Introduction

Nanoparticle delivery systems have attracted much attention as a drug delivery strategy. It has the advantages of unique drug targeting, slow controlled release features, and protection function, especially in the transfer of hydrophobic drugs. Nanotechnology Initiative (NNI) defines nanotechnology as a size of approximately 1 to 100 nanometers (nm), but it can be extended to 1000 nanometers over a wide range.1 The ideal nanoparticle carrier should have the following properties: specific targeting, drug release controllability, carrier non-toxic, and biodegradable.

Albumin is a very soluble protein, which is consisting of 585 amino acid residues. The relative molecular weight of albumin is 66,500 Da, and it can maintain biological activity under conditions of pH 4–9, 40% ethanol, or heating at 60 °C for 10 hours.2 The three-dimensional structure of albumin, including hydrophilic and hydrophobic domains and charged amino acids, enables it to deliver drugs with different physicochemical properties. Meanwhile, the amino and carboxyl groups on the surface of albumin provide binding sites for polymers or ligands.3 In addition, albumin is more suitable as a carrier for drug delivery owing to its ready availability, nontoxicity, biodegradability, immunogenicity, and preferential accumulation and uptake in tumors and inflamed tissue.4 Many therapeutic drugs and metabolic compounds in blood plasma are transported by human serum albumin (HSA), one of the smallest proteins and abundant in blood plasma.5 HSA affects the drug metabolism process and therapeutic effect in vivo, which has attracted researchers to explore the mechanism and influencing factors of drug albumin binding.6–8 Large amount of clinical trials on albumin-based nanoformulations have been described elsewhere.9 Here, we listed marked albumin nanodrug in Figure 1. In fact, it has been a long history for albumin being used for drug delivery, reported as early as 1960s, initially as a diagnostic agent.10 Nano-sized albumin drug carrier emerged in 1970s11 and 1980s,12 whereas the latter one was developed into Nanocoll®, [99mTc]-labeled nanocolloidal albumin, approved for oncology rheumatology in 1995.13 Some other albumin nanoformulations were developed during 1990s, including hydrocortisone-albumin, which was reported for treating eye inflammation,14 and methotrexate–albumin conjugate, which was conducted in a Phase I trial for cancer treatment.15 The latter one was reported capable of rheumatoid arthritis treatment in 2003.16 Among others, due to the fast development of nanoparticle albumin bound (nab) technology, one useful and proven drug delivery platform that poorly water-soluble active pharmaceutical ingredients can be encapsulated into nanoparticles,17 Abraxane® (nab-paclitaxel) was approved by the Food and Drug Administration (FDA) in 2005. It showed the advantage of avoiding solvents/solubilizers during the formulation process, high tolerated dose, long drug residence time in the tumor, short infusion time, and it reduced risk of hypersensitivity reactions. Abraxane® (~130nm) is considered to dissociate into a 10 nm albumin bound paclitaxel (PTX) complex during intravenous administration18 and widely used for clinical tumor treatment due to its accumulation in tumors, increasing the concentration of PTX in the tumor stroma and enhancing its anti-tumor activity.19 The successful launch of Abraxane®, is a milestone in the development of albumin drug delivery systems and is also seen as a turning point in nanomedicine. In 2007, just two years after its launch, annual sales reached approximately $300 million. Based on the nab technology, Fyarro® (nab-sirolimus) was approved in November 2021.20 Fyarro® is able to inhibit the mammalian target of rapamycin (mTOR), which controls key cellular processes such as cell survival, growth, and proliferation, and is often dysregulated in human cancers. Compared to oral mTOR inhibitors, intravenous administration of nab-sirolimus demonstrated higher intratumoral accumulation, stronger mTOR inhibition, and higher tumor growth inhibition. Fyarro® was approved for the treatment of unresectable or metastatic malignant perivascular epithelial cell tumors (PEComa) in adults.21 Besides, Nanozora®, an albumin-binding nanobody directed against human TNF-α, was approved in Japan for the treatment of rheumatoid arthritis in 2022. Other drug formulations that bind to albumin in vitro or in vivo are currently at different clinical stages.22

Figure 1 Marketed albumin nanodrug products.

Albumin nanoparticles as drug delivery systems have been mainly used for drug delivery and targeted therapies in the last two decades. Figure 2 shows the numbers of publications in PubMed combining the key word “albumin nanoparticle” with different applications from 2004 to 2023, with anti-tumor being a major area of research. However, with the increasing health impact of inflammatory diseases globally, research on albumin nanoparticles in the field of anti-inflammation is on the rise. Great potential of using albumin nanoparticles for different applications remains covered. Therefore, in this review, we first discuss various albumin nanoparticle fabrication methods, from both pros and cons. Then, modification strategies used for the functionalization or stabilization of albumin nanoparticle will be introduced, including organic albumin nanoparticles (targeted and non-targeted modification with organic building blocks), metal albumin nanoparticles, inorganic albumin nanoparticles, and albumin nanoparticle-based hybrids. Finally, further application on albumin nanoparticles used for various critical diseases will be discussed.

Figure 2 Number of peer-reviewed publications from 2004 to 2023 combining key words “albumin nanoparticle” and different applications (PubMed database).

