Contents
2 Dendrimer-based SPECT imaging agents with 99mTc labelling
3 Dendrimer-based SPECT imaging agents with 111In labelling
4 Dendrimer-based SPECT imaging agents with 125I labelling
5 Dendrimer-based SPECT imaging agents with 131I labelling
1 Introduction
Cancer is a malignant tumor, which is caused by the uncontrolled proliferation of cells. Based on the characteristics of local invasion and distant metastasis of cancer, early detection and treatment is the most effective way to cure cancer
[1⇓⇓⇓~5]. Molecular Imaging (MI) is one of the most effective methods for the early diagnosis of cancer. It is a hot spot of imaging research and cancer treatment in the 21st century, which uses imaging methods to conduct qualitative and quantitative research on living organisms at the cellular and molecular levels, and uses existing medical imaging technology to conduct non-invasive real-time imaging of internal physiological or pathological processes at the molecular level without interfering with the physiological conditions of the body
[6⇓⇓⇓~10].
At present, molecular imaging techniques include two categories, namely structural imaging and functional imaging. Structural Imaging is mainly composed of Ultrasonic Imaging (US), Magnetic Resonance Imaging (MR), X-ray Computed Tomography Imaging (CT) and Bioluminescence Tomography (BLT), which can provide anatomical information of tumors
[11⇓~13]. Functional imaging mainly includes positron Emission Tomography (PET) and Single Photon Emission Tomography (SPECT) in nuclear medicine imaging, which have high sensitivity and can distinguish the metabolic differences between tumors and normal tissues
[14⇓~16]. Among many imaging technologies, SPECT imaging technology, as a functional imaging technology, has the advantages of simple method, high diagnostic accuracy, high sensitivity, no penetration depth limitation, and can find and monitor pathological tissues at the molecular level
[17,18]. As a necessary part of SPECT imaging, radionuclides play a vital role. By monitoring the distribution, flow and metabolism of radionuclides in the body, SPECT imaging can monitor the physiological and pathological changes of the lesion site at the molecular level
[19]. However, due to the relatively short circulation time of small molecular radionuclides in the body, it is easy to metabolize out of the body, and it has no tissue specificity, so it is easy to adsorb or remain in normal tissues and organs, and has radiation damage to normal tissues
[20,21]. In order to make up for the shortcomings of small molecule radionuclides, radionuclides are often combined with nano-carriers to construct SPECT imaging contrast agents, which can prolong their circulation time in vivo and achieve their specific distribution in vivo through functional modification. As an essential element of SPECT imaging,Common radionuclides include technetium -99(
99mTc), indium -111(
111In),Gallium -67(
67Ga), thallium -201(
201Tl), iodine -125(
125I) and iodine -131(
131I), etc.Among them, the
99mTc has the physical properties of shorter half-life (6 H) and gamma emission energy (140 keV). Based on its high SPECT imaging resolution, it can be used for short-term in vivo SPECT monitoring. And based on the underlying chemical properties of the
99mTc, a variety of chemotherapeutic agents can be labeled to monitor the progress of radiation therapy
[22]. The half-life of the
111In is 2.83 days (2.83 d), and the γ emission energies are 173 keV (89%) and 247 keV (95%), respectively. At present,
111In has been used to label various carriers, such as monoclonal antibodies (MAb) and lymphocytes, and as a contrast agent for SPECT imaging of specific tumors or lymphatic infections
[23]. The half-life of
67Ga is 78.3 H, which is often used to locate and differentiate tumors and inflammation. The SPECT imaging of the lesion site mediated by it can distinguish the normal tissue overlapped with it to reduce the error caused by image overlap and increase the sensitivity of detection
[23,24]. The
201Tl has a relatively long radioactive half-life (74 H) and can be used for long-term SPECT imaging. Moreover,
201Tl labeled SPECT imaging contrast agents can quantify the degree of malignancy of gliomas and distinguish areas of radiation necrosis from areas of tumor recurrence
[23,24]. As isotopes of radioactive iodine,
125I and
131I have long radioactive half-lives. The half-life of the
125I is 60 d and the gamma-ray emission energy is 28 keV. The half-life of the
131I is 8.01 d, and it can simultaneously emit gamma rays (364 keV, 81.7%) for SPECT imaging and beta rays (0.606 MeV, 89.9%) for radiotherapy (Table 1)
[23,24].
In order to construct SPECT imaging contrast agents based on nanomaterials, a variety of nanocarriers have been selected, such as liposomes, mesoporous silica, dendrimers, micelles, ferritin, albumin, iron oxide, gold nanoparticles, superparamagnetic rare earth ions and quantum dots
[25⇓⇓~28]. Among many nanocarriers, dendrimers have precise structure and excellent physical and chemical properties, which can make up for the inherent defects of small molecular radionuclides in vivo, improve their stability and specificity, and prolong their circulation time in vivo to achieve efficient SPECT imaging. The dendrimer has a precise molecular structure, which is composed of a small molecule core, an internal cavity formed by multiple branches, and a peripheral functional group. The structure is shown in Figure 1. The peripheral groups can realize the modification and assembly of functional molecules to realize the biological functionalization; The inner cavity formed by the multi-branch structure can be used for loading various nanoparticles and small molecule drugs
[29⇓⇓~32]. In addition, based on the excellent physical and chemical properties, good water solubility, strong biocompatibility and stability of dendrimers, it can achieve long-term blood circulation and high tissue specificity in vivo. Therefore, dendrimers are often used as ideal carriers to construct SPECT imaging contrast agents.
