. The elimination half-life (T 1/2λz ) of 188 Reliposome and 188 Re-BMEDA was 185.33 h and 34.72 h, respectively, in a C26 ascites tumor model [32]. T 1/2λz of 188 Re-liposome was 5.3-fold longer than that of 188 Re-BMEDA in blood. The AUC (0→∞) of 188 Re-liposome and 188 Re-BMEDA was 763.03% ID/g × h and 81.28% ID/g × h, respectively. The AUC (0→∞) of 188 Re-liposome in blood was 9.4-fold larger than that of 188 Re-BMEDA. The total body clearance of 188 Re-BMEDA (1.23 mL/h) was higher than that of 188 Re-liposome (0.13 mL/h). Furthermore, The AUC (0→∞) of 188 Re-liposome and 188 Re-BMEDA was 800.6% ID/g × h and 172.6% ID/g × h, respectively, in a C26 solid tumor model [30]. The AUC (0→∞) of 188 Re-liposome in blood was 4.65-fold larger than that of 188 Re-BMEDA. The AUC (0→∞) of 188 Re-liposome and 188 Re-BMEDA was 488.97% ID/g × h and 62.73% ID/g × h, respectively, in an NCI-H292 solid tumor model [40]. The AUC (0→∞) of 188 Re-liposome in blood was 7.8-fold larger than that of 188 Re-BMEDA. These pharmacokinetic parameters indicate 188 Re-liposome has significantly greater MRT with longer T 1/2λz and the greater AUC compared with those from 188 Re-BMEDA.
All biodistribution showed 188 Re-liposome could accumulate significant amounts in the tumor, liver, spleen and kidneys [30,32,36,40,54]. Liposome could be captured in the liver and spleen by the reticuloendothelial system [2]. The highest uptake of 188 Reliposome in tumors was found at 24 h after administration, and was steadily maintained until 72 h after administration. The highest tumor to muscle ratio (Tu/Mu) was up to 25.8 and 14.4 at 24 h in the C26 murine colon ascites model [36] and HT-29 human colorectal carcinoma solid tumor model [31], respectively. Very low uptake of 188 Re-liposome has been reported in the organs of the central nervous and musculoskeletal systems [30,36]. Biodistribution studies suggested that 188 Re-liposome exhibits high retention in blood circulation and tumor accumulation in vivo. Our study also showed 188 Re-liposome could accumulate in brain tumors. The mechanism may be due to the neoangiogenesis properties of glioma, with newly formed vasculatures overpassing the blood-brain barrier (BBB). Besides, glioma cells can degrade tight junctions by secreting soluble factors, leading to BBB disruption [33]. The excretion of 188 Re-liposome has been studied [36,37]. The fraction of 188 Re-liposome excreted in the urinary and gastrointestinal tract is derived from accumulative urine and feces using metabolic cages [37,55]. The total excreted fraction of 188 Re-liposome by urine and feces was about 47-87% in different tumor models. The 188 Re-liposome was mostly excreted via feces (29-61%), suggesting the importance of hepatobiliary excretion for these compounds.

Longitudinal MicroSPECT/CT Imaging of Tumor Targeting for 188 Re-Liposome
Several tumor-bearing mouse models demonstrated longitudinal microSPECT/CT could be used as the imaging of tumor targeting by using 188 Re-liposome [30,32,34,35,38]. The accumulation and localization of nanotargeted 188 Re-liposomes has been studied in C26 solid mouse models [30,34]. The images revealed 188 Re-liposomes ( Figure 4B) and 188 Re-DXR-liposomes ( Figure 4C) remained in the tumor for up to 72 h post injection, while the corresponding images for free 188 Re-BMEDA revealed it could not accumulate in the tumor at 4 h post injection ( Figure 4A). Although uptake of 188 Re-liposome in the spleen and liver are the common features of nanoparticles following intravenous injection into mice, the images revealed a higher uptake in tumors up to 72 h after intravenous injection. A positive correlation (r = 0.663) of tumor uptake of 188 Re-liposome by biodistribution and microSPECT semi-quantification image analysis was obtained using Pearson correlation analysis ( Figure 4D) [30]. MicroSPECT/CT imaging also revealed a higher uptake of 188 Re-liposome in tumors up to 72 h post injection in a human LS-174T-colorectal cancer tumor-bearing mice model [38]. Whole-body autoradiography results also confirmed the correlation between microSPECT/CT imaging and biodistribution data [38]. Our results indicated molecular imaging is a non-invasive imaging modality that can longitudinally monitor the behavior of 188 Re-liposome radiotherapeutics in the same animal across different time points.  Figure 4A). Although uptake of 188 Re-liposome in the spleen and liver are the common features of nanoparticles following intravenous injection into mice, the images revealed a higher uptake in tumors up to 72 h after intravenous injection. A positive correlation (r = 0.663) of tumor uptake of 188 Re-liposome by biodistribution and microSPECT semi-quantification image analysis was obtained using Pearson correlation analysis ( Figure 4D) [30]. MicroSPECT/CT imaging also revealed a higher uptake of 188 Re-liposome in tumors up to 72 h post injection in a human LS-174T-colorectal cancer tumor-bearing mice model [38]. Whole-body autoradiography results also confirmed the correlation between microSPECT/CT imaging and biodistribution data [38]. Our results indicated molecular imaging is a non-invasive imaging modality that can longitudinally monitor the behavior of 188 Re-liposome radiotherapeutics in the same animal across different time points.

