Nanoscale bioconjugates: A review of the structural attributes of drug-loaded nanocarrier conjugates for selective cancer therapy.

Nanobioconjugates are nanoscale drug delivery vehicles that have been conjugated to or decorated with biologically active targeting ligands. These targeting ligands can be antibodies, peptides, aptamers, or small molecules such as vitamins or hormones. Most research studies in this field have been devoted to targeting cancer. Moreover, the nanostructures can be designed with an additional level of targeting by being designed to be stimulus-responsive or "smart" by a judicious choice of materials to be incorporated into the hybrid nanostructures. This stimulus could be an acidic pH, raised temperature, enzyme, ultrasound, redox potential, an externally applied magnetic field, or laser irradiation. In this case, the smart capability can increase the accumulation at the tumor site or the on-demand drug release, while the ligand ensures selective binding to the tumor cells. The present review highlights some interesting studies classified according to the nanostructure material. These materials include natural substances (polysaccharides), multi-walled carbon nanotubes (and halloysite nanotubes), metal-organic frameworks and covalent-organic frameworks, metal nanoparticles (gold and silver), and polymeric micelles.

Introduction

Advanced drug delivery

Nanomaterials with an exciting and exceptional surface area refer to a vast spectrum of nanoscale materials, with at least a dimension limit of 1–100 nm [1]. Nanomaterials' first and foremost merit is to precisely adjust and direct the final nanomaterial's characteristics from the initial steps through focusing on synthesis methods (top-down and bottom-up), proper functionalization and modification, and monitoring the size and shape [2]. Various nanomaterials owning breathtaking features are synthesized considering the mentioned variables. Zeolites, carbon allotropes (quantum dots, carbon nanotubes (CNTs), fullerene, nanodiamonds, etc.), silicone, layered double hydroxides, nanoporous materials, MXenes, and many others are some instances of synthesized nanomaterials [3, 4]. According to the convenient synthesis approaches, high specific surface area, exciting physicochemical properties, easy functionalization and conjugation, nanomaterials have been applied as catalysts [5, 6, 7, 8], magnetically separable catalysts [9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24], photocatalyst [25, 26], solar cells [27, 28], supercapacitors [29, 30], drug carriers [31, 32, 33], tissue engineering [34], biological facets [35], water treatment [36, 37], etc., with high efficiencies.

Conjugating the nanomaterials to the biological sections is the most significant stage of prosperous application of nanomaterials because of the various feasible reaction in case of internalizing into the biological ambiance. The NBCs have developed the nanomaterial attachment to the target biological cell, confirmed therapeutic effects, and diminished the toxicity to normal tissue. Such developments make the NBCs highly applicable in targeted drug delivery [38]. For instance, enhanced photodynamic treatment of lung cancer stem cells was conducted by administrating gold nanoparticles (NPs)-antibody NBC with synergistic therapeutic impacts [39].

The recent decade can be regarded as the era of “nano-biotechnology” due to the impressive number of achievements that have been made in the fields of targeted drug delivery, nanomedicine, and theranostics [40]. The rational combination of biotechnology and nanotechnology has developed novel high-tech pharmaceutical preparations with hitherto inconceivable features and properties. Many advantages of drug delivery vehicles have been reported in recent years, including stability in blood and serum, reduced drug side effects, extended stability and storage lifetime, improved pharmacokinetics and pharmacodynamics, targeting specific biological markers, on-demand, and controlled release, and various synergistic combinations [41]. One common point in all of the reported advantages is “biological engineering”, through which the structure of a biologically active component is manipulated by effective chemical conjugation [42].

In this topic, many important aspects could be discussed. For instance, the design of an efficient strategy to carry out a stable conjugation between the chemical functional groups present on the nanoscale drug carrier and a biologically active ligand or targeting agent [43, 44, 45, 46, 47, 48, 49]. The high stability of the conjugation will result in a sufficiently long lifetime within the internal body environment. Moreover, they can be designed to be sensitive to a specific internal factor (pH, redox, or temperature) or external factor (laser irradiation, magnetic field, or ultrasound), which can provide an on-demand drug release strategy [50, 51, 52, 53]. In conventional pharmaceutical finished products, an enteric coating can be used for controlling the drug release process after oral administration [54, 55, 56]. Other strategies including surface-modification of the microscale and nanoscale drug carriers [57, 58], using specific organic structures (as linkers) [59, 60], can provide irradiation-triggered drug release [61, 62]. In drug delivery studies, there are many options to design an efficient strategy for targeting a specific type of tissue and control the drug release process. Recently, significant advances have resulted in better-targeted drug delivery (especially for cancer therapy), in which the main basis is chemical conjugation between the surface of the nanoscale drug carrier and a biologically active component which could be a protein or other moiety [63, 64].

Generally, three important components of the total structure of the final carrier can be identified: 1) Drug carrier: the tiny size of the carrier helps to allow cellular uptake (internalization) [65], while the shape and architecture are important for the prevention of drug leaching during circulation in the bloodstream [66, 67]. 2) Linker: stable organic structures that are responsive or sensitive to a specific condition or agent can be used as linkers to prevent premature drug release until the target is reached [68, 69]. Moreover, the correct degree of hydrophobicity is essential for designing a suitable linker [68, 69, 70]. 3) Biologically active portion: this section includes both synthetic and natural molecules, such as cell-penetrating peptides (CPPs) [32], monoclonal antibodies (mAbs) [71], folic acid (FA, vitamin B9) [72], or aptamers [73]. The biologically active section can be designed as a target ligand and facilitate cell penetration and uptake.

Advantages and limitations

The main advantage of these advanced drug delivery systems (DDS) is their ability to increase the maximum tolerated dose (MTD) and decrease the minimum effective dose (MED) in cancer patients to enlarge the therapeutic window [74, 75]. These nanobioconjugates (NBCs) (here defined as a nanoscale carrier attached to a biologically active component) allow selective targeted delivery to the intended cells or tissues [76]. The nanocarriers can incorporate an anticancer cytotoxic drug, such as docetaxel, paclitaxel, doxorubicin (DOX), etc., which are used in conventional chemotherapy but can have undesirable side effects, including signaling pathways blockage, liver cancers cells proliferation inhibition, bone marrow, and hair follicle cell growth deterrence when the drug accumulates in healthy tissues [77]. Targeted nanocarriers loaded with these drugs can significantly reduce their side effects by protecting them until they reach the tumor. When most of the administrated dose of medicine is specifically delivered to the target tissue, drug accumulation in healthy tissues is decreased, and there would be no need to increase the overall dose to a toxic level [78, 79].

On the other hand, the patient's tolerance to the toxicity of these agents is increased, thereby providing a higher margin of biosafety. As a result, the MTD will be increased, and MED will be decreased. Another essential advantage of these NBCs is their ability to release the loaded drug with high spatial and temporal control in the target tissue [80].

Recently, many drug delivery systems have been designed in which a triggering agent or stimulus is employed to control the drug release. These stimulus-responsive drug delivery vehicles have been called “smart” nanostructures. Briefly, a specific pH or temperature or irradiation with a particular light wavelength could trigger the drug release from the NBC [32, 62]. One limitation is that in the preparation process, the temperature should be kept below 4 °C to preserve the integrity of proteins and other sensitive biological molecules. One limitation is that in the preparation process, the temperature should be kept below 4 °C to maintain the integrity of proteins and other sensitive biological molecules [81]. One of the most sensitive types of these molecules is antibodies, which can be purified at 25 °C but should be kept at 4 °C for long-term storage. The formulation plays a pivotal role in this aspect [82, 83]. Other limitations of NBCs are assigned to abundant affinity biomolecules that could be bound to nanostructure resulting in many undesired phenomena, including barricading biochemical activity changing the target and biomolecule features [84].

Aim of this review

We have collected some recent publications discussing targeted drug delivery, concentrating on those instances in which a nanoscale cargo carrier is conjugated to a biologically active domain. These carriers can be made of natural clay, carbon nanotubes, micelles, metals and metal oxides, natural polymers, or metal-organic and covalent-organic frameworks (MOFs and COFs). Moreover, there are many other advantages of this approach, such as easy internalization into cells (due to the tiny scale), reduction in drug side effects (due to protection of the normal tissues from the cytotoxic drug), and physical direction of the cargo to the target tissue (for instance by using a magnetic field on magnetic carriers). We attempt to provide a logical classification for these NBC systems based on the type of nanoscale carrier. Secondly, a brief review of the preparation routes and therapeutic effectiveness is provided.

Classification of nanomaterials

The nanomaterials are a significant part of science and technology with highly-developed growth in a wide application spectrum with at least one dimension in the 1–100 nm range. While possessing this size range in three dimensions is pertinent to nanoparticles (NPs). Hence, nanomaterials are derived from NPs and progressed a distinguished material class from a historical viewpoint. Nanomaterials are attentive because of their distinct magnetic, mechanical, optical, thermal, and many other characteristics. In nano-scale size, materials demonstrate exceptional features because the bulk to atom/molecule transition occurs. For instance, nanocrystals have a low melting point, and the lattice constant reduction is allocated with increasing the superficial atoms/ions compared to the total atoms/ions.

The unknowing applicability of nanomaterials by people goes back to history. However, in 1857, the nanoscale gold particles in the form of colloid were synthesized by Michael Faraday as the first reported study on nanomaterials [85]. Richard Phillips Feynman in 1959 opened a new insight for modern nanotechnology's concept by his famous phrase “There's Plenty of Room at the Bottom” [86].

Moreover, the ongoing research on nanocatalysts has expanded from 70 years ago [85]. The unprecedented properties of nanoscale materials like high surface area, fast charge transfer, developed mechanical strength, and convenient functionalization favors them to be employed in many reactions and applications. Accordingly, nanomaterials have witnessed attention in drug delivery due to numerous unique properties. As a proof of concept, nanomaterials with mesoporous silica basis have been utilized in sustained DDSs, immediate DDSs, targeted DDSs, and stimuli-responsive controlled DDSs. Such materials have the adjustability of drug release rate by conjugating to biological species, such as polymers, or by functional group introduction. The latest trend in NBCs is to design stimuli-triggered drug release systems to premature drug release reduction, localized drug delivery, and diminish the amount of the loaded drugs [3]. The prepared NBCs later be applied in various biological areas, including sensing, imaging, drug delivery, etc.

