Currently, the disadvantages of BTZ (e.g., high hydrophobicity, poor stability, low bioavailability, high toxicity to normal tissues, and drug resistance) limit its clinical applications and efficacy. In addition, BTZ binds nonspecifically to proteins in serum and is rapidly cleared in the liver, impairing its accumulation and penetration into sol-id tissues. This limits the use of BTZ for the treatment of solid tumors.

With the rapid development of nanotechnology, drug delivery systems (e.g., lipo-somes, polymer micelles, inorganic nanoparticles, and biomimetic nanoparticles) have become a research hotspot. The nano-delivery platform can increase the ac-cumulation of drugs at the targeted site and realize controlled drug release, thereby improving the therapeutic effects and reducing the occurrence of side effects. Thus far, numerous BTZ-based nanoformulations have been developed and investigated to fur-ther improve druggability and enhance the therapeutic effects. In this section, research progress regarding BTZ-based nanoformulations is summarized.

1. Liposomes

Liposomes are closed vesicles with a bilayer structure, which are composed of phospholipid and cholesterol [114]. Owing to the similar biochemical structure to that of the cell membrane, liposomes have a favorable safety profile and biocompatibility [115]. Thus far, liposomes have been widely utilized for the delivery of drugs, and nu-merous products have become commercially available. Hydrophilic and hydrophobic drugs can be wrapped into the hydrophilic core and phospholipid bilayer, respectively [116].

Vijay et al. prepared D1X-encapsulated BTZ (D1XB) liposomes, in which BTZ and dexamethasone were loaded. Dexamethasone, a cholesterol analogue, can be modified on the surface of the liposomes to strengthen and stabilize the lipid bilayer. In addition, dexamethasone is a synthetic ligand of glucocorticoid receptors that can specifically bind to tumor cells with high expression of these receptors [117]. Studies have shown that the loading in liposomes improves the accumulation of BTZ into tumor sites. D1XB has demonstrated strong cytotoxicity against WM cells.

Zuccari et al. prepared asparagine-glycine-arginine (NGR) peptide active-targeting liposomes, termed NGR-SL (amino-lactose-BTZ [LM-BTZ]) [26]. BTZ and LM were firstly complexed via a borate ester bond, and subsequently encapsulated into the liposomes. By forming pH-sensitive borate ester bonds, LM can bind the poorly water-soluble BTZ to the hydrophilic core and prevent leakage of free BTZ from the lipo-somes. This process improves the encapsulation efficiency and achieves specific re-lease of the drug. In a mouse model of neuroblastoma, NGR-SL (LM-BTZ) exhibited a higher therapeutic index and lower toxicity than the free drug.

2. Polymeric Micelles

Polymer micelles are a type of nanoparticle with a core-shell structure formed by the self-assembly of amphiphilic block copolymers in aqueous solution. The hydro-phobic core can accommodate the insoluble substances to improve solubility. Besides the physical encapsulation manner, the drug can also be chemically conjugated to the amphiphilic block copolymers to realize the drug loading and delivery [118].

Liu et al. designed a dual pH-sensitive drug delivery system termed BTZ-polymer conjugates (BTZ-PC). The copolymers were synthesized by catechol-functionalized PC conjugation with polyethylene glycol (PEG) via acetal bonds, followed by formation of the micelles after self-assembly [119]. BTZ can be encapsulated into the hydrophobic core of the micelles by conjugation with catechol via a borate ester bond. Both acetal and borate ester bonds are acid-unstable and can be broken in the acidic tumor micro-environment to achieve pH-sensitive drug release. In a xenograft mouse model of hu-man breast cancer, BTZ-PC micelles exerted enhanced antitumor effects and reduced toxicity compared with the free drug.

Liu et al. modified dopamine methylacrylamide to the end of PEG-block-poly(D,L-lactic acid) (PEG-b-PCL) using the “graft” method, leading to the formation of the PEG-b-PCL-b-PDMA co-polymer. Afterwards, BTZ was grafted on the polymer chain by conjugation with the catechol group in dopamine to form a pH-sensitive borate ester bond. Subsequently, BTZ-loaded copolymer micelles were formed under self-assembly [121]. The borate ester bond is a dynamic covalent bond, which improves the therapeu-tic effects of the drug. In a mouse model of hepatocellular carcinoma, the micelles were associated with stronger antitumor effects and a lower incidence of adverse reactions compared with the free drug.