Nowadays, a variety of dendrimer-based SPECT imaging contrast agents have been developed and utilized, which can achieve accurate and efficient SPECT imaging of tumors, and can effectively monitor the occurrence and development of cancer and evaluate the prognosis. The types of the dendrimer-based SPECT imaging contrast agents include a dendrimer SPECT imaging contrast agent labeled with 99mTc, a dendrimer SPECT imaging contrast agent labeled with 111In, a dendrimer-SPECT imaging agent labeled with 125I, and a Dendrimer-SPECT imaging agents labeled with 131I, which have different functional imaging effects respectively. Therefore, this review summarizes the construction and biomedical applications of radionuclide-labeled dendrimer-based SPECT imaging contrast agents in recent years, and provides a reference for the development of new and intelligent SPECT imaging contrast agents for early cancer diagnosis.
2 99mTc labeled dendrimer SPECT imaging contrast agent
As the most commonly used gamma radionuclide in SPECT imaging,
99mTc has relatively short half-life (6 H), low injection volume and low production cost (molybdenum -99(
99Mo)/
99mTc). The labeling efficiency on the surface of the functionalized dendrimer is high, the radioactive stability in vivo and in vitro is good, the small molecular
99mTc is not easy to fall off from the labeling agent, and the labeling agent can be used for monitoring the pharmacological and pharmacokinetic behaviors of drugs and the metabolic distribution in vivo
[33].
In order to obtain a stable
99mTc labeled dendrimer for SPECT imaging, Parrott et al synthesized the first to eighth generation (G1 ~ G8) aliphatic polyester dendrimers with p-toluenesulfonyl ethyl ester (TSe) as the core
[22]. Subsequently, by removing the protective agent TSe, the G5, G6 and G7 generation aliphatic polyester dendrimers (COOH-G5-(O
2Bn)
32, COOH-G6-(O
2Bn)
64 and COOH-G7-(O
2Bn)
128) with carboxyl (COOH) as the core and an average of 32, 64 and 128 benzyl dioxide (O
2Bn) groups on the surface were obtained. The benzyl group is further removed, then the tridentate bis (pyridyl) amine (BisPy) ligand is introduced into the carboxyl core of the polyester dendrimer by an amidation reaction, and finally the radionuclide
99mTc is chelated by the ligand to obtain the G5/G6/G7 generation functionalized polyester dendrimer ([TcBisPy-G5-(OH)
32]
+, [TcBisPy-G6-(OH)
64]
+ and [TcBisPy-G7-(OH)
128]
+)) which is functionalized by the BisPy group and 32/64/128 hydroxyl groups and is labeled with the
99mTc. Studies have shown that G5-G7 generation polyester dendrimers labeled with
99mTc can be used for real-time and non-invasive SPECT imaging in vivo. By studying its biodistribution in vivo, it can be seen that the G5-G7 generation dendrimers labeled with
99mTc can be quickly and effectively removed from the blood through the kidney, almost completely removed within 15 minutes and excreted from the body through the bladder, and there is no specific binding with normal tissues and organs. The rapid clearance of these three polyester dendrimers may be due to the fact that the molecular weights of the G5, G6, and G7 generation dendrimers (4.1, 7.8, and 15.2 kDa) are much lower than the renal filtration cut-off molecular weights (40-60 kDa), and their hydrodynamic size (< 3 nm) is lower than the renal pore radius (5 nm). The G5/G6/G7 generation polyester dendrimer marked by the
99mTc constructed by the method mainly takes a core formed by amidation of BisPy ligand as a single high-affinity binding site for chelating the
99mTc, overcomes the defect of multi-site binding of the dendrimer and radionuclide, obtains the polyester dendrimer marked by the
99mTcstably, and improves the practical value in SPECT imaging and clinical application.
Wen et al. Constructed two multifunctional
99mTc labeled dendrimer-encapsulated gold nanoparticles (Au NPs) complexes (
99mTc-Au DENPs)
[34]. The fifth generation polyamidoamine dendrimer (G5.NH
2) was modified by cyclodiethylenetriaminepentaacetic anhydride (cDTPAA) and methoxy polyethylene glycol (mPEG) in turn.The dendrimer-coated gold nanoparticles were synthesized by sodium borohydride (NaBH
4) reduction method, and the residual terminal amino groups on the surface of the dendrimer were acetylated or glycidylated. Finally, the
99mTc was labeled with cDTPAA ligand to obtain acetylated
99mTc-Au DENPs(
99mTc-Au-Ac DENPs) and glycidylated
99mTc-Au DENPs(
99mTc-Au-Gly DENPs (fig. 2). Within a certain concentration range ([Au]=0~80μmol/L),
99mTc-Au DENPs), it has good colloidal stability, radiation stability and biocompatibility.
Based on the unique chemical structure of the G5.NH2, the inner cavity is wrapped with Au NPs, and the periphery is marked with 99mTc by a chelating agent cDTPAA, so that the constructed 99mTc-Au DENPs has the function of SPECT/CT dual-modality imaging. In vivo SPECT imaging showed that 0.5 H after injection, 99mTc-Au-Ac DENPs mainly existed in the lungs, while 99mTc-Au-Gly DENPs were detected in the heart, liver, kidney and posterior vena cava, and 99mTc-Au-Gly DENPs mainly accumulated in the blood. Over time, the two 99mTc-Au DENPs are eventually excreted from the body through the urinary system. The difference in distribution in vivo may be due to the different hydrodynamic radii of the 99mTc-Au-Ac DENPs(95.6±5.8 nm) and the 99mTc-Au-Gly DENPs(50.4±3.2 nm), as well as the different functional groups modified on the surface. Sentinel lymph node (SLN) imaging plays an important role in guiding the clinical treatment of cancer patients because the lymphatic system is the common pathway of most cancer metastasis. Therefore, in order to explore the possibility of SPECT/CT dual-modality imaging of the two 99mTc-Au DENPs in lymph nodes, the two 99mTc-Au DENPs,SPECT were injected into the left and right feet of rabbits respectively, and the imaging results showed that the 99mTc-Au DENPs mainly accumulated in the popliteal lymph nodes 1 hour after injection. As time went on, the SLN area became clear and bright, and was easy to detect, which indicated the effective diffusion of the two 99mTc-Au DENPs from the injection area to the SLN area. The results of CT imaging also showed that the SLN area became clear with the passage of time, and the SLN boundary could be accurately delineated 2 hours after injection, and the SLN area could be easily detected even 4 hours after injection. Compared with the traditional blue dye and tantalum oxide nanoparticles, the constructed 99mTc-Au DENPs has higher imaging resolution and sensitivity, and can realize SPECT/CT dual-modality imaging of different organs and cancer metastasis monitoring.
