Albumin Nanoparticle-Based Drug Delivery Systems.

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 1970s 11 and 1980s, 12 whereas the latter one was developed into Nanocoll ® , [ 99 mTc]-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 administration 18 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. 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). Number of peer-reviewed publications from 2004 to 2023 combining key words “albumin nanoparticle” and different applications (PubMed database).

In some studies, the long circulating half-life of ~19 days of albumin is associated with Megalin Cubilin receptor-mediated renal rescue 223 and its binding to endothelial and epithelial cell cycle neovascularized Fc receptors (FcRn). 224 HSA level is maintained by FcRn through the intracellular sorting mechanism that protects from lysosomal degradation: 225 HSA is internalized into cells through pinocytosis and then combines with FcRn at low pH within the acidic endosome, recycles to the extracellular chambers and dissociates at a physiological pH, which avoids degradation in the lysosome. 226 However, other non-receptor bond plasma components are degraded ( Figure 7 ). Figure 7 Recycling model of HSA through binding FcRn receptors. Abbreviations : FcRn, the neonatal Fc receptor; HSA, human serum albumin. Recycling model of HSA through binding FcRn receptors. The enhanced tumor accumulation of HSA NP is due to the EPR effect passively mediated uptake enhancement. In brief, the extensive angiogenesis of tumor tissue can lead to leakage of the vascular system, and larger molecules can enter the tumor stroma through the vascular system. Another pathway is to rely on active receptor transport. The most well-known albumin receptor, gp60, a 60 kDa glycoprotein (albondin), which mediates the endocytosis of natural HSA in endothelial cells, is widely expressed and involved in vascular endothelial cells except for the brain. Albumin combined with gp60 causes internalization and transcytosis: Albumin binds gp60 receptors and activates caveolin-1 and cell phagocytosis through endothelial cells. 74 , 227–229 Besides, gp18 and 30 can be found in a wide variety of cells, such as macrophages, fibroblasts, and brain cells. However, gp18 and gp30 do not preferentially bind to native albumin and only have affinity for modified albumin. The entered albumin drug complex binds to the overexpressed secretory acidic cysteine-rich protein (SPARC) in tumor cells, which promotes drug entry into the tumor cells. 229 However, few clinical studies have suggested that the concentration of SPARC in tumor tissue has no significant impact on treatment efficacy. 230 Figure 8 shows the HSA-NPs delivery strategy. Figure 8 HSA-NPs delivery strategy. Abbreviations : HSA-NPs, human serum albumin nanoparticles; gp60, glycoprotein 60; SPARC, secretory acidic cysteine-rich protein. HSA-NPs delivery strategy. K-Ras is one of the driver genes for pancreatic cancer, and tumor cells with mutated K-Ras exhibit high levels of endocytosis. HSA is its main source of nutrition. 231 Lu et al 232 prepared BSA – polycaprolactone (PCL) nanoparticles loaded with albendazole (ABZ) for the treatment of pancreatic cancer by desolvation method, and in the cytotoxicity experiment, the IC50 value of BSA-PCL nanoparticles was 1.32 ± 0.35 μg/mL, and the IC50 value of free ABZ was 3.17 ± 0.36 μg/mL, which was about 2.4 times the concentration of BSA-PCL nanoparticles, indicating that the uptake of BSA-PCL nanoparticles by cancer cells was higher than that of free ABZ. In this literature, it is shown that the optimal particle size of albumin nanoparticles is between 100 and 200 nm, and it may be that the larger the particle size, the larger the area where the nanoparticles contact the cell membrane. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME), which promote tumor growth and metastasis. Therefore, eliminating TME can achieve anti-tumor efficacy. 233 Previous studies have shown that palmitic acid (PA)-modified albumin can target the scavenger receptor-A of polarized macrophages such as TAM. 234 Feng et al 235 prepared palmitic acid-modified HSA NPs (PSA NPs) loaded with pirubicin (THP) to achieve double targeting of tumor cells and TAMs, and the inhibition rate of THP-PSA NPs on tumor volume in mice reached 81.0%, without causing damage to normal tissues. The mechanism of action is shown in Figure 9 . Figure 9 THP-PSA NPs mechanism of action. 235 (Reprint with permission from Feng J, Xiang L, Fang C, et al. Dual-targeting of tumor cells and tumor-associated macrophages by palmitic acid modified albumin nanoparticles for antitumor and antimetastasis therapy. ACS Appl Mater Interfaces. 2022;14(13):14887–14902. Copyright (2022) American Chemical Society. 