^99mTc is accessible in the 7⁺ oxidation state by reducing ^99mTcO₄^– to a lower oxidation state using the stannous chloride. Ascorbic acid, as an antioxidant, is usually added to the solution of ^99mTc complex to keep it stable. The pH at which the complex is stable is 7.0 because it must be used in biological tissues. Then, Tc can tightly bind to a single specific atom or a small part of the chelating group. For instance, 2-hydrazinonicotinic acid (2-HYNIC), a bifunctional chelator suitable for Tc, easily attaches to bioactive molecules through an amidification reaction [30]. Since the coordination of HYNIC is possible from the nitrogen atom of its hydrazine moiety, it cannot saturate the coordination capacity of ^99mTc, thus, the use of co-ligands is essential to complete the coordination sphere. Tricine, ethylenediaminediacetic acid (EDDA), nicotinic acid, pyridine dicarboxylic acid (PDA), glucamine, mannitol, and glucoheptonic acid are commonly used co-ligands to construct ^99mTc-HYNIC complexes [31]. Figure 1 illustrates the process of binding HYNIC to a bioactive molecule and subsequently forming complexes with ^99mTc using co-ligands. Studies showed that tricine and EDDA give the most stable radiochemical complex even up to one day post-incubation [32]. Therefore, HYNIC has been recently considered to bind to antibodies, fatty acids, proteins, and peptides via the formation of amide bonds, followed by coordinating with ^99mTc to be used for radio imaging, diagnosing, and monitoring various targets and diseases, including activated T lymphocytes, pancreatic neuroendocrine neoplasms, cancer, and carcinoma cells [33–40].

BBN is a 14-amino-acid peptide (pGlu-Gly-Arg-Leu-Gly-Thr-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂) that binds to G protein-coupled receptors (GPRs). The latter is overexpressed in different kinds of human cancer cells, especially breast, lung, and prostate cancers (PCs) [41]. Faintuch et al. [42] prepared a series of ^99mTc-HYNIC-linker-BBN to compare their biodistribution and scintigraphy imaging in mice bearing PC-3 tumor cells. They found that ^99mTc-HYNIC-βAla-BBN is rapidly produced during the radiolabeling step with no need for purification, bears high radiochemical efficiency with more with internalization (12% within 30 min) and tumor uptake [32, 42–44]. Inspired by the mentioned research, de Barros et al. [45] prepared a pH-sensitive liposome encapsulating ^99mTc-HYNIC-βAla-BBN_(7–14) and used it to detect human breast cancer. The prepared nano-liposome with an approximately 165 nm diameter displayed a strong signal in the tumor tissue, showing a good tumor-to-muscle of 9.31% injected dose (ID)/g. It was also enclosed that ^99mTc-HYNIC-βAla-BBN_(7–14) can recognize Capan-1 pancreatic adenocarcinoma at its early stage with an uptake of 0.47% ID/g [46], and LNCaP prostate tumor [47]. Another research revealed the applicability of ^99mTc-HYNIC-BBN or imaging women’s breasts with malignant tumors [48]. Later, Faintuch et al. [49] could design a ^99mTc-mercaptoacetyltriglycine (MAG3) complex coupled to BBN via a 6-aminohexanoic acid (6-Ahx) linker (^99mTc-MAG3-Ahx-BBN). The RCP of this complex was approximately 96% at neutral pH, exhibiting high internalization (75% within 30 min) and great affinity for BBN receptors [49]. Following their research, Faintuch et al. [50] compared the ability of ^99mTc-MAG3-Ahx-BBN and ^99mTc-MAG3-Ahx-DUP1 to diagnose prostate carcinoma. DUP1 is a synthetic peptide (Phe-Arg-Pro-Asn-Arg-Ala-Gln-Asp-Tyr-Asn-Thr-Asn) with high affinity for DU-145 prostate and PC-3 cells [51]. The mentioned study demonstrated that the DUP1 tracer was more hydrophilic than the BBN one, with greater kidney uptake. However, due to its higher specificity to receptors of gastrin-releasing peptide, the BBN tracer displayed superior internalization (78%), while tumor uptake for both tracers was comparable [50]. Many other BBN-based ^99mTc-radiotracers have been developed through similar protocols to improve stability and high uptake in target tissues [52–59]. Moreover, the focus of some research is on the preparation of multifunctional systems containing BBN-based ^99mTc-radiotracers conjugated to the surface modified nanoparticles (NPs) with target-specific molecular recognition [60–64].

