Epitope Imprinted Nanoparticles as Customized Synthetic Antibodies for Biorecognition in Theranostics

Vol.:(0123456789) Regenerative Engineering and Translational Medicine https://doi.org/10.1007/s40883-025-00478-x PERSPECTIVE Epitope Imprinted Nanoparticles asCustomized Synthetic Antibodies forBiorecognition inTheranostics SimãoP.B.Teixeira1,2,3 · RuiL.Reis1,2 · RuiM.A.Domingues1,2,3 Received: 17 January 2025 / Revised: 7 August 2025 / Accepted: 18 August 2025 © International Iberian Nanotechnology Laboratory (INL) 2025 Abstract As medicine advances towards an era of personalized and targeted therapies, the need for precise and reliable molecular recognition elements becomes increasingly critical. These elements are essential for directing therapeutic agents to specific cells or tissues, thereby maximizing efficacy while minimizing off-target effects. Among the emerging technologies, epitopeimprinted nanoparticles (EINPs) have demonstrated exceptional potential as “synthetic antibodies” for the recognition of biomarkers. These nanoparticles are designed to mimic the binding capabilities of natural antibodies but with enhanced stability, specificity, and ease of production. Unlike traditional antibodies, which can be costly, labile, and prone to significant side effects, EINPs offer a cost-effective, robust, and customizable platform for targeted sensing and therapeutic applications. This Perspective Review explores the design, synthesis, and application of epitope-imprinted nanoparticles, highlighting their advantages and potential to revolutionize the landscape of abiotic molecular recognition in disease detection and targeted therapies. Finally, we present perspectives on foreseeable breakthroughs and advances, offering insights into how molecular imprinting techniques can be expanded to new biomedical fields, such as tissue engineering. Lay Summary Personalized medicine requires precise tools to identify and target diseased cells while avoiding off target effects. Epitope-imprinted nanoparticles (EINPs) are emerging as synthetic alternatives to antibodies, designed to recognize disease markers with high stability, low cost, and fewer limitations than natural antibodies. By mimicking biological recognition, EINPs show promise in applications such as disease detection, drug delivery, and tissue engineering. This perspective review focus on how these nanoparticles are created and applied, and highlights their potential to transform regenerative medicine and targeted therapies through more reliable, customizable molecular recognition systems. Keywords Biomarkers· Epitope imprinting· Molecular recognition· Nanoparticles· Targeted therapies· Theranostics Introduction As society evolves progressively into an era of precision medicine, targeted therapeutic and diagnostic approaches have been acquiring a more central role in order to enhance the specificity and efficacy of medical treatments and disease prevention [1]. Many of these strategies rely on the molecular recognition of cell surface biomarkers—molecules expressed on the outside of cell membranes that are unique to certain cell types or disease states. Biomarkers are crucial for identifying diseased cells, such as cancer cells, allowing for accurate profiling of disease phenotypes, precise drug delivery, and real-time monitoring of treatment effects. It is worth noting that, in the context of targeted therapies, these cell surface molecules are typically designated as targets. By exploiting these molecules, tailor-made theranostics tools can selectively deliver therapeutic agents to target cells * Rui L. Reis [email protected] * Rui M. A. Domingues r[email protected] 1 3B’s Research Group, I3Bs – Research Institute onBiomaterials, Biodegradables andBiomimetics, Headquarters oftheEuropean Institute ofExcellence On Tissue Engineering andRegenerative Medicine, University ofMinho, AvePark – Parque de Ciência e Tecnologia, Rua Ave 1, Edifício 1 (Sede), Barco, Guimarães4805-694, Portugal 2 ICVS/3B’s–PT Government Associate Laboratory, Braga/Guimarães, Portugal 3 INL – International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga S/N, 4715-330Braga, Portugal Regenerative Engineering and Translational Medicine while sparing healthy tissues, thus reducing undesired side effects and improving treatment outcomes. This approach not only enhances the efficacy of treatments but also allows for personalized interventions tailored to the unique molecular profile of each patient. Molecular recognition of cell markers is also seen as essential for the deployment of effective biomaterials for tissue regeneration, promoting selective cell adhesion, proliferation, and differentiation, as well as allowing the fabrication of functional scaffolds capable of triggering or responding to specific cellular cues [2]. Over the past few decades, biomedical strategies involving molecular targeting have leveraged the use of antibodies, particularly monoclonal antibodies (mAbs) owing to their high affinity and selectivity toward biomolecules of interest [3]. However, their lengthy and expensive production processes [4], short shelf-lives and half-lives (preand postadministration, respectively), and significant immunogenic risks [5] make them suboptimal tools. These limitations have generated interest in the development of alternative binding partners with a similarly specific molecular recognition capability, for example, peptides or aptamers [5, 6]. These molecules present several advantages over traditional antibodies, such as easier chemical modification, predicable structures, programmability, automated synthesis, and higher stability. Yet, their nature as small biomolecules also makes them prone to fast clearance and degradation. Perhaps more importantly, their discovery and development procedures are long, costly, and somewhat inefficient, making them impractical for a large number of research applications and laboratories [7, 8]. Therefore, there is a pressing need for alternatives, which have similar molecular recognition capabilities, but are simultaneously stable, scalable, and cost-effective, rendering them more widely accessible [9]. In this