Albumin Human Serum Albumin (HSA)

HSA is abundant in plasma protein (35–50 g/L human serum), which has the inherent characteristics of ligand binding, wide tissue distribution, and long cycle half-life. There is a total of 35 cysteine residues within the HSA molecule, of which 34 residues form 17 disulfide bonds with each other. These 17 disulfide bonds make the structure of proteins more stable and can be modified under certain conditions.23,24 The three structural domains I, II, and III of Albumin have flexibility. Further, the major three structural domains and their subdomains stabilized with disulfide bonds in the albumin molecule offer the different binding sites of the drug, which can not only bind to multiple compounds to improve the solubility of hydrophobic compounds, but also bind to specific ligands and antibody molecules to achieve targeted drug accumulation.7 There are two ways for drugs to bind to albumin: one is by covalent bond, and the other is by hydrophobic embedding in the albumin hydrophobic cavity. For Abraxane®, PTX and albumin are combined by hydrophobic interaction.8,25

Bovine Serum Albumin (BSA)

BSA and HSA exhibit high homology in structure, making them widely studied as model proteins. BSA is a water-soluble spherical protein containing a polypeptide chain composed of 583 amino acid residues,26,27 with an isoelectric point of ~4.7. It makes up approximately 60% all proteins animal serum.28 BSA has a unique hydrophobic region in its molecular structure27,29 and is also used to study the interaction between albumin and drugs.30,31 The properties of rich, low cost, easy to purify, unusual ligand-binding properties, and widely accepted characteristics in the pharmaceutical industry (easy process ability and scalability) have been widely used in drug delivery systems.32,33 BSA is rich in functional groups, such as carboxyl and amino groups, and has a strong affinity34 with metal nanoparticles. It is often used as a coating agent for metal nanoparticles to improve their colloidal stability and biocompatibility, and endow them with the characteristics of easy functionalization and low toxicity, which is conducive to the application of metal nanoparticles in biomedicine and other fields.35,36 Compared to HSA, the limitation of BSA is its potential immunogenic response.33

Preparation Techniques Desolvation

The preparation of HSA nanoparticles by desolvation is reproducible and relatively simple.37 This method has a short preparation cycle without surfactants and widespread applications in the encapsulation of hydrophilic and hydrophobic drugs.2 In this method, acetone or ethanol as a desolvation agent is added to the aqueous solution of albumin with the condition of a constant stirring and it continues until the solution becomes turbidity.38,39 The effect of desolvating agents is to gradually alter the tertiary structure of albumin, leading to phase separation and aggregation of proteins.40,41 A crosslinking agent, such as a glutaraldehyde solution, is necessary to stabilize the unstable particles. In order to completely crosslink the amino acid residues in the protein, the suspension is stirred continuously. The guanidino side chains in the arginine residues and amino moieties in lysine residues of albumin are solidified with the aldehyde group of glutaraldehyde by a condensation reaction.42–44 And then, the nanoparticles are purified with centrifugation. The preparation process is shown in Figure 3A. Langer et al38 confirmed that the rate of ethanol addition, the pH of the solution and the concentration of the albumin had an impact on the particle size of nanoparticles in this method. The smaller nanoparticles were formed with the smaller the ethanol addition rate and the high pH levels achieved proper particle size. At an albumin concentration of 50mg/mL, the smallest particles were obtained.

Figure 3 Albumin nanoparticle preparation. (A) Desolvation; (B) Emulsification; (C) Nab technology; (D) pH-condensation; (E) Thermal gelation; (F) Nano spray drying; (G) Self-assembly; (H) Microfluidic technology.

A tubing pump is an essential instrument to carefully control desolvation agent addition speed. Some studies reported manually adding ethanol using a syringe. Bax et al45 optimized the design of the device and reported a method for simplifying the program by controlling the addition of ethanol to the device. It is important to note that, in this study, N-(3-Dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC) as a cross-linker was used, which is a zero space cross-linker and can be easily removed instead of glutaraldehyde, resulting in reducing the preparation time of nanoparticles to 3 hours. Although reports of albumin nanoparticles used for drug delivery have used glutaraldehyde for stabilization, in vivo studies have shown that residual aldehydes have a certain toxicity, which restricts their use considerably.46,47 Luna-Valdez et al48 reported the formation of nanoparticles from wheat bran water extract by a cold setting desolvation approach, which avoided using glutaraldehyde (toxic) and organic solvent. Besides, physical crosslinking methods include drying, heating and ultraviolet radiation27 and other potential crosslinking agents such as glucose49 are developed.

Emulsification

The emulsion-solvent evaporation method is more suitable for producing nanoparticles with a smaller size and lower polydispersity index, and it is a reliable method with good repeatability and amplification potential.50,51 Compared to pH-condensation technology or microfluidic methods, this method is less time-consuming, less complex, and uses fewer chemicals. An albumin solution (aqueous phase) is stirred with a non-aqueous solution (oily phase) containing an appropriate amount of emulsifier to obtain a crude emulsion. The organic solvents such as dichloromethane or chloroform are usually used as oil phase. The emulsion can be homogenized by ultrasonic treatment or homogenization. Then, the emulsion droplets are solidified by chemical crosslinking or heating deformation, and finally, the residual organic solvent is removed to collect albumin nanoparticles. This method is suitable for hydrophobic drug encapsulation by binding with hydrophobic cavities on HSA molecules. However, the use of toxic organic solvents is major setbacks of this method.52 Besides, surfactants are required for emulsion stabilization. Figure 3B shows the preparation process. Alfagih et al53 prepared nanoparticles encapsulating model antigen and BSA via double emulsion solvent evaporation. In addition, by adding chitosan hydrochloride (CHL) into the outer phase of the lotion solvent, a hybrid cation CHL nanoparticle was formed, which led to surface adsorption on the nanoparticle. It may be used to transport proteins to the lungs for immune stimulation applications such as vaccines.

Figure 4 The formation of targeted modified albumin nanoparticles and the drug release in the cancer cells.

Abbreviations: HA, hyaluronic acid; CD44, surface antigen differentiation group 44; FR, folate receptor; TfR, transferrin receptor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TNF, tumor necrosis factor.