Pretargeting involves the use of high affinity and specificity biomarkers to obtain a high contrast of target to the background, improving the tumor-to-nontumor ratio [65–67]. Morpholino oligomers (MORFs) contain DNA bases in their scaffold attached to morpholine rings through phosphonodiamidite groups. These synthetic oligomers are widely employed for nuclear-pre-targeted imaging [68–72]. Since binding MORF to a carrier through a covalent bond is difficult, the utilization of streptavidin (SA) as a linker makes it easy to attach a biotinylated carrier to biotinylated MORF via simple mixing. Consequently, Faintuch et al. [73] prepared ^99mTc-MAG3-cMORF as well as Biotin-βAla-BBN and Biotin-MORF and mixed them in the presence of SA to have a ^99mTc-labeled nano-peptide to image lymph nodes bearing tumor cells.

SST is a kind of cyclic peptide hormone, containing 14 or 28 amino acids, which plays its role in different ways, including interaction with G protein-coupled SST receptors to control cell proliferation, regulation of cellular functions, and inhibiting the release of other hormones, such as insulin and glucagon secretion [74–76]. The peptidases distributed in plasma and tissues rapidly degrade the natural SST, therefore, its highly short life (1–3 min) makes it useless in clinical cases [77]. Consequently, many analogs of SST have been developed regarding the clinical approaches. Lanreotide or somatuline is an SST-analogue octapeptide, containing 8 amino acids (D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂) cyclized through a disulfide bond between two cysteine moieties. Lanreotide can manage the symptoms resulting from neuroendocrine-active tumors [78]. In the early 2000s, its ^99mTc complex was considered to be used in radio imaging of neuroendocrine tumors. Consequently, ^99mTc-lanreotide was prepared in a tartrate-phthalate buffer solution, containing maltose, glycine, and SnCl₂ solution, followed by the addition of ^99mTc. This complex was stable for 6 h, mainly distributed in the gastrointestinal tract, showing its applicability in radiodiagnosis [79]. However, the study of this complex was of interest only in those years and forgotten later.

Octreotide (OCT) and octreotate (TATE) are more attractive than lanreotide in radio imaging studies for the detection of unrespectable neuroendocrine tumors. To aim for this, Melo et al. [80] developed the synthesis of ^99mTc-HYNIC-Tyr³-OCT and ^99mTc-HYNIC-Tyr³-TATE (Figure 2) and investigated their biodistribution. They found fast blood clearance and high biodistribution of OCT and TATE in the pancreas, intestine, stomach, lung, and blood. The great uptake of these radio-drugs in the kidney and pancreas is due to the high density of SST receptors. Moreover, low uptake was observed in bones, liver, spleen, heart, brain, and thyroid [80, 81]. In another research, OCT was loaded on Au NPs coated with Lys³-BBN or mannose (Figure 3). The prepared radio-agents were useful to detect sentinel lymph nodes [82, 83]. The time-consuming preparation of such loaded radio drugs is one of the drawbacks of this method, which limits its clinical application.

Exendin-4 is an analog of glucagon-like peptide 1 (GLP-1), which can attach to GLP-1 receptors, expressed in patients who suffer from insulinomas, a kind of small pancreatic endocrine tumor [84–86]. Currently, exendin-4 is consumed for the treatment of type 2 diabetes [87, 88]. Because radiolabeled exendin_(9–39) has the potential to detect GLP-1 receptors and also OCT can identify SST receptors, a combination of HYNIC-exendin_(9–39) and OCT (Figure 4) can detect malignant insulinomas pancreatic tumors, which express high and low densities of GLP-1 and SST receptors, respectively. Biodistribution studies of ^99mTc-HYNIC-exendin_(9–39)-OCT showed suitable uptake in target tumor cells (2.71% ID/g), blood (1.5% ID/g), and kidney (95.0% ID/g) and it was rapidly eliminated from renal after 2 h [89–92].