235 Abbreviations : THP-PSA NPs, palmitic acid-modified human serum albumin nanoparticles; M2-TAMs, M2-tumor-associated macrophages; IL-10, interleukin-10; TGF- β1, transforming growth factor-β1; IL-12p70, interleukin-12p70; TNF- α, tumor necrosis factor α; IFN- γ, Interferon γ; MDSCs, Myeloid-derived suppressor cells. THP-PSA NPs mechanism of action. 235 (Reprint with permission from Feng J, Xiang L, Fang C, et al. Dual-targeting of tumor cells and tumor-associated macrophages by palmitic acid modified albumin nanoparticles for antitumor and antimetastasis therapy. ACS Appl Mater Interfaces. 2022;14(13):14887–14902. Copyright (2022) American Chemical Society. 235 In 2020, the rapid spread of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) worldwide was associated with reduced albumin levels, cytokine levels, etc., and more recently, there have been reports of a complete recovery in patients with severe COVID-19 infused with immune serum rich in albumin, antibodies, and plasma proteins. 236 The mechanism of action should be similar to that of convalescent plasma, ie, albumin prevents histone H4-mediated platelet activation and aggregation. 237 Park et al 238 proposed drug-loaded albumin nanoparticles as therapeutic agents to address the clinical outcomes observed in patients with severe SARS-CoV-2. Polyethylene glycolated nanoparticle albumin binding can alter the pharmacokinetic behavior of ginsenoside soap, such as reducing clearance and prolonging blood circulation time. Analysis of plasma samples from COVID-19 patients revealed decreased serum albumin levels, increased coagulation and NETosis associated with elevated histone H4, and severe cytokine storms and inflammation in blood and lung tissue. Therefore, albumin was chosen as a vehicle for ginsenoside delivery to simultaneously address blood clotting, NETosis, and cytokine storms. It is hypothesized that another effect of albumin is to inhibit platelet aggregation, pulmonary hemorrhage, and endothelial necrosis by binding to extracellular histones secreted by viral infections in patients with severe COVID-19. Steroid glycosides, found in ginsenosides, can inhibit severe inflammation and prevent death from hypotension due to septic shock in critically ill patients. 239 HSA can combine a variety of endogenous and exogenous compounds, these properties can affect the distribution and efficacy of compounds in the body, HSA can store many compounds and can greatly increase the solubility of compounds. 240 Li et al 241 studied the interaction of ribavirin and lamivudine with HSA using fluorescence spectroscopy and X-ray crystallography, and the X-ray structure showed that ribavirin, lamivudine and lamivudine bind to the IIA subregion of HSA mainly through the formation of hydrogen bonds and hydrophobic interaction forces, which provided guidance for the subsequent albumin delivery system of ribavirin and lamivudine. Inflammatory diseases are caused by the adhesion of polymorphonuclear neutrophils to the lining of the circulatory system or vascular endothelium, and transmigration of the uncontrolled neutrophils. 242 Therefore, it is a useful therapeutic strategy to target neutrophils through nanodrug delivery systems. 243 Albumin nanoparticles are reported to be internalized by neutrophils and have effects on acute inflammation 244 and chronic inflammation. 245 BSA nanoparticles can internalize into neutrophils through Fcγ receptors on the surface of neutrophils. Recently, Liu et al 44 prepared an RGD peptides-modified BSA NPs loading with Celastrol (CLT) (CBR NPs) using a desolvation method to induce apoptosis of circulating inflammatory neutrophils to treat rheumatoid arthritis, while CBR NPs could decrease the toxic side effects of CLT. The results showed that CBR NPs effectively inhibit the recruitment of inflammatory neutrophils in inflammatory joints. Besides, neutrophils could be like “a bus” to deliver therapeutic nanoparticles across the blood vessel barrier for inflammatory treatment. Zhu et al 245 reported BSA NPs loaded with glucose oxidase (BSA-GOx-NPs) to treat endometriosis, which is a chronic inflammatory disease, by the neutrophil hitchhiking strategy. BSA-GOx-NPs internalized by neutrophils in vivo and neutrophils consistently enriched in ectopic lesions. GOx could be released to deplete glucose and induce apoptosis. The results provided a promising direction for the treatment of endometriosis. BBB is a major challenge in brain cancer treatment, and the characteristics of brain microvascular endothelial cells are high expression of tight junction proteins and poor cross cell endocytosis. The BBB is not a static barrier, in fact, there is a large exchange of substances on the BBB through nutrient transporters, 246 transporters in the brain barrier can be used for the delivery of brain drugs. Albumin is an important source of nutrition for the body, but it is excluded by the brain, due to the rapid growth of tumors and