Dosimetry of 188 Re-Liposome
Radiation dosimetry analysis is an important aspect of evaluating the safety and efficacy of internal emitters for radionuclide therapy [56]. Regulatory agencies (e.g., the US Food and Drug Administration, FDA) approved the use of animal models to estimate the dosimetry before clinical trials [57]. Radiation dosimetry was conducted to estimate the absorbed doses in non-target organs, as well as in target tumors for 188 Re-liposome, to extrapolate the animal data to humans. Radiation dose estimates for normal tissues and tumors were calculated using the OLINDA/EXM program [58].
Various studies from colon carcinoma tumor-bearing mice models indicate the absorbed dose of 188 Re-liposome in liver, kidneys and red marrow are all about 0.24-0.40,

Dosimetry of 188 Re-Liposome
Radiation dosimetry analysis is an important aspect of evaluating the safety and efficacy of internal emitters for radionuclide therapy [56]. Regulatory agencies (e.g., the US Food and Drug Administration, FDA) approved the use of animal models to estimate the dosimetry before clinical trials [57]. Radiation dosimetry was conducted to estimate the absorbed doses in non-target organs, as well as in target tumors for 188 Re-liposome, to extrapolate the animal data to humans. Radiation dose estimates for normal tissues and tumors were calculated using the OLINDA/EXM program [58].
Various studies from colon carcinoma tumor-bearing mice models indicate the absorbed dose of 188 Re-liposome in liver, kidneys and red marrow are all about 0.24-0.40, 0.09-0.20 and 0.033-0.050 mGy/MBq, respectively [36][37][38]55]. Either liver or red marrow may be the dose-limiting organ for radionuclide PEGylated nanoliposome therapy [59]. High absorbed doses are also observed in the gastrointestinal tract, including the lower large intestine, upper large intestine and small intestine. This suggests 188 Re-liposome was mostly excreted via feces. Very low absorbed doses of 188 Re-liposome are noted in the organs of the central nervous and musculoskeletal systems. Bone marrow toxicity is also generally dose-limiting for β-emitting radiopharmaceuticals. Those results indicated the nanoliposome formulation did not cause higher absorbed doses in normal tissue than nontargeted formulation, but it did cause higher absorbed doses in tumors. Systemically targeted radionuclide therapy using 188 Re-liposome is feasible, promising, and may have the advantage and benefit of reduced toxicity and improved therapeutic efficacy.

Acute Toxicity of BMEDA and 188 Re-Liposome
Non-clinical safety studies are necessary to assess the safety and toxicity profiles of drug compounds under development and before clinical trials [60]. Acute toxicity studies in various animal models aiming to provide pharmaceutical information intended for human use, and the information is useful for providing preliminary identification of target organs of toxicity. Since BMEDA (C 10 H 24 N 2 S 2 ) is a new chemical entity for chelating 188 Re in 188 Re-liposome, the acute toxicity studies of BMEDA should be evaluated. The LD 50 value of BMEDA is estimated to be 8.13 and 8.68 mg/kg in rodents for male and female mice, respectively. No difference in body weights and no observable gross lesions are observed among 3-and 6-mg/kg BMEDA-treated and control mice [45]. Extended acute toxicity studies of BMEDA have also been confirmed in non-rodents, with beagles receiving a BMEDA dose of 1 mg/kg that had no adverse effect and no doses causing life-threatening toxicity [46].