context, molecular imprinting (MI) has been increasingly demonstrating its potential to generate fully synthetic materials with abiotic affinity for biomolecules aiming at various biological applications [10, 11]. Synthetic polymers that can be designed to recognize and bind biologically relevant molecules are of great importance and influence a number of emerging technologies [9], either as single systems or incorporated into other platforms that can assist in the identification of particular compounds or cell markers related with a number of diseases, for example, specific cancer types [12] or autoimmune conditions [13]. The potential presented by the recognition of these molecules by polymeric systems is significant for both diagnostic (e.g., bioimaging) and therapeutic purposes, which can be further combined into integrated theranostics platforms [14, 15]. This Perspective Review covers essential aspects of MI for biomolecular targets using the epitope template approach, mainly focusing on key parameters of epitope selection and design, and some of the latest applications that show the efficacy and versatility of this technology. Moreover, it offers prospects for future developments based on MI polymers in the biomedical field, particularly foreseeing its large-scale adoption for regenerative medicine strategies. The Concept ofMolecular Imprinting MI is a technique used to create specific recognition sites within polymer networks by crosslinking monomer(s) in the presence of a template molecule, which corresponds to the recognition target [16]. During this process, a monomer solution is mixed with the template, forming temporary interactions between them. After crosslinking and polymerization, the template is removed, leaving behind a polymer structure with cavities that are complementary to the shape and chemical structure of the imprinted molecule. Importantly, these nanocavities retain the precise spatial organization of molecular interactions necessary for recognizing the target. As result, molecularly imprinted polymers (MIPs) can selectively recognize and bind to their targets through a “lock and key” mechanism similar to those found in natural systems like antibodies and enzymes. Thus, these biomimetic polymer networks should be created by carefully designing the interactions between the network’s building blocks and the specific ligand in order to maximize the recognition potential. MIPs have garnered increasing attention due to several advantages, including their high affinity and selectivity, which enable them to rival natural biomolecule analogs such as antibodies [11], while simultaneously exhibiting greater chemical stability over time and under various environmental conditions. These properties allow for prolonged transport and storage without cold chains [17] and increase the expected function and circulation time in the human organism. Moreover, their production is comparatively simpler, faster, and less costly, making them more widely accessible to a wider variety of applications [18]. These characteristics, along with the tailor-made construction allowed by a large library of available building blocks, make MIPs particularly suitable for the generation of artificial receptors for biomolecules with no natural binding partners or with incompletely solved structures [7, 19]. Although synthetic recognition of small compounds is now standard practice, MI of proteins traditionally presented significant challenges due to their environmental sensitivity and chemical incompatibility [20]. This required the development of new bulk imprinting methods using water-soluble monomers, essentially based on acrylate and acrylamide derivatives [21] that resulted in materials essentially behaving like hydrogels [14]. However, the production of densely crosslinked polymer structures was necessary in order to ensure the retention of effective binding sites in these networks, greatly hindering protein rebinding Regenerative Engineering and Translational Medicine since their diameter is often larger than the hydrogel mesh sizes. Additionally, common washing methods for template removal could compromise the integrity of imprinted sites, also contributing to a lower imprinting efficiency. These limitations have been, to a large extent, smartly bypassed by the emergence of surface imprinting techniques, in which MI occurs only at the surface of thin polymer layers, instead of in bulk [22, 23]. This not only allows free template diffusion in solution but also potentially reduces the amounts needed for imprinting procedures. Despite these developments, other relevant limitations of direct biomacromolecule imprinting remain unsolved. For example, the predominance of strong hydrogen bonds with water often disrupts template-monomer interactions, thus decreasing imprinting effectiveness [24]. This presents a particularly high barrier when targeting molecules rich in hydroxyl and other strongly hydrophilic groups, particularly saccharides. Moreover, the structural complexity of proteins further contributes to these problems, adding an extra layer of difficulty to their MI [25]. The large number of functional groups spread over different regions of protein structures, along with their conformational flexibility, strongly contribute to producing poorly fit binding sites for later recognition. Lastly, but also critically, is the prohibitive cost of most biologically relevant proteins (e.g., growth factors, cellular receptors), which makes them unreasonable options as imprinting templates in large scale [14, 26]. Epitope‑Imprinting Emerges asSolution The described list of limitations made biomacromolecule imprinting an inefficient concept, setting back the development of materials for synthetic recognition of proteins until the proposal of the epitope imprinting approach [27]. This strategy is inspired by the functioning mechanisms of natural antibodies, which recognize only a small, exposed domain out of a larger molecule, termed an epitope [25]. Similarly, this imprinting approach consists in selecting a portion of the target molecule, which is used as the template for MI instead of the full macromolecule. After