Nanoscale drug carrier

Physical and biochemical properties

The ability of different nanoscale materials to be loaded with drugs or biologically active molecules has attracted much attention from researchers. For instance, many systems based on metal or metal oxide nanoparticles have been designed that can form hybrids or composites with other types of materials (such as polymers), in which the drug molecules are immobilized into the polymeric matrix [87, 88]. Reportedly, a 5-fluorouracil (5-FU) anticancer drug was administered against colon cancer. The target-specific oral administration is preferred compared to an intravenous-administrated 5-FU cytotoxic drug with earnest side effects on the digestive system. Thus, the encapsulated 5-FU drug into the Zn-based metal-organic framework (5-FU@MOF-5) was coated with carboxymethylcellulose (CMC), a natural polymer with high water uptake capacity, which has stability in acidic pH that resembles gastric pH, to be protected from stomachic degradation and carry the drug to the colon [88]. Other nanostructures possess a porous structure (mesoporous or microporous) inside which large amounts of drug molecules could be tightly encapsulated and successfully delivered to the target tissue [89, 90]. Examples of these nanostructures include, silica nanoparticles [34], halloysite nanotubes (HNTs) [91], and MOFs [92], or COFs [93].

HNTs, aluminosilicate minerals with large cavity volumes, could entrap the cargo not only in their lumen but on their surface. Significantly, the cargo entrapment in HNT lumen is demanded to diminish the burst release effect (Figure 1a). The hydrogen peroxide (H2O2) existence in the environment caused the hydrogel's B–C linkage breaking, leading to structural degradation and controlled H2O2-triggered release. This bond-breaking operated the alteration of arylboronates into phenols as if the formed fluorescein activated the hydrogel's fluorescence effect [94].

Figure 1

a) H2O2-triggered release procedure of HNTs@PVA@PA. Figure a was adapted by permission from: Chemical Engineering Journal, 2020, 380, 122474 [94]. b) The illustration of the ultrasound-responsive drug release using a vibration enhancer. (i) Hydrogel microbeads contained vibration enhancers and drug models. (ii) Vibration enhancers in the hydrogel microbeads under the applied ultrasound irradiation. (iii) Drug models were released from hydrogel microbeads by the vibration of hydrogel microbeads and vibration enhancers. Figure c was adapted by permission from: Materials & Design, 2021, 203, 109580 [99].

Meng et al. have designed a zirconium MOF (Zr-MOF) with novel azobenzene-containing ligands, which protruded from the MOF's surface. The azobenzene groups of the cargo-loaded MOF were further bound to β-cyclodextrin (β-CD), which entrapped the cargo in the pores and prevented the immediate withdrawal of cargo. The strength of this study was related to the UV light-induced trans-to-cis isomerization of azobenzene groups, which act as stimuli-responsive linkers in the framework. Since the β-CD demonstrated enhanced binding affinity to trans azobenzene than cis azobenzene in an aqueous environment, the azobenzene's isomerization from trans-to-cis compelled β-CD ring separation, leading to MOF's nanopores opening and cargo release [95].

Besides, enzymes, which are categorized in endogenous stimuli-responsive substances, are obtained from physiologic and pathologic alterations viz. enzyme concentration. Polymers, phosphor esters, inorganic materials, etc., have been introduced as nanomaterials with enzyme-stimuli drug delivery procedures. In pathological situations, such as inflammations or tumors, stimuli-triggered cargoes containing ester bonds or peptide chains are prone to breaking according to different enzymes, resulting in the localized release of loaded drugs or proteins to exploit the therapeutic effects of the targeted zone. For instance, aqueous micelles preparation based on hydrophobically modified alginate natural polymer (HMA) modified with dodecyl glycidyl ether applied for DOX as a model drug in hydrophobic DDS. Notably, the drug release pattern of DOX-HMA boosted in acidic pH and Alpha-L-fucosidase enzyme presence confirming the pH- and enzyme-triggered drug release behavior [96]. Bixenmann et al. have provided a DDS regarding amphiphilic poly(esteracetal) homopolymers, and the polymeric pH- and enzyme-triggered degradation profiles were studied. Hydrophobic drug solubilization was displayed by hydrophobic dye- and an amphiphilic immune Toll-like receptor (TLR)7 agonist. The ester groups in the structure are susceptible toward pH˃7 hydrolysis or degrading by solid-supported lipase enzyme. Also, the acidic pH-responsive depolymerization is conducted by acetal groups in tumor and endolysosomes [97].

On the other hand, the drugs will rapidly release when exogenous stimuli-triggered nanocargoes encounter physical stimuli signals. Ultrasound exogenous stimuli with high-frequency sound waves could physically lead to an efficient drug release. In the case of lower than 20 kHz ultrasound frequencies, they are utilized for imaging. In contrast, higher ultrasound frequencies lead to nanocarrier fracture and cargo release or cancerous cell membrane penetrance improvement. Although the commercialization of many microbubbles has been investigated (Albunex, Optison, Levovist, and Sonazoid, etc.), they have some applicable restrictions to reach tumor's vascular compartments and profound permeation, such as the large size of 1–10 micron, short half-life, and less stability [98]. An efficient ultrasound-triggered DDS was designed based on encapsulation of release enhancer tungsten particles with high acoustic impedance (102 × 106 [N s m−3]) in calcium alginate hydrogel microbeads expediting the drug release rate (Figure 1b). The tungsten particles and ultrasound wave synergistic effects promoted the delivery efficiency due to cavitation occurring outside the hydrogel and by the complex and drug carrier's vibration. Tungsten particles have more priorities than microbubbles, making them consistent in stimuli-triggered DDS for long-term controlled release. The advantages are stability in the body environment after exposure to ultrasound irradiation, indicating tungsten's slow dissolution [99]. However, the stimuli-responsive DDS comprises more than two stimuli agents [100].

In some cases, the drug molecules are covalently bonded to the nanocarrier's surface. In this case, an appropriate strategy for breaking the bonds and releasing the drug should be designed as the structural attributes matter the most in delivering the drug to the cancerous cells or tissues [101]. The structural stability of the nanocarriers to prevent rapid degradation in the blood serum is another critical consideration. If the structure is degraded before accomplishing the drug delivery process, the loaded drug will be released into the circulation in the blood vessels. The targeting ability will be lost if the loaded cytotoxic drug is gradually leached from the system (or ultimately released if the system collapses). Recently, we carried out an in vitro evaluation of drug leaching from a magnetic nanosystem designed for cancer therapy by employing a simulated circulatory system [102].

The most crucial biophysical property of nanomaterials for cancer therapy is their ability to undergo the enhanced permeability and retention (EPR) effect, by which the nanoscale drug carriers preferentially accumulate in tumors, but not in normal tissues [103, 104]. The EPR relies on the fact that the newly formed blood vessels in cancerous tumors are different from normal mature blood vessels because molecules and nanostructures with the correct sizes, such as macromolecular proteins or nanoparticles, can leak out of the porous vessels, accumulate in the tumor, and are then internalized into the cancer cells. Moreover, the renal excretion of these nanoparticles after accomplishing the drug release process depends on them having the correct size. One type of nanomaterial called “quantum dots (QD)” has attracted attention in recent years [105, 106]. To address the issue of nanoparticle excretion and avoid the requirement to use nanoparticles <15 nm, efforts have been made to employ biodegradable nanomaterials in drug delivery [107, 108]. These nanomaterials can be decomposed into their building blocks after the drug delivery process is released back into the bloodstream. From the toxicology aspect, it should be noted that the concentrations of the degradation products from the nanoparticles should not exceed the permitted values [109].

Surface functionalization of the nanomaterials may allow effective conjugations to many different types of biologically active structures. For instance, metal oxide nanoparticles which include hydroxyl functional groups on their surface can be activated and used as the sites for covalent bonds [11, 110, 111, 112, 113, 114]. Moreover, micelles and polymeric nanospheres can contain aliphatic hydroxyl groups [107, 108, 115], while clay [116] and carbon nanotubes [117] include silanol and carboxylic acid groups, respectively. The negative charge on the surfaces of gold nanoparticles can be enhanced through the surface plasmon resonance (SPR) effect via irradiation with a specific wavelength of light [102, 118]. Gold nanoparticles can be functionalized with thiol-containing biomolecules such as reduced antibodies [119]. MOFs and COFs include metallic atoms in their structure that could be used as sites for binding to the anionic groups (amine and carboxyl) present in the native structure of proteins [120]. Generally, stable and effective NBCs require firm bonds, which are cleavable in the specific conditions about the target tissue or organ.

Overall, the conjugation of the natural or synthetic nanomaterials and biological sections produce a unique and novel hybrid type of molecular drug carrier that could synergistically incorporate the components' individual physical and biochemical properties, yielding prompt and exceptional properties. Conjugating specific proteins to nanoparticles (NPs) has initiated a new improvement in molecular and cellular biology, propelling a comprehensive development of in vivo gene delivery, medical/cancer imaging, and receptor-targeted delivery. The NBC procedure intensely relies on components' physical and biochemical properties and binding stability. Also, environmental conditions, such as temperature, pH, concentration, etc., affect the process. Paying attention to polymer's cross-linking agents, surface functional groups, and the nature of precursors straightly impress the interaction between nanomaterials and biological sections and defines the drug loading amount and stimuli-responsive release behavior due to the stability and size of the formed pores. The DDS design with minimum drug leakage from the NBCs carrier before targeted cells and smart conducting the drug to the cancer cell is possible by optimizing the mentioned parameters.

Engineered biological structures

The biologically active components that are to be used in the NBC structure may need to be manipulated or modified to form chemical bonds to the nanoscale drug carrier. Some of the well-known species suitable for this purpose are discussed below. Antibodies are secreted by the B-cells as part of the immune defense system and are one of the most valuable biological components in NBCs. Antibodies function with a key-lock paradigm that binds to antigens on the target cell surface [121]. The antibody binds to the surface antigen through this key-lock function, triggering its uptake into the target cell by endocytosis. Each antibody can exclusively attach to its cognate antigen using its variable amino acid regions at the tip of the Y-shaped structure, which is the basis of selective recognition [122]. All IgG antibodies have this Y-shaped structure, containing two heavy chains and two light chains joined together by disulfide bonds [121, 122]. The antibody's lysine and glutamate amino acids provide amine and carboxyl groups to form chemical conjugation bonds [123]. Moreover, there are in total 16 disulfide bonds in the antibody structure, of which four can be reduced to thiol groups by using a mild reducing agent such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol. These thiol groups could also be used as an appropriate site for the covalent binding and conjugation of antibodies onto the surface of the nano-carrier [124].