A 28-day extended acute toxicity study for 188 Re-liposome was performed in Sprague Dawley rats via a single intravenous injection, obtaining the "no observed adverse effect level (NOAEL)" estimated to be greater than 185 MBq (5 mCi) per rat (weight of a rat estimated to be 200 g) [48]. None of the rats died and no clinical sign was observed during the 28-day study period. No differences in the biochemistry parameters, gross lesion and histopathological damage were found between the 188 Re-liposome treated and control groups.

Therapeutic and Combination Effects of 188 Re-Liposome
The treatment of colorectal cancer patients remains a challenging problem [61]. We demonstrated the therapeutic efficacy of 188 Re-liposome in C26 [22] or LS-174 [38] colorectal tumor bearing mice. Mice receiving various doses of 188 Re-liposome (from 22.2 to 37 MBq) can significantly increase overall survival time by more than 60% compared with control saline injection. The results showed the dose-dependent therapeutic efficacy and prolonged survival time of 188 Re-liposome in colorectal tumor-bearing mice [22,38]. Liposomal drugs such as pegylated liposomal doxorubicin (DXR) can be designed to improve the pharmacological and therapeutic index for cancer therapeutics [2,13], but the limited distribution of doxorubicin in solid tumors causes drug resistance and a lower chemotherapy response [62]. The developments for improving therapeutic efficacy, reducing side effects and overcoming the drug resistance of multiplex nanoliposomes are of considerable interest.
For the therapeutic efficacy in a C26 murine colon carcinoma solid tumor e model [34], the tumor volume inhibition and the survival curves following various treatments are shown in Figure 5A,B, respectively. Two (25%) of the mice treated with 188 Re-DXRliposome ( Figure 5C) or 188 Re-liposome were completely cured after 120 days. The bimodality radio-chemotherapeutics of 188 Re-DXR-liposome and radiotherapeutic of 188 Reliposome showed better mean growth inhibition (MGI) rates (MGI = 0.048; 74 days) and (MGI = 0.134; 60 days), respectively, than those treated with chemotherapeutics of Lipo-DOX (MGI = 0.413; 38 days) ( Figure 5D). The therapeutic efficacy of 188 Re-liposome and the synergistic effect of the combination of 188 Re-DXR-liposome point to the potential benefit and promise of the co-delivery of nanoliposome radio-chemotherapeutics for adjuvant cancer treatment on oncology applications [34]. and the synergistic effect of the combination of 188 Re-DXR-liposome point to the potential benefit and promise of the co-delivery of nanoliposome radio-chemotherapeutics for adjuvant cancer treatment on oncology applications [34]. For several decades, 5-Fluorouracil (5-FU)-based regimens have been the first choice of the primary or adjuvant chemotherapy for colorectal cancer [63,64]. We compared the therapeutic efficacy by single equivalent dose (80% maximum tolerance dose, MTD) of 188 Re-liposome (80% MTD; 29.6 MBq) with that of 5-FU (80% MTD; 144 mg/kg) in different tumor models [22,[36][37][38]. All therapeutic results including median survival time and tumor volume inhibition demonstrated better therapeutic 188 Re-liposome efficacy (80% MTD) than that of 5-FU (80% MTD) treatment in mice. For the comparative therapeutic study of 188 Re-liposome and 5-FU in a C26 murine colon carcinoma solid tumor mice model [22], the tumor volume inhibition and the survival curves following various treatments are shown in Figure 6A,B, respectively. With respect to therapeutic efficacy, the tumor-bearing mice treated with 188 Re-liposome showed a better mean tumor growth inhibition rate (MGI) and longer median survival time (MGI = 0.140; 80 days) than those treated with the anti-cancer drug 5-FU (MGI = 0.195; 69 days) and untreated control mice (48 days) ( Figure 6C). For several decades, 5-Fluorouracil (5-FU)-based regimens have been the first choice of the primary or adjuvant chemotherapy for colorectal cancer [63,64]. We compared the therapeutic efficacy by single equivalent dose (80% maximum tolerance dose, MTD) of 188 Re-liposome (80% MTD; 29.6 MBq) with that of 5-FU (80% MTD; 144 mg/kg) in different tumor models [22,[36][37][38]. All therapeutic results including median survival time and tumor volume inhibition demonstrated better therapeutic 188 Re-liposome efficacy (80% MTD) than that of 5-FU (80% MTD) treatment in mice. For the comparative therapeutic study of 188 Re-liposome and 5-FU in a C26 murine colon carcinoma solid tumor mice model [22], the tumor volume inhibition and the survival curves following various treatments are shown in Figure 6A,B, respectively. With respect to therapeutic efficacy, the tumor-bearing mice treated with 188 Re-liposome showed a better mean tumor growth inhibition rate (MGI) and longer median survival time (MGI = 0.140; 80 days) than those treated with the anti-cancer drug 5-FU (MGI = 0.195; 69 days) and untreated control mice (48 days) ( Figure 6C).  [22].