its removal, the resulting MIP can rebind the full target by specifically recognizing this particular segment (Fig.1). While initially explored for the recognition of small oligopeptides [28, 29], it did not take long before the first applications targeting full proteins were reported [30, 31]. Not long after, the concept of epitope-imprinted nanoparticles (EINPs) as plastic antibodies was introduced, showing that such materials could compete in functionality and effectiveness with their biological counterparts, having their bioactive potential Fig. 1 The concept of epitope imprinting implies the recognition of a macromolecule through the imprinting of a small domain selected from its full chemical structure—the epitope Regenerative Engineering and Translational Medicine demonstrated for invitro cell culture [32] and invivo as antitoxins [33]. Opting for smaller compounds as templates helps bypass the structural and financial challenges typically associated with larger molecules while still preserving selective recognition and affinity for the larger targets. Their reduced size results in less intramolecular flexibility and fewer functional groups, which contributes to more uniform and consistent imprinted cavities. Unlike proteins, small peptides do not denature because they lack complex tertiary and quaternary structures, and they can also be synthesized to dissolve in a variety of solvents beyond water, allowing their use in different MIP synthesis conditions [25]. However, even though smaller epitopes reduce diffusion-related issues during template removal, their bulk imprinting for later full protein recognition still faces limitations because polymer mesh size hinders subsequent diffusion and rebinding of the targeted larger macromolecules. As a result, epitope imprinting is particularly advantageous for strategies involving surface imprinting, for example, on nanoparticles, where the binding sites are more readily accessible [28]. Additionally, smaller templates are more cost-effective and easier to produce through chemical synthesis [34], allowing the recognition of biologically relevant molecules at much lower costs. Due to these impactful imprinting concept improvements, research into protein MI has grown exponentially, showing promise for a wide array of applications, including biosensing [35], separation technology [36], targeted drug delivery [37], or tissue engineering [38]. This has prompted a number of excellent reviews on the topic, covering relevant points regarding MI synthesis methods, standardized evaluation parameters, and applicability [11, 14, 20, 35, 39, 40], including our own, in which we have comprehensively reviewed the most crucial features of epitope imprinting technology [22]. Here, we provide a brief overview and update on the basic but relevant concepts of epitope imprinting, with particular focus on the crucial element of epitope design, contextualized by concentrating on its application for the development of systems recognizing cell surface biomarkers and targets for theranostics purposes. It covers the most recent works on this field, highlighting key breakthroughs and presenting a critical perspective on our vison for future research directions. Looking fortheRight Epitope Arguably, the most important step for the success of each new epitope imprinting process is the epitope selection. Replacing the full target biomolecule with a smaller alternative template requires an adequate selection procedure to ensure a good correspondence between the imprinted cavities and the final target. This means that the epitope must correspond to a monomer sequence unique to the parent macromolecule, while simultaneously being suitable for the MI synthesis procedures and located in an accessible region in its native form for later rebinding. Recent progress in the field has led to increased interest in saccharide MI, aiming to identify specific glycoproteins [41, 42] or aberrant glycosylation sites on cell surfaces [43, 44], often linked to particular illnesses. Nonetheless, peptides remain largely the most common type of epitope used in MI protocols. Figure2 provides a concise overview of the available strategies for this selection. Briefly, these strategies can be categorized into empirical vs. rational; terminal vs. internal; immunogenic/functional vs. structural; linear vs. structured. Empirical Selection Processes Terminal Protein Sequences The most straightforward and traditionally used empirical method for epitope selection is the use of a linear peptide corresponding to the Nor C-terminal sequence of the target protein [27, 31]. The main advantages are (i) since these sites are not commonly targeted for post-translational modifications, the template peptide is very likely to directly correspond to the target protein portion, and (ii) these sequences are frequently exposed on protein surfaces, thereby increasing the probability of target recognition by EINPs [45]. Moreover, this selection can be quickly performed by simply looking at the target protein sequence in commonly accessible databases [46], such as UniProt or RCSB protein databank (PDB) [47–49]. Using linear peptides as MI templates is also a less expensive option compared to conformational ones, avoiding the complexity associated with three-dimensional structures, while also facilitating modification or conjugation reactions that increase the peptide functionality (e.g., oriented immobilization) [50, 51]. In a remarkable example of this strategy, Canfarotta etal. prepared dual-imprinted nanoparticles for drug delivery to specific cancer cells, by simultaneously imprinting them against the cytotoxic drug doxorubicin and the C-terminal linear sequence (SLNITSLGLRSLKEISDG) of the extracellular portion of the epidermal growth factor receptor (EGFR), which is exposed at the protein surface. A cysteine residue was added to the epitope to allow its oriented immobilization on the solid phase used in the MI synthesis procedure. The prepared EINPs attained an equilibrium dissociation constant (KD) of 3.6nM for the binding to the extracellular domain of the EGFR protein and were able to selectively recognize