Gu et al. synthesized a novel single copolymer HA-P(TMC-co-DTC) and prepared the biodegradable micelles (HA-curcumin) by self-assembly. In this process, the shell of HA is crosslinked to the core by disulfide bonds to increase the stability of the mi-celles [122]. BTZ-loaded micelles (HA-core-disulfide-crosslinked biodegradable mi-celles-BTZ-pinanediol [HA-CCMS-BP]) were prepared by encapsulating alcohol-esterified BTZ in the carriers. HA can actively target solid tumors overexpressing CD44, while disulfide bonds can selectively release the drug in tumor sites with high gluta-thione expression. The results showed that the cytotoxicity of HA-CCMS-BP was lower than that of the free drug. In a mouse model of MM, HA-CCMS-BP targeted therapy for MM was better tolerated than the free drug.

3. Dendrimers

Dendrimers refer to dendritic macromolecules that start from core molecules and repeatedly grow outwards to form highly branched structures [136] The cargoes en-capsulated in dendrimers can stably exist due to the spatial effects. Furthermore, the groups on the surface of dendrimers can control the solubility of hybrid nanocompo-sites and be linked with other molecules for further modification. Dendritic materials, such as polyamidoamine (PAMAM) and branched polyetherimide, have been widely used as carriers for drug delivery [137].

Wang et al. used PEG as a linker to connect cyclic arginine-glycine-aspartic acid (cRGD) to fifth-generation PAMAM and performed modifications with catechol to ob-tain the nanocarriers DPA-G5-PEG-cRGD [124]. BTZ was subsequently conjugated with catechol by a pH-sensitive borate ester bond to obtain a novel DPA-G5-PEG-cRGD/BTZ formulation. cRGD can target integrin αvβ3, which is highly expressed dur-ing tumor angiogenesis, thus achieving effective drug internalization. In healthy mice, the blood toxicity associated with this formulation was lower than that observed with the free drug. Interestingly, cRGD-PEG-PAMAM-Cat/BTZ also showed significant anti-tumor activity in a mouse model of MM.

Similarly, Wang et al. prepared a novel nanoformulation (G5-KAC-Cat-BTZ), in which dopamine (Cat) functionalized with a neutral shell (acetylated lysine, KAC) was grafted onto G5 PAMAM. Next, BTZ was loaded into the dendrimers through reaction with catechol [125]. In the acidic tumor microenvironment, the borate ester bond formed by BTZ and Cat realized effective drug release.

4. Nanogel

Nanogel is a type of water-soluble, three-dimensional, gelatinous nanoformula-tion formed after the crosslinking of the polymers. The network structure of the nano-gel provides convenient space for accommodating a variety of compounds, such as mo-lecular drugs, luciferin, proteins, peptides, and nucleic acids. Advantages of the nano-gel include adjustable particle size, large surface area, high water content, and degra-dability [138,139]. In recent years, the construction of a controlled drug release system using the nanogel has become a research hotspot.

Liu et al. developed a novel method for the encapsulation of BTZ, in which BTZ was loaded with dopamine-grafted HA nanogel via a borate ester bond [126]. Owing to the location of dopamine on the skeleton structure of the nanogel (NSS-BTZ), more BTZ loading was achieved (≤8.58%). In this nanoformulation, HA can actively target CD44, while the borate ester bond is pH-sensitive. These advantages enable accurate and efficient drug release in the acidic tumor microenvironment with high CD44 ex-pression. In an animal model, free BTZ was linked to severe systemic toxicity. In con-trast, at the same dosage, BTZ loaded into the nanogel exhibited better antitumor effi-cacy and was associated with a significantly lower rate of side effects.

In a study performed by Amin et al., the highly efficient photothermal agent pNIPAAm-co-pAAm was synthesized. Afterwards, dopamine nanoparticles (DP) as the carriers of BTZ were incorporated into pNIPAAm-co-pAAm to prepare the tem-perature-responsive nanogel, termed NIPAM-AM-DP [127]. The nanogel directly kills the tumor cells through photothermal therapy, as well as localizes and releases BTZ for combination therapy.