RGD is a tripeptide comprising Arg-Gly-Asp residue, which is responsible for cell adhesion to the extracellular matrix [93]. RGD peptides have the potential to be used in tissue engineering, therapy, and imaging [94, 95]. It has been validated that some kinds of cyclic peptides containing RGD residue can bind integrin α_vβ₃, which is linked to the progress of various diseases [96]. It must be noted that a cyclic RGD-pentapeptide, comprising of Arg-Gly-Asp-Tyr-Lys [c(RGDyK)], is a derivative of RGD with a high affinity for α_vβ₃ integrin, which is expressed on the cell membrane of many cancerous cells [97, 98]. Decristoforo et al. [99] synthesized c(RGDyK) to make ^99mTc-HYNIC-c(RGDyK), as depicted in Figure 5. High uptake of the prepared radiotracer was observed in α_vβ₃-integrin-receptor-positive M21 melanoma cells up to 2.73% ID/g, while tumor-to-organ ratios were equivalent to that observed for [¹⁸F]Galacto-RGD.

^99mTc-MAG3 is frequently employed for renal and kidney function imaging [100, 101]. The carboxyl group of MAG3 can bind to a linker molecule, carrying a biomolecule. As shown in Figure 6, ^99mTc-MAG3 is attached to c(RGDyK) via a polyethyleneglycol linker, forming ^99mTc-MAG3-PEG8-c(RGDyK). It was revealed that ^99mTc-MAG3-PEG8-c(RGDyK) traces malignant melanoma cells, SK-MEL-28, after 30 min of incubation with an internalization of 96.13%. Due to its specificity for human SK-MEL-28 cells, this radiotracer is efficient for the early diagnosis of melanoma [102, 103]. Moreover, this tracer was employed for imaging diverse tumor models, including blood, lungs, kidneys, spleen, stomach, pancreas, liver, intestine, muscle, and bone carcinoma. The results revealed its high biodistribution in lung cancer cells, presenting the applicability of ^99mTc-MAG3-PEG8-c(RGDyK) tracer to detect lung tumors in addition to melanoma cells [104]. It must be noted that polyethyleneglycol protects peptides from enzymatic degradation and thus decreases proteolysis, also increases overall hydrophilicity of the compound, leading to the increased peptide T_½ and stability [105]. Schiper et al. [106] prepared HYNIC-E-[c(RGDfK)]₂ (Figure 7) and made its complex with ^99mTc to test its biodistribution on Swiss mice infected with osteonecrosis. This radiotracer showed the highest bone uptake after 15 days and could detect bone infarction efficiently [106]. In another research, c(RGDfK) was grafted on the surface of gold NPs modified with ^99mTc-HYNIC-GGC, where GGC is Gly-Gly-Cys residue. This NP showed high specificity for detection of α_vβ₃-integrin-receptor-positive M21 melanoma cells [107].

Based on the previously published method [108, 109], Caporale et al. [110] synthesized a small cyclic pentapeptide of c(RGDfV), containing cyclized Arg-Gly-Asp-D-Phe-Val, which is an analogue of c(RGDyK). Then, two complexes of ^99mTc-c(RGDfV) were prepared using nitrido nitrogen atoms (Figure 8). These complexes were stable after incubation for 4 h at 37°C in biological serum [110].

GX1 (Cys-Gly-Asn-Ser-Asn-Pro-Lys-Ser-Cys) is one of the high-affinity peptides with angiogenesis that can inhibit it. Accordingly, GX1 has been widely used for targeting and imaging cancer cells because of its binding specificity to integrin α3β1 receptors [111–113]. Correspondingly, binding the RGD peptide to GX1 may improve tumor cell affinity in radio imaging. In this regard, cyclized GX1 was introduced in two complexes with ^99mTc, i.e. ^99mTc-HYNIC-PEG4-c(GX1) and ^99mTc-HYNIC-E-[c(RGDyK)-c(GX1)] (Figure 9). Both prepared tracers showed considerable stability after 4 h remaining in human serum under physiological conditions. They had great hydrophilic features with great renal excretion, however, the clearance of ^99mTc-HYNIC-PEG4-c(GX1) from the blood was faster. After 5 min of incubation (0.41%), about 51% of the latter was internalized, while ^99mTc-HYNIC-E-[c(RGDyK)-c(GX1)] showed the highest binding value after 2 h of incubation (0.35%). Comparable biodistribution was observed for both tracers in most organs [114, 115]. Later, their ability in radio imaging was established, revealing better visualization, favoring tumor uptake (2.96% at 1 h), and highest binding (1.14%) at 2 h for ^99mTc-HYNIC-E-[c(RGDyK)-c(GX1)] in glioma U87MG cells [116]. Moreover, magnetic resonance imaging (MRI) confirmed the specific binding of these tracers to human U87 glioblastoma in the brain [117]. Another research signified that a remarkable uptake can be obtained by injecting ^99mTc-HYNIC-PEG4-c(GX1) into mice bearing B16F10 and SK-MEL-28 melanoma cells (1.41% and 2.42%, respectively) at 1 h [115].

In another study, a hexapeptide of Gly-Arg-Gly-Asp-His-Val (GRGDHV) was synthesized as an RGD analog, and labeled with a tricarbonyl complex of ^99mTc, as illustrated in Figure 10. However, the sites of complexation of peptide with ^99mTc have not been determined. This complex was stable for 24 h in human serum under biological conditions. Moreover, after 1 h of incubation, ^99mTc(CO)₃-GRGDHV showed distinct binding in C6 tumorigenic cells with bound and internalized fractions of 22% and 34%, respectively. The accumulation of this radiopeptide was observed in the brains of glioblastoma allograft tumor-bearing rats and normal rats, showing biodistribution up to 1.57 and 0.6, respectively, at 4 h. The results showed promising application of this radiolabeled peptide in the clinical diagnosis of glioblastoma [118].

NT is a neuropeptide, containing 13 amino acids of pyroGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu, playing its role in the central nervous system through interaction with dopamine receptors, smoothing muscle contraction, and regulation of luteinizing hormone and prolactin release [119–121]. Regarding the published results for the application of NT analogues in tumor imaging [122], Teodoro et al. [123] investigated the bioactivities of NT_(8–13) analogue (Arg-NMe-Arg-Pro-Tyr-Ile-Leu) using its complex ^99mTc-HYNIC-βAla-NT_(8–13). Considerably high uptake of this tracer was observed for the lung, blood, and kidneys, while it was rapidly eliminated from the blood. Moreover, low uptake of this tracer was detected in the liver and intestine, showing its potential to be used in tumor imaging due to its high uptake and fast clearance from the blood [123].

The cationic peptide LyeTx I is a natural 25 amino acid peptide, isolated from Lycosa erythrognatha venom, exhibiting antimicrobial activities [124–126]. Fuscaldi et al. [127] designed two chelating agents for ^99mTc using C- and N-terminus of LyeTx I and HYNIC to give LyeTx I-K-HYNIC and HYNIC-LyeTx I, respectively. Then, the bioactivities of these agents were determined against Staphylococcus aureus and Escherichia coli, and it was discovered that the latter couldn’t inhibit the growth of bacterium, while the former LyeTx I-K-HYNIC showed antibacterial properties with minimum inhibitory concentration (MIC) values of 5.05 μmol/L and 10.01 μmol/L against the mentioned species, respectively. Accordingly, ^99mTc-LyeTx I-K-HYNIC complex was prepared with high purity to study infected tissues [127, 128]. In that study, researchers evaluated the biodistribution of two peptidoglycan aptamers, Antibac1 and Antibac2, which were labeled with ^99mTc. These aptamers were specifically designed for bacterial infection diagnosis. The results exhibited that these tracers can easily recognize a bacterial infection focus [129].

Recently, ubiquitin (UBI) peptide is a cationic, synthetic antimicrobial peptide fragment. It has been considered a versatile antibacterial agent to be labeled with ^99mTc [130–132], which is useful for distinguishing bacterial infections. The use of radiolabeled peptides for infection imaging in humans has been an area of active research. Here are some key findings from clinical studies involving ^99mTc-UBI_(29–41) as an infection-imaging agent [133]. In 2005, a 29–41 fragment of UBI with the sequence of Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg was labeled with ^99mTc through coordination using its Lys and Arg7 amino acids. The obtained ^99mTc-UBI_(29–41) was injected into patients with osteomyelitis, diabetes, and fever of unknown origin. Imaging showed infected tissues, which were comparable with the results obtained by biopsy [134–136]. In another research, it was determined that the imaging results of infected tissues obtained by ^99mTc-UBI_(29–41) were in agreement with those recorded by ⁶⁷Ga-citrate. Moreover, after 24 h, nearly 85% of ^99mTc-UBI_(29–41) is eliminated by renal clearance [137]. Vallejo et al. [138] used this radiotracer to detect mediastinitis after cardiac surgery. Furthermore, ^99mTc-UBI_(29–41) was applicable to diagnose musculoskeletal [139] and postsurgical spinal infections [140].