Li et al. Modified cDTPAA on the surface of G2 by amide bond using the second generation (G2) polyamidoamine dendrimer (PAMAM) as a template, then modified the functional molecule PEG-FA, and used it as a template to encapsulate Au NPs, and finally used cDTPAA ligand to label radionuclide
99mTc
[35]. The obtained FA-targeted multifunctional dendrimer-coated gold nanoparticles ({ (Au
0)
6-G2-DTPA(
99mTc)-PEG-FA}DENPs)) can realize SPECT/CT imaging of tumors with high folate receptor expression (FAR) in vivo and in vitro. The results showed that the particle size of the obtained nanoparticles was 1. 6 nm, the nanoparticles had good water dispersibility and colloidal stability, and the cell compatibility was good in the concentration range of 0 ~ 4 000 nmol/L. Based on the targeting specificity of FA, the constructed folate (Au
0)
6-G2-DTPA(
99mTc)-PEG-FA}DENPs can specifically bind to cancer cells and tumors with high folate receptor expression to achieve targeted SPECT/CT imaging in vivo and in vitro. In vivo SPECT imaging showed that, at the same time point after injection, the SPECT images of the constructed (Au
0)
6-G2-DTPA(
99mTc)-PEG-FA}DENPs at the tumor site were brighter and the signal intensity and tumor/muscle signal ratio were significantly higher than those of the non-modified multi-functional dendrimer-coated gold nanoparticle (Au
0)
6-G2-DTPA(
99mTc)-PEG-FA}DENPs. At 90 min after injection, the SPECT signal intensity and tumor/muscle signal ratio were 1.2-fold and 1.6-fold higher, respectively, than those of the non-targeted (Au
0)
6-G2-DTPA(
99mTc)-mPEG}DENPs contrast agent (Figure 3). Therefore, based on the functional modification of FA, { (Au
0)
6-G2-DTPA(
99mTc)-PEG-FA}DENPs can be used as a targeted nano-probe to achieve SPECT/CT dual-modality imaging of ovarian cancer, lung cancer, glioma and other tumors with high folate receptor expression.
Xing et al. modified 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), PEG-duraycin, methoxy-polyethylene glycol-carboxyl (mPEG-COOH) and fluorescein isothiocyanate (FI) successively on the surface of the fifth generation (G5) PAMAM to form multifunctional dendrimers
[36]. Subsequently, the dendrimer was used as a template to encapsulate Au NPs, and then the remaining amino groups on the surface of the dendrimer were acetylated, and finally the
99mTc was labeled by DOTA chelating agent to obtain the
99mTc labeled duramycin functionalized dendrimer-encapsulated gold nanoparticles (
99mTc-duramycin-Au DENPs). The results showed that the
99mTc-duramycin-Au DENPs had good radiostability, colloidal stability and cell compatibility in a certain concentration range ( [Au] = 20 ~ 100 μmol/L). Most importantly, this multifunctional nanoprobe enables targeted SPECT/CT imaging of apoptotic cancer cells in vitro and tumor apoptosis after doxorubicin (DOX) treatment. By establishing a subcutaneous transplantation tumor model in vivo, the apoptosis of tumor cells after DOX treatment was observed, so as to evaluate the SPECT/CT imaging ability of
99mTc-duramycin-Au DENPs after tumor cell apoptosis in vivo. It was found that no dendrimer-encapsulated gold nanoparticles (
99mTc-Au DENPs) labeled with
99mTc-duramycin-Au DENPs or
99mTc and not modified by duramycin were found in the tumor sites of tumor-bearing mice before DOX treatment, indicating that duramycin has no specificity for normal tumor cells. However, after 3 days of DOX treatment, the SPECT images of mice injected with
99mTc-duramycin-Au DENPs and
99mTc-Au DENPs showed that the SPECT signal intensity of
99mTc-duramycin-Au DENPs tumors was significantly higher than that of
99mTc-Au DENPs at each time point after 2 hours of injection, and reached the peak at 8 hours after injection, and clear SPECT images could still be obtained at 12 hours. The tissue distribution of the tumor-bearing mice injected with
99mTc-Au DENPs and
99mTc-duramycin-Au DENPs was studied 8 hours after the injection, and it was found that the functionalized dendrimers were mainly accumulated in the liver and spleen, while the accumulation in the lung, heart, kidney, intestine, stomach and soft tissue was relatively low. Compared with the
99mTc-Au DENPs,
99mTc-duramycin-Au DENPs, the dendrimer modified with duramycin has a higher tumor concentration, which further proves that the dendrimer modified with duramycin has a specific targeting effect (fig. 4). In vivo CT imaging results also confirmed that
99mTc-duramycin-Au DENPs can be used as a targeted nanoprobe to monitor early chemotherapy of tumors. In conclusion, the constructed
99mTc-duramycin-Au DENPs can be used as a nano-probe to monitor the early response to chemotherapy for SPECT/CT imaging after apoptosis of tumor cells.