Other types of biologically active species are aptamers [73] or cell-penetrating peptides (CPPs) [32] that contain nucleic acid or amino acid sequences that can recognize specific proteins on the cell surfaces. In comparison with antibodies, these biomolecules show less selectivity in cell attachment. Aptamers are short oligonucleotide sequences composed of either DNA or RNA bases with a three-dimensional folded structure [73]. Compared to antibodies, working with aptamers is more convenient due to their lower sensitivity to temperature [125]. CPPs are composed of a short amino acid sequence that facilitates cell attachment by creating an effective electrostatic interaction with the phosphate or sulfonate groups present in the cell membrane [32]. CPPs do not act as a recognition agent but substantially assist the cell penetration and internalization. Recently, there have been many reports in which aptamers or CPPs have been tagged onto the surface of nanocarriers via covalent binding [32, 126, 127, 128, 129]. Since these molecules have different free functional groups, it is easy to design a conjugation strategy.

FA is another biologically active compound used to prepare targeted NBCs. In comparison with antibodies, FA is more stable (less sensitive to temperature) and also less expensive [130]. As an agent for specific cell targeting, FA is recognized by its cell-surface receptor (folate receptor). In most tissues, the folic receptor is expressed at low levels. Still, it is over-expressed in many cancer cells, which has led researchers to widely use FA for targeted drug delivery [130, 131].

One point in the preparation of the NBCs is to be careful not to carry out a conjugation procedure that disturbs the biologically active recognition site. In this case, the specific function of the NBC could be lost. For example, suppose an antibody is being used. In that case, it is essential to choose a conjugation site different from amino acids in the complementarity-determining region (CDRs) since this is the leading site for antigen binding [132]. Figure 2 illustrates some structures of nanoscale drug carriers and the biologically active components, which researchers in recent years have used.

Figure 2

The sketch of the shape and morphology of some of the drug nanocarriers and the conjugated biological sections, utilized by the researchers in the last decade. Photothermal therapy (PTT) defines as a cancer treatment approach using electromagnetic irradiation. The conjugation of nanomaterials with biological sections (aptamers, antibodies, CPP, FA) creates various nanocarriers for drug loading and transferring the cargo to the target cells.

To deeply concentrate on biological sections in the architecture of NBCs, a concise yet comprehensive debate on the latest papers seem crucial. In a recent study, Maleki et al. designed an anti-infection NBC based on silica NPs with high porosity, Au NPs, and polyvinyl alcohol (PVA) as an efficient carrier to deliver cefixime (CFM). Regarding the high importance of the carrier's cell adhesion, a dipeptide comprised of cysteine–arginine (CR) was conjugated to the carrier's surface. The drug carrier was smartly designed to incorporate Au NPs in a PVA polymeric substrate, which results in controlled drug release setting off via localized surface plasmon resonance (LSPR) heating. According to the confocal microscopy, the CFM@SiO2/PVA/Au–CR cargo demonstrated better bacterial cells (Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae)) internalization compared to bare CFM. Moreover, the optical density measurements authenticated that this NBC system has shown elevated antimicrobial activity against K. pneumoniae and E. coli cells with (93.0 ± 1.5) % and (86.8 ± 1.0) %, respectively. One of this study's strengths is its enhanced antibacterial characteristic at lower drug dosage [133]. Another study has utilized D-amino acid-containing peptides to conjugate with bendamustine (BEN) drug, a DNA-alkylating chemotherapeutic with a short half-life, unstable and incompatible structure. D-amino acid-contained D-peptides compared to L-peptides have durable biostability against endogenous enzymes. The prepared NBC (BEN–FF–peptide 5) benefits from concurrent BEN and peptide 5 conjugations to self-assembled peptide backbone for human breast cancer treatment (MCF-7). This strategy has approved higher cellular internalization, structural stability in human serum, and improved MCF-7 growth inhibition. Also, the MCF-7 cells viability impedes through p53 (target genes in MCF-7 cells) signaling regulation [134]. Advanced cancers in patients require specific therapeutic strategies. In this case, a group of scientists has investigated a mesothelin-directed antibody-drug conjugate (ADC), BMS-986148, alone or combined with Nivolumab. Due to the preclinical research, the combination of BMS-986148 with anti-programmed death-1 mAb nivolumab upgrades the antitumor impacts as the PD-1 expression hinders the T-cell activating process and expanding prior activated cells. Hence, the surrounding PD-1 route through nivolumab reinstates and progresses antitumor T-cell activity and enhances cytokine production, resulting in progressed clinical results in advanced solid tumors [135]. Tanner et al. have profoundly overviewed the latest improvements in aptamers (single-stranded nucleic acids) and aptamer-enabled materials. Aptamers have the ability to bind to the cell surface and other molecules; therefore, they can act as biological drug carriers for improved therapy or in imaging fields. These nucleic acid carriers provide convenient cellular internalization, drug release, and improved drug efficiency. New preparation routes augmented the controlled physicochemical characteristics for many desired applications [136].

Decidedly, antibodies, aptamers or CPPs, and FA are different categories of biologically active compounds with specific characteristics to form engineered biological structures. In general, the smartness of engineered biological systems owes to applying the cell-responsive components, namely proteins, peptides, etc., that perform the cellular behavior regulations with high affinity.

Nanobioconjugates

Based on the nature and structure of the applied nanoscale drug carriers, we have attempted to make a logical classification for some recently reported NBCs, including some advantages of each cargo, biologically active components, and the drug nano-carriers.

Natural materials

Polysaccharides have been widely used as drug delivery vehicles in recent years. These natural polymers, such as carrageenan [137], dextran [138], chitosan [138], hyaluronic acid [138], or pullulan [139], are ideal resources for preparing biopolymer-based anticancer drug delivery systems due to their beneficial physical and biological characteristics such as biodegradability, nontoxicity, and presence of reactive groups. Their chemical structures are demonstrated in Figure 3a-d, respectively.

Figure 3

The chemical structures of a) carrageenan. Figure a was adapted by permission from: Marine Drugs, 2020, 18, 658 [137], b) dextran, c) chitosan, d) hyaluronic acid. Figures b–d was adapted by permission from: International Journal of Molecular Sciences, 2020, 21, 9159 [138].

κ-Carrageenan (κ-Car) is a high molecular weight sulfated polysaccharide obtained by the extraction of certain red seaweed species [140]. This natural linear polymer consists of galactose and hydrogalactose units connected by glycosidic linkages. Its natural origin, biodegradability, and hydrophilicity have led to its wide application in the food and pharmaceutical industries. Its structural features, such as a negative charge and its gel-forming ability, have led to this polysaccharide being used as a gel-forming and viscosity-enhancing agent. In the past few years, κ-Car has been used in the pharmaceutical industry as a polymeric matrix for sustained-release oral pills and also as an emulsifying and stabilizing agent [140, 141]. Recently, a κ-Car grafted graphene oxide nanocarrier conjugated to biotin (GO-κ-Car-biotin) was prepared, and its application in the targeted delivery of DOX to cervical cancer cells was investigated [142]. Graphene oxide (GO) is a two-dimensional material composed of a network of carbon atoms with sp2 hybridization (polyaromatic network) with a large number of reactive functional groups (such as carboxylic, hydroxyl, and epoxide) on its surface. GO derivatives have been of interest to researchers to prepare efficient drug carriers. The high surface area and the presence of reactive groups on the surface of the GO allow it to interact with other molecules such as targeting agents or drugs through covalent and non-covalent bonding for targeted drug delivery. GO, and its derivatives have had several impressive biological and medical applications, such as encapsulation of drug molecules, gene delivery, and photothermal therapy (PTT) [143, 144]. One serious challenge of GO drug carriers is their possible toxicity to cells in culture. A practical solution to alleviate or minimize this disadvantage is to modify GO with biodegradable and non-toxic natural polymers [145]. Drugs could be loaded onto the GO surface by hydrogen-bonding or electrostatic interactions (GO is charged in some particular cases) with its functional groups.

Furthermore, the aromatic network of the GO allows hydrophobic π-π interactions with several types of anticancer drugs [146, 147]. The combination of κ-Car with GO in a nanostructure provides features such as high dispersion, low toxicity, and good biocompatibility. Moreover, the combination of the GO and κ-Car offers a very high specific surface area and includes some reactive functional groups for the covalent conjugation to drugs [142].

Drug carriers conjugated to some vitamins, such as FA, vitamin B12, biotin, or riboflavin, are a suitable type of NBCs that have been used for the targeted delivery of cytotoxic agents into cancer cells, resulting in better biosafety [148]. Three fluorescence-labeled biotin conjugates were designed to monitor the tumor-targeting drug delivery progression of the biotin-linker-SB-T-1214 (taxoid) conjugates. As depicted in Figure 4a, the receptor-mediated endocytosis (RME) was considered for biotin-fluorescein conjugate, and the biotin-linker-coumarin conjugate was prepared for RME internalization authentication and coumarin release detection by fluorescence of free coumarin molecules through disulfide breaking. Coumarin derivatives are fluorogenic substances that have been applied for drug release detection. The biotin-linker-taxoid-fluorescein conjugate was synthesized to prove the RME internalization and drug release procedure. The released fluorescent molecules were bound to the cancerous cell's protein or microtubule target [148].

Figure 4

a) The schematic illustration of the receptor-mediated endocytosis (RME) for drug conjugate, drug release process, and drug attachment to the target protein. Figure a was adapted by permission from: Bioconjugate chemistry, 2010, 21, 979–987 [148]. b) The schematic illustration of the suggested hypothesis of utilizing AS1411 derivatives for carrying C8 ligand into nucleolin-overexpressed cancer cells. Figure b was adapted by permission from: Scientific reports, 2019, 9, 1–12 [154]. c) The schematic illustration of the internalization of GEM-conjugated polyamino acid-based micelles and their reduction-responsive drug release. Figure c was adapted by permission from: Polymer Chemistry, 2017, 8, 2490–2498 [157]. d) The schematic illustration of the structure and suggested mechanism for the targeted impact of CSKSSDYQC-dextran-poly (lactic-co-glycolic acid) nanoparticles (CSK-DEX-PLGA-NPs = CDPs). Figure d was adapted by permission from: Molecular Pharmaceutics, 2019, 16, 518–532 [158].