In the comparative therapeutic study of 188 Re-liposome and 5-FU in a C26 murine colon peritoneal ascites and tumor mice model [36], significant inhibitions of hemorrhagic ascites formation ( Figure 7A) and the inhibition of tumor growth ( Figure 7B) were observed in the 188 Re-liposome treated group (83.3%, 63%) in comparison with the 5-FU (44.9%, 9.1%). The survival curves after various treatments were showed in Figure 7C. With respect to therapeutic efficacy, the tumor-bearing mice treated with 188 Re-liposome showed better mean survival time and increased life span (32.8 days, 34.6%) than those treated with the anti-cancer drug 5-FU (26.7 days, 9.6%) and untreated control mice (24.3 days) ( Figure 7D). In the comparative therapeutic study of 188 Re-liposome and 5-FU in a C26 murine colon peritoneal ascites and tumor mice model [36], significant inhibitions of hemorrhagic ascites formation ( Figure 7A) and the inhibition of tumor growth ( Figure 7B) were observed in the 188 Re-liposome treated group (83.3%, 63%) in comparison with the 5-FU (44.9%, 9.1%). The survival curves after various treatments were showed in Figure 7C. With respect to therapeutic efficacy, the tumor-bearing mice treated with 188 Re-liposome showed better mean survival time and increased life span (32.8 days, 34.6%) than those treated with the anti-cancer drug 5-FU (26.7 days, 9.6%) and untreated control mice (24.3 days) ( Figure 7D).
We also worked on studies of combination therapy using 188 Re-liposome and chemo therapeutic agents. 188 Re-liposome was combined with sorafenib (Nexavar) [42], or Lipo-Dox [43] in C26 colorectal tumor mouse models. Hsu showed mice treated with 188 Reliposome and Lipo-Dox pretreatment experienced limited tumor growth ( Figure 8A). Seventy-five percent of the mice could survive with the combination of 188 Re-liposome and Lipo-Dox. Groups with Lipo-Dox or 188 Re-liposome treatment showed a survival rate of less than 25% at the same time point [43]. With respect to therapeutic efficacy, the tumor-bearing mice treated with 188 Re-liposome and pretreated with Lipo-Dox showed increased mean survival time and increased life span (more than 120 days, 242.9%) compared to those treated with 188 Re-liposome (57 days, 62.9%) or Lipo-Dox (47.5 days; 35.7%) and untreated control mice (35 days) ( Figure 8B). 188 Re-liposome combined with Nexavar achieved higher survival rates (75%) compared with the 188 Re-liposome (62.5%) or Nexavar (0%) alone groups at the end of the study ( Figure 8D) [42]. The therapeutic effect of the chemotherapeutic agent for the tumor significantly improved by 188 Re-liposome demonstrated its delivery and effectiveness. We also worked on studies of combination therapy using 188 Re-liposome and chemotherapeutic agents. 188 Re-liposome was combined with sorafenib (Nexavar) [42], or Lipo-Dox [43] in C26 colorectal tumor mouse models. Hsu showed mice treated with 188 Re-liposome and Lipo-Dox pretreatment experienced limited tumor growth ( Figure  8A). Seventy-five percent of the mice could survive with the combination of 188 Re-liposome and Lipo-Dox. Groups with Lipo-Dox or 188 Re-liposome treatment showed a survival rate of less than 25% at the same time point [43]. With respect to therapeutic efficacy, the tumor-bearing mice treated with 188 Re-liposome and pretreated with Lipo-Dox showed increased mean survival time and increased life span (more than 120 days, 242.9%) compared to those treated with 188 Re-liposome (57 days, 62.9%) or Lipo-Dox (47.5 days; 35.7%) and untreated control mice (35 days) ( Figure 8B). achieved higher tumor growth inhibition (53%) compared with the 188 Re-liposome (25%) or EBRT (30%) alone groups at 21 days post-injection ( Figure 8C) [44]. Combination treatment had no additive adverse effects or significant biological toxicities on white blood cell counts, body weight, or liver and renal functions. According to combination studies of 188 Re-liposome with chemotherapeutics or EBRT, the combination strategy may be a potential treatment modality for cancers.  [42][43][44].

