Folic acid (FA) is a well-known molecule used for treatment and prevention of different diseases. FA (or folate) receptors (FR) are expressed in all cells of living organisms but overexpressed in some types of cells of malignances, particularly various types of cancer cells [1, 2], osteosarcoma [3], acute myeloid leukemia [4], as well as tumor-associated macrophages, which are often present in the microenvironment of both epithelial and non-epithelial tumors and affect tumor progression, immune evasion, and metastasis [5], and activated macrophages associated with autoimmune diseases such as rheumatoid arthritis [6]. For example, in epidermal carcinoma, FR overexpression can be approximately 100–300 times higher compared to normal cells [7]. There is a positive correlation between the level of FR expression in tumor cells and their resistance to standard chemotherapy. In most cases, FR overexpression on the surface of cancer cells is associated with a severe disease course, for instance, in the case of breast cancer, FR overexpression is linked with worse outcomes including decreased disease-free survival rates [8, 9]. In the case of primary and metastatic head and neck squamous cell carcinomas patients with FR overexpression have a lower survival rate [10].

Using targeting ligands, that generally can be called biovectors (molecules of natural or synthetic origin that are able to target cells) is a rather common approach in modern nanobiotechnology. The conventional approaches in cancer therapy such as chemotherapy and radiotherapy are known to lead to dramatic nontargeted effects on the human body including a decrease in the effective cytotoxic dose effect of therapeutic agents on cancerous cells and growth of inhibitory effect on normal cells [11, 12]. Hybridization of FA residue as a vector to other molecules and confirmation of its effectiveness as a targeting ligand comes back to the 1940s [13]. Then in 50 following years, there were numerous works devoted to FA conjugation to other molecules in PubMed database (only approximately 9% belongs to these 50 years compared of all amount of works devoted to this area of research from the 1940s to nowadays), while later the number of works grows dramatically, and their number in the recent 10 years have reached approximately 55% of the total number.

There are several FA-linked applications for targeted delivery of therapeutic agents that have been tested in the clinic so far. In some cases, the authors call the systems obtained for targeted delivery via FR “FA-targeted”. So, the applications include FA-targeted protein toxins (such as use of FA-Pseudomonas exotoxin conjugate to kill FR-positive cancer cells), FA-targeted chemotherapeutic agents (such as FA-paclitaxel and FA-camptothecin), FA-targeted immunotherapy (such as use of FA/anti-T cell receptor to train the immune system to recognize tumor activating mutations), FA-targeted gene therapy (such as FA-nonviral gene therapy vectors) [14, 15], folate-targeted antisense oligodeoxyribonucleotides (ODN) and small interfering RNAs (siRNA) delivery (such as use of ODN or siRNA to knockout or modulate gene expression) [16], radioimaging and radiotherapic agents [17, 18], and folate-targeted systems for magnetic resonance imaging (MRI) and other. Some of the therapeutic systems including FA residue have been tested in preclinical [19] and clinical studies up to now (for instance NCT04241315 completed in 2023).

Nanosized and submicron particles, or nano- and submicroparticles (NSPs), have been widely engineered for targeted cancer therapy and diagnostics in recent decades. The NSPs can be core-shall, dendrimeric, layer-by-layer structures, mono- and multicomponent particles, micelles, etc. [20–22]. Non-organic, organic, and hybrid particles with components of varied molecular weight have been evaluated. Among natural (albumin, gelatin, chitosan, dextran, hyaluronic acid, lactoferrin) and synthetic [polyethylene glycol, polyamidoamine, polylactic acid, polyethyleneimine, poly(L-lactide)-b-poly(ethylene glycol)] macromolecules, albumin as a component of NSPs possesses a great interest due to its biocompatibility, biodegradability, low immunogenicity, the long half-life, minimal toxicity, good stability, good reactivity, and high compatibility with drug molecules [23]. It has been shown also that incorporation of albumin in the structure of nanoparticles prolongs their circulation [24], reduces the thrombogenic activity [25], and improves their colloidal stability, etc. [26, 27]. Moreover, human serum albumin (HSA) is extremely robust towards pH (this protein is stable in the pH range of 4–10), and temperature (can be heated at 60°C for up to 10 hours) [28].