active metabolism, it is very thirsty for nutrients, in this case, the intake of albumin in tumor tissues will be greatly increased and used as an energy source. Albumin-binding proteins, such as SPARC and gp 60, are the main mechanisms by which tumors take up albumin, 247 and SPARC is overexpressed in brain tumors. Lin et al 248 provided a green method for the synthesis of BBB-penetrating albumin nanoparticles with the ability to co-encapsulate different drugs and does not require cross-linking agents ( Figure 10 ). The hydrophobic drugs PTX and fenreta amine produce a synergistic effect to induce albumin self-assembly, forming double-loaded nanoparticles. The albumin nanoparticles were modified by the cell-penetrating peptide LMWP (low molecular weight protamine), which offers a promising pathway of biomimetic targeted administration of brain tumors in combination therapy. T807 is a novel tau positron emission tomography agent for Alzheimer’s disease that has a low molecular weight and can effectively cross the BBB. Hahn et al 249 prepared erythrocyte membrane (ETm) coating and T807-modified HSA nanoparticles (T807-ETm/HSA NPs), red blood cell membrane coating can prolong the circulation time of nanoparticles in the blood, and T807 ligand can target the brain. In mouse in vivo imaging experiments, T807-ETm/HSA NPs had the largest distribution in the brain, so this vector can cross the BBB and be used to transport drugs to treat brain diseases. Figure 10 Schematic diagram of Albumin NP entering the BBB. 248 (Reprint with permission from Lin T, Zhao P, Jiang Y, et al. Blood-brain-barrier-penetrating albumin nanoparticles for biomimetic drug delivery via albumin-binding protein pathways for antiglioma therapy. ACS Nano. 2016;10(11):9999–10012. Copyright (2016) American Chemical Society. 248 Abbreviations : NP, nanoparticle; BBB, Blood-Brain-Barrier; LMWP, low molecular weight protamine; Gp60, glycoprotein 60; SPARC, secretory acidic cysteine-rich protein; PTX, paclitaxel; 4-HPR, fenreta amine. Schematic diagram of Albumin NP entering the BBB. 248 (Reprint with permission from Lin T, Zhao P, Jiang Y, et al. Blood-brain-barrier-penetrating albumin nanoparticles for biomimetic drug delivery via albumin-binding protein pathways for antiglioma therapy. ACS Nano. 2016;10(11):9999–10012. Copyright (2016) American Chemical Society. 248 LNs are attractive therapeutic targets for treating various unmet clinical needs. However, due to the anatomical structure and nature of LNs, high concentrations of therapeutic agents in these tissues are difficult to achieve through the administration of free drugs or conventional formulations. Albumin-hitchhiking approach is attributed to the drug/vaccines targeting towards LNs and enhancing immunogenicity ( Figure 11 ). Antigens-bond albumin is taken to the lymphatic vessels and then flowed into the LN until it has been processed to the surface of dendritic cells. 250 Zhang et al 251 designed a novel HSA nanoformulation loaded with an immunosuppressant tacrolimus (TAC) (TAC-HSA-NPs), which had high lymphatic targeting efficiency to further enhance the efficacy of lymphatic immunosuppression. Wherein, hydrophobic TAC triggers the self-assembly of albumin and enhances the adhesion between HSA molecules without crosslinking. TAC-HSA NP could target and drain LNs via cell surface Fc γ receptors and inhibit the proliferation of immune cells via cell surface Fc γ receptors, which was considered consistent with the reported targeted delivery of albumin nanoparticles to inflammation site infiltrated with adherent neutrophil. 242 In order to further improve the efficiency of LN targeting, albumin nanoparticles coupled with antibodies that could recognize lymphocytes or LN vasculature have also been widely studied. Tumor-draining lymph nodes (tdLNs), including Tregs and MDSCs, are the first site of metastasis and abound with immunosuppressive factors, which play a crucial role in generating and regulating tumor-related immune responses. 252 They experience antigen priming during lymphatic drainage of tumor-associated antigens (TAAs). Albumin nanoparticles modified with TAAs targeted tdLNs by lymphatic drainage as an anticancer vaccine will also be a field worth studying. Figure 11 Albumin-hitchhiking approach to target vaccines to the lymph node and enhance immunogenicity. 250 Reprinted from Journal of Controlled Release, 327, Abdallah M, Mullertz OO, Styles IK, et al. Lymphatic targeting by albumin-hitchhiking: applications and optimisation, 117-128. Copyright 2020 with permission from Elsevier. 250 Albumin-hitchhiking approach to target vaccines to the lymph node and enhance immunogenicity. 250 Reprinted from Journal of Controlled Release, 327, Abdallah M, Mullertz OO, Styles IK, et al. Lymphatic targeting by albumin-hitchhiking: applications and optimisation, 117-128. Copyright 2020 with permission from Elsevier. 