Clinical Studies of 188 Re-Liposome
The FDA guidelines for an exploratory IND (eIND) published in 2006 [65] give the ability to conduct a human trial for obtaining early pharmacokinetic and pharmacodynamics information according to the microdosing concept. The term microdosing is defined as less than 1/100th of the dose calculated to yield a pharmacological effect of the drug candidate based on primary in vitro and in vivo data and administered at a maxi- External beam radiotherapy (EBRT) can deliver high-energy radiation beams to cover both gross tumors and potential microscopic tumor cells in the vicinity of a tumor, including esophageal cancers. Our studied showed 188 Re-liposome combined with EBRT achieved higher tumor growth inhibition (53%) compared with the 188 Re-liposome (25%) or EBRT (30%) alone groups at 21 days post-injection ( Figure 8C) [44]. Combination treatment had no additive adverse effects or significant biological toxicities on white blood cell counts, body weight, or liver and renal functions. According to combination studies of 188 Reliposome with chemotherapeutics or EBRT, the combination strategy may be a potential treatment modality for cancers.

Clinical Studies of 188 Re-Liposome
The FDA guidelines for an exploratory IND (eIND) published in 2006 [65] give the ability to conduct a human trial for obtaining early pharmacokinetic and pharmacodynamics information according to the microdosing concept. The term microdosing is defined as less than 1/100th of the dose calculated to yield a pharmacological effect of the drug candidate based on primary in vitro and in vivo data and administered at a maximum dose of ≤100 µg [24,66]. Due to the BMEDA being the first to be used in humans, the eIND is an approach to evaluate the imaging, pharmacokinetic and safety of 188 Re-liposome.
An open-label, single-arm, phase 0 clinical trial with a 111 MBq microdose of 188 Reliposome injected in patients with metastatic cancer was carried out. The study proved the safety of 188 Re-1iposome in 12 patients refractory to current standard/available therapies. None of the subjects showed any serious adverse effects following 188 Re-liposome injection, and no clinically significant abnormalities in physical exams or laboratory exams were found [29]. Patient 14, who had a nasopharyngeal tumor (NPC) over the left nasal cavity and lung metastases, showed high uptake (3.99% IA/kg) at the targeted area at 24 h after injection with 188 Re-liposome, corresponding to the soft tissue lesion (blue arrow) seen on the corresponding MRI ( Figure 9A,B). Anterior planar images of Patient 14 were obtained at different time points following injection with 188 Re-liposome and reveal a discernible uptake of radioactivity in the left nasopharyngeal region ( Figure 9C). This nasopharyngeal tumor shows a clear response to the low radioactivity of 188 Re-liposome, as verified by nasopharyngoscopic examinations at two and twelve months after injection ( Figure 9D-F) [29]. mum dose of ≤100 µg [24,66]. Due to the BMEDA being the first to be used in humans, the eIND is an approach to evaluate the imaging, pharmacokinetic and safety of 188 Re-liposome.