Biotin (vitamin H or B-7) is needed for tumor growth and proliferation. The specific biotin receptors on cancer cells are significantly higher than normal cells (e.g., fibroblasts). Several reports have demonstrated the excellent performance of biotin-conjugated drug carriers for targeting and internalization into cancer cells [149, 150]. In one report, κ-Car-grafted GO was conjugated to biotin in a vitamin-receptor targeted drug delivery approach. The synthetic procedure for GO-κ-Car-biotin NBCs consisted of three steps [142]. The conjugation of the biotin to the κ-Car-grafted GO was achieved by a strong ion-ion interaction between the amine groups of positively charged biotin and the OSO3- groups of κ-Car. According to the characterization studies, DOX molecules were entrapped on the surface of the GO sheets by π-π interactions. UV-vis spectroscopy verified the successful encapsulation of DOX in the biotin-functionalized GO/carrageenan carrier system. The high efficiency of drug encapsulation was attributed to the large specific surface area of the carrier.

Furthermore, the presence of π-π interactions and hydrogen bonds between the drug and the GO was suggested to be important in the drug-loading process. It was observed that the highest amount of drug release occurred at acidic pH values. It was proposed that the hydrogen bonds, electrostatic and π-π interactions were weakened, resulting in the released entrapped DOX molecules. Bioassay tests were performed to investigate the cytotoxic effect of the biotin-functionalized GO/carrageenan DOX carrier compared to free DOX on cell lines, human dermal fibroblasts (HDF, normal cells), and human cervical carcinoma (HeLa, cancer cells). Briefly, it was found there was no significant toxicity to normal fibroblasts by the GO/carrageenan DOX delivery system, in contrast to high cell death and changes in morphology for the HeLa cells at the same dosage. They concluded that biotin as a selective ligand could distinguish between HeLa and HDF cells and serve as a particular cancer treatment [142].

Dextran (DEX) is a natural hydrophilic polysaccharide consisting of glucose monomers, with properties such as biodegradability, low toxicity, and good colloidal stability. In one report, a DOX-loaded DEX-spermine magnetic nanocarrier was prepared and tested as a vehicle for targeted drug delivery of DOX to breast cancer cells. The nanocarrier was conjugated to a monoclonal antibody (anti-HER2 mAb) that recognized a breast cancer marker. When DEX natural polymer was combined with spermine (SP) as an amine-containing molecule, the resulting compound showed biocompatibility and biodegradability and a large number of reactive functional groups to react with a broad range of cargos. In that work, the DEX–SP–DOX magnetic nanocarrier was conjugated to the anti-HER2 as a targeting agent [151]. HER2 receptors are over-expressed on the surface of invasive breast cancer, so the drug is precisely targeted to the cells via the antibody. The functionalized magnetic DEX–SP nanocarrier was prepared in four stages. 1) A reductive amination approach was used to prepare the DEX–SP compound. To form an imine bond, DEX was converted to a dialdehyde intermediate by oxidation and then reacted with SP in alkaline conditions (Schiffs base). 2) The DEX–SP conjugate was then obtained by treatment with a reducing agent. 3) The magnetic DEX–SP–DOX nanocarrier was prepared via ionic gelation (in the presence of tripolyphosphate, TPP as a cross-linking agent). 4) The DOX-loaded magnetic DEX–SP nanocarrier was conjugated to the anti-HER2 mAb using 1-ethyl-3-(3-dimethyl amino-propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form amide bonds. The binding efficiency of the antibody to the magnetic nanocarrier was estimated at around 24%, using the Bradford spectrophotometric assay [151].

In another study, an aptamer-functionalized DEX-grafted nanographene was designed to enhance the targeted delivery of curcumin (CUR), which is a natural polyphenol with valuable medicinal properties (antimicrobial, anti-inflammatory, and anti-cancer effects) to breast cancer cells [152]. Different ratios of the loaded DOX within the magnetic DEX-SP nanocarrier were prepared, and the highest entrapment efficiency (EE%) was found at 1:10 (DOX: DEX-SP). When the carrier was functionalized with the mAb, the particle size increased from 62 to 84 nm, while its zeta potential decreased because of the effect of the mAb. The chemical modification of the GO with the natural dextran significantly reduced its cytotoxicity on 4T1 and MCF-7 cells. In addition, the prepared GO-DEX-Apt-CUR had higher cytotoxicity towards 4T1 and MCF-7 cells, compared to free CUR and GO-DEX-CUR, especially at low concentrations of CUR [152].

AS1411 is a single-stranded G-rich DNA aptamer that can bind to the protein nucleolin, which is over-expressed on the tumor cells' surface and then taken up into the cells by nucleolin recognition and endocytosis, as shown in Figure 4b [153, 154]. Nucleolin is a nuclear glycoprotein translocated onto the surface of tumour cells while it remains in the nucleus in normal cells. DEX possesses numerous reactive hydroxyl groups that can be used for GO attachment. The conjugated aptamer improves the cellular uptake and internalization by receptor-mediated endocytosis. The AS1411-functionalized DEX-grafted nano-graphene was prepared in several steps. 1) Functionalization of the DEX with 1,2-ethylenediamine (EDA) through reductive amination to produce amine-terminated dextran (EDA-DEX). 2) Creation of a covalent bond between the activated GO using EDC and EDA-DEX to produce DEX-grafted GO. 3) Conjugation of GO–DEX to the AS1411 aptamer using carbonyl diimidazole (CDI) as an activation reagent. 4) Loading the CUR into the GO–DEX–AS1411 by mixing an aqueous GO–DEX– AS1411 and CUR solution and then removing any unloaded CUR [152].

Chitosan is another polysaccharide, which has been extensively used in recent years with applications in nutrition, biology, catalysis, and environmental remediation [155]. This natural polymer has many advantages for researchers in various fields, such as biocompatibility, biodegradability, the ability to form a gel matrix, and an overall cationic charge. Many amine groups and hydroxyl groups lead to interactions with different molecules, polymers, and nanoparticles without the need for complicated reactions. Therefore, in recent years, diverse composites, hydrogels, and films have been prepared from chitosan. Conjugation reactions between the chitosan chains and other materials like peptide chains have been carried out for drug delivery purposes. Chitosan has a high loading capacity for drug molecules and valuable mucoadhesion properties that can be used in topical drug delivery. The ability of chitosan to bind to anionic cell junction proteins leads to the opening of tight intercellular junctions, increasing the drug penetration. Peptide-conjugated N-trimethyl chitosan was synthesized and tested for increasing the oral bioavailability and antitumor effects of gemcitabine in breast cancer [156]. Gemcitabine is a nucleoside analog with antitumor activity against different tumors such as breast cancer, pancreatic cancer, bladder cancer, and ovarian cancer. For example, as could be observed in Figure 4c, the spherical micelles of self-assembled GEM-conjugated polyamino acids formed in the aqueous media had a receptor-mediated endocytosis internalization, wherein by increasing cancer cell's GSH concentration, the disulfide linkage cleavage occurred, which led to GEM fast release [157]. A particular oligopeptide chain (CSKSSDYQC) was used as a targeting ligand for goblet cells and conjugated to a chitosan nanocarrier. The internalization of the cargo into the goblet cells was enhanced by this conjugation strategy to inquire mucus' impact on the targeting process. Due to the suggested mechanisms in Figure 4d, three different destinies occurred for nanoparticles. First, the convenient attachment of neutral NPs to the mucus layer has brought them to the vicinity of the intestinal epithelial cells. Second, some NPs' identification by intestinal goblet cells was accomplished via CSK peptides, and their absorption into the blood flow happened. Third, other NPs were absorbed into the blood flow by unlatching the narrow cellular bypass route [156, 158].

In many cases, the conjugation between the drug nanocarrier and the biologically active agent (here CSKSSDYQC peptide) involves the formation of amide peptide bonds. The CSKSSDYQC peptide was activated by EDC reagent and reacted with the primary amino groups in chitosan [156]. Afterward, the gemcitabine molecules were loaded onto peptide-chitosan conjugate by an ionic gelation method. The in vitro cytotoxicity of the gemcitabine-loaded peptide-chitosan nanocarrier was confirmed on 4T1 breast cancer cells using a methyl tetrazolium (MTT) test. Still, there was no toxicity for the drug-free peptide-chitosan conjugate. Table 1 presented hydrogels utilized for drug loading into the NBC systems considering their polymers’ advantages and disadvantages.

Table 1

The polymeric hydrogels applied in NBC systems.

Entry	Hydrogel	Loaded drug	Advantages and disadvantages	Ref.
1	Chitosan–quercetin (CHITQ)	Caffeine	Chitosan has biocompatible, biodegradable, bioadhesive structure, N-isopropylacrylamide (NIPAAm) functional monomers cause thermally-triggered DDS, flavonoid quercetin (Q)-modified chitosan provides high derivation degrees.	[159]
2	DOX-conjugated polyethyleneglycol/polycaprolactone (PEG/PCL hydrogels)	Doxorubicin (DOX)	Convenient miscibility with drug according to the low viscosity of the aqueous nature of polymeric hydrogel, gelation/solidification at body temperature after injection allowing targeted drug delivery, DEG hydrophilic polymer aids retaining the water content of hydrogel, while the strong hydrophobicity of PCL impedes drug release.	[160]
3	graphene oxide (GO)–hybrid supramolecular hydrogel (HSH)	DOX	The ureidopyrimidinone (UPy) groups donate self-healing features and provide dimers through quadruple hydrogen bonding, UPy-modified nanocellulose forms cell-adhesive macroporous hydrogels.	[161]
4	Methoxypolyethylene glycol conjugated to arginine-functionalized poly(L-lysine) dendron and encapsulated MMP-9 shRNA plasmid (MPEG-PLLD-Arg/pMMP-9)	MMP-9 shRNA plasmid (pMMP-9)	Biocompatibility of MPEG, α-CD, PLLD-Arg, Desirable size, zeta potential, and low molecular weight of MPEG-PLLD-Arg makes this conjugated supramolecule hydrogel possess safety in gene sustained release.	[162]
5	PEG hydrogel with a patterned DNA aptamer	Proteins	The 3D hydrogel is capable of monitoring the proteins' simultaneous or successive immobilization and release with distinguished time and concentration.	[163]
6	Fmoc-phenylalanine fibril hydrogels	Anticancer drug	Fibrillary hydrogel coating with BSA-based proteins layers or stabilization of hydrogel via a microporous system increases the mechanical strength of the hydrogel to overcome the innate disadvantage of fiber-based materials' low mechanical strength. The N-terminally fmoc-protected phenylalanine hydrogel indicates stability and biocompatibility.	[164]
7	Naphthalene-conjugated FFc/HAd composite hydrogels	curcumin	The hydrogel scaffold's thin nanofibers and honeycomb morphology resulted in extensive, sustained drug release. The HA donates biocompatibility and enhanced mechanical properties to the composite hydrogel.	[165]

aPhosphatase, besterase, cdiphenylalanine, dhyaluronic acid.