Albumin in composition of NSPs can also be considered a targeting agent due to the fact that Glycoproteins (Gp18, Gp30, and Gp60) receptor and albumin-binding glycoprotein [secreted protein acidic and rich in cysteine (SPARC)] receptor secreted on the surface of tumor cells can promote preferential uptake of albumin [29, 30] and albumin nanoparticles [31]. SPARC as a major modulator of extracellular matrix proliferation and cell migration was showed to be associated with increased tumor invasion and metastasis in various tumor types [30]. As an efficient energy and nutrition source, albumin can be actively consumed by growing tumors to support amino acid metabolism of cells [32]. Moreover, albumin NSPs can penetrate and concentrate in cancer cells due to enhanced permeability and retention (EPR), what can be called passive targeting. Accumulation of NSPs takes place at tumor sites with leaky vessels, and tumor tissues show selective extravasation and retention of macromolecules and NSPs > 10 nm in a tumor [30]. The sum of these data albumin can be considered an excellent potential carrier for the drug delivery to tumor cells [33]. Albumin is considered an effective carrier also due to that albumin consumption is increased at active sites of inflammation (as well as cancer cells, synovial cells use albumin as nitrogen and energy source), therefore hypoalbuminemia develops [34].

The pull of works in PubMed database on conjugates and ligands, NSPs for targeting (3,722 works by the end of 2024) contains approximately 4% of studies devoted to albumin and approximately 8% of studies addressed to folate (Figure 1A). Among the works of this pull, in the last 10 years, the amount of works devoted to folate and albumin simultaneously accounts for approximately 15% of the total number of papers devoted to albumin and approximately 9% of the total number of papers devoted to folate (Figure 1B). There is an increasing trend of research related to folate-albumin-NSPs compared to research with and without relation to NSPs, i.e. in 2024 the portion of the papers related to folate-albumin-NSPs compared to research with and without relation to NSPs was 67% in PubMed (or 77% in Google Scholar), while the amount of works devoted to folate-albumin-NSPs is approximately 1% (or approximately 6% in Google Scholar) of the total number of papers devoted to conjugates and ligands, NSPs for targeting.

In the present review, we demonstrate a variety of folate-functionalized albumin-containing nano- and submicron delivery systems for therapy and diagnostics in terms of their application, composition, and pharmacokinetic parameters.

Conjugation of FA residue to serum albumin is used for functionalization of albumin-containing systems with three different types of complexity. The examples of the conjugation for obtaining folate-albumin-NSPs are presented in Figure 2.

The simplest systems consist of individual albumin molecules coupled with therapeutic, visualizing, or other agents with specific action without any other modifications of albumin [35]. Another type of system includes cross-linked albumin nanoparticles with chemicals [36–40]. In the third type of system, albumin is used as coverage (coating, shell) for nanoparticles (particularly inorganic). The last type currently receives great interest due to the fact that it offers various functions of NSPs to combine the merits of all components and to mitigate the possible drawbacks of bare nanoparticles, particularly magnetic [41–43].

There are plenty of studies devoted to FA non-covalent binding to serum albumin, but it was demonstrated that systems with non-covalent bound FA are not stable enough in physiological conditions [44], while covalent binding of FA to albumin results in significantly better extent of binding and uptake of systems to cancer cells than physical binding [45].

Conjugation of FA to albumin is usually carried out via carbodiimide reaction. Carbodiimides belong to the kind of zerolength crosslinkers [46] which induce the formation of a covalent bond between carboxylates and primary or secondary amines without becoming part of the final amide-bond crosslink between the target molecules. The carbodiimide reaction takes place between the folate carboxy group and the amino groups of proteins [36–39, 45, 47–58], in some cases this is a reaction between folate amino groups and free carboxylic groups of proteins [35, 59]. In the carbodiimide reaction different diimide reagents [N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) [36, 39, 40, 45, 49–51, 60]; N,N'-dicyclohexylcarbodiimide (DCC) [37, 52–56]; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) [38] or (EDCI) [58], etc.] are used. There are several main ways of conjugation between FA and albumin leading to different products. According to our analysis, the most popular ways are when in the carbodiimide reaction FA in the form of N-hydroxysuccinimide (NHS)-ester, is conjugated with albumin (Figure 3A). Interaction of NHS-esters with albumin firstly leads to the formation of a bond between the carboxylic group of the NHS-ester and amine group of protein [40] via acylation of nucleophilic residues of albumin, in particular its N-terminus and lysine residues [61, 62]. The less common but feasible way of conjugation of FA to serum albumin—a carbodiimide reaction between amino groups of FA and carboxylic groups of albumin—takes place with formation of folate-albumin conjugate of another type (Figure 3B). In this way, albumin can be taken in the form of NHS-ester [59]. Since L-glutamic acid of FA can stay at the FR pocket entrance, α-carboxylic of the glutamic acid is involved in coupling with FR [63]. Thus, it should be noted that binding affinity between FR and folate-albumin conjugate will depend on type of conjugate between FA and serum albumin [64].