and deliver doxorubicin to cells overexpressing this surface target. The use of N-termini is not as frequent as C-termini, mostly because the N-terminus may be hydrolyzed or truncated due to signal peptide removal [52]. Nevertheless, Qin Regenerative Engineering and Translational Medicine etal. showed the efficacy of this approach by producing biodegradable MIP nanocapsules against the N-terminal epitope of CD59 glycoprotein (YNCPNPTADCK) [53]. Using N,N′- diacrylylcystamine as the cross-linker, these structures were selectively degraded in the more acidic tumor microenvironment, leading to the release of encapsulated doxorubicin and the selective killing of MCF-7 cells (which overexpress CD59). Additionally, by embedding carbon quantum dots in MIPs, they also functioned as bioimaging tools, thereby demonstrating the theranostic potential of EINPs. The Z. Liu group has been particularly active in exploring this approach, demonstrating its versatility in combination with multiple synthesis routes. For example, the N-terminal nonpeptide of transmembrane glycoprotein NMB (GPNMB), a marker of triple negative breast cancer (TNBC), was used as template to imprint silica-based nanoparticles by a novel method termed reverse microemulsion-confined epitope-oriented surface imprinting and cladding (ROSIC). These EINPs demonstrated simultaneously impressive selectivity and affinity for the target protein, allowing invivo targeted imaging of TNBC-bearing mice using GPNMB-specific NIR797-doped nanoparticles [54]. EINPs targeting this protein were further redesigned to work as “nanobeacons,” displaying numerous haptens that helped to recruit endogenous antibodies, thereby effectively directing innate immune mechanisms towards GPNMD-positive tumors invivo, constituting a promising targeted cancer immunotherapy [55]. Fig. 2 Overview of the different strategies for selection of epitopes for molecular imprinting Regenerative Engineering and Translational Medicine These authors also developed a remarkable anti-cancer therapeutic strategy based on EINPs termed “molecularly imprinted lysosomal nanodegraders” (MILND), using as templates the N-terminal epitopes of different cancer-associated immunosuppressor proteins, namely, signal regulatory protein alpha (SIRPα) and programmed death–ligand 1 (PD-L1) [56, 57]. These EINPs could specifically bind the target protein, either SIRPα on tumor-associated macrophages or PD-L1 on cancer cells, signaling these molecules for lysosomal degradation, thereby reversing the immunosuppressing CD47-SIRPα or programmed cell death protein 1 (PD-1)/PD-L1 signaling and restoring the Regenerative Engineering and Translational Medicine recognition capability of macrophages and T cells, respectively, towards cancer cells. This resulted in substantial tumor growth inhibition in tumor-bearing mouse models, proving the effectiveness of this technology for improved immunotherapy (Fig.3A). The same group also synthesized EINPs against the N-terminus of vascular endothelial growth factor (VEGF), which allowed these particles to recognize the two major pro-angiogenic isoforms of this growth factor (VEGF165 and VEGF121) with high affinity [58]. These synthetic nanomedicines could thus block VEGF signaling in the cancer microenvironment, showing a potent anti-angiogenic effectthat led to tumor growth suppression. Both strategies constitute major improvements compared to current anti-cancer therapies, demonstrating great promise for drug-free approaches that can achieve effective tumor suppression with few to no side effects. Altogether, several studies have concluded that neither Nnor C-termini have proven to lead to inherently superior imprinting performance compared to one another [59, 60]. The choice for each particular target appears to be more complex and dependent on several physicochemical criteria, such as solubility in adequate solvents (porogens), compatibility with imprinting method, or peptide length. The length of the template sequence is particularly crucial, being Limited in two ways. On the one Hand, peptides shorter than 8 amino acids (a.a.) hardly function as idiotypic, with a higher likelihood of corresponding to multiple protein targets [31]. This is why a large portion of epitope imprinting works use 9-mer templates, considered to represent a near-unique code in unstructured domains for the identification of specific proteins. On the other Hand, sequences beyond 16 a.a. can become too flexible and undergo intramolecular interactions, thus acquiring three-dimensional conformations different from the native protein that reduce the imprinting efficiency [61]. Between these limits, research data has shown that longer sequences generally lead to increased specificity and better recognition by EINPs, by providing more molecular interactions between the imprinted nanoparticle and the target. But, interestingly, the molecular weight of the epitope seems to correlate more strongly with the resulting EINP affinity than its a.a. number [62]. This was exemplified by Tai etal., who demonstrated that a heavier 9-mer peptide generated EINPs with a KD of 10nM, compared to 20nM using a Lighter 10-mer peptide. Probably, heavier peptides tend to retain a more stable conformation during polymerization, resulting in more uniform imprinted cavities. Moreover, the compared affinities of each MIP reveal differences when tested against the epitope template vs. against the full target protein. Thus, we can conclude that there is no ideal template peptide length within the 8–16 a.a. range and that it is therefore important to analyze the success of imprinting experimentally with the whole protein in an ad hoc manner. Advanced Experimental Selection Methods Considering that terminal sequences are not always the best epitope for each protein, more complex methodologies started being adopted to select adequate imprinting templates. One of those methods involves the construction of a.a. sequences with certain physicochemical properties of interest for a particular MI protocol or application. For example, in a study with the goal of detecting natriuretic peptide hormones in blood samples, 9-mer peptide