As a result, natural-based NBCs provide a massive family of biopolymeric cargo-loaded nanocarriers. Other nanocarriers inherit highlighted characteristics, including inexpensiveness, natural-derived raw materials, and convenient functionalization and facile applicability in cargo loading and on-demand release [166].

All-inclusive, there are recent anticancer therapeutics designed for tumor cell targeting. Hyaluronic acid, chitosan, pullulan, heparin, Chondroitin Sulfate, dextran, inulin, polysialic ACID (PSA), alginate, curdlan, xyloglucan, etc., are some natural moieties mainly derived from polysaccharide family incorporated in NBCs. The micelles formed by self-assembly of copolymers with amphiphilicity have attracted much attention in the drug delivery facet to carry the anticancer drugs to the target cell. Various structural attributes of DDS are engaged, like the core-shell polymeric structures with a hydrophobic core to efficiently encapsulate the hydrophobic drug and a functionalized hydrophilic shell with active biological moieties to conveniently bind to the cell. These NBC strategies enhance the drug delivery process and drug release.

Carbon nanotubes

Multi-walled carbon nanotubes (MWCNTs) are nested cylindrical carbon-only structures with diameters ranging from nanometers to micrometers [167]. MWCNTs have been investigated for tumor drug delivery, biological imaging, and photothermal tumor ablation due to their capacity for drug encapsulation and their ability to absorb near-infrared radiation (NIR) [168, 169, 170]. MWCNTs are considered to be more effective than single-walled carbon nanotubes (SWCNTs) [167]. After exposure to NIR, MWNTs produce a localized heating effect resulting in thermal destruction of the tumor, known as PTT [170]. In one study by Zhang et al. [171], MWCNTs were modified by polyethylene glycol (PEG) chains to solubilize them. Then, cysteine-arginine-glutamic acid-lysine-alanine (CREKA) peptide was attached as a targeting agent, and finally, two fluorescent dyes, Cy3 (greenish-yellow emission) and IR 783 (NIR emission), were linked. Via a simple preparation route, the MWCNTs were initially oxidized by sulfuric and nitric acids and then coated with maleimide-modified PEG via H-bonding. Then, the CREKA peptide was attached by reacting the thiol group of cysteine amino acid with PEG maleimide groups. When PTT is used for tumor destruction or growth inhibition, it has been reported that the tumor temperature depends on the concentration of the MWCNTs inside the tumor blood vessels. The illumination generates a large amount of fibrin that causes coagulation within the tumor vessels. Illumination-generated fibrin combined with the presence of the CREKA peptide chain increased the specific accumulation in the tumor. In vivo studies using size-matched tumor xenograft mouse models showed that MWCNTs-PEG plus laser gave only a 31.8% growth inhibition rate. In contrast, due to the targeting property of CREKA-functionalized MWCNTs-PEG, the tumor was eradicated after NIR laser exposure [171].

Another study used MWCNTs modified by an antibody against P-glycoprotein (Pgp) as described by Ming and co-workers [172]. They conjugated the Pgp antibody to the carbon nanotubes for targeted PTT application. Sulfuric and nitric acids initially oxidized the MWCNTs to reduce the size and increase the aqueous dispersibility. Next, the Pgp antibody was activated with N-succinimidyl-S-acetylthioacetate (SATA) in phosphate-buffered saline (PBS) and then was deacetylated with hydroxylamine to generate sulfhydryl groups after purification (Figure 5a). When the Pgp-MWCNT NBC was incubated with tumor spheroids of Pgp-expressing drug-resistant cancer cells and activated with a NIR laser, there was significant cancer cell death. Due to the hypoxic core of tumor spheroids, any oxygen-dependent therapeutic methods face serious difficulties. At the same time, the PTT strategy does not depend on oxygen and can still be effective in the hypoxic regions in the center of solid tumors. Significant apoptosis has occurred with Pgp-MWCNT under NIR irradiation in comparison with the non-irradiated control. As a desirable result, there is substantial therapeutic synergy between the Pgp-targeted drug delivery and NIR irradiation with ∼73% cell death in one hour [172].

Figure 5

a) Preparation route of Pab-functionalized MWCNTs. (DSPE-PEG: Distearoyl-sn-glycero-3- phosphoethanolamine conjugated to polyethylene glycol5000 terminated with a methoxy group, Mal: maleimide, FITC: fluorescein isothiocyanate). This figure was adapted by permission from: ACS applied materials & interfaces, 2018, 10, 33464–33473 [172]. b) A schematic of the preparation strategy of cRGD-functionalized polylipopeptide micelles (cRGD-Lipep-Ms). (PEG-PAPA: poly(ethylene glycol)-b-poly(α-aminopalmitic acid), MMAE: Monomethyl auristatin E, cRGD: cyclic RGD (arginine–glycine–aspartic) peptides, cRGD-Lipep-Ms: cRGD-functionalized polylipopeptide micelles). This figure was adapted by permission from: Molecular Pharmaceutics, 2018, 15, 4854–4861 [173]. c) The illustration of MWCNT/γ-Fe2O3/PEI-PEG-FA/DOX synthesis. This figure was adapted by permission from: Journal of Biotechnology, 2021, 341, 51–62 [174].

In another example from 2018, You and colleagues reported the targeted delivery of oxaliplatin using MWCNTs [175]. They loaded the oxaliplatin drug by contraction of the walls of the MWCNTs at shallow temperatures. Then, the surface was functionalized with biotin as a targeting agent. The nanotubes were activated by acid treatment, and the shorter carboxylated MWCNTs were conjugated to the TAT-polyethyleneimine (PEI) copolymer (a cell-penetrating peptide). The release of oxaliplatin was studied in PBS at pH 7.4 and 5.3 to emulate the blood and lysosomal environments. They found that oxaliplatin release was faster in an acidic environment because the H-bonds between the nitrogen groups of the PEI and oxaliplatin were dissociated by protonation of the amino groups at the acidic pH. The efficacy of the oxaliplatin-containing TAT/biotin-functionalized MWCNTs in treating malignant glioma in vivo was evaluated by an MRI method. The tumor volume after treatment with the NBC was 3.6 mm3 (after 21 days), while the volume was 18.3 mm3 for the oxaliplatin-containing TAT/biotin-free MWCNT, at the same dosage [175].

Halloysite nanotubes (HNTs) possess a hollow tubular structure and are found as a naturally occurring aluminosilicate mineral [176]. HNTs have a 200–1000 nm length and a diameter of 50–70 nm. The chemistry of the Al–OH groups on the inside and the Si–OH groups on the outside of the tubes allows easy modification with biomaterials like chitosan, PEG, or levodopa [177]. Moreover, the porous structure of the empty tubes can be used as an eco-friendly, available, and bio-adaptable carrier for drug delivery [178]. The drug molecules can be encapsulated into the lumen or adsorbed and intercalated onto the external walls of the rolled tubes. The release of the encapsulated drug molecules depends on the interactions between the walls and the external conditions such as pH and temperature.

Moreover, the amount of loaded drug mass and its solubility could also affect the release behavior. In one example of the application of the HNTs in targeted drug delivery, a doxorubicin-loaded folate-functionalized HNT was described by Wu and colleagues in 2017 [179]. PEG initially coated the HNTs to prolong the circulation time in the bloodstream and improve their solubility. Folate residues were then grafted onto the PEG/HNTs to prepare a tumor-targeted system. The length of the HNTs was shortened via ultrasonication to improve the EPR effect. It was found that ca. 38% of the loaded doxorubicin was released in acidic conditions, while these values were only ca. 9% at neutral pH (pH 7.4). This result is because the H-bonds between the rolled walls were weakened via the protonation of the oxygen atoms present in the internal network (alumina and silica) under acidic conditions. As a result, the tightly rolled-tubular structure was expanded. After tail vein injection, the antitumor effects of the doxorubicin-loaded folate-functionalized HNTs in 4T1-bearing mice showed 65% tumor growth inhibition. In contrast, the inhibition was only 35% for free doxorubicin at the same dosage [179].

Another approach to target tumors using drug delivery vehicles relies on the direction of magnetic nanoparticles using an externally applied magnetic field. Magnetic direction can be combined with biologic targeting of NBCs synergistically [180]. Magnetic direction can shorten the circulation time required of the NBCs within the bloodstream to reduce the likelihood of drug leaching. For instance, in the case of porous HNTs, tiny magnetic nanoparticles could be incorporated within the pores. A magnetic NBC constructed of PEG-modified HNTs, iron oxide nanoparticles, β-cyclodextrin, and folate was described in 2019 [181]. The magnetic separation of cancer cells that had bound to the magnetic NBC was performed.

Magnetically-directed drug delivery was administrated by applying mixed acid-oxidized MWCNTs with further γ-Fe2O3 depositing through the atomic layer deposition (ALD) approach. The magnetic MWCNTs functionalized with polyethyleneimine (PEI) group. Afterward, folic acid (FA) was attached to the PEI via polyethylene glycol (PEG) for DOX anticancer drug delivery to the Human liver cancer cells (LCLPI 11) cells. The schematic illustration is depicted in Figure 5c. The interactions between the DOX drug and carrier were through π-π stacking. The magnetic saturation of the drug carrier was measured at 3.74 emu/g. Its reduction in comparison with the individual γ-Fe2O3 with about 60 emu/g magnetic saturation is allocated to the non-magnetic moieties in the FA conjugated carrier. Although the magnetization is negligible, it is high enough to deliver DOX-loaded cargo to the tumor site using an external magnetic field. The combination of MWCNTs with enhanced loading capacity and magnetic drug carriage precisely and localized to the cancer cell is suggested in the case of delivering high drug dosages [174].

Another HNT-based magnetic composite material incorporating chitosan oligosaccharides was reported for selective targeting. The magnetic HNTs were used as a drug carrier for camptothecin with high loading capacity. The magnetic HNTs were modified by chitosan via solid-liquid interactions, including H-bonding and electrostatic interactions. An in vitro investigation of camptothecin release at acidic pH confirmed the structural expansion of the HNTs, resulting in ca. 83% drug release in 24 h [182].