HSA contains 59 lysine residues (more than 10 of them are available on the surface [65]), while bovine serum albumin (BSA) contains 58 lysines. Varanko and coauthors noticed that Lys199 is the most reactive lysine residue for conjugation of NHS-esters with human albumin [65], while for ketoprofen-N-hydroxysuccinimidyl ester (equivalent to glucuronide) in a model experiment, there were easily identified 11 ketoprofen-adduction sites (at Lys137, Lys195, Lys199, Lys212, Lys351, Lys402, Lys432, Lys436, Lys525, Lys536, and Lys541) [66]. Using mass spectrometry, it was shown that 6 lysine residues (Lys261, Lys350, Lys413, Lys431, Lys471, Lys474) located on BSA molecule surface are involved in interaction with NHS-ester of FA [67]. After formation of amide bonds through carbodiimide coupling the other amino acids located in the neighborhood of lysins and N-terminus are involved in reaction with NHS-ester [65]. In this reaction serine, tyrosine and threonine, arginine, histidine are reported to be involved [61] if they are available on the protein surface. For a mixture of bovine albumin and NHS-ester of FA, it was shown an engagement in interaction via different mechanisms of the following listed in the brackets amino acid residues: hydrogen bonding (Tyr149, Tyr340, Glu152, Glu339, Gln220, His289, Val342), π-π interaction (Tyr156), hydrophobic interaction (Lys187, Arg194, Arg256) [40].

Systems formed after coupling of NHS-ester of FA and albumin (BSA or HSA) in different forms (individual albumin; albumin in form of protein nanoparticles; albumin on the surface of organic, inorganic or in hybrid particles) are often characterized using methods of UV-Vis spectroscopy [36, 38, 40, 50, 52, 54, 58] and Fourier transform infrared spectroscopy (FTIR) [37, 40, 49, 51, 58, 68]. Methods of size exclusion chromatography [45], high-performance liquid chromatography (HPLC) [69], and gel-filtration chromatography [50, 58] followed by UV-Vis spectroscopy; differential scanning calorimetry (DSC) [40, 51]; ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS) [35] are also used. Seldom studies include quantification of conjugating via additional chemicals and calculation of: amino groups of albumin through UV-Vis spectroscopy [45] and defined amino acids using fluorescence [70, 71]. Direct biological confirmation of FA-albumin conjugation is also used when NSPs are created for subsequent biomedical usage. The synthesized systems can be tested on biological in vivo and in vitro models, particularly cell lines. For example, folate-modified systems are compared with ones without folate or the differences of an effect of folate-modified systems on cells with and without FR overexpression are compared. The main ways of biological confirmation of folate-albumin conjugation we given below.

Among NSPs containing serum albumin and FA residue (folate-albumin-NSPs), in most cases, FA residue is covalently bound to NSPs or albumin. The variety of applications of NSPs with albumin and folate in covalently bound form is listed in Table 1 and the advantages of different folate-albumin systems (conjugates, albumin particles, and shell@core structures) are listed in Table 2. The therapeutic agents could be bound covalently (for example, β-carbolinium derivates [35], chlorin e6 (Ce6) [59, 72], 5-fluorouracil (5FU) [69], doxorubicin (DOX) [58]) and non-covalently (for example, DOX [72], paclitaxel [54]) bound to NSPs.

Analysis of different studies helps to reveal that biomedical effectiveness of FA residue as targeting agent is confirmed mainly through in three ways: (1) comparison of folate-albumin-NSPs and non-modified albumin-NSPs on FR-positive cells [35–40, 49–51, 53–58, 69, 72, 75, 76]; (2) analyzing of folate-albumin-NSPs influence on cells with different amount of FRs [59, 68]; (3) comparison of folate-albumin-NSPs containing drug with the individual drug on FR-positive cells [52, 58, 71]. In all considered studies, FA residue acts as a targeting agent, which significantly improves targeting properties of NSPs by FR-specific endocytosis [77]. Nevertheless, it is worth noting that the NSPs without folate also can be characterized by enhanced targeting resulting from EPR effect, while retention effect is due to glycoprotein trap in cancer cells [78]. Combination of the EPR effect with active targeting is one of the important advantages of folate-albumin-NSPs in cancer diagnosis and therapy.