At present, 99mTc is the most commonly used radionuclide in nuclear medicine imaging. Because of its high sensitivity, effective imaging and low radiation hazard to patients, more than 80% of radiopharmaceuticals contain 99mTc. According to the above summary of 99mTc labeled dendrimer SPECT imaging contrast agents, there are common points in their construction methods. First of all, they all use dendrimers as nano-templates to effectively regulate the size of imaging contrast agents for effective metabolism from the body. Secondly, the biomedical distribution of SPECT imaging contrast agents in vivo can be effectively regulated by modifying various functional molecules (such as :O2Bn, mPEG, acetyl, glycidyl, folic acid, etc.), thus realizing the functional imaging of SPECT imaging contrast agents in vivo. Finally, in order to label the radionuclide 99mTc on the functionalized dendrimers, ligand chelating agents (such as BisPy, cDTPAA, DOTA) are often used to achieve efficient and stable labeling of radionuclide 99mTc. At present, 99mTc labeled dendrimer SPECT imaging contrast agents have been constructed, which can effectively improve the enrichment of radiolabels in tumor sites by modifying functional groups such as targeting molecules on the surface of dendrimers, so as to carry out real-time and sensitive tumor SPECT imaging monitoring. At the same time, it also reduces the radiation damage to normal tissues and organs. Among many radionuclides, 99mTc is the most commonly used radionuclide for SPECT imaging, which is due to its potential chemical properties and unique physical properties, such as short half-life (6 H) and low energy gamma rays (140 keV), which is more conducive to short-term and effective SPECT imaging in vivo, and has good radiation safety and can decay quickly. However, due to the short radioactive half-life of the 99mTc labeled complex, it can only be used for short-term SPECT imaging in vivo, and it is difficult to achieve long-term monitoring and prognosis evaluation after treatment. Therefore, how to achieve better long-term imaging effect and improve the blood circulation time of dendrimer-based SPECT imaging contrast agents still needs further study.
3 111In labeled dendrimer SPECT imaging contrast agent
111In is a radioisotope used in SPECT imaging with a half-life of 2.83 days. Because of their similar coordination chemistry to yttrium (
90Y),
111In labeled radiotracers are often used as imaging surrogates to determine the biodistribution and dose of the corresponding
90Y analogs.
111In labeled vectors, such as bleomycin, lymphocytes and monoclonal antibodies (MAb), have been studied as imaging agents for targeting tumors or for lymphocyte infection
[23].
Biricova et al. compared PAMAMs of first generation (G1) and fourth generation (G4) with 10- [ (4-carboxy-1-oxypyridyl) methyl] -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (H
4do3a-py
NO-C) is covalently bound through a pyridine-4-carboxylic acid group,And labeling the radionuclide
111In to prepare the G1-PAMAM and the G4-PAMAM(G1-[
111In(do3a-py
NO-C) which are modified with the H
4do3a-py
NO-C group on the surface and labeled with the
111In] and the G4-[
111In(do3a-py
NO-C)])
[37]. In order to optimize the radiolabeling conditions, the effects of buffer type, pH value and reaction temperature on the yield and purity of the radiolabeled products G1-[
111In(do3a-py
NO-C)] and G4-[
111In(do3a-py
NO-C)] were investigated. It was found that the radiochemical purity of G1-[
111In(do3a-py
NO-C)] and G4-[
111In(do3a-py
NO-C)] was higher than 95% at 30 ℃ in pH = 5. 0 acetic acid buffer (0. 2 mol/L). The kinetic stability study of the radioactive compounds showed that the G1-[
111In(do3a-py
NO-C))] and the G4-[
111In(do3a-py
NO-C))] were stable within 48 H after preparation, and the chemical purity of the two radioactive compounds only decreased by about 4% -7% within 48 H even in the presence of competitive ligands ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA), which indicated that both the G1-[
111In(do3a-py
NO-C))] and the G4-[
111In(do3a-py
NO-C))] had high radiochemical purity and radiostability. By evaluating the pharmacokinetics and biodistribution of the two drugs in rats, it was found that G1-[
111In(do3a-py
NO-C)] mainly accumulated in the kidney within the shortest time interval (5 min) after administration, and showed relatively rapid blood clearance. At 2 H after injection, only 0.51% ± 0.07% of the radiation dose remained in the blood. However, G4-[
111In(do3a-py
NO-C)] showed significant long-term uptake of radioactivity in the liver and kidney, with 20.56% ± 2.59% and 5.85% ± 0.58% of the radiation dose remaining in the liver and kidney at 48 H after injection (Figure 5). It was found that G1-[
111In(do3a-py
NO-C)] was rapidly removed from the body mainly through urine, while only a small part of G4-[
111In(do3a-py
NO-C)] was excreted through urine 48 hours after injection, and most of the radiopharmaceuticals remained in the body. In contrast, G1-[
111In(do3a-py
NO-C)] has better biodistribution and metabolic characteristics, while the accumulation of radioactivity in liver and kidney of G4-[
111In(do3a-py
NO-C)] limits its application in nuclear medicine imaging.