An open-label, single-arm, phase 0 clinical trial with a 111 MBq microdose of 188 Re-liposome injected in patients with metastatic cancer was carried out. The study proved the safety of 188 Re-1iposome in 12 patients refractory to current standard/available therapies. None of the subjects showed any serious adverse effects following 188 Re-liposome injection, and no clinically significant abnormalities in physical exams or laboratory exams were found [29]. Patient 14, who had a nasopharyngeal tumor (NPC) over the left nasal cavity and lung metastases, showed high uptake (3.99% IA/kg) at the targeted area at 24 h after injection with 188 Re-liposome, corresponding to the soft tissue lesion (blue arrow) seen on the corresponding MRI ( Figure 9A,B). Anterior planar images of Patient 14 were obtained at different time points following injection with 188 Re-liposome and reveal a discernible uptake of radioactivity in the left nasopharyngeal region ( Figure 9C). This nasopharyngeal tumor shows a clear response to the low radioactivity of 188 Re-liposome, as verified by nasopharyngoscopic examinations at two and twelve months after injection ( Figure 9D-F) [29].   Two recurrent ovarian cancer patients received cohort 1 therapeutic activity of 188 Reliposome in phase 1 clinical trials. Four months after the treatment, the CA-125 started to decline. Chest film follow-up also showed a partial resolution of right pleural effusion [25]. These results suggest 188 Re-liposome may reverse from a drug resistance status to a drug sensitive status and may be a novel strategy in overcoming drug resistance in ovarian cancer. Although the two cases do not prove the therapeutic efficacy of 188 Re-1iposome, the extended life after treatment of the two cases is more than expected.

Conclusions
The advantages of a longer shelf-life radionuclide generator, higher energy β-emitter for radiotherapeutics and suitable gamma ray energy and half-life for nuclear imaging provide 188 Re with suitable characteristics for internal radiotheranostics application. However, the main disadvantage of 188 Re nuclear imaging is the presence of a high-energy background signals (478 keV, 633 keV, and 829 keV), with high-energy ultra-high resolution multi-pinhole collimators being required to optimize both image quality and quantitative accuracy for 188 Re SPECT nuclear imaging in diagnostic applications.
The effectiveness and safety of personalized medicine involves multiple factors that contribute to cancer disease types and development, and resistance to radio/chemotherapy. We demonstrated nanoliposome is a suitable passive delivery system for 188 Re radionuclide in various cells and tumor animal models, which include prolonging the radioactivity of 188 Re in tumor sites with longer biologic half-life through EPR effects, suppressing tumor growth and increasing the survival rate in various tumor animal models. However, enhanced specificity could be further considered through active targeting nanoliposome [67][68][69]. Although combinational use of internal nanotargeted 188 Reliposome with chemotherapy in clinical settings is still in its infancy, it has heralded the dawn of a new era in future cancer treatment through our experimental achievements in preclinical animal models investigation and radio/chemo-combination therapeutic effect and survival comparison results. Nevertheless, two issues need to be overcome in those novel therapeutic strategies' design. First, the exploration of the optimal protocol of active nanotargeted radionuclide therapy in combination with nanotargeted chemotherapy to decrease the nanotargeted drugs that accumulate in the reticulaendothelial system, such as the liver, spleen, and red marrow. Second, each cancer type possesses specific clinical characteristic properties, such as the tumor microenvironment, and invasion time periods, etc. Increasing preclinical and clinical investigated results showed the effect of radiation in terms of modulating immunity could increase its therapeutic efficacy role in systemic anti-cancer treatment [67]. Local external radiation therapy in combination with chemotherapy of tumor-triggering systemic response is described as an abscopal effect. Our systemic investigations and comparison of nanotargeted 188 Re-liposome pharmacology as well as the therapeutic effect in various animal models and translational clinical research results point to a new novel formulation of nanotargeted 188 Re-liposome combining radio/chemo/EBRT/immuno-combination therapy. This could potentially enhance the nuclear imaging quality and accuracy in addition to the systemic tumor lesions treatment effectiveness.
In conclusion, this paper provides an overview and highlights our multifunctional and multi-institutes collaboration of nanotargeted 188 Re-liposome from preclinical tumor and ascites animal models to preliminary clinical translation studies. The results demonstrate 188 Re-liposome is both capable and favorable in vivo tumor targeting and nuclear imaging, biodistribution, pharmacokinetics, dosimetry, radiotherapeutic effectiveness, and has a good synergistic radio/chemo-combination effect. However, further efforts and challenges in preclinical and clinical efficacy and toxicity studies are required to translate the new novel nanotargeted 188 Re-liposome with the radio/chemo/immuno-combination magic bullet therapeutic concept to further advance new technologies to clinical applications for the healthcare benefits of suitable cancer patients.