Additional nanosized cores in the biomedical delivery systems can improve their ways of application. Particularly, magnetic cores can make the systems applicable in MRI, magnetic hyperthermia (MH) (heating under the action of alternating fields) to tumors, and make possible magnetic concentrating in tumor tissues (due to magnetic characteristics of particles).

Most of the elaborated delivery systems including serum albumin and FA residue are developed for cancer therapy [including chemotherapy, photothermal therapy (PTT), photodynamic therapy (PDT), and combined therapy] and imaging. A minor part of the work is devoted to treatment of other diseases, particularly rheumatoid arthritis.

All the examples will be given below.

Nano- and submicron delivery systems including conjugate of FA and serum albumin (folate-albumin-NSPs) are widely used in research devoted to medical purposes, particularly for disease treatment and diagnostics. The prevailing number of these systems are aimed at cancer therapy through additional chemical substances and/or physical effects (i.e. chemotherapy, PDT, PTT, and MH). Fewer studies have focused on the detection and visualization of tumor nodes and singular cells via MRI and/or additional labelling of NSPs with dyes. It is worth noting that such complex hybrid particles as folate-albumin-NSPs are vital for tumor types with FR overexpression which often is associated with worse outcomes of disease (i.e. breast cancer, metastatic squamous cell carcinomas). Among such upgraded systems, we can list the systems with dual targeting (when one of the biovectors is FA residue), stimuli-response systems [which change their state according to different characteristics of microenvironment (i.e. pH, composition, etc.)]. Furthermore, in recent years the number of complicated systems for combined cancer therapy is growing. In these modern systems, the sum of different therapy approaches synergistically enhances the effects of each one, which results in a significant increase in total effectiveness. Additionally, there are systems for theranostics allowing identifying the disease in its early stages, avoiding side-effects of drugs, and monitoring the effectiveness of the therapeutic agents and treatment process. Substantially lower amounts of studies have been focused on the development of treating systems for inflammatory diseases. In all these systems FA residue is used as a targeting agent which allows to enhance targeting of systems and decrease their toxicity and side-effects. Serum albumin is used as a biocompatible component carrying out drug transport and bringing additional targeting to delivery nanosystems. Using of folate residue and serum albumin contributes to advantages of NSPs. The use of additional nanosized cores in the biomedical systems can improve their applications. Particularly, magnetic cores can make the systems applicable in MRI, MH to tumors, magnetic concentrating in tumor tissues.

So, design of the new complex systems aimed at cancer treatment is currently actively progressing and the grade of complexity of systems is constantly increasing.

However, there are only a few FA-linked applications for targeted delivery of therapeutic agents that have been tested in the clinic, despite hybrid systems containing FA conjugated to albumin (particularly supramolecular systems) are promising agents for medicine, from diagnostics and therapy of cancer to treatment of inflammatory diseases. A number of underlying issues can be considered. First of all, complex hybrid particles consist of multiple components: carrier (or platform), targeting ligand, and pharmaсeutically active compounds. These complex systems are hardly reproducible when transferred from laboratory testing to commercial manufacturing. Secondly, clinical studies of FR-targeting have shown limited sensitivity and sensitivity as diagnostic tools, thus FR-targeting using FA residue in the folate-albumin NSPs is less specific than some other possible ways of targeting (that is confirmed by a constant level of studies devoted to folate-albumin NSPs compared to all NSPs for targeting: approximately 1% in PubMed or approximately 6% in Google Scholar).

The most common form in which FA is covalently bound with albumin is NHS-ester form, and we have briefly shown in the review the ways of folate-albumin conjugate obtaining by this and other paths of carbodiimide chemistry. Confirmation of FA residue conjugation to NSPs in the prevailing number of research was received through testing the synthesized systems on in vivo and in vitro models, including usage of cell lines. Additionally, some researchers characterized hybrid systems and/or folate-albumin conjugates with physical/physicochemical methods: UV-Vis spectroscopy, FTIR, DSC and X-ray diffraction, UV-MALDI-TOF MS, high-performance liquid and size exclusion chromatography. We suppose that a thorough analysis of modern studies (including those, characterizing quantitative parameters of binding) is undoubtedly needed for characterization of both non-covalent and covalent binding between FA and serum albumin. Such an analysis could reveal more patterns for design of folate-albumin NSPs that we consider rather important due to two reasons. Firstly, there are some examples of successful biomedical usage of FA and serum albumin both with and without their conjugation (hybridization), so further engineering of folate-albumin NSPs definitely makes sense. Secondly, conjugation between FA and albumin is just an example of ligand conjugation with albumin applicable for hundreds targeting agents and drugs.