Kobayashi et al. combined 2- (p-isothiocyanatobenzoyl) -6-methyldiethylenetriaminepentaacetic acid (1B4M) with G2-PAMAM to obtain a dendrimer PAMAM-1B4M modified with an average of 14.3 1B4Ms on the surface, reacted it with an excess of sulfosuccinimidyl 6- [3- (2-pyridyldithio) propionamide] hexanoate (sulfo-LCSPDP) and solid dithiothreitol, filtered and purified
[38]. At the same time, sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfoSMCC) was reacted with the humanized antibody HuTac to obtain sulfoSMCC-modified HuTac (SMCC-HuTac). Then the PAMAM-1B4M with thiol group was coupled with SMCC-HuTac, and the humanized antibody and 1B4M-modified dendrimer (HuTac-PAMAM-1B4 M) were obtained by high performance liquid chromatography (HPLC) and size exclusion chromatography (SEC). The final labeling of PAMAM-1B4M or HuTac-PAMAM-1B4M with radionuclide
111In and yttrium -88(
88Y) results in an unsaturated formulation containing less than one radionuclide (
111In/
88Y) label per dendrimer. Subsequently, saturated PAMAM-1B4M and HuTac-PAMAM-1B4M labeled with
111In/
88Y were prepared by the same method using cold indium and yttrium chloride, and the radiolabeling efficiency was higher than 85%. Studies have shown that the biodistribution of saturated and unsaturated agents in tumor-bearing mice is different. Normal mice, A431 tumor-bearing mice and ATAC4 tumor-bearing mice injected with
111In labeled saturated/unsaturated HuTac-PAMAM-1B4M compounds were studied with
111In-HuTac-1B4M as control. It was found that the accumulation of unsaturated PAMAM-1B4M labeled with
111In was significantly higher than that of saturated PAMAM-1B4M in liver, kidney, spleen and bone at most time points. The liver uptake of the
111In labeled HuTac-PAMAM-1B4M saturated formulation (27% ± 2% ID/G) was lower than that of the unsaturated formulation (32% ± 2% ID/G), but higher than that of the
111In-HuTac-1B4M control (10% ± 0% ID per gram). The blood clearance of saturated/unsaturated HuTac-PAMAM-1B4M labeled with
111In was higher than that of HuTac-1B4M labeled with
111In. The biodistribution of HuTac-PAMAM-1B4M in tumor-bearing mice was similar to that in normal mice, but the accumulation of HuTac-PAMAM-1B4M in specific tumor ATAC4 was significantly higher than that in non-specific tumor A431. The accumulation of radiolabeled PAMAM-1B4M and HuTac-PAMAM-1B4M in the liver, kidney, and spleen decreased as the chelate was saturated with the stabilizing element, suggesting that the properties of PAMAM itself contribute to differences in biodistribution.
At present, the constructed 111In labeled functional dendrimers are often used as SPECT imaging tracers, which can be used in biomedical applications in vivo based on their high radiolabeling efficiency and radioactive stability. The construction methods of dendrimers labeled with 111In for SPECT imaging contrast agents are similar. Based on a dendrimer nano platform, various functional molecules and labeling carriers are modified on the surface of the dendrimer nano platform, and then radionuclides are labeled to obtain the corresponding SPECT imaging contrast agent through 111In. SPECT imaging contrast agents with different functions can be obtained based on the differences in biodistribution and metabolism between dendrimers of different generations. The modification of specific antibodies (e.g., HuTac) allows the functionalized dendrimers to be efficiently enriched in specific tumors (e.g., ATAC4). In general, the introduction of dendrimers as a nano-platform to label radionuclide 111In not only makes it have higher radiolabeling efficiency and stability, but also can regulate its distribution and metabolism in vivo and give it functionalization. Since the half-life of radionuclide 111In is 2.83 days, which is relatively moderate, in order to maximize the imaging effect of SPECT in vivo.In the design of 111In labeled dendrimer SPECT imaging contrast agents, nano-carriers (such as dendrimers of different generations) with moderate blood half-life in vivo are often selected, and PEG-modification is reduced to avoid long-term retention in vivo, so as to effectively perform SPECT imaging and biomedical monitoring in vivo. Although functional modification and the selection of dendrimers with different generations can optimize the SPECT imaging effect, there are still many defects, such as the inability to be excreted from the body quickly after metabolism, which leads to the decline of imaging quality. In addition, the difference of structure and physical properties of different functionalized dendrimers will lead to the difference of biological distribution of functionalized dendrimers labeled by 111In in various organs in vivo, thus affecting the imaging effect of SPECT. Therefore, it is very important to improve the specific distribution of 111In labeled dendrimer SPECT imaging contrast agent in specific tissues in vivo, reduce the accumulation and uptake of non-specific organs, and then improve the SPECT imaging efficiency of target tissues. At present, the construction of new functional dendrimer labeling carriers still needs further study, which will lay the foundation for the construction of precise and efficient SPECT imaging contrast agents in the future.
4 125I labeled dendrimer SPECT imaging contrast agent
Compared with other radionuclides,
125I has a very long half-life (60 d) and a lower gamma-emitting energy (27. 4 ~ 35.4 keV), which is often used as a marker in animal radioimmunoassay or preclinical in vivo experiments. Similar to other iodine-based radioisotopes (e.g.,
123I,
124I),
125I has a wide range of applications, based on its own low-energy internal conversion electrons, which can be used for autoradiography and biopsy of thyroid tumors
[23,24].