Applying the CNTs in biomedical facets has become a growing attitude since they are widely utilized in biological imaging, photoluminescence and photoacoustic labels; sensors, and drug delivery. One conventional way to create new structural features through CNT functionalization, is to sonicating CNTs in phospholipid-polyethylene glycol solution. The innate optical features of CNTs sustained through non-covalent functionalizing strategies than covalent bonded functionalization. Further, the conjugation between target ligands (CPPs, antibodies, etc.) and functionalized CNTs facilitated in vitro and in vivo studies of targeting cancer cell. Besides, these carriers could be applied to deliver chemotherapy drugs and to conjugate with RNA (siRNA).

Micelles

Polymeric micelles are self-assembled nanostructures composed of amphiphilic chains, which have a hydrophobic region at one and a hydrophilic region at the other end [183]. The hydrophobic groups comprise the core, while the hydrophilic groups are the shell, which can be dispersed in the aqueous medium. Micelles are used for drug delivery since they can entrap hydrophobic drugs within the core and be functionalized or modified at the surface. Micelles were first observed as colloidal aggregates of molecules such as surfactants or detergents. Another advantage of micelles is that they can be constructed to be sensitive to temperature, light, or ultrasound waves to provide on-demand controlled drug release [184].

Moreover, polymeric micelles can be conjugated to pH-sensitive ligands for controlled drug release [185]. One crucial factor in the formation of micelles is the critical micelle concentration (CMC), which can be influenced by the chemical structure, temperature, pressure, etc. Micelles are formed when the surfactant concentration becomes higher than the CMC value [186].

Generally, it has been estimated that about 40% of small molecule drugs are hydrophobic and require a carrier to solubilize them for delivery to the target tissue [187]. Therefore, micelles have been mainly used for drugs with a hydrophobic nature. Compared with antibody-drug conjugates (ADCs), micelles can be preferable due to more straightforward production, non-immunogenicity, and high capacity for drug loading. Moreover, they have lower costs than ADCs, essential in large-scale production. Importantly, it has been studied that polymeric micelles [188] and liposome [189] NBCs emerge an increased cargo delivery efficiency in comparison with inorganic bioconjugation routes. One example of micelles for targeted drug delivery was reported by Qiu et al., who used cRGD-functionalized polylipopeptide micelles (cRGD-Lipep-Ms) for delivery of monomethyl auristatin E as an anticancer drug [173]. This system was better tolerated in mice than the free monomethyl auristatin E. It was found that the monomethyl auristatin E-containing cRGD-micelles had an MTD over 2.0 mg/kg, which was 10 times higher than the free drug, and also accumulated better in the cancer cells. The designed route for the preparation of the cRGD-Lipep-Ms and the chemical structures of the used ingredients are shown in Figure 5b.

Several methods can be used to encapsulate drugs in micelles [190]. 1) Dialysis bag method. This route involves adding a small amount of water to the polymer solution and adding the drug in a water-soluble organic solvent such as methanol [191]. The unencapsulated drug is removed by dialysis with a semipermeable membrane. 2) Solvent emulsion method. In this method, the drug and the polymer are dissolved in a water-immiscible solvent such as chloroform. The solution is slowly added to stirred water to produce an emulsion. 3) Solid distribution method. In this method, the drug is dissolved with the polymer in an organic solvent, and a solid matrix is obtained by evaporation of the solvent under reduced pressure. Then, the micelles are prepared by adding water to the dry solid followed by ultrasonication. 4) Two-phase separation method. In this method, the drug and polymer are dissolved in a water-miscible organic phase such as tetrahydrofuran and added dropwise to water with vigorous stirring.

As discussed above, the acidic environment of the tumors can be used for drug delivery with pH-sensitive micelles. Tang et al. reported a reversibly activatable CPP-conjugated drug delivery system connected to a pH-sensitive polyglycine linker called HE [192]. The HE-CPP conjugate was fused to the surface of polyethyleneglycol-polylactic acid (PEG-PLA) micelles, and the anticancer drug paclitaxel was loaded into the nanostructure. This was confirmed by zeta potential, where a reversible charge conversion was found depending on the surrounding environment, resulting in pH-responsive drug release. The zeta potential was negative at neutral pH ∼7.5 and changed to a positive value in acidic conditions (pH ∼6.0, typical of cancer cells). Controlled drug release can also be achieved by changing the temperature with heat-sensitive micelles. The drug is loaded into a temperature-sensitive polymer matrix, and the release occurs at a specific temperature. MNPs (particularly AuNPs) can be included in the polymeric micelle network to provide a temperature-sensitive drug release by heating the MNPs by laser irradiation using the LSPR effect. Table 2 summarizes recent reports about targeted drug delivery using advanced NBC systems based on micelles.

Table 2

Summary of recent reports of micelle-based advanced NBCs, for targeted drug delivery in cancer.

Delivery system	Targeted cell	Bioassay tests	Results	Ref.
SA–Dex–OA/DOXa	Bel-7402 (cervical)	In vitro & in vivo	Selective accumulation of SA–Dex–OA/ICG micelles into tumors, 79.2% tumor inhibition	[193]
FA-HA-TOSb	MCF-7 & H22 (breast)	In vitro & in vivo	Strong anticancer effects against MCF-7, good targeting	[194]
cRGD-rPTM/DOXc	MDA-MB-231 (breast)	In vitro& in vivo	Efficient DOX loading, redox-triggered drug release, high selectivity, good antiproliferative activity	[195]
SA-PEG/DOXd	Bel-7402	In vitro and in vivo	pH-triggered sustained drug release, good tumor growth inhibition	[196]
RG5-CPSOe	GBM (brain)	In vitro and in vivo	Enhanced crossing of blood-brain barrier, improved cellular uptake	[197]
PIC/siRNAf	BxPC3 (pancreatic)	In vitro and in vivo	Higher cellular uptake, enhanced binding affinity to BxPC-3 cells	[198]

Doxorubicin (DOX)-loaded sialic acid-dextran (Dex)-octadecanoic acid (OA) micelles.

Paclitaxel (PTX)-loaded folic acid (FA) and α-tocopherol succinate (TOS)-hyaluronic acid (HA) micelles.

Doxorubicin (DOX)-loaded cRGD-decorated poly(ethylene glycol)-b-poly(l-tyrosine) (PTM)-lipoic acid micelles.

Doxorubicin (DOX)-loaded sialic acid (SA)-decorated poly(ethylene glycol)–hydrazone micelles.

(RG)5-loaded cholesterol-conjugated polyoxyethylene sorbitol oleate (CPSO) micelles.

siRNA-loaded antibody fragment (Fab')-installed poly(ethylene glycol) and poly(l-lysine) (PIC) mice.

In conclusion, the targeted anticancer treatment based on nanomedicine, which refers to applying nanotechnology in medicine, were administrated. As one of the well-known nanostructures forming in the case of surfactant's more concentration than CMC, micelles could be loaded by drugs or conjugated to the ligands via tailored bindings through amide thioether, disulfide, acetyl-hydrazone and polycyclic groups. The interactions between drug-nanocarrier (physically or chemically) depend on the functionalization groups, elucidating the drug release behavior.

Metal-organic frameworks (MOFs) and covalent-organic frameworks (COFs)

MOFs are a new class of mesoporous materials formed by the self-assembly of organic linkers and metal ions. MOFs have gained considerable attention in various research fields, such as proton conduction, gas storage, catalysis, and drug delivery. MOFs possess high biocompatibility and are not recognized as foreign by the host immune system. MOFs can be functionalized with different biomaterials and used for targeted drug delivery [199, 200, 201]. COFs like zeolites are a new class of crystalline porous materials that can be either be two-dimensional or three-dimensional in morphology. In COFs, organic building blocks are linked together by strong covalent bonds. COFs have a large accessible surface area, good biocompatibility, and high porosity, providing a high loading capacity inside the large pore volume. Several studies have recently reported optimizing the pore size, biocompatibility, and drug release characteristics of MOFs, COFs, and the MOFs/COFs combination with polymers is a helpful strategy to widen the applicability of MOFs and COFs in targeted drug delivery [202, 203]. In one example, MIL-101 (chromium terephthalate MOF) was used for the targeted delivery of doxorubicin by Wang et al. in 2015 [199]. After encapsulating the doxorubicin into the MIL-101, the exterior surface was modified with β-cyclodextrin using a strain-promoted [3 + 2] azide-alkyne cycloaddition to provide a suitable substrate for the next step of the functionalization. The primary purpose of the surface modification with β-cyclodextrin was to offer effective interactions with hydrophobic molecules (like adamantane) based on a host-guest interaction. In the final step, the surface was functionalized with K(ad)RGDS-PEG1900, which is a pegylated peptide with the structure Lys(adamantane)-Arg-Gly-Asp-Ser-bi-PEG1900 (bi stands for a benzoic-imine bond), used as a targeting agent. The benzoic-imine bond between the PEG and the K(ad)RGDS peptide was sensitive to acidic conditions and rapidly dissociated inside the cancer cells.

Moreover, the link between the β-cyclodextrin and MIL-101 (a disulfide bond) was cleaved in the same conditions. As a result, ca. 80% death of HeLa cells was achieved in 4 h, whereas only 20% cell death was found with free doxorubicin at the same dosage. The synthetic route, confocal microscopy images of the cells (good internalization and cellular uptake), and the tumor tissues from sacrificed mice after treatment with the MOF-based drug delivery system are discussed in another study [199].

In another exciting report, two different types of zirconium-based MOFs called NH2-UiO-66 and MOF-808 were functionalized with FA and used for targeted delivery of 5-fluorouracil as an anticancer drug [200]. In MOF-808, a Zr6 cluster on the nanoparticle's surface was coordinated to the terminal carboxyl group in the FA molecule in place of formate or hydroxyl ligands. The maximum encapsulation efficiency for FA-MOF-808 and FA-NH2-UiO-66 was 38.42 wt% and 30.26 wt%, respectively. Using an MTT assay, 80–90% cell death was observed for both systems.