Nab Technology

Nab technology can mostly not alter the physiological properties of HSA among these techniques.54 Nab technology (Figure 3C) is an albumin nanoparticle preparation technology that uses albumin as a matrix and stabilizer. Under high shear forces, an oil phase containing an aqueous insoluble drug and an albumin-containing aqueous phase are mixed to prepare an O/W emulsion in which the drug is in the absence of any conventional surfactant or any polymer core. First, the drug is dissolved in an organic solvent (usually chloroform, dichloromethane) at a high concentration as an oil phase. Secondly, albumin is dissolved in an aqueous medium to obtain an aqueous phase. Then, the oil and water phases mix under high-pressure homogenization, followed by vacuum evaporation of the solvent quickly to obtain a colloidal dispersion system composed of ultra-fine nanoparticles. The cavitation during homogenization makes the free sulfhydryl group of albumin cross-linked to form a disulfide bond. During the process, the drug is wrapped inside the nanoparticles, preserving the physiological properties of HSA. Compared with the traditional preparation method, nab technology has no conventional surfactants or any polymer core and no special infusion equipment.9 Furthermore, the albumin acts as a lyophilization agent without adding the other conventional freeze-dried protective agent. He et al55 assessed the risk of nab-PTX-related adverse events (AEs) compared with traditional taxanes in various primary solid organ malignancies. Although nab-PTX increases the risk of general hematological and non-hematological AEs, allergic reactions are significantly reduced, and the neurotoxicity is easier to recover. Compared to the traditional PTX, lower doses of nab-PTX administered weekly had better tolerance. However, it still carries ocular adverse effects.56 A significant decrease in visual acuity was observed in patients receiving nab-PTX treatment. In clinical, when the treatment cannot be stopped due to the patient’s general condition, the effective alternative treatment is topical dorzolamide or steroidal treatment.

In a recent study, the palmitate albumin nanoparticles loaded with PTX were prepared based on the nab technology: anhydrous ethanol and chloroform are used as the solvents for emulsification and homogenization under high pressure without surfactants. Albumin was not easily denatured at low temperatures during the preparation process. Finally, the organic solvents were removed through ultrafiltration.57 In 2016, Furedi et al58 prepared Voriconazole nanoparticles (VCZ-NPs) by nab technology. The concentration of HSA in water was controlled at 2% because during high-pressure homogenization processes, higher HSA concentration can cause sample foaming in the samples, resulting in unstable and unpredictable VCZ concentration. An acceptable PDI value (below 0.3) for VCZ-NPs was obtained under the condition of six homogenization cycles at 1800 bar. And the optimized particles met the requirements for intravenous administration with an average particle size of 81.2 ± 1 nm. Furthermore, VCZ-NPs showed a good encapsulated concentration of 69.7 ± 4.2% and increased the water solubility of VCZ greatly.

pH-Condensation

The pH-condensation method (Figure 3D) is to dissolve the drug in an HSA solution at room temperature and incubate it in the dark with the adjusted pH value. The solution is stirred or sonicated to accelerate the coagulation process of albumin followed by crosslink with glutaraldehyde. Then, HSA nanoparticles (HSA-NPs) are obtained by centrifugation, washing, and freeze-drying. However, it is not convenient to control the particle size of the nanoparticles by adjusting the pH value. It usually controls the particle size by adjusting the salt concentration or adding other organic solvents to obtain uniform particle size and spherical nanoparticles.

Lin et al34 reported the preparation of HSA-NPs with approximately 100 nm in diameter using the pH-condensation method without surfactants. The particles were prepared by dropping acetone into an HSA aqueous solution with a pH of 7–9, then cross-linking with glutaraldehyde and purification by gel permeation chromatography. The study showed that as the pH value of the HSA solution increased, the particle size decreased, which was clear as the ionization of HSA increased, and the repulsion of HSA molecules occurred during particle formation. HSA nanoparticles with sizes between 90 and 250 nm were obtained through the control of the pH and the addition of acetone. Merodio et al43 added ganciclovir and a cross-linking agent in an albumin aqueous solution. Then, the pH of the solution was adjusted to the isoelectric point of the protein. The aqueous phase is washed with ethanol (Vwater/Vethanol=1:2) to obtain albumin nanoparticles. The main drawback of this method was that the pH value was adjusted under salt-free conditions, while the glass electrode had limited reliability, especially in high protein concentrations.

Thermal Gelation

In brief, protein conformational changes and unfolding can be induced by heating the albumin solution, subsequent protein–protein interactions (hydrogen bonding, electrostatic and hydrophobic interactions, and disulfide sulfhydryl exchange reactions) and aggregation of albumin particles. This method avoids the potential toxicity caused by adding organic solvent. The properties of the obtained nanoparticles depend on the process conditions, such as pH, protein concentration, and ionic strength.9 However, this method is not suitable for heat-sensitive drugs. Figure 3E shows the preparation process. Li et al59 proposed a novel self-assembly method through thermal-driven to prepare BSA-NPs. BSA and vanillin (non-toxic, as a crosslinking agent) were dissolved in deionized water at 37 °C. Then, the solution was incubated at 70 °C for 2 hours to form BSA-NPs. During the heating process, a large number of covalent bonds were formed, such as amide and disulfide bonds between BSA molecules, which greatly improved the stability of nanoparticles.

Nano Spray Drying

Spray drying is a mature method of producing dry powder from liquid phases commonly used in the pharmaceutical industry with the advantage of integrated particle formation and drying.60,61 In this method, particle formation occurs in a continuous, single-step process. Moreover, through the simple operation of process parameters or configuration changes, the ideal particle properties, such as particle size, flow property, and bulk density, can be adjusted. Compared with liquid formulations, the solid products through this method have the advantages of physicochemical properties and stability.62,63 Therefore, nano spray drying may be a general and commercially feasible technology for preparing peptide and protein drugs. A conventional spray drying process includes the following steps: Liquid raw materials are atomized into small spray droplets, and then contact with hot dry gas at high temperature to evaporate water. When water evaporates from a droplet, it forms a solid product and recovers the powder from the dry gas.64,65 However, albumin is prone to deformation and inactivation.66 The preparation process is shown in Figure 3F.