epitopes were chosen based on two premises: possessing an isoelectric point (pI) close to the target molecule and boasting a balanced ratio between hydrophobic and hydrophilic residues [63]. This simple method helped to generate MIPs with impressive dissociation constants (2–20pM), as well as low reported cross-reactivity. In other works, aiming to detect Regenerating Protein 1B, a biomarker of Pancreatic ductal adenocarcinoma, researchers scanned the target protein sequence and selected seven oligopeptides from different subregions considering, among other properties, their solubility in dimethyl sulfoxide. In one of the reports, the best performing peptide owed its success to the good solubility provided by 6 charged (EDREDD) and 3 polar (NTY) residues [64]. These results demonstrate that considering the physicochemical properties of epitope templates is a good practice during their selection to heighten the performance of produced EINPs. Nonetheless, in their second work, the authors show that one Fig. 3 Empirical epitope selection methods. A Imprinting of N-terminal epitope of tumor surface target SIRPα for anti-cancer immunotherapy. (i) Illustration of the preparation route of molecularly imprinted lysosomal nanodegrader (MILND) and immunotherapy approach by MILND via blocking the SIRPα-CD47 signaling pathway. (ii) Fluorescence images demonstrating the uptake of MILND by tumor-associated macrophages (green: fluorescein-labelled MILND, red: lysosomes stained by Lyso Tracker Red, blue: DAPIstained nuclei). (iii) Relative protein expression of MILND-targetedSIRPα protein degradation in macrophages measured by western blot. (iv) Relative protein expression of SIRPα, phosphatidylinositol 3-kinase, catalytic subunit alpha (PI3K-p110α), phosphorylated protein kinase B (p-Akt) and myosin IIa in control, MILND, or nonimprinted NILND treated cells. (v) Relative tumor volume growth curves of CT26 syngenetic tumor mice over 15days treatment. (vi) Photograph of the dissected tumors from the CT26 syngenetic tumor mice after 15 days of therapy in each treated group. (vii) Average tumor weight resected from the CT26 syngenetic tumor mice after different treatment. Adapted with permission [56]. Copyright 2024, Elsevier. B Advanced empirical epitope selection by snapshot imprinting. (i) Diagram depicting the snapshot imprinting process, wherein cell surface proteins are imprinted, the resultant EINPs are collected, and the template peptides eluted and sequenced in order to identify novel biomarkers. (ii–iv) Extracellular region of epidermal growth factor receptor (EGFR); regions considered to be epitopes highlighted in orange, remainder in blue. Epitopes selected from: (ii) label-free deep proteome analysis; iii) snapshot imprinting. (iv) Depiction of epitopes ultimately selected for imprinting highlighted in red. Adapted under CC BY 4.0 [12] ◂ Regenerative Engineering and Translational Medicine of the chosen peptides improved sensor accuracy probably because of its better exposure at the protein surface compared to the other epitopes [61]. Thus, a more rational epitope selection method that also accounts for the structure of the full protein can likely improve the quality of synthesized MIPs, reducing the time and costs associated with testing numerous alternative templates. In alternative, Bagán etal. have reported a promising experimental method to directly derive surface-accessible epitopes from the target protein. This involves the immobilization of the protein on a solid phase support, namely, silica nanoparticles, followed by its enzymatic digestion. After washing away the digested portions, only the surface-exposed peptides, which were covalently bound to nanoparticle supports remained. These surfaceimmobilized epitopes could then be used to produce MIPs, which were able to recognize the target protein [46]. The resulting MIPs can be compared to polyclonal antibodies, with affinity towards an undetermined variety of epitopes. This approach significantly reduces the development time, bypassing the epitope selection step while ensuring the imprinting of surface-accessible peptides. On the downside, it requires the use of full proteins during the synthesis process, nullifying some of the advantages sought by epitope imprinting, particularly concerning costs of implementation. Additionally, the MIPs prepared by this method demonstrated a relatively unsatisfactory degree of selectivity, indicating the need for further experimental refinement before considering a widespread use of this protocol. In a comparable development, boronate-affinity glycanoriented surface imprinting represents a step forward for this strategy [65]. In this innovation, while the rationale remains similar, glycans are imprinted instead of peptides. The working principles of this concept restrict its use to glycated proteins, but these correspond to more than 50% of the total proteins in mammalians, representing a large portion of extracellular membrane-associated proteins [66]. Moreover, glycoproteins are often used as disease biomarkers and therapeutic targets [67]. This strategy relies on the reversible covalent interactions that boronic acids can establish with cis-diol-containing molecules, such as sugars. In alkaline conditions, these species form stable cyclic esters, dissociating when the pH switches to acidic values. Thus, this ability can be leveraged for facile template immobilization and removal post-imprinting. The studies which first pioneered this concept achieved MIPs with remarkable selectivity, particularly when compared to the previous peptide-imprinting approach [41, 68], demonstrating the potential of this technique. On the other hand, the affinity displayed for the target saccharide is low compared to other imprinting approaches (KD in the micromolar range), although of similar magnitude to natural sugar-binding molecules such as lectins. Further refinements (e.g., monomer mixture) might improve the quality of MIPs prepared by this technique. Alternatively, target protein digestion can be applied as a first step to identify and isolate proteotypic peptides for subsequent imprinting procedures [69, 70], having