5. Inorganic Nanoparticles

The term inorganic nanoparticles refers to a category of nanocarriers based on in-organic materials. Their advantages include good size control, low toxicity, and a large specific surface area [140,141]. In addition, inorganic nanoparticles have general prop-erties, such as broad availability, easy functionality, good biocompatibility, and con-trolled release. Many inorganic materials have been widely used in the development of nanodrugs for the diagnosis and treatment of tumors, such as calcium phosphate, gold nanoparticles, carbon materials, mesoporous silica, etc. [142].

Sabine et al. prepared mesoporous silica nanoparticles (MSNs) for the encapsula-tion of BTZ [130]. The heptapeptide (HP) was combined with anti-biotin protein to block the pores of MSNs, thus yielding a new formulation termed MSNsHP. HP can be specifically recognized and cleaved by matrix metallopeptidase 9 (MMP9), which is highly expressed in tumor tissue, thereby opening the MSN channel to release the drug. The results showed that MSNsHP could remain stable under normal conditions, while it responsively released BTZ in the acidic tumor microenvironment. As the nanocarrier for BTZ, MSNsHP reduced the systemic toxicity of BTZ and selectively re-leased the drug.

Gozde et al. prepared chitosan-coated superparamagnetic iron oxide nanoparticles (CS-MNP) for the encapsulation of BTZ. The nanoparticles were formed by ionic cross-linking in the presence of chitosan and tripolyphosphate molecules [131]. It was found that CS-MNP improved the therapeutic efficacy of BTZ. Because the protonated amino group of chitosan produces a swelling of osmotic pressure at a low pH, CS-MNP has the advantage of pH-responsive release.

6. Biomimetic Nanomaterials

Biomimetic nanomaterials are “living” materials with similar functions to those of living organisms. Unlike allogenic compounds (e.g., chemically synthesized polymers or inorganic nanoparticles), biomimetic materials are associated with low toxicity and not recognized by the immune system. This may be beneficial for improving the phar-macokinetics and biodistribution of the drugs [143,144]. Biomimetic nanoparticles are generally developed by coating or mixing the biomimetic nanomaterials with organic or inorganic nanoparticles [145]. At present, biomimetic nanomaterials (e.g., protein polypeptides, DNA, RNA, exosomes, phages, various cells, and cell membranes) have been extensively studied. Among those, albumin, exosomes, and cell membranes are the most widely investigated materials. Specifically, it has been shown that a variety of cell membranes (erythrocyte, platelet, tumor, macrophage, neutrophil, etc.) are useful for drug encapsulation.

Yu et al. synthesized a copper sulfide/carbon dot nanocomposite (CuSCD) with a hollow structure. CuSCD could be utilized as a multifunctional nanocarrier for both the photothermal conduction and encapsulation of BTZ [134]. Furthermore, the macro-phage membrane hybridized with the T7 peptide was coated on the surface of BTZ-loaded CuSCD to form a novel biomimetic drug delivery system termed CuS-CDB@MMT7. The T7 peptide binds to the ferritin receptor, which is overexpressed on tumor cells, and mediates the endocytosis of the nanoparticles. In addition, the macro-phage membrane avoids immune recognition, thus ensuring that the therapeutic drugs safely reach the target.

CuSCDB@MMT7 exerts both phototherapeutic and chemotherapeutic effects and has exhibited greater antitumor efficacy in the breast cancer mouse model.

Min et al. obtained phage P22 virus capsids by high-temperature treatment [135]. Catechol ligands were chemically attached to the interior surface of the P22 viral cap-sid for subsequent encapsulation of BTZ. A novel nanoplatform was obtained by con-necting hepatocellular carcinoma-targeting peptide SP94 (SFSIIHTPILPL) as a ligating agent to the outer surface of the P22 virus capsid nanocomposite. This biomimetic sys-tem is characterized by high efficiency for the loading of BTZ. Owing to the presence of SP94, the biomimetic nanoparticles can achieve a localized release of BTZ.

This entry is adapted from the peer-reviewed paper 10.3390/biom12010051