However, due to the product loss in the drying chamber wall and the separation of fine particles by the cyclone separator (<2 μm), the capacity of spray drying is low, and the yield of traditional spray drying on a laboratory scale is not optimal (20–70%). In order to improve the technology and expand the application range, B ü chi (Switzerland Labortechnik AG) has developed the fourth laboratory-scale spray dryer B-90 (following previous generations).67 This equipment is suitable for producing fine particles (300 nm – 5 μm) at a satisfactory, even a small amount of sample (milligrams).68 The latest development in technology and the successful launch of the nano spray dryer B–90 have produced submicron spray drying particles.69 Although the nano spray dryer B–90 has significant advantages, the mini spray dryer B–290 with dual fluid nozzles can more directly expand the process to the initial pilot stage, and then to the industrial production stage. The feed flow rate is 1.41 kg/h, much higher than the previous 0.60 kg/h, which greatly improves production efficiency.

Self-Assembly

Self-assembly technology (Figure 3G) increases the hydrophobicity of albumin molecules through certain methods, such as the reduction of disulfide bonds within protein molecules by the addition of denaturants (β-mercaptoethanol, dithiothreitol and cysteine),27 and the reduction of primary amine groups on protein surfaces by the addition of lipophilic drugs.70 The drug molecules can combine with the hydrophobic region of albumin under stirring conditions, thus inducing the formation of albumin nanoparticles. Self-assembly technology mainly forms a clear and stable structure through non-covalent interaction between molecules. Nanoparticles have the characteristic of small particle size and good flexibility due to the solidification of nano micelles. However, this method is only used for lipophilic drugs and has difficulties in scaling up.71 In addition, the addition of reducing agents has incidences of potential toxicity. In the self-assembly method, glutathione (GSH) can be used as a reducing agent to prepare a redox-sensitive albumin nanoparticle. GSH disrupts disulfide bonds in albumin molecules, exposing hydrophobic cavities and binding with hydrophobic drugs, which then re-oxidize to form disulfide bonds. This nanoparticle can rapidly release drugs in tumor tissue, as tumor tissue result in higher levels of GSH compared to normal tissue.72,73 Safavi et al74 reported a strategy for preparing BSA nanoparticles loaded curcumin (CCM-BSA-NPs) by self-assembly with high ionic strength of buffer solution instead of reducing agents at room temperature. Compared to CCM-BSA-NPs with DTT, the CCM-BSA-NPs had better antioxidant and more effective biological activity. Their group also synthesized piperine-loaded HSA-NPs (PIP-HSA-NPs) with self-assembly and desolvation methods, respectively. The results indicated that the self-assembled nanoparticles had significantly higher drug encapsulation efficiency and drug loading efficiency than the particles obtained by the desolvation method. And the self-assembly method maintained the secondary structure of HSA. Besides, the NPs by self-assembly method exhibited more cumulative drug release, making them have better therapeutic effects on tumor cells.71

Microfluidic Technology

Recently, studies have shown that microfluidic systems can accelerate clinical translation of nanoparticles due to their ability to generate nanoparticles in a well-controlled and reproducible manner.75 The microfluidic device can produce nanoparticles from milliliters or even several liters by viscously mixing nanofluids. Compared to the traditional preparation methods, the liquid is in the laminar flow state in the microfluidic chip, and the flow rate of the liquid is consistent, with a high mixing efficiency. Therefore, the size distribution of nanoparticles is uniform, with good repeatability and high efficiency.76 The preparation process is shown in Figure 3H, where the aqueous phase and the organic phase flow into the mixture from multiple inlet ports on the chip. Samples are collected within specified time intervals. Nanoparticles with different requirements were prepared by adjusting the flow rate and the shape of the chip. For example, Sun et al77 prepared cabazitaxel-HSA NPs with an inverted W-type microfluidic chip through microfluidic technology without any cross-linking agent and toxic solvents. The nanoparticle showed a higher efficiency and higher drug loading than that prepared by the traditional preparation.

Albumin Nanoparticles Modification Organic Albumin Nanoparticles

Albumin is typically negatively charged at a pH of 7 (PI=4.7),78–80 which makes it carry different types of chemotherapy drugs. Although albumin nanoparticles reduce drug side effects and improve the curative effect, because of the antibody targeting or protein adsorption in plasma, easily results in the mononuclear macrophage system identification and consumption, thus, the nanoparticles are cleared in the systemic circulation.81,82 In this section, we briefly introduce albumin-based organic nanoparticles for efficient delivery. The high content of carboxyl and amine groups and different binding sites offer a great possibility that albumin nanoparticles can be easily coated with different polymers or ligands, through covalent coupling to change surface properties (such as PEG modification to improve the hydrophilic, avoid the macrophage phagocytosis), binding with ligands (such as folic acid modification of tumor cells with rich folate receptors targeting, immune antibody interacts with antigen (antibody-mediated system)), and other measures to realize the active target of drugs.