Malik et al. Used several common dendrimers, including PAMAM, polypropyleneimine dendrimer with 1,4-butanediamine (DAB) or ethylenediamine (DAE) as the core, and carbosilane grafted ethylene oxide dendrimer (CSi-PEO) (Figure 6), to systematically study the influence of the generation of dendrimers and surface functional groups on their in vitro biological properties by labeling radioactive
125I
[39]. The results showed that PAMAM dendrimers, usually with an amino terminus, exhibited concentration-dependent and algebra-dependent hemolysis, and morphological changes of erythrocytes were observed after 1 H even at low concentrations (10 μg/mL). At concentrations below 1 mg/mL, CSi-PEO dendrimers and dendrimers with carboxyl (COOH) termini showed neither hemolysis nor cytotoxicity. In conclusion, PAMAM cationic dendrimers with amino groups on the surface were cytotoxic after incubation for 72 H, and the median lethal dose (IC
50) was about 50~300μg/mL,IC
50, depending on the type of dendrimer, the generation and the cell type used. After intravenous injection of the third and fourth generation of cationic PAMAM (10 μg/mL) labeled with
125I into rats, it was observed that the dose in the blood was less than 2% 1 H after injection, indicating that it could be quickly cleared from the blood circulation.
However, the anionic PAMAM dendrimers modified with carboxyl groups on the surface of the 2.5, 3.5 and 5.5 generations had longer circulation time in vivo, and the dose in blood reached 20% -40% 1 H after injection. The blood clearance rate of anionic PAMAM was related to the generation of dendrimers, and the lower the generation, the longer the blood circulation time. The concentration of anionic and cationic dendrimers in blood was related to the liver uptake (about 30% -90%) of the dendrimers in rats 1 H after injection. Therefore, to reduce liver uptake when it is necessary to target other focal sites (such as tumors), the structure of dendrimers needs to be carefully designed to optimize their distribution in vivo.
Wiwattanapatapee et al. studied the effects of the size, charge and concentration of cationic and anionic PAMAM dendrimers labeled with
125I on the absorption and transport of the rat small intestine in vitro using the everted rat intestinal sac system
[40]. Firstly, the third and fourth generation cationic PAMAMs (G3 and G4) with surface modified amino groups and the second, third and fifth generation anionic PAMAMs (G2.5, G3.5 and G5.5) with surface modified carboxyl groups were labeled with radioactive
125I by using the Bowton-Hunter reagent hydroxyphenylpropionic acid N-hydroxysuccinimide ester to obtain various types of PAMAM labeled with radionuclide
125I. Subsequently, these
125I labeled dendrimers (10 to 100 μg/mL) were incubated in vitro with the adult rat in vitro gut pouch eversion system for 2 H, and radioactivity was transferred to tissues and serosal fluid over time. The results showed that all generations of anionic PAMAMs had very high serosal transfer rates, with the volume of medium containing dendrimers transferred per mg protein per hour ranging from 3.4 to 4.4 μL (EI = 3.4 to 4.4 μL/mg protein/H). When the concentration of dendrimers was in the range of 10 ~ 100 μg/mL, the serosal transfer rate was linear with the concentration of dendrimers. However, the tissue uptake rate was higher than that of anionic G2.5 labeled with
125I and anionic G5.5 labeled with G3.5(EI=0.6~0.7μL/mg protein/h),
125I (EI = 2.48 ± 0.51 μL/mg protein/H). The tissue uptake rate (EI = 3.3-4.8 μL/mg protein/H) of
125I labeled cationic PAMAMs (including G3 and G4) was higher than the serosal transfer rate (EI = 2.3-2.7 μL/mg protein/H), which may be due to the nonspecific adsorption of cationic PAMAMs on the mucosal surface. Anionic PAMAM labeled with
125I has great potential to be developed as an oral drug delivery system because of its higher serosal transfer rate than other synthetic macromolecules in the everted intestinal sac system of rats.
At present, the constructed dendrimer SPECT imaging contrast agent marked by the 125I is mainly used for analyzing and researching the biomedical performance and the distribution of the dendrimer in vivo, and has good imaging guidance and tracing effects. At present, the known 125I labeled dendrimer SPECT imaging contrast agents often compare the influence of different generations and surface groups on their biological properties, and evaluate their blood circulation time in vivo, organ distribution and absorption. In the construction of contrast agents, tyrosine and its analogues (such as hydroxyphenylpropionic acid N-hydroxysuccinimide ester) are often used as chelating agents for 125I labeling, and the labeled products have high radiolabeling efficiency and good radioactive stability. Based on the long radioactive half-life (60 days) of 125I, it can support long-term radioactive imaging tracing in vivo. Therefore, in order to effectively monitor the distribution of functionalized dendrimers labeled with 125I in vivo, different surface groups are modified and different types and generations of dendrimers are selected to improve their biocompatibility in vivo, and their blood circulation time in vivo is regulated to prolong their SPECT monitoring time in vivo as much as possible. The 125I labeled dendrimer SPECT imaging contrast agent constructed by this design strategy can be used for long-term monitoring and prognosis evaluation after treatment in vivo. However, there are also some defects, such as the high price of 125I, which is 3 to 4 times that of 131I; Its half-life is as long as 60 days, which has a long-term radiation effect on various tissues and organs in vivo, and it takes longer time to completely decay. Therefore, in order to reduce the radiation damage of 125I and optimize its biodistribution in vivo to achieve specific imaging monitoring, further research is needed to construct new 125I labeled dendrimer SPECT imaging contrast agents to improve their biodistribution in vivo and reduce radiation damage.
5 131I labeled dendrimer SPECT imaging contrast agent
The radioactive 131I can simultaneously emit gamma rays and beta rays for SPECT imaging and radiotherapy respectively, and the SPECT imaging tracing is used as a guide while the radiotherapy is carried out. However, due to the nature of small molecular radioactive 131I, it only has strong affinity for thyroid, but has no tissue specificity for other tissues and lesions. Therefore, it is often used to monitor and treat thyroid cancer, which greatly limits the scope of application of radionuclide 131I. Dendrimers, as an excellent nano-platform, have the characteristics of good biocompatibility and functional modification, which can endow 131I with certain tissue specificity and improve their radiostability, and then realize SPECT imaging and radiotherapy of different types of tumors in vivo, so as to broaden their biomedical applications.