been employed, for example, on the precise identification of signature peptides of pro-gastrin-releasing peptide in serum samples. More recently, the Piletsky group introduced an ingenious advance in this approach [12, 71, 72], which was termed “snapshot imprinting” (Fig.3B). In this technique, MIPs are first synthesized in the presence of the target protein(s), generating “protein-MIP complexes,” with non-reacted and low-affinity material being washed out of the mixture. Then, these complexes undergo enzymatic or chemical digestion, where non-bound peptides are also washed. In this manner, only the peptides corresponding to the imprinted cavities remain bound to the polymer complex. Lastly, these peptides are separated and sequenced, allowing the identification of surface-exposed portions of the target protein with ideal properties to be used as epitopes in following imprinting processes. This concept has been applied, for example, in the development of MIPs to serve as allosteric modulators of acetylcholinesterase activity [71]. These presented excellent functionality, with a particular formulation increasing the enzyme activity by 47 × and recovering its activity from 11% (following exposure to an inhibitor) to 73%. In a similar application, this procedure was repurposed for efficient proteomic mapping of cancer cell surfaces, aiming to identifyaccessible diagnostic markers and potential therapeutic targets [12]. In the tested cancer cell Lines, this allowed the classification of 438 proteins, with 283 considered to be transmembrane or extracellular proteins, creating a “proteomic fingerprint” of the surface of each cell type. Additionally, three previously unknown epitopes of EGFR were generated and used to successfully produce EINPs for selective recognition of this cancer marker. Another method for epitope identification leverages sequence similarity to design versatile EINPs capable of recognizing multiple peptides or glycans within the same family. This has been applied to create imprinted “nanotags” for labelling certain glycosylation patterns of interest [73]. Using a clever combination of imprinting strategies, Li etal. engineered improved surface-enhanced Raman scattering (SERS) platforms for precise diagnosis of hepatocellular carcinoma based on the relative glycosylation of the biomarker alpha fetoprotein (AFP). First, the C-terminal epitope of AFP was used to prepare an imprinted substrate that captured the protein from samples. To detect the total level of captured protein, EINPs against the N-terminal epitope and decorated with Raman reporter PATP (4-aminothiophenol) were used. Then, EINPs imprinted with the A2G2S2 glycan structure and tagged with 4-NTP (4-nitrothiophenol) were used to quantify the proportion of AFP modified with Regenerative Engineering and Translational Medicine different glycans containing this specific structure, which is associated with malignancy. This dual detection strategy enabled simultaneous quantification of both total and A2G2S2-glycosylated AFP, allowing for accurate ratiometric analysis of clinical samples. Monomers andSmall Molecules asEpitopes In a more simplified approach, smaller molecules such as a single amino acid have also been proven to be effective templates for targeting specific proteins. Given that a single monomer cannot be as idiotypic as a whole sequence, this strategy compromises selectivity and affinity for a faster and less costly development with a more practical implementation. For instance, fluorenylmethyloxycarbonyl (Fmoc)-protected phosphorylated single amino acids have been successfully investigated as template epitopes for the targeting of phosphorylated peptides, focusing particularly on phosphotyrosine (pY) [74, 75], but also demonstrating applicability for phosphoserine [76] and phosphohistidine [77] residues. Phosphorylation is a key post-translational modification that regulates various biological processes, including cell growth, division, and apoptosis. Abnormal phosphorylation patterns are often associated with diseases such as cancer, diabetes, and neurodegenerative disorders. Thus, identifying phosphorylation patterns can prove an important step to identify certain diseases or predict responses to particular therapies, helping in the construction of personalized interventions. This approach has been successfully validated against the benchmark methods for phosphorylated amino acid enrichment, involving either titanium oxide (TiO2) or anti-pY antibodies [78]. Moreover, besides functioning only as an alternative to traditional methods, it has also been combined with TiO2 to selectively enrich protein extracts from cancer cells in phosphopeptides before mass spectrometry analysis [74], suggesting its potential to improve phosphoproteomics research. As an alternative to Fmoc-pY, other groups have instead explored the use of phenylphosphonic acid (PPA) as a dummy template for identification of pY residues, owing to the extremely similar molecular structures of PPA and pY [79, 80]. This method takes advantage of a lower cost template molecule to obtain increasingly impressive results in terms of affinity, adsorption capacity and selectivity, stretching the boundaries of the epitope concept. Besides amino acids, monosaccharides like glucuronic acid and N-acetylneuraminic acid have proven effective as epitope templates. Nanoparticles imprinted with these sugars were successfully used to recognize and fluorescently label specific glycosylation sites in macromolecules like (proteo)glycans, for example, hyaluronic acid, considering the abundance of these specific sugar monomers in their structures [81–83]. Notably, these nanoparticles exhibited no cross-reactivity with other common monosaccharides found at the terminal ends of proteoglycans or glycoconjugates on cell surfaces, proving their remarkable selectivity despite the simplicity of this approach. These pioneering saccharide EINPs thus demonstrate tremendous potential for the development of new theragnostic tools, since altered glycosylation levels or particular glycan patterns are indicative of pathological conditions such as infections or cancer malignancy [82]. Additionally, other studies have also tested the combination of a single monosaccharide with a single amino acid (mannose and tryptophan, respectively) to imprint microspheres, showing a satisfactory recognition ability for the glycopeptide antibodies telavancin and teicoplanin [84, 85]. It remains to be seen if this strategy can improve recognition of more complex macromolecules, such as cell surface glycoproteins, compared to saccharide imprinting only. Despite the encouraging advances in empirical epitope identification methods, they still show significant performance limitations. For proteins with a real interest as biomarkers or therapeutic targets, which inevitably have prohibitive costs, methods based on their direct and disposable usage in digestion protocols present a notable obstacle. On the other hand, approaches relying on single monomers or small molecules have limited scope of target proteins, relying on specific structural properties. This invites the development of more efficient, rational approaches that can be universally used to identify proteotypic epitopes for any kind of target, widening the range of potential applications. Rational Strategies forEpitope Identification The shortcomings presented by empirical epitope selection prompted the development of new rational approaches. These methods aim to maximize the affinity and selectivity of MIPs by carefully designing the imprinted template so that its structure and properties best mimic the epitope in the native protein. These strategies have been progressively improved and standardized into workflows based on bioinformatic tools, allowing the comparative analysis and alignment of protein sequences, 3D visualization of protein structures, search for naturally occurring epitopes, and simulation of molecular interactions, with continuously improving outcomes. Learning fromNature—Immunogenic andFunctional Epitopes An attractive strategy for rational epitope selection draws inspiration from the original concept of epitope as a fraction of a protein that is recognized by an antibody. Thus, EINPs can be conceived as synthetic equivalents of antibodies and be imprinted against already known immunogenic sequences. Bioinformatic platforms such as the Immune Regenerative Engineering and Translational Medicine off-target species. Besides the epitope selection, monomer choice, synthesis method, and nanoparticle design all critically influence EINP performance. Significant progress in these areas has also been made recently. For instance, employing mixtures of functional monomers tailored to the chemical nature of the template residues can enhance binding affinity by optimizing interactions between the target and the imprinted cavities. Both acrylate/acrylamidebased and silane-based monomer mixtures have demonstrated promising outcomes [55, 73, 123, 124], although there is a need for more representative invivo assays to further validate these approaches. In vivo administration raises additional concerns regarding nanoparticle biodistribution, clearance pathways, and long-term biocompatibility, challenges faced broadly by nanomedicine platforms, which are equally relevant for EINPs [125, 126]. However, very few studies have directly focused on these aspects. For example, Kassem etal. report that EINPs prepared by solid phase imprinting and administered in mice either intravenously or orally were able to reach every organ tested, including the central nervous system, being excreted through both fecal and urinary routes [127]. Additionally, the study noted dose-dependent toxicity as well as signs of inflammatory and immune reactions when EINPs were administered orally. While these results established a relatively safe dose threshold, they underscore the importance of incorporating nanoparticle design and engineering strategies for modulation of pharmacokinetics. These include both materials design (e.g., steric repulsion, size tuning, endosomal escape) and bioengineering (e.g., reticuloendothelial system blockade, microenvironmental reprogramming, permeability enhancement) approaches. In parallel, development of fully biodegradable MIPs based on natural polymers is another emerging strategy that can help to overcome some of these barriers, namely, long-term toxicity and invivo persistence [128]. Other major barriers to clinical application of new MIPbased medical products are production scaleup and alignment with regulatory standards. While epitope imprinting offers a theoretical edge in cost efficiency over traditional techniques using large biomacromolecules, bioreactors, or live animals, this has not yet translated into off-the-shelf products. This could be related to the lack of scalable, reproducible production technologies with high yields and attaining narrow, well-defined size distributions, allowing full control over EINP properties [129]. Nevertheless, some studies are finally attempting to bridge this gap, focusing on more automated, innovative reactor systems that allow a continuous production routine more compatible with industrial standards [130, 131]. Combining these advances with increasingly powerful computational approaches and machine learning/artificial intelligence could significantly accelerate the development of clinically viable EINPs. Table 1 (continued) Target/biomarker Epitope selection method Epitope(s) Monomers, crosslinker(s) Synthesis technique KD (nM)* Reference TNFα Rational selection, bioinformatic workflow, conformational TPEGAEA AB, NIPAm, TBAm, MBAm SPI NA [119] ERα Rational selection, functional relevance, immunogenic SHSLQKYYITGEAEGFPATV AA, APMAm, NIPAm, TBAm, FMA, MBAm SPI 10.8–14.7 [123] *Dissociation constant (KD) is noted whenever it is available in the cited reference. Imprinting factor (IF) is provided whenever KD could not be found Target/biomarker: AFP alpha-fetoprotein,B2M beta2 microglobulin,EGFR epidermal growth factor receptor,ERα estrogen receptor alpha,GPNMB glycoprotein NMB,HAVCR-1 Hepatitis A Virus Cellular Receptor 1,HER2 human epidermal growth factor receptor-2,PD-L1 programmed death-ligand 1,ProGRP progastrin-releasing peptide,REG1B regenerating protein 1B,TNFα tumor