Non-Targeted Modification

Circulating albumin nanoparticles can passively accumulate in tumor tissue through enhanced penetration and retention (EPR). In addition, the properties of nanoparticles can be controlled by changing particle size, surface coating, and binding with ligands to achieve tissue-specific targeting. These approaches enable the capacity of HSA NPs avoiding the uptake of macrophages mainly located in the reticuloendothelial system (RES) of the liver.83Table 1 shows the examples of albumin nanoparticles modified with non-targeted modification polymers.84–99

Table 1 The Examples of Albumin Nanoparticles Modified with Non-Targeted Modification Polymers

Polyethylene Glycol (PEG)

Since the albumin nanoparticles are cleared in a few seconds or several minutes after intravenous administration, they cannot be applied to tumor therapy. PEG is a hydrophilic, non-toxic, non-immunogenic Polymer material, and has good biocompatibility.100,101 It can be covalently linked to albumin to modify nanoparticles. Studies have shown that albumin nanoparticles coated with PEG could reduce the recognition of RES, resulting in a prolonged plasma-circulating half-life.102 Then, they can passively penetrate malignant tissue, where they are retained and accumulated to improve therapeutic efficacy. In addition, PEG also can be used as a linker to connect albumin and ligands, making it targeted.103,104

Fahrländer et al84 developed several PEGylated HSA NPs and the PEGylation extent was characterized and quantified. Actually, this study aims to develop a quantitative PEG method and monitor the effect of different PEG reagents on the amount of PEG attached to the surface of HSA NPs. PEGylation is a potential way to improve the blood circulation time of nanoparticles. Results confirmed that modifying HSA NPs covalently with a suitable PEG derivative could enhance plasma circulation in vivo. In Sharma’s study, 5-FU conjugated PEG-HSA-NPs were developed to improve the pharmacokinetic of 5-FU and therapeutic profiles. The results indicated that the IC50 value of PEG-modified nanoparticles was significantly lower than that of unmodified nanoparticles, and the half-life was significantly prolonged.105

Chitosan

Chitosan is a cationic, alkaline polysaccharide polymer, which has unique biological activity. It has excellent biocompatibility and biodegradability, less toxic, meantime low prices.106 The deacetylation levels and molecular weight of chitosan have a certain impact on its toxicity. Lower toxicity is related to less molecular weight.107 Chitosan-coated albumin nanoparticles have been studied for delivery of DNA,108 the antitumor drug,90 the nose to brain drug,109 and the ocular drug.88 The surface charge of nanoparticles has a significant impact on its interaction and absorption with the cell membrane.107 Positively charged nanoparticles demonstrate an enhanced internalization on account of the ionic interactions with the negative-charged cytomembrane, and then they are able to escape from lysosomes. So, positive-charged nanoparticles exhibit perinuclear localization, whereas the negatively charged and neutral nanoparticles prefer to colocalize with lysosomes.110,111 Modified by chitosan coating, albumin nanoparticles can shift the original negative charges to positive charges, which exhibits the potential for drug delivery.112 The use of water-soluble chitosan modification can effectively improve the surface hydrophilicity of nanoparticles and adjust the surface charge to near neutrality, thus avoiding the adsorption of plasma protein and macrophage phagocytosis, and extending the cycle time of the nanoparticles in the blood.113,114 However, excessive positive charge brings greater toxicity. In Esfandyari-Manesha’s studies, chitosan-coated HSA-NPs showed more cytotoxic effects than the negatively charged HSA-NPs.112

Polyethylenimine (PEI)

PEI is a cationic polymer, which can provide positive charges to the albumin nanoparticles’ surface to facilitate the transport of negatively charged small molecules as well as facilitate transport across the Blood-Brain Barrier (BBB).96,115

Mohammad-Beigi et al78 developed polyethyleneimine-coated HSA (PEI-HSA) NPs applied to different neurodegenerative disorders. The aggregation of the 140-residue protein α-synuclein (αSN) shows an important role in the pathogenesis of these diseases. However, αSN could interact strongly with PEI-HSA NPs, resulting in significant changes in the secondary structure of αSN. These findings emphasized the potential role of PEI-HSA NPs in reducing the pathogenicity of αSN in vivo. In 2016, the group prepared cationic PEI-HSA NPs loaded with the antioxidant gallic acid (GA) through adsorption which could inhibit αSN aggregation. They concluded that PEI-HSA-GA NP is a potential delivery system for transporting GA to the brain.116

PEI is also an effective carrier for nucleic acid delivery.117 Rahimi et al92 developed PEI-coated BSA NPs (BSA-PEI NPs) for efficient delivery of CRISPR/Cas9 system in both ribonucleoprotein and DNA (px458 plasmid) forms. Through electrostatic interactions, PEI with a positive charge binds to the surface of BSA and increases cellular uptake.

Polycation/Polyanion Deposition

In cancer-targeting therapy, layer-by-layer nanoparticles (LbL-NPs) displayed their growing important positions, which were prepared by depositing polycation and polyanion on the surface of colloids.118 LbL NPs offer unique benefits, including improved stability of nanocarrier in circulation, controlled release of drug, and storage stability. The electrostatic effect is the main driving force for multi-layer assembly, and other forces include hydrophobic interaction, hydrogen bond, covalent bond and so on. Ruttala et al97 reported that polyarginine and poly(ethylene glycol)-block-poly (L-aspartic acid) deposit alternately onto an albumin conjugate for the formation of the LbL-based nanoparticle albumin-bound PTX (nab-PTX). The colloidal stability was improved because of the presence of polyamino acids, which prevented the dissociation of nab-PTX even in the case of high dilution.