250 Besides, sentinel lymph node (SLN) resection is one of the traditional methods for tumor treatments. SLNs are the first regional LNs that receive lymph flow from primary tumors. The detection of SLN is crucial for estimating tumor staging and treatment decisions. SLN localization has been used for the diagnosis of solid tumor metastasis. 99mTc-labeled Mannose-based HSA (MSA), which binds to a blue dye, has been reported as an SLN imaging agent by binding to macrophages in LNs. 253 Indocyanine green loaded HSA (ICG-HSA) nanoparticles which radiolabeled with technetium-99 m (99mTc) were prepared to locate SLNs and inhibit tumor metastasis. The photothermal treatment of SLN enhanced the inhibitory effect on lung metastasis in mice and significantly prolonged their survival time. 254 Immunotherapy combined with chemotherapy is one of the current options for cancer treatment. LNs play a crucial role in initiating the progression and metastasis of cancer. 255 The therapeutic strategies for regulating the immune suppression microenvironment of LNs are receiving increasing attention. Various immune modulators have been developed to reshape the tumor immunosuppressive microenvironment and improve treatment effectiveness. However, currently, most immune modulators have limited accumulation in tumor sites or LNs after intravenous or oral administration, which hinders their clinical efficacy. Albumin nanoparticles show great potential in the delivery of immune modulators. Song et al 256 developed an albumin nanoparticle to encapsulate PTX and PI3Kγ inhibitors which increased the drug delivery to macrophages in both tumors and LNs. The Regeneration of complex structures after injury requires different processes, such as wound healing, cell death, dedifferentiation, and stem cell (or progenitor cell) proliferation. Tissue regeneration is related to the apoptotic program and inflammation. 257 Research on model organisms has begun to characterize the source of regenerated cells, determine their efficacy, and determine the molecules required for supplementary events. 258 Albumin nanoparticles have shown great potential for application in the delivery of regeneration promoters in regenerating bone tissue, 259–261 wound healing, 262–264 repairing spinal cord injury, 265 , 266 and so on. Lin et al 89 provided a nanofiber scaffold containing chitosan-stabilized BSA nanoparticles for controlling the delivery of dual drugs to create an osteogenic microenvironment for bone regeneration. The nanoparticles were prepared through desolvation with BSA as the nano carrier to maintain the bioactivity of drugs. Then, a chitosan stable layer was prepared by the electrostatic self-assembly method. The results indicated that the scaffold promoted the differentiation of osteoblasts, which could provide a new approach for designing functional biomaterials for bone tissue engineering that can be used in regenerative medicine. The basic fibroblast growth factor (bFGF) is one of the key components that promotes dermal cell proliferation and migration for wound healing. However, due to the rapid decomposition of bFGF in physiological microenvironments, its unstable nature greatly limits its clinical application. As shown in Figure 12 , Son et al 262 presented the bFGF-loaded HSA NPs (HSA-bFGF NPs) to improve their stability with a simple desolvation and crosslinking method. Due to the short half-life of bFGF, the strong cross-linking of bFGF and HSA to form nanoparticles is basically beneficial for the long-term supplementation of bioactive bFGF in tissue regeneration. The HSA-bFGF NPs retained the complete characteristics of the bFGF protein, especially improving tissue regeneration in terms of microstructure and functional recovery in vivo. Figure 12 Schematic illustrations depicting cellular delivery of bFGF, followed by wound healing cascades of HSA-bFGF NPs. Reprint with permission from Son B, Kim M, Won H, et al. Secured delivery of basic fibroblast growth factor using human serum albumin-based protein nanoparticles for enhanced wound healing and regeneration. J Nanobiotechnol. 2023;21(1):310. http://creativecommons.org/licenses/by/4.0/ 262 . Abbreviations : bFGF, the basic fibroblast growth factor; HSA, human serum albumin; TGF- β, transforming growth factor-β; MEK, mitogen-activated protein kinase; PI3K, Phosphatidylinositol 3-kinase; ERK, extracellular regulated protein kinases; AKT, protein kinase B. Schematic illustrations depicting cellular delivery of bFGF, followed by wound healing cascades of HSA-bFGF NPs. Reprint with permission from Son B, Kim M, Won H, et al. Secured delivery of basic fibroblast growth factor using human serum albumin-based protein nanoparticles for enhanced wound healing and regeneration. J Nanobiotechnol. 2023;21(1):310. http://creativecommons.org/licenses/by/4.0/ 262 .