necrosis factor alpha Monomers/crosslinkers: 4-VPy 4-vinylpyridine, AA acrylic acid, AAB (N-acrylamido)-benzamidine, AB (4-acrylamidophenyl)(amino)methaniminium acetate, AAm acrylamide, APMAm N-(3-aminopropyl)methacrylamide hydrochloride, APTES 3-aminopropyltriethoxysilane, BAC N,N′-diacrylylcystamine, BnTES benzyltriethoxysilane, DMAEMA dimethylaminoethyl methacrylate, DVB divinylbenzene, EAMA N-(2-aminoethyl) methacrylamide hydrochloride, EGDMA ethylene glycol dimethacrylate, EVAL poly(ethylene-co-vinyl alcohol), FAm N-fluoresceinylacrylamide, FMA fluorescein-O-methacrylate, HEMA 2-hydroxyethyl methacrylate, IBTES isobutyltriethoxysilane, MAA methacrylic acid, MAm methacrylamide, MBAm N,N′- methylene-bis-acrylamide, NIPAm N-isopropylacrylamide,PAm N-phenylacrylamide, PTES phenyltriethoxysilane, TEOS tetraethyl orthosilicate, TFMAA trifluoromethacrylic acid, TFU N−3,5bis(trifluoromethyl)-phenyl-N′−4-vinylphenylurea, UPTES 3-ureidopropyltriethoxysilane Synthesis techniques: BACOSI boronate-affinity-controllable oriented surface imprinting, BOSIC boronate affinity–anchored epitope-oriented surface imprinting and cladding, ROSIC reverse microemulsion-confined epitope-oriented surface imprinting and cladding, SPI solid phase imprinting Regenerative Engineering and Translational Medicine Finally, progress toward regulatory approval will also depend on standardization efforts and compliance with established quality management frameworks. This includes adherence to good manufacturing practices (GMP), implementation of robust quality control protocols, and alignment with international regulatory standards (e.g., ISO—International Organization for Standardization). Without consistent protocols for characterization and batch validation, clinical translation remains difficult. Regulatory bodies such as the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) will require comprehensive data on product reproducibility, safety, and efficacy demonstration before MIPor EINP-based systems can enter the clinical pipeline [126]. Conclusions andPerspectives Epitope molecular imprinting technology has rapidly advanced, addressing many challenges associated with imprinting entire macromolecules like proteins in polymeric matrices. Compared to biological alternatives—such as antibodies or aptamers—EINPs offer greater versatility, lower production costs, scalability, and long-term stability. These properties position them as strong candidates for theranostics applications. Additionally, the synthetic nature of EINPs is well-aligned with the growing ethical pressure to reduce animal use in R&D, a priority gaining momentum among policymakers and the public. Recent innovations have propelled epitope imprinting toward high-value molecular targets, including circulating biomarkers, cancer cell surface proteins, and growth factors. These advancements pave the way for real-world applications in diagnostics, regenerative medicine, and targeted therapies. The rise of bioinformatics and molecular modeling tools together with increased computational power accelerates epitope selection and polymer optimization, streamlining MIP development while reducing their costs. As the accessibility of this technology improves, more research groups globally are contributing to the field, expanding its application scope in a wide range of the biomedical areas such as drug delivery [51, 132], tissue engineering [22, 114], drug-free anti-cancer therapies [100, 119], and precision diagnostics [133, 134]. Despite this progress, significant challenges remain. Continued development of automated, large-scale production systems will be essential for the democratization of EINP access, allowing present and future companies to quickly upscale the tailor-made synthesis capabilities of these materials for commercial purposes. The appearance of new companies using this technology (e.g., MIP Diagnostics Ltd, SEMOREX), particularly leveraging the advantages offered by the solid phase imprinting method, is an encouraging sign that a turning point is being reached for MIPs. We foresee a future where clinicians and researchers can swiftly request EINPs tailored for specific proteins or diseases, receiving customized materials within weeks. Standardized protocols for design, testing, and quality control will be essential to support this transition from lab to clinic. These firm advances in recent years provide confidence in our vision of EINPs as real plastic antibodies, enabling novel solutions across biomedicine and clinical care. Acknowledgements Figures1 and 2 were created with BioRender. com. Author Contribution S. P. B. T. and R. M. A. D. conceived the review framework. S. P. B. T. conducted literature review and wrote the original draft. All authors participated in the discussion and writing of the paper. All authors read and approved the final manuscript. Funding This work was supported by the European Research Council (ERC) under the European Union’s Horizon Europe research and innovation programme (Grant agreement No. 101171765 – Consolidator Grant FORTIFy); by FCT - Fundação para a Ciência e a Tecnologia, I.P. & Ministry of Education, Science and Innovation of the Portuguese Republic through project Wi-Pi (DOI: 10.54499/2022.05526. PTDC), individual contract for R.M.A.D (DOI: 10.54499/2020.03410. CEECIND/CP1600/CT0013), and funding of Associate Laboratory ICVS/3Bs (DOI: 10.54499/UIDB/50026/2020; 10.54499/ UIDP/50026/2020; and 10.54499/LA/P/0050/2020). The authors would also like to thank the contributions to this research from the project “TERM RES Hub – Scientific Infrastructure for Tissue Engineering and Regenerative Medicine”, reference PINFRA/22190/2016 (Norte-01-0145-FEDER-022190), funded by FCT in cooperation with the Northern Portugal Regional Coordination and Development Commission (CCDR-N), for providing relevant lab facilities, state-of-the art equipment and highly qualified human resources. Data Availability The authors confirm that the data supporting the findings of the study are available within the article. Declarations Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. 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