Targeted Modification

In addition to passive targeting, the active targeting of nanoparticles further improves the therapeutic effect of tumors. Tumor cells differ from normal cells in their biology and physiology and generally express specific markers on the cell surfaces that can be exploited for drug-targeting approaches. The clinical treatment of the tumor is not ideal; the main reason is that anti-tumor drugs produce a variety of serious adverse reactions. Receptor-mediated targeting agents are therefore developed to deliver the drug to tumor cells specifically, reduce damage to normal cells, and reduce side effects.119 Active targeting can be achieved by a specific modification (Figure 4). The ligand modified on the surface of the nanoparticle has specific targeting and high affinity, which leads to specific targeting of the tumor cells.120,121Table 2 shows the examples of albumin nanoparticles modified with targeted ligands.122–141

Table 2 The Examples of Albumin Nanoparticles Modified with Targeted Ligands

Hyaluronic Acid (HA)

Surface antigen differentiation group 44 (CD44) is a widely distributed glycoprotein on the cell surface.142,143 As one of the important receptors of hyaluronic acid, CD44 is overexpressed on the surface of malignant cells, it therefore can be used as an active target by HA and CD44 receptor, mediating tumor-targeted therapy.144 The active amino group on the surface of albumin nanoparticles can be coupled with hyaluronic acid to achieve the targeting modification.

Huang et al145 prepared HA-coated FITC-Cx43 MP-HSA NPs, while Connexins (Cx) are transmembrane proteins and Cx43 mimetic peptide (Cx43 MP) has the effect to reduce inflammation, edema, and vascular leakage. Experimental results showed that HA-coated HSA NPs led to higher cellular uptake in vitro and enhanced in vivo tissue penetration through ligand–receptor interactions between CD44 receptors and HA, as compared to uncoated HSA NPs. In addition, HA-coated HSA NPs were also a sustained-release system with better cell biocompatibility, which reduced injection frequency in clinical practice.

Folic Acid (FA)

Elevated expression of the folate receptor (FR) was found in tumor cells.146 Based on the research, 97% of investigated ovarian carcinomas overexpressed FR. Hence, FA becomes an attractive targeting molecule besides its small molecular weight (Mw441.4 g/mol), low immunogenicity, as well as its good biological compatibility. Albumin nanoparticles decorated by FA can activate targeting to tumor cells, which helps to reduce drug side effects, and improve therapeutic effect.147–149 Usually, EDC and N-hydroxy succinimide (NHS) are used to activate the carboxyl group of folic acid and prepare an active ester intermediate (FA-NHS), which can couple with albumin.

Chen et al150 prepared a FA-conjugated HSA magnetic nanoparticle (FA-CDDP/HSA MNP). Characterization indicates that FA-CDDP/HSA MNPs could be prepared successfully with an average size of 79 nm. Wang et al151 developed FA-decorated HSA loaded with nanohydroxycamptothecin (nHCPT) nanoparticles (FA-HSA-nHCPT-NPs), and determined the antitumor activity of FA-HSA-nHCPT-NPs adopting the MCF-7 cells with FR overexpressed on the surface. In the meantime, BALB/c mice were inoculated with human MCF-7 cells in situ and ectopic, respectively. FA-HSA-nHCPT-NPs showed more effective antitumor activity than that of raw HCPT. Meanwhile, the concentration of HCPT in tissue distribution analysis was much higher in the mice treated with FA-HSA-nHCPT-NPs. The group of FA-HSA-nHCPT-NPs also showed a higher tumor inhibitory rate than the raw HCPT. In conclusion, FA-HSA-nHCPT NPs can serve as a feasible delivery system with significant targeting effects on tumors. The examples above indicate that HAS-NPs can play a different role by modifying different polymer materials in cancer treatment. Based on previous research,152 our group developed FR-targeted HSA NPs of cabazitaxel (FA-NPs-CTX) by a self-assembly method. FA-NPs CTX did not affect the drug release of NPs and has good biocompatibility and physicochemical properties, such as sustained release and colloidal stability. Compared with FA-unmodified NPs-CTX, FA-NPs-CTX showed a greater inhibitory effect on tumor cells overexpressing FR in the cytotoxicity experiments.153

Transferrin (Tf)

Endocytosis mediated by membrane Tf receptors (TfR) is an effective cellular uptake pathway during the administration of anticancer drugs. The TfR is overexpressed on a great many tumor cells,154 so that Tf and the antibodies against the TfR have been widely studied. Due to the presence of TfR in the BBB, Tf-coupled solid lipid nanoparticles can enhance drug delivery to the brain.155 The mechanism by which nanoparticles bound to Tf enhance therapeutic efficacy seems to be that cells have a higher absorption of drugs.133,156

Ulbrich et al133 prepared the Tf-coupled HSA NPs and the TfR-antibody-coupled HSA NPs. NHS-PEG-MAL was used as a cross-linking agent to couple transferrin or TfR-mAb to connect HSA-NPs. Loperamide-loaded HSA NPs coupled with transferrin or TfR-mAb had a strong anti-nociceptive effect, while IgG2a-modified HSA NPs could not transport the drug through the BBB. Therefore, these TfR mAb or Tf-mediated nanoparticles are effective carriers for drugs to cross the BBB.

Monoclonal Antibodies (mAb)

Tumor-specific ligands can be coupled to the surface of nano-carrier systems to achieve targeted drug delivery.157 Among them, mAbs have great potential. Hasan et al developed a targeted delivery system of anti-human epidermal growth factor receptor 2 (anti-HER2) mAbs using polyethylene glycol albumin nanoparticles for breast cancer cells. A new mAb (1F2) targeting the HER2 extracellular domain connected to the surface of albumin nanoparticles was proved to have the highest cell uptake rate.158

Wagner et al136 covalently coupled DI17E6 to the surface of HSA NPs loaded with doxorubicin. DI17E6 is a humanized and de-immunized mAb against av integrin, which can inhibit the growth of melanoma in vitro and in vivo, and can inhibit angiogenesis by interfering with avb3 integrin. They demonstrated that compared to free doxorubicin, targeted HSA NPs loaded with the cell-inhibitory drug doxorubicin showed increased cytotoxic activity. In addition, the modification of DI17E6 also improved the therapeutic effect of the nanoparticles.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)