Elsewhere, a MOF constructed of 5,10,15,20-tetrakis(4-carboxylphenyl) porphyrin, benzoic acid, N, N-dimethylformamide, and Zr4+ ions that are known as “PCN-224”, was functionalized with a DNA aptamer that recognized A549 lung cancer cells and tested for targeted delivery of doxorubicin to A549 cells [201]. The aptamer was modified with carboxyl and fluorescein groups from the structural design aspect at either end. The carboxyl was linked to Zr4+ ions by a coordination bond, producing DNA-functionalized PCN-224. The UV-vis absorption spectrum showed that the drug loading efficiency of the PCN-224 was about 50 μg/mg due to hydrophobic interactions and π-π stacking between the MOF and doxorubicin. The morphology and size of the MOFs affect the drug loading and whether they can be taken up by the cells. This report was based on 5,10,15,20-tetrakis(4-carboxylphenyl) porphyrin is a highly effective photosensitizer for photodynamic therapy, which causes tumor cell death after illumination with visible light. In vitro studies compared A549 cells (specific to the aptamer) and MCF-7 cells (irrelevant cancer cells), showing ca. 72% apoptosis in A549 cells but only 18% for MCF-7 cells, confirming the specificity of the targeting approach.

Although MOF porous structures have limitations in cargo-loading size, the one-pot synthesis method has overcome this downside. So, even molecules with larger sizes than MOF's voids could be loaded without undesired burst release [204]. Unfortunately, based on three main reasons, small molecules are delivered more efficiently with MOFs than macromolecules, viz. peptides and proteins. 1) Protein-conjugated nanomaterials via covalent bonding cause a poor loading efficiency [205]. 2) The delivery efficacy diminishes due to the low biologically stable structure of protein-MOF conjugates [206]. The protein's superficial charge could prevent the internalization into the cells [207]. Even though MOFs are nanophotosensitizers (nPSs) candidates in photodynamic therapy (PDT), some shortages, such as low permeation depth to the tissue due to the light's short wavelength and insufficiency of oxygen-dependent procedures in case of hypoxic tumors, should be eliminated. Hence, the core-shell upconversion nanoparticle@porphyrinic MOFs (UCSs) toward hypoxic tumor therapy was designed. The heterogeneous structural attribute permits boosted energy transfer from upconversion nanoparticles (UCNPs) to the core to the MOF shell leading to near-infrared (NIR) light-responsive cytotoxic reactive oxygen species formation. In addition to that, the tirapazamine (TPZ) prodrug, which was hypoxia-activated captured in MOF's pores to produce TPZ/UCSs. Concludingly, the tumor therapy process of this work not only renders the cancer cell growth inhibition via NIR-responsive PDT and through hypoxia-activated chemotherapy with a synergistic effect [208]. Table 3 demonstrates the in vitro and in vivo findings of some of the recently applied MOF-based conjugated systems for drug delivery.

Table 3

The in vitro and in vivo studies of MOF-based conjugated DDS.

Entry	MOF-based conjugated systems	Targeted cell	In vivo results	In vitro results	Ref.
1	MAa-HfMOF-PFCb-Ni-Zn,MA-HfMOF-PFPc-Ni-Zn	MDA-MB-231d, MIAPaCa2e, and HeLaf, MCF-10ag	MA-HfMOF-PFC-Ni-Zn demonstrated stronger PDT impacts compared with MA-HfMOF-PFP-Ni-Zn	80 μg/mL of MA-HfMOF-PFP-Ni-Zn killed the cancer cell lines, while 20 μg/mL of MA-HfMOF-PFC-Ni-Zn applied to kill the cell lines. MAHfMOF-PFP-Ni-Zn and MA-HfMOF-PFC-Ni-Zn displayed low toxicity toward the MCF-10a cell lines compared with cancer cell lines.	[209]
2	Fe3O4@MOF-DOXh-CDsi-Aptj	MDA-MB-231	Treated HUVEC cells exhibited high viability (∼90%) and no toxicity. Fe3O4@MOF-DOX-CDs-Apt prevented the cell proliferation, more than 77% selective apoptosis of MDA-MB-231 at 24h and less than 10% apoptosis of normal HUVEC cells.	-	[210]
3	pEGFP-C1@ZIF-8, pEGFP-C1@ZIF-8-PEI 25 kD	MCF-7 cells	Improved cellular uptake and endosomal escape of the protected pDNA with the superior ZIF-8-PEI 25 kD vector, resulting in efficient gene expression. pEGFP-C1@ZIF-8-PEI 25 kD showed a higher transfection efficacy (10%) ​in every dosage of nanocarrier compared with pEGFP-C1@ZIF-8.	-	[211]
4	UiO-66@AgNCs@Apt@DOX	MCF-7 cells	UiO-66@AgNCs@Apt shows low cytotoxicity to MCF-7 cells, enhanced antitumor impact through efficient DOX delivery and its intracellular controlled release.	DOX can be delivered to the nucleus, and the one-pot encapsulated UiO-66@AgNCs@Apt@DOX has great potential as in vivo targeted drug delivery system.	[212]
5	cell penetrating peptides-zeolitic imidazolate frameworks	HeLa cells, HeLa puLc 705 cells, and U-87 MG-luc2 cancer cells	The carrier did not show any toxicity and was biocompatible toward kidney, skin, breast, blood, bones, and connective tissue cell lines.	Most of the oligonucleotides internalized the cells through endocytosis, and the gene was released after degrading in the cytosol.	[213]
6	Mineralized MOF coupled with a lysosome-targeting aptamer (CD63-aptamer)	T cell	Improved antitumor capability with 35.4% tumor cell apoptosis.	Tumor suppression rate reached 70.9% in 30 days.	[214]
7	BSAl@ZIF-8/DNA NPs	mRNAs	The survivin DNA initiated a PERm-based DNA machine in the presence of hairpin, primer, dNTPs, and KFPn.	-	[215]

amaltotriose bChlorine-based MOF, cPorphyrin-based MOF, d(triple negative breast cancer) cells, e(pancreatic cancer) cells, f(cervical cancer), g(an immortalized human breast epithelial cell line derived from non-tumorigenic breast epithelium), hDoxorubicin, iCarbon dots, jNucleolin-binding aptamer, AS1411, kCiprofloxacin, lBovine serum albumin, mPrimer exchange reaction, nKlenow (exo-)fragment polymerase.

Collectively, the examples in this section addressed the latest advances in the field of MOFs and their structural attributes, including COFs and MOFs functionalization routes, properties, and the MOF-based NBCs with focusing on stimuli-responsive drug release behavior. Also, the DDS containing the MOF-based NBCs with multi-stimuli-responsiveness (viz. chemo-photodynamic therapy, etc.) demonstrated the more rapid drug release profile, yet possessing the selectiveness over specific targets. The synergistic behavior of multi-stimuli-triggered release was inquired in vitro and in vivo. Still, there are several possibilities and optimizations of the reaction variants to be noticed.

Metal nanoparticles (MNPs)

Metallic nanoparticles (MNPs) can be prepared using physical, chemical, or biological methods. Generally, the synthetic approaches to MNPs can be divided into two categories. 1) Top-down approaches in which the nanostructures are produced from bulk material, e.g., grinding or microfabricating large pieces of metal to reach the nanoscale size. These methods are labor-intensive, costly, and less suitable to nano-bio systems due to limits on scaling up. 2) Bottom-up approaches in which the procedure starts from molecular precursors, e.g., metal salts. Metal nanoparticles have less toxicity and increased biological effects than bulk metal salts. Targeted delivery and controlled release of encapsulated drugs mean they can be used as nanocarriers.

As biologically compatible and FDA-approved MNPs, gold nanoparticles (AuNPs) have been the leading choice in medical applications over the last years. The advantageous properties of AuNPs include their high stability, well-developed synthetic methods, size-dependent optical properties, and good capability for surface modification. AuNPs have been extensively used in biomedical applications such as genetics, surface plasmon-resonance biosensing, in vivo photoacoustic imaging, laser anti-cancer phototherapy, photodynamic therapy, surface-enhanced Raman spectroscopy diagnosis, and for the targeted delivery of drugs, DNA, and antigens. AuNPs can be synthesized in various shapes and sizes, such as spheres, rods, shells, triangles, cubes, and stars [216]. Currently, the main emphasis in advanced applications of AuNPs involves the replacement of various ligands on the surface of the nanoparticles. DNA, enzymes, antibodies, and polymers can all be conjugated to AuNPs, and in many cases, this does not influence their activity. AuNPs have been used as drug carriers, and if targeting by antibodies or other ligands is employed, they can deliver drugs to targeted cells or tissues.

Moreover, the photophysical properties of the AuNPs can be used as a trigger for controlled drug release [217]. A recent report described an efficient drug delivery system including iron oxide magnetic nanoparticles as the core and coated with AuNPs dispersed inside a polymer shell [102]. In this report, MCF-7 breast tumors were grown in mice and targeted using a docetaxel-containing iron oxide/AuNPs nanocarrier (Au/Fe3O4/PVA-10%DXL). The LSPR (localized surface plasmon resonance) effect of the AuNPs was used for controlled drug release in the tumor, with synergy observed between the magnetic properties and the plasmonic-mediated photothermal release of the encapsulated docetaxel. Around 80% apoptosis was observed in MCF-7 breast cancer cells (Figure 6a) and good biosafety in vivo and ex vivo studies (Figure 6b). Green LED light at 526 nm wavelength was used for plasmonic photothermal activation of the 38 nm average diameter AuNPs. In vivo experiments, near-infrared (NIR) irradiation (808 nm) was used because the green LED light could not penetrate the skin. Dynamic light scattering (DLS) showed that the AuNPs tended to aggregate together. The agglomeration of the AuNPs within the polymeric matrix improved the LSPR effect of the AuNPs when irradiated with NIR light (Figure 6c).

Figure 6

(a) MTT test using a docetaxel-containing iron oxide/AuNPs nanocarrier on the MCF-7 breast cancer cell line (C: MCF-7 control, DXL: individual docetaxel, P4: mercaptopropyl-modified Fe3O4/PVA particles, P6: Fe3O4/PVA-10%DXL, and P7: Au/Fe3O4/PVA-10%DXL), (b) the ex vivo fluorescence images after the major organs were excised and tetramethylindocarbocyanine iodide (DiR) fluorescence distribution was studied using a Maestro in vivo imaging system, and (c) a schematic presentation of the LSPR heating by AuNPs agglomerated in the magnetically delivered Au/Fe3O4/PVA-10%DXL nanomedicine composite. This figure was adapted by permission from: Small, 2020, 16, 2002733 [102].

