1. Introduction
As gastrin-releasing peptide receptor (GRPR) is well acknowledged to be overexpressed in cancers, it attracts the attention of scientists dealing with novel delivery systems based on active targeting, where GRPR ligands are used as moieties able to lead to preferential accumulation of the nanoparticles in the sites of interest. These nanoparticles may act as drug delivery vehicles, so they deliver cytotoxic biological activity in the tumor site. On the other hand, detectable nanoparticles can be tracked in the human body using different imaging modalities, hence helping with cancer localization and staging. Finally, both these activities can be incorporated into one construct and act as theranostic, the idea derived from the personalized medicine approach, which connects oncological diagnostics and treatment at the same time. During the design of a targeted nanoformulation, fundamentally, three vital points need to be initially considered: the choice of the proper targeting ligand, the type of nanoparticles, and the means of bringing those together (Figure 1). These three aspects need to be fine-tuned to allow the successful development of nanosystems with anticipated properties and activity, particularly with respect to the desired mode of operation: therapeutic, diagnostic, or, combining both, theranostic (Figure 1).
Figure 1. Targeted nanosystems may perform different roles, but, fundamentally, three major aspects, carrier nanoparticle, targeting ligand, and means of bringing them together, must be fine-tuned to yield successful formulation. Created with BioRender.com.
There are certain trends that are noticeable in GRPR nanotargeting research. This receptor is overexpressed in numerous cancers, i.e., breast, colon, glioblastoma, head and neck squamous cell, lung, neuroblastoma, pancreatic, and prostate
[1]. However, the vast majority (ca. 70%) of the published studies concerning the use of nanosystems focus on applications in prostate or breast cancer research. It seems coherent, though, with the public health data pointing out that those malignancies are (next to colon and rectum) in the top three of cancer prevalence by type worldwide
[2]. However, when considering the type of nanoparticles exploited in the GRPR targeting research, almost half of the formulations are based on either gold nanostructures or liposomal/micellar vesicles, which are predominantly exploited in imaging and therapy, respectively. Those trends also seem well grounded, since, out of 25 clinically approved nanoformulations (U.S. Food and Drug Administration (FDA) and/or European Medicines Agency (EMA)), 10 were based on liposomes, proving the clinical potential of those structures
[3]. Gold nanostructures may have not reached such clinical success yet, but the combination of relatively simple, cheap, and well-understood synthesis, beneficial biological half-life, convenient and stable modification due to universal gold–sulfur interactions, and a wide range of activity (photothermal therapy, contrasting agent for photoacoustic imaging, radiosensitization, etc.) make them appear very promising and worth exploring.
2. Targeting Ligands
Ligands of GRPR can fall into one of two groups: agonists and antagonists. Agonists share the receptor activation sequence (Trp-Ala-Val-Gly-His-Leu-Met-NH
2) with GRP and activate the GRPR signaling pathways, leading to downstream effects such as cell growth, proliferation, increased motility, and invasiveness. It was pointed out, though, that these effects are not desired in clinically viable formulations. Moreover, some bodies of evidence suggest that antagonists may perform better as targeting ligands
[4]. Despite those flaws, surprisingly, most of the research published in the field of nanostrategies for targeting this receptor concerns the use of agonists. One of the possible reasons might be that internalization of the nanoparticle delivering the cargo, together with the receptor upon its activation, may be beneficial for some drug delivery applications and is convenient to track in vitro. However, actual reasons for this state of the matter remain to be elucidated and may be much more trivial, e.g., easy access and broad awareness of bombesin (BBN), the best known GRPR ligand, which happens to be an agonist.
The wide range of agonistic ligands has been shown in the literature. The relatively easiest approach is to use the commercially available native sequence of bombesin
[5]. Kulhari and co-workers exploited it in combination with biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles to successfully target both breast and prostate cancer cells and improve docetaxel (DTX) anticancer activity
[6][7][8]. The same ligand was also used to target the [
64Cu]CuS nanoparticles toward prostate cancer by Cai et al.
[9]. Interestingly, using native bombesin, Du, and Li investigated if the synthetic strategy, namely pre/post-functionalization of nanostructured lipid carriers, affects the performance of the nanosystem for targeted lung cancer combination therapy
[10].
Variations on the ligand structures start with exchanging single amino acids to engineer the sequence towards facilitated conjugation, for example, replacing the third amino acid from the native sequence with lysin
[11][12][13], therefore providing additional -NH
2 groups—such ligands are also commercially available
[5].
Nevertheless, much more sophisticated structures were also developed. One of the most prominent alterations in the bombesin architecture is to shorten the peptide chain down to an essential receptor binding sequence, Trp-Ala-Val-Gly-His-Leu-Met-NH
2, which can lower the price of the custom peptide synthesis. These short peptide chains can also be further engineered to provide convenient means of condensation with nanoparticles
[14][15][16], which will be discussed in more detail in the next subchapter. Frequently, the ligand is combined with poly(ethylene glycol) (PEG) to improve the pharmacokinetics of the construct and minimize the reticuloendothelial system uptake upon in vivo administration
[17][18].
Adding extra functionality to the structure represents a very attractive strategy for bombesin structure alterations. An example of such modifications is introducing the radioisotope chelation functionality to the ligand. Two main chelators used in such research are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and hydrazinonicotinic acid (HYNIC), and the isotopes used are
177Lu and
99mTc, respectively
[19][20]. Another interesting idea is to combine the bombesin binding motif with another peptide, creating heteromultimeric ligands, which can improve the construct targeting ability (e.g., RGD sequence against integrin α
υβ
3 [21][22], prostate-specific membrane antigen (PSMA)-inhibiting peptides for targeting of prostate cancer) or cellular penetration (e.g., Tat(49–57), HIV-1-derived sequence
[23][24]).
Regarding the antagonistic ligands, GRPR antagonists can be categorized into six classes, out of which, five are peptides/peptoids, while the last class comprises low-molecular flavone derivatives
[25]. Classes 2, 3, and 4 (in contrast to classes 1 and 5) are based on the modifications of the binding region of BBN. Class 2 is D-Phe12 analogs; class 3 focuses on modifications in positions 13–14 (or 26–27 when considering GRP sequence), while class 4 comprises analogs devoid of Met14 (desMet14). Among all the papers hereby reviewed, only six deal with the nanosystems exploiting antagonist ligands. Lahooti and colleagues used a class 2 antagonist with D-Phe12 substitution to successfully target their ultrasmall paramagnetic iron oxide nanoparticles (USPION) to GRPR-positive breast cancer xenograft in vivo
[26]. Particular attention was given to class 3 antagonist D-Phe-Gln-Trp-Ala-Val- NMeGly-His-Sta-Leu-NH
2 (BBN-AA1) by the group of Accardo. They successfully incorporated this sequence into amphiphilic derivative monomer, which was used to form liposomes loaded with doxorubicin (DOX)
[27]. The same monomer was also used to form sterically stabilized micelles as a targeted carrier of poorly water-soluble anticancer drug—gold(III) dithiocarbamate
[28]. The BBN-AA1 sequence with some spacers and maleimide-reactive cysteine was used to obtain the kit for targeted DOX-loaded liposomes, compliant with Good Manufacturing Practice (GMP), with satisfactory stability
[29]. Li and co-workers exploited another class 3 antagonist sequence: [D-Phe6-Sta13-Leu14-NH
2]bombesin(6–14)
[30]. In their research, this sequence was further modified with Alexa Fluor 750 to obtain a carrier system for molecular imaging of oral squamous cell carcinoma. Tagliviani and co-workers exploited Demobesin-1—a GRPR peptide antagonist that falls into the class 4 analogs
[31]. They have shown that, upon adding a trioxatridecan-succinamic acid spacer, Demobesin-1 provided successful cell recognition functionality to the polyoxometalate clusters.
Accardo et al. analyzed the performance of seven different GRPR antagonists based on DOTA and BBN derivatives coupled with PEG—formulations with a very simple design but offering an insight into the mechanisms by which they interact with GRPRs and, hence, provide varying targeting yield and diagnosis options
[18]. From their systematic analysis, they concluded that most of the examined BBN derivatives serve equally well for targeting purposes (which was confirmed with gamma camera recordings), but those with NMeGly11 and Sta13-Leu14 would be most suitable for imaging in vivo, since their plasma half-lives are over 15 days, and the latter also provides a favorable absorption mostly into the tumor tissues and no other organs of the body.
As mentioned above, GRPR is the mammalian bombesin receptor family member, comprising three distinct receptors. Basically, each of these receptors is expressed in different neoplastic conditions and has its own set of ligands, however, the universal binding sequence was discovered (D-Tyr6-Gln7-Trp8-Ala9-Val10-βAla11-His12-Phe13-Nle14), sometimes referred to as pan-bombesin. This finding is particularly attractive because with such a ligand it is possible to target tumors expressing various bombesin receptors patterns while improving the construct’s tumor-targeting efficiency and applicability. Heidari and co-workers used a peptide based on a universal binding sequence (Lys-Gly-Gly-Cys-Asp-Phe-Gln-Trp-Ala-Val-bAla-His-Phe-Nle) to target breast cancer in vitro and in vivo with gold nanorods for photothermal therapy
[32] as well as imaging
[33], and they pointed out its several advantages in comparison to native bombesin sequence, such as overcoming the issues related to receptor heterogeneity in tumors and ameliorating renal clearance due to the negatively charged hydrophilic aspartic acid residue. Similarly, Salouti et al.
[34] and Jafari et al.
[35] proposed the application of this peptide in breast cancer imaging using gold and superparamagnetic iron oxide nanoparticles, respectively. Pan-bombesin was also shown to improve prostate cancer imaging
[36][37].
3. Ligand Incorporation
One of the biggest challenges in the synthesis of particles targeting GRPR or any other receptor is the incorporation of the targeting ligand in the structure of the vehicle. Various approaches can be found in the literature. A state-of-the-art technique based on the formation of an amide linkage between the ligand and the nanoparticle is most commonly used. Chemistry of 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC, EDAC) in combination with N-hydroxysuccinimide (NHS)
[13] or sulfo-N-hydroxysuccinimide (sulfo-NHS)
[33][38] is most commonly used to create the bond. Usually, this amide is formed between the N-terminus of the peptide ligands and carboxyls on the nanoparticles. Tang and co-workers successfully decorated their poly(acrylic acid)-functionalized lanthanide nanoparticles with BBN analog using EDC/sulfo-NHS chemistry
[39]. It was even shown to be feasible to prepare ready-to-use kits with, e.g., stable NHS esters. EDC/NHS reactions are usually led in water, which is beneficial in terms of biocompatibility and purification procedures. However, the reaction, for instance, in tetrahydrofuran (THF) or dimethylformamide (DMF) is also possible, as shown by Du and Li
[10] and Mansour et al.
[40], respectively.
Nevertheless, with no carboxylic groups available on the surface of the nanoparticle, or no amine group available in the ligand, the other way around can be equally efficient
[9][41]. Poly(amidoamine) (PAMAM) dendrimers were conjugated with DOTA-modified BBN using the amine group on the dendrimer and carboxylic group of the DOTA moiety
[42][43]. Hajiramezanali and co-workers used this approach to exploit amine groups on chitosan and conjugate succinylated BBN derivative
[44]. However, organic solvents and water-insoluble substrates are also used, such as 2-(1H-7-azabenzotriazol-1-yl)-1.1.3.3-tetramethyluroniumhexafluorophosphate) (HATU)
[43][45][46] or N,N′-dicyclohexyl carbodiimide (DCC)
[31][47] in DMF with the addition of N,N-diisopropylethylamine (DIPEA)
[43]. In addition, click chemistry such as thiol-maleimide
[29][48][49] or copper-catalyzed azide-alkyne cycloaddition was useful
[36][37][50][51]. Dash and colleagues used cysteine groups incorporated in the peptide structure to click the GRPR-binding peptide with maleimide-containing PEGylated magnetic reduced graphene oxide
[52].
In some cases, the ligand can be incorporated into the structure of particle-forming substrates before the particle is formed
[53]. Accardo et al. have used solid-phase peptide synthesis with Fmoc/tBu chemistry to synthesize the ligand peptide [DOTA-bAla]BBN(7–14) and combine it with the amphiphilic monomers
[54]. Subsequently, these monomers were cleaved from the Rink amide resin, purified, and assembled into targeted supramolecular aggregates. A similar approach was adopted by Accardo and co-workers to prepare DOX-loaded targeted liposomes
[27]. Kanazawa et al. used this approach to combine bombesin with stearic acid directly in solid-phase peptide synthesis, and they used it for polymeric micelles
[55]. Yang and co-workers incorporated the GRPR binding motif of BBN into the structure of their engineered peptides able to form a stable coiled-coil nanostructure
[56]. An interesting approach was also presented by Zhang and colleagues—they have genetically engineered
Escherichia coli bacteria to express elastin-like peptides capable of coassembly into micelles
[57]. One of the peptides was engineered to contain a GRP-derived binding sequence.
In some instances, the application of a linker is necessary due to the configuration of functional groups present in both ligand and particle; both homo- and heterobifunctional linkers are available. For example, Achilli et al. used glutaraldehyde as a homobifunctional amine-reactive crosslinker to functionalize human serum albumin (HSA) protein corona of biohybrid gold nanoparticles with [Lys1-Lys3(DOTA)]BBN
[58]. Kim et al. tethered bombesin ligand to amine-modified poly(ethylene glycol) block of amphiphilic copolymer using a heterobifunctional linker, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
[59]. Montet et al. have used amine- and thiol-reactive succinimidyl iodoacetic acid to ligate the BBN derivative with amine-containing iron oxide nanoparticles
[60]. Lee and co-workers used N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to ligate cysteine-terminated derivative with glycol chitosan nanoparticles
[61].
In addition, noncovalent bonding may be useful in this context. Successful attachment of the BBN analog to gold nanoparticles can be achieved by exploiting the interaction between nitrogen atoms in free amine groups and the gold surface
[12][34]. Ocampo-Garcia et al. have developed a shelf-storage stable kit based on gold nanoparticles operating on this principle
[11]. On the other hand, Chanda and co-workers used the thioctic-acid-modified bombesin derivative to exploit thiol–gold interactions for stable functionalization (stable even in a reducing environment of dithiothreitol)
[62]. Hosta-Rigau et al. used additional cysteine to provide thiol for interaction with gold
[63]. Combining functional linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and gold–sulfur interactions can be an interesting alternative for gold nanoparticles
[15][61]. Li and colleagues exploited hydrogen bonds and π–π stacking to functionalize nano-graphene oxide with a Sta-BBN ligand
[30]. Interestingly, Trujillo-Benítez and co-workers used the DOTA moiety incorporated in the BBN derivative structure to achieve stable chelation of Sm atoms and, therefore, ligation of the ligand with metal oxide nanoparticles
[22]. Young et al. have used one of the strongest known noncovalent interaction systems, biotin–streptavidin, to bind biotinylated bombesin to quantum dots decorated with streptavidin, resulting in efficient GRPR-selective fluorescent label operating in vivo
[64].
Finally, a ligand can also be protected inside the nanoparticle. De Barros et al. postulate that, due to natural plasma and tissue peptidases, peptides should be protected inside the nanocarrier; therefore, they have encapsulated radiolabeled bombesin derivatives inside the long-circulating pH-sensitive liposomes. This approach led to the successful targeting of GRPR-positive breast cancer and Ehrlich tumor cells
[65][66][67].