Zhu et al. modified the surface of G5.NH
2 with 3- (4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester (HPAO) and PEG-FA, acetylated the remaining amino groups on the surface of the dendrimer, and finally labeled the
131I on the surface of the functionalized dendrimer through HPAO to prepare a radioactive
131I labeled functionalized dendrimer
131I-G5.NHAc-HPAO-PEG-FA (fig. 7)
[41]. The results showed that the average number of HPAO on the surface of each functionalized dendrimer was 9.4. The obtained functionalized dendrimer has good biocompatibility, has no cytotoxicity when the concentration is as high as 20μmol/L, and can specifically bind to cancer cells with high folate receptor expression. Based on the phenol structure of HPAO, the radioactive
131I can be effectively labeled at the ortho position of the phenolic hydroxyl group, thereby indirectly labeling the surface of the functionalized dendrimer. The obtained
131I-G5.NHAc-HPAO-PEG-FA has higher radioactive stability and radiochemical purity, and has good effects of SPECT imaging and radioactive therapy of targeted high folate receptor expression tumors. This strategy of combining dendrimer nanotechnology with radionuclides provides a new direction for the development of nanoplatforms for SPECT imaging and radiotherapy for different types of cancer.
Cheng et al prepared G5.NH
2-mPEG by EDC coupling reaction of G5.NH
2 and methoxy-polyethylene glycol-carboxyl (mPEG-COOH), and then modified maleimide-polyethylene glycol-succinimidyl valerate (MAL-PEG-SVA), Buthus martensii Karsch chlorine toxin (BmK CT-SH) and HPAO on the surface of G5.NH
2 to obtain functional dendrimer G5.NH
2-HPAO-(PEG-BmK CT)-(mPEG)
[42]. And then that remain amino groups on the surface of the dendrimer are acetylate, and finally the radionuclide
131I is labeled by a chloramine trihydrate (ch-T) method to obtain the product
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG). The radiochemical purity of the obtained
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) was 99. 5% ± 0.7% by instantaneous thin layer chromatography (ITLC). Its radiochemical purity was higher than 91% even when it was dissolved in PBS or FBS for 24 H. The results show that the
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) has good radiochemical purity and radiostability. NHAc-HPAO- (PEG-BmKCT)- (mPEG) was found to be cytotoxic to rat lung epithelial cell line (RLE-6 TN) and C6 glioma cells in a certain concentration range (0. 1-50 μmol/L), and the cytotoxicity of G5.NHAc-HPAO- (PEG-BmKCT)- (MPEG) was over 90%. BmK CT can specifically bind to the matrix metalloproteinase-2 (MMP-2) receptor on the surface of glioma cells, so the
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) has good targeting specificity on glioma cells in vitro and in vivo
[43⇓~45]. In particular, the in vivo SPECT imaging results showed that the concentration of the constructed
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) in the tumor site reached a peak at 15 H after injection, and the constructed
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) showed significantly stronger SPECT signal in the tumor site than the non-BmK CT modified functionalized dendrimer
131I-G5.NHAc-HPAO-(PEG-MAL)-(mPEG). At each time point after injection, the SPECT signal value of the
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) at the tumor site was higher than that of the non-BmK CT-targeted
131I-G5.NHAc-HPAO-(PEG-MAL)-(mPEG) (fig. 8). In addition to the function of SPECT imaging,
131I also has the function of radiotherapy. By evaluating the tumor growth rate, it was found that the tumor growth rate of C6 tumor-bearing mice treated with
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) was significantly lower than that of C6 tumor-bearing mice treated with other reagents (Na
131I,
131I-G5.NHAc-HPAO-(PEG-MAL)-(mPEG)) (fig. 9), reflecting that the modification of BmK CT greatly enhanced the specific enrichment of
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG)) at the tumor site, thereby enhancing its anti-tumor effect. The constructed
131I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) can not only realize the function of targeted glioma SPECT imaging, but also has the efficacy of targeted radiotherapy.
Zhao et al. Also used G5.NH
2 dendrimer as a nano platform, used the functionalized dendrimer G5.NH
2-(PEG-MAL)-(mPEG) modified by mPEG-COOH and MAL-PEG-SVA as a template, indirectly connected the targeting reagent chlorotoxin (CTX) to the surface of the functionalized dendrimer through the maleimide group (MAL) in MAL-PEG-SVA, and then further modified HPAO, and finally labeled the radioactive
131I to obtain the CTX-targeted radioactive diagnostic and therapeutic reagent
131I-G5.NHAc-HPAO-(PEG-CTX)-(mPEG)
[46]. The results showed that the average number of CTX and HPAO loaded on each dendrimer was 7. 7 and 21. 1, respectively, and the dendrimers had good cytocompatibility in a certain concentration range (0-20 μmol/L). Based on the surface modification of CTX, the obtained
131I-G5.NHAc-HPAO-(PEG-CTX)-(mPEG) can specifically bind to cancer cells with overexpression of MMP2, thereby realizing targeted glioma SPECT imaging and radiotherapy.