Albumin is an attractive protein for preparing nanoparticles with potential therapeutic applications. However, current reports indicate that various albumin-based nanocomplexes are usually synthesized by strategies such as electrostatic adsorption, chemical conjugation and biomineralization. 267 These methods may lead to the presence of multiple components in the complexes, and there is a lack of comprehensive studies on the biocompatibility and toxicity of these complexes, which has seriously hindered the further development and widespread clinical application of albumin-based complexes. In response to these problems, measures could also be taken to focus on the reduction of impurities and more comprehensive toxicity testing of the complexes during the process of design and synthesis. The use of glutaraldehyde as a crosslinking agent or UV irradiation 9 for crosslinking during the preparation of albumin nanoparticles can increase the potential toxicity of the obtained albumin nanoparticles. Therefore, some natural cross linking agents such as tannic acid, ascorbic acid, municipal acid, and glucose 268 have been studied. Besides, for the drug-loaded albumin nanoparticles, physical instability and premature release during a long-term storage are still the challenges. Further, whether albumin nanoparticles can maintain uniform and stable particle size and high drug loading in large-scale production and the reproducibility are urgent issues that need to be considered in the clinical translation process. Careful design and formulation optimization are necessary. Albumin as a drug carrier also has the limitations, such as albumin variability, high cost, limited sources of HSA, and mild immune reactions when BSA is used for injection. Some studies have reported that proteins isolated from other species can also be used as drug delivery carriers. For example, soybean protein-based nanoparticles are considered as potential natural delivery carriers because of their structural characteristics. Gong et al 269 enumerated a variety of preparation methods for soy protein-based nanoparticles and described the improvement of functional properties of bioactives after encapsulation. It provides a theoretical basis and reference for the subsequent preparation of novel soy protein nanoparticles. Besides, it offers a potential pathway for the application of soy proteins and natural bioactives in the food and pharmaceutical fields. Silk protein has been widely used as scaffolds for vascular, skin, bone, cartilage and neural tissues. 270 Silk fibroin nanoparticles (SFN) have the advantages of simple preparation method, good biocompatibility, good degradation, and easy chemical modification. Lozano-Perez et al 271 prepared SFN loaded with hydrophobic platinum precursor (PtBz), which successfully improved the delivery of insoluble PtBz and showed high selectivity for tumor cells, reducing the killing of normal cells. Albumin has potential in delivering antibacterial, analgesic and antifungal drugs as well as active substances for obesity prevention. In future studies, more attention could be paid on chemical modifications of albumin, which could broaden the application of albumin nanoparticles. Besides, albumin nanoparticles can passively target tumor sites through the EPR effect; however, albumin is also an essential protein for tumor maintenance and growth. Therefore, how the administration of albumin formulations affects the development of tumors and the changes in the immune environment of albumin carriers after treatment of tumors, as well as the effects of subsequent tumor metastasis and recurrence are the interesting studies. 272 The field of nanomedicine is attracting more and more attention as it offers efficient and smart therapeutic options for the treatment of cancer, inflammatory and other diseases. In this field, albumin nanoparticles are attracting attention due to their high affinity for hydrophobic drugs, surface modification possibilities and high loading capacity, which allows us to overcome the formidable hurdles posed by many of the compounds currently on the market. 9 Although there is a lot of research on nanotechnology, some synthetic polymers, metal nanoparticles, etc. are limited in clinical applications due to their own toxicity. With the continuous development of the biomedical field and the increase of application demand, the market potential of albumin nanoparticles will be further released, and it is expected to become an area with greater market potential and commercial application prospects. Especially with the rise of personalized medicine and precision therapy, the targeted delivery advantage of albumin nanoparticles will be more valued. We expect albumin nanoparticles to be promising drug delivery systems in the clinical development of disease.

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 al 38 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. 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 al 45 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 al 48 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 radiation 27 and other potential crosslinking agents such as glucose 49 are developed. 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 al 53 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. The formation of targeted modified albumin nanoparticles and the drug release in the cancer cells. 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 al 55 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 al 58 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. 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 al 34 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 al 43 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 ( V water / V ethanol =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. 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 al 59 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. 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 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 al 74 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 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 al 77 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.