TRAIL belongs to the Tumor necrosis factor (TNF) receptor superfamily, which is a type II membrane protein.159,160 In humans, death receptors (DR4 or DR5) overexpress on neoplastic cells, while healthy normal cells do not significantly express death receptors.161 Therefore, TRAIL is considered a safe and selective anticancer and apoptotic drug with minimal toxicity to normal cells. The combination of chemotherapy drugs and TRAIL is a method for treating cancer. Due to the positive charge of TRAIL, it usually combines with HSA through electrostatic adsorption, as HSA is negatively charged under neutral pH conditions.139

Min et al139 developed a formulation of PTX-bound HSA NPs with embedded TRAIL (TRAIL/PTX HSA-NP) to treat pancreatic cancer. They investigated a series of characteristics of the nanoparticles, including surface morphologies, particle sizes, zeta potentials, loading efficiency, cytotoxicity, and apoptotic activity. Moreover, TRAIL-coupled nanoparticles showed stronger anti-tumor activity. Choi et al141 designed and prepared HSA-NPs with surface modification with TRAIL, meanwhile containing doxorubicin. The results showed that the nanoparticles had significant cytotoxicity in most types of tumor cells with general or drug-resistant characteristics, especially on CAPAN-1 cells, which are human pancreatic cancer cells. These studies have proved that it is a useful targeting agent that can affect tumor cells in various tissues and organs.141 Recently, the regulation of TRAIL-mediated exogenous apoptosis pathways in vitro through nanoparticles has also been reported as a strategy for cancer treatment. In Ahmed’s162 studies, F. cretica methanolic extracts based albumin nanoparticles, liposomes and silver nanoparticles were prepared. F. cretica albumin and silver nanoparticles upregulated the in vitro TRAIL, DR4, DR5, and FADD gene expression in Hep-2 cell lines and exhibited high anticancer activity.

Metal and Inorganic Albumin Nanoparticles

Metal and inorganic nanoparticles provide intrinsic advantages to biomedical fields due to their unique physical properties, controllable structure, and diverse surface chemistry. Metal nanoparticles are wildly applied to inhibit bacterial tolerance growth with expansion of the range of antimicrobial activity163 and antitumor treatment which can combine with radio- and chemotherapy.164 However, the low hydrophilicity of metal nanoparticles can lead to aggregation and their therapeutic potential remains to be justified. Inorganic nanoparticles can be divided into metal-based and non-carbon sources. Their physiochemical properties and released metal ions/trace elements contribute to various therapeutics without acute toxicity. Functionalized inorganic albumin nanoparticles can be generated with inorganic NPs through chemical conjugation, coating, or biomineralization. The coupling of albumin and inorganic nanoparticles makes full use of the benefits of albumin (high hydrophilicity, large amount of binding sites, and performance as a reduction reagent) in photothermal therapy and immunotherapy.165–167 In this section, we outline the main metal albumin NPs (gold, silver, copper) and main inorganic albumin nanoparticles (magnetic albumin and albumin-template nanoparticles). We further elaborate on their applications in drug delivery and biomedical treatment (Figure 5A).

Figure 5 (A) Illustration of albumin metal nanoparticles fabrication and application; (B) Docking studies of C1–C3 and HSA using Autodock. a) The overall structure of the HSA complex; b) C1–C3 located within the hydrophobic pocket in subdomain IIA of HSA; c) the interaction mode between C1–C3 (showing stick representation) and HSA (cartoon form).168 (License Number: 5778641204720. Reprinted from Journal of Inorganic Biochemistry, 153, Gou Y, Zhang Z, J. Q, et al. Folate-functionalized human serum albumin carrier for anticancer copper(II) complexes derived from natural plumbagin, 13–22. Copyright 2015 with permission from Elsevier.168 (C) a) Synthetic process and therapeutic mechanism of BSA-IrO2 NPs. b) TEM image and photograph (inset) of BSA-IrO2 NPs. c) HAADF-STEM image and EDX elemental mapping. d) UV/Vis absorption spectrum of BSA-IrO2 NPs in aqueous solution. (Reprinted with permission from Zhen W, Liu Y, Lin L, et al. BSA-IrO(2): catalase-like nanoparticles with high photothermal conversion efficiency and a high X-ray absorption coefficient for anti-inflammation and antitumor theranostics. Angew Chem Int Ed Engl. 2018;57(32):10309–10313.© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.166

Abbreviations: FRET, fluorescence resonance energy transfer; PTT, Photothermal therapy; MRI, magnetic resonance imaging; BSA, bovine serum albumin; PDT, Photodynamic Therapy; PAT, Photoacoustic tomography; CT, Computed Tomography; CAT-like, Catalase-like; ROS, reactive oxygen species.

Silver Albumin Nanoparticles

Among metal nanoparticles, silver nanoparticles (AgNPs) are widely used due to their excellent antimicrobial and photoelectron behavior.169–172 Unmodified AgNPs will form aggregates due to their poor stability in aqueous media. In Korolev’s study,173 AgNPs were modified by the addition of either HSA or Tween-80 (Polysorbate-80) to increase the stability of AgNPs in aqueous suspensions. AgNPs with Tween-80 showed significant hemolysis incubation, while non-modified and albumin-coated AgNPs had minimal hemolytic activity. The cytotoxicity of the albumin-coated AgNPs on human adipose-tissue-derived mesenchymal stem cells was lower than that of the native and Tween-80-covered AgNPs. Meanwhile, albumin-coated AgNPs and non-modified AgNPs were equally effective in terms of bactericidal activity against pathogens. The targeting abilities, cellular uptake, and toxicity of nanoparticles can be influenced by a protein coating,174 which provides a new strategy for drug delivery. In earlier research, the protein coating may decrease the toxicity and cellular uptake of AgNPs.175 Recently, Park et al176 studied the role of protein coatings on the toxicity of AgNPs to liver hepatocellular carcinoma (HepG2) cells. The protein on the surface forms a protective coating that inhibits AgNPs dissolution. However, HSA-coated AgNPs are more toxic to HepG2 cells than uncoated AgNPs, and that the mechanism of toxicity cannot be simply explained by AgNP dissolution. The observed differences in toxicity reactions may be caused by various factors, including the size and concentration of nanoparticles, different cell types, exposure time, and the selection of toxicity measurements.

There are also scholars using silver nanoparticles of quantum dot immunoassay on HSA competitive fluorescence immunoassay. If the antibody-loaded CdSe quantum dots are aggregated with HSA-coated silver nanoparticles, fluorescence resonance energy transfer (FRET) will be caused by the distance between the two nanoparticles being reduced enough.177 In this case, the Ab-QD fluorescence detector is overcome. However, if HSA is added to Ab-QD, their surfaces will be blocked, and they will no longer be polymerized with HSA-AgNPs. The decrease in fluorescence intensity (peak 570 nm) was negatively correlated with HSA concentration in the sample. HSA concentration in the range of 30–600 ng/mL can be determined with this method. The data show that anisotropic silver nanoparticles and quantum dots as energy donors can be successfully applied to various competitive immunoassays for sensitive detection of any analyte based on resonance energy transfer.

Gold Albumin Nanoparticles

Gold and silver nanoparticles are hotspots in chemistry, condensed matter physics, nanomaterials, and clinical immunology research in recent years.178–181 The AuNP albumin system could be used in various biomedical areas, including photo-thermal therapy, drug delivery, diagnostics, theranostics and immunoassays.182 AuNPs could be capped with albumin molecules or encapsulated into albumin nano capsules to deliver drugs to target sites.183 Mocan et al184 used BSA bound to gold nanoparticles (GNPs) as active vectors to target liver cells with laser treatment. Compared with GNPs, BSA-GNPs showed their high affinity to liver cancer cells. Murawala et al185 provided methotrexate (MTX) loaded Au-BSA NPs, which were extremely stable under strong electrolyte and pH conditions. And Au-BSA NPs showed better proficiency in inhibiting human breast cancer cells (MCF-7) compared with free MTX. Chen et al186 designed albumin-based cisplatin-conjugated AuNPs with radiation therapy (RT), in which Au can increase RT-induced immunogenic cell death and potentiate the abscopal antitumor immunity. Compared with cisplatin, albumin AuNPs loaded cisplatin showed reduced side effects which can be safely administered concurrently with ablative RT.

Copper Albumin Nanoparticles

The interaction of metal compounds with HSA applied in anti-cancer research is increasing. According to the research, HSA can increase the solubility of the metal compound copper(II), and enhance its anti-tumor effect187,188 Fluorescence spectra and molecular docking show that the Cu(II) complex binds to the IIA subdomain of HSA (Figure 5B).168 Cu(II) compounds produce intracellular reactive oxygen species (ROS) in tumor cells, and the complexes of HSA and copper(II) compounds are, to some extent, able to selectivity accumulate in cancer cells compared to the individual Cu(II) complex, but do not improve the normal cell cytotoxicity levels in vitro. Gou et al189 prepared a component by the reaction of CuCl2/ Cu(NO3)2/CuBr2 with HL to obtain a novel phenoxy-bridged binuclear copper(II) compound C3. (C3) and mononuclear copper compounds (C1 and C2) showed stronger anticancer effects. The HSA nano carrier functionalized with folic acid (FA) was combined with Cu(II) to study its anti-cancer properties and mechanisms. FA-HSA-metal drug complexes exhibited high cytotoxicity to tumor cells with high expression of FA to a certain extent and showed the inhibition of the activity of proliferative Bcl-2 family proteins and CDK1/cyclin B1.

Jiang et al190 developed a Cu(II) reagent based on the specific residue of HSA NPs, which was used for multitargeted tumor microenvironment (TME) to inhibit cisplatin resistance. The structure of the HSA-Cu complex indicated that two Cu compound (C4) molecules bind and hide in the hydrophobic cavities of the IB and IIA subdomains of HSA, forming a stable HSA-C4 complex that effectively protected C4 from damage by endogenous ions/macromolecules/compounds. In addition, HSA NPs could serve as an effective carrier for C4 and transport them to tumor tissue.

Magnetic Iron Oxide Albumin Nanoparticles

Magnetic nanoparticles, especially Fe3O4 nanoparticles, are widely studied nanomaterials with special magnetic properties, easily synthesized, and low toxicity.191–193 It has been reported that there is a great potential in biomedical, biological separation, DNA hybridization, and detection.194–197 However, the characteristic of being easily oxidized by pure Fe3O4 may affect magnetic properties. In order to overcome this problem, various coatings have been added to its surface, but due to their toxicity, resulting in low biocompatibility and high immunogenicity. BSA protein can be immobilized on the surface of magnetic nanoparticles by physical adsorption or covalent binding. Among them, covalent conjugation can make the BSA coated magnetic nanoparticles more stability. He et al198 used BSA as a biosensor to protect Fe3O4 nanoparticles. In addition, iron oxide nanoparticles loaded with cytotoxic drugs were injected intravenously into blood vessels, and accumulated in the tumor tissue through a gradient magnetic field, the drug bioavailability effectively improved and side effects reduced, respectively, in MDT. Zaloga et al199 proposed a method for magnetic drug targeting w