At present, the dendrimer SPECT imaging contrast agent marked by 131I has broadened the scope of its application in nuclear medicine to a certain extent, and can achieve good SPECT imaging and radiotherapy effect for different types of tumors. The construction method and the idea are similar to those of the dendrimer SPECT imaging contrast agent labeled by 125I, and tyrosine analogue HPAO is covalently bonded on the surface of the dendrimer after functional modification so as to effectively and stably label the 131I on the phenol structure of HPAO. Dendrimer-based nanotemplates are often modified with biocompatible molecules (such as PEGs) and targeting agents (such as FA, BmK CT, CTX, etc.) To improve their biocompatibility in vivo and give them tumor targeting specificity. The 131I can be effectively and stably brought into the lesion site for SPECT imaging and radiotherapy by using the nano-medium such as dendrimers. At present, based on the physical and chemical properties of 131I, the relatively long radioactive half-life (8.01 days) and the characteristics of emitting gamma rays and beta rays simultaneously for SPECT imaging and radiotherapy, respectively, G5.NH2 dendrimers are used as a nano platform.Functionalized PEGs and targeting agents (such as FA, BmK CT, CTX, etc.) Are commonly used to modify the nanoparticles to prolong the blood circulation time in vivo and improve the enrichment of the lesion site, thereby prolonging the imaging time of SPECT in vivo and improving the efficiency of radiotherapy. The longer the PEGs with higher molecular weight are used, the longer the retention time of the functionalized dendrimers in the body will be. The functional modification method can be used for designing and constructing the SPECT imaging contrast agent which effectively meets the application requirements of the 131I. Nanotechnology based on dendrimers can achieve long-term circulation in vivo, which is conducive to improving its enrichment in tumor sites. However, there are still some imperfections, most of the nanoparticles still can not escape the uptake of reticuloendothelial system (such as liver, spleen, etc.), and will still be enriched in normal tissues and organs, which reduces their retention in tumor sites, thus affecting the efficiency of SPECT imaging and radiotherapy. Therefore, how to reduce the uptake of reticuloendothelial system to further improve the accuracy of SPECT imaging and the efficiency of radiotherapy still needs further study, which also provides ideas for the subsequent construction of novel intelligent SPECT imaging contrast agents.
6 Conclusion and prospect
This review summarizes the construction of dendrimer-based SPECT imaging contrast agents and their biomedical applications, and elaborates on the development of dendrimer-based SPECT imaging contrast agents labeled with 99mTc, 111In, 125I and 131I, as well as their unique nuclear medical properties in vitro and in vivo. Through the comparison of these radionuclides, it can be seen that they have different radioactive half-life and emission energy, and according to the difference of radioactive half-life, the appropriate functionalized dendrimer nano-carrier can be selected to control the SPECT imaging time in vivo. For example, 99mTc is the most commonly used radionuclide for SPECT imaging, which has a short radioactive half-life (6 H) and a low gamma-ray energy (140 keV). As a result, only short-term SPECT imaging can be performed in vivo, and long-term SPECT monitoring and prognosis evaluation after treatment can not be carried out. However, the short-term nuclear medical decay in the body also ensures the safety of radiation in the body. Although 111In has a moderate radioactive half-life (2. 83 d), its basic research and clinical application are less in China, because of its high cost of preparation, difficulty in obtaining it from China, and high price of importing it from abroad, which is far less widely used than 99mTc. Secondly, the emission energy of 111In is higher, and the safety of internal radiation is not as good as that of 99mTc. Although 125I and 131I are both isotopes of radioactive iodine, their physical and chemical properties are quite different. The radioactive half-life of 125I (60 days) is much longer than that of 131I (8.01 days), which means that 125I is more suitable for long-term imaging monitoring and prognosis in vivo, but its radiation damage to organisms lasts longer, which has both advantages and disadvantages. Secondly, the 125I has better SPECT imaging resolution than the 131I, and the imaging effect is relatively good. The 131I is based on the fact that the SPECT imaging function and the radiotherapeutic effect can be achieved at the same time, and the SPECT imaging is often used as a tracing guidance while the radiotherapeutic treatment is carried out, so that the treatment is monitored at the same time, and the use is convenient. However, the resolution of SPECT imaging in 131I is low, and it is difficult to perform accurate imaging. Although all kinds of radionuclides have unique physical and chemical properties and application scope, if we want to achieve the best in vivo SPECT imaging effect, we also need to combine the functional dendrimer nano-platform loaded with the radionuclide.The radioactive half-life of the matched nuclide and the in vivo blood half-life of the functionalized dendrimer are discharged out of the body after the optimal SPECT imaging in the body is completed.
By summarizing the functional characteristics of various radionuclide-labeled dendrimer SPECT imaging contrast agents, we can see that based on dendrimer nanotechnology and its excellent physical and chemical properties,Can be used as a nano platform to efficiently mark various radionuclides, and can improve the radioactive stability, the biocompatibility and the enrichment amount of the radionuclide marker at a focus part through functional modification. At the same time, the circulation time and tissue distribution of various SPECT imaging contrast agents should be adjusted according to their needs, so as to improve the efficiency of SPECT imaging. At present, various radionuclide-labeled dendrimer SPECT imaging contrast agents have been developed, which have significantly improved the shortcomings of small molecule nuclides in biomedical applications in vivo. However, there are still some shortcomings, although SPECT imaging has high sensitivity, its spatial resolution is still low compared with other imaging modes, so it needs to cooperate with other imaging modes to make up for its lack of imaging resolution by building multi-modal imaging. Therefore, the construction of more accurate and sensitive dendrimer-based multi-modality imaging contrast agents to make up for the defects of SPECT imaging mode has become the focus of future research, which will lay the foundation for accurate and efficient diagnosis.
the National Natural Science Foundation of China(21807059)
the Natural Science Foundation of Jiangsu Province(BK20180711)
the Natural Science Foundation for Colleges and Universities in Jiangsu Province(17KJB350005)
and the Postgraduate Research & Practice Innovation Program of Jiangsu Province(KYCX20_1122)
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