Another valuable type of MNPs is silver nanoparticles (AgNPs). AgNPs have advantages of high conductivity, broad-spectrum SPR effect, chemical inertness, and increased stability. Many studies have shown that AgNPs can be used as anti-tumor agents with anti-proliferative and pro-apoptotic properties. In comparison with AuNPs, AgNPs are less expensive for plasmonic photothermal applications under NIR irradiation. For example, a magnetic drug delivery system constructed of iron oxide MNPs, AgNPs, carbon nanotubes, and FA was reported for the targeted delivery of doxorubicin by Wang et al. [218]. Briefly, cancer cells labeled with calcein-AM were incubated with the hybrid nanostructure and largely killed after NIR laser irradiation.

Iron oxide MNPs (Fe3O4) have been applied for targeted drug delivery. Iron oxide MNPs are biocompatible and biodegradable, non-toxic, and stable against temperature and acidic conditions [219, 220]. The iron oxide MNPs is the magnetic behavior that allows them to be manipulated with an external magnetic field [9]. Moreover, the surface of iron oxide MNPs can be conveniently functionalized with other molecules due to numerous hydroxyl groups, which can be used for covalent binding [6, 10]. Recently, we have investigated the ability of iron oxide MNPs combined with organic polymers to be magnetically directed to the target tissue [32, 102]. They can also be surface functionalized with targeting agents, such as FA or antibodies. Tumor accumulation and penetration can be achieved by applying a strong external magnetic field. After that, cell attachment is induced by the biologically active targeting agents tagged onto the surface. One example was reported by Ma et al., who suggested a smart drug delivery system constructed of the iron oxide MNPs and the FA targeting agent [221].

Concludingly, the utilization of metal NPs has spread into a broad spectrum of biological applications, namely, imaging, sensing, drug delivery, etc. The conjugation of metal NPs to the biomolecules includes metals like gold NPs to magnetic NPs. Peptides are capable of permeating to plasma membrane were employed for intracellular delivery of metallic NPs. Moreover, biological sections that identify the particular cell receptors are exerted to target the NPs. These bioconjugates are applied in drug delivery, cancer therapy, curing Alzheimer's disease, in vitro diagnosis. The low cytotoxic and biocompatible nature of the metallic NPs-based bioconjugates is another upside of these systems.

Preparation routes of nanoscale drug carriers

A concise investigation on preparation approaches of some nanoscale drug carriers, i.e., iron oxide, metal-based NPs, micelles, and unnatural species is presented.

Iron oxide

Iron oxide preparation methods are divided into physical and chemical categories. Ball milling is a physical method with two distinct approaches, ordinary ball milling, and high-energy ball milling. The first method is related to crushing large NPs of Fe3O4 to obtain ultrafine particles due to steel ball movement into the ball mill tank. Time and frequency are two influential factors in this method. The latter ball milling approach applies high-energy ball mill the materials to form nanoscale spinel ferrites. From chemical processes, co-precipitation is one of the conventionally used approaches. The Fe3O4 NPs are synthesized by adding a base into the solution containing Fe2+/Fe3+ salts. The hydrothermal method is attributed to pouring the aqueous solution of the precursors into a Teflon-lined autoclave and placing it in an oven under high temperature and pressure for dissolution of insoluble species. The resultant further will be separated and dried to obtain NPs. Pyrolysis is considered as one of the magnetic NPs’ preparation methods, where metal compounds underwent thermal decomposition at high temperatures. Then, the precursors oxidized to form Fe3O4 NPs. The sol-gel route mentions the mixture of chemically-active compounds in a liquid phase. It should be noted that the stable, transparent sol in the solution is prepared through hydrolysis or polycondensation. Afterward, the formed sols are directed into the gel, followed by drying to prepare nanoscale magnetic NPs. Also, other preparation routes, namely microemulsion, sonochemical approach, electrodeposition, polyol method, etc., are suggested for magnetic NPs preparation. Each of these methods has individual merits and demerits for being assessed [222].

Metal-based NPs

Many top-down and bottom-up approaches have been applied for metal-based NPs synthesis. In the thermolysis method, the organometallic ingredients underwent dissociation in organic solvents at an upper than 100 °C temperature and inert environment to inhibit NPs’ oxidation. The chemical reduction approach referred to dissolution of metal ingredients in a solvent and stirred with a reducing agent and a surfactant. The biochemical method represents biological species, i.e., plants, algae, yeasts, fungi, bacteria with chemical reagents. The NPs growth process depends on enzyme-mediated or non-enzyme-mediated reduction procedures intracellularly or extracellularly.

Moreover, in the wave-assisted or sonochemical method, the general procedure depends on the cavitation produced by ultrasound waves in the solution of metal ingredients, reducing agents, and surfactants. The localized hot spots in the solution producing over microcavities’ formation and explosion have very high temperature and pressure, which can prompt the chemical reactions. Furthermore, electrochemical routes with more beneficial features than chemical methods, cementation, biological, and other approaches should be considered. The well-matched one with our desired preparation process should be selected [223].

The most applied process for micellization of core-shell nanocarriers is the self-assembly of amphiphilic block copolymers conveniently in water or aqueous solutions. The core block should possess a hydrophobic feature to ease forming the micelles and monitoring their colloidal stability. In contrast, the corona block should be hydrophilic in non-ionic or zwitterionic forms. In the case of reactive functionalities on the core block, bifunctional cross-linking agents were applied for polymeric micellization. Notably, the micelle formation mainly relies on the CMC. It means that turning the amphiphilic block copolymers happens only in a concentration above the CMC threshold. Contingent upon surfactant-based micelles with low molecular weight, the CMC is very high, causing some side effects and a decreased stability compared to the polymeric micelles. There are some techniques to prepare the drug micellar nanocarriers. The precipitation/evaporation is based on the dissolution of copolymers and drugs in volatile solvents, followed by adding gently to an aqueous phase for organic solvent removal. The ultrasonication method depends on the simultaneous dispersion of amphiphilic copolymers and hydrophilic drugs in an aqueous media.

In contrast, the dissolution of hydrophobic drugs occurred in the organic solvent, which a rotary evaporator will further take out. The resultant undergoes sonication to drug-loaded micelles. In the thin-film hydration method, the dissolution of copolymers and hydrophobic drugs in organic solvents occurs. Then, a thin film is prepared by evaporating the solvent. By adding the aqueous medium to the flask, the drug-containing micelles are prepared. Other drug-loaded micelle preparation routes should be investigated, including emulsification/evaporation, direct dissolution, dialysis, etc. [190].

Ceramic-based NPs

The ceramic-based hybrid NPs, such as hydroxyapatite (HA), calcium chloride (CaCO3), zirconia (ZrO2), silica (SiO2), and titanium dioxide (TiO2), have extensive use in tissue technology and drug delivery. The sol-gel procedure is one of the most applied methods for silica NPs synthesis, rendering highly pure and homogeneous NPs. Mesoporous NPs are a result of introducing surfactants to the sol-gel procedure. It has been reported that calcium phosphate NPs are synthesized through a simple co-precipitation route. The calcium chloride (CaCO3) is synthesized via spray-drying approach, which is the atomization of a solution comprising favored product components into droplets through spraying and fast droplet evaporating into solid powder via hot air. The temperature and pressure of the procedure should be maintained constant [224].

Future perspectives

The DDS has lately paved the way for a noteworthy revolution in nanomedicine toward engineered and smart nanocargoes. Accordingly, the nanoscale bioconjugates designed with various biologically active ligands (antibodies, peptides, aptamers, vitamins or hormones) demonstrate a higher penetration rate to the cell compared to huge carriers. The drug release behavior of these NBC mainly depends on the structural features (porosity, hydrophilicity, hydrophobicity, etc.) of nanocarrier and targeting cell or issue, the environmental conditions (pH, temperature, concentration, etc.), and the interactions between nanocargo and cells’ active sites (chemical or physical), all of which should be optimized based on the targeted cell or tissue. As a future approach, the delivery of radiolabeled nanomaterials and nucleic acids for gene therapy and siRNA delivery is significant for NBC systems. NBCs can be designed to contain therapeutic radioisotopes and incorporate multivalent cancer-targeting molecules for more selective treatment. The main problems that need to be overcome are to optimize the physical and chemical properties, the targeted specific binding, and their stability. More studies should be dedicated to developing biomaterial NBCs with more safety and effectiveness against various diseases in the coming years.

Conclusion

In brief, the drug delivery field is persistently developing and unveils new research routes as our perception about cancer therapy and its nuances elevates. Nanotechnology has a vital role in DDS and treatment, and the nanomedicine-based therapies impressively affect cancer cells. Nevertheless, an appropriate selection of the nanocarriers’ structure, properties, and binding affinity to the targets is challenging in the targeted delivery of pharmaceutical agents. Hence, many nano-based targeted therapies have made it difficult to accurately differentiate cancer cells from normal cells. Understanding that tumor cells with specific receptors smartly attach to the individual ligands on the drug-loaded NBC through which the drug with various release profiles leaves the nanocarrier opens exciting opportunities for the researchers to observe the role of NBC structural attributes in initiating, progressing, and terminating cancer. The ideal characteristics that NBC systems should provide are divided into three five categories, including (1) facile NPs and NBC synthesis, preparation, and functionalization, drug loading, and delivery to the target cells, (2) NBC stability and biocompatibility (without drug), (3) pursuing the drug-loaded NBC in real-time drug delivery detection, (4) capability of mediating the stimuli-responsive drug release, and (5) controllably adjusting the loaded drug toxicity. This survey presents a deep and concise study on the NBC drug carriers of different kinds, such as natural materials, MOFs, COFs, MNPs, and micelles. Meanwhile, all of the highlighted reports provided by scientists have been briefly mentioned in this study. The synergistic cargo delivery with the magnetic field or NIR irradiation from when it enters the body to reach the target tissue has been debated. Besides, various on-demand drug release stimuli have been discussed.

Comprehensively, NBC's technology is rapidly moving onwards, and researchers have focused on reducing the utilized cargo-loaded NBC amounts and enhancing the site-specific targeted delivery. Furthermore, it is equally important to pay attention to the drug delivery mechanism and target cell or tissue type. Table 4 summarizes the most utilized expression abbreviations through the context and definitions.

Table 4

Abbreviations & definitions used in the context.

Abbreviation	Definition
Amphiphile	A chemical compound possessing both hydrophilic and lipophilic properties.
CMC	In colloidal and surface chemistry, the critical micelle concentration is defined as the concentration of surfactants above which micelles form and all additional surfactants added to the system will form micelles.
cRGD	A tripeptide Arg-Gly-Asp consists of arginine, glycine, and aspartate.
EPR	Enhanced permeability and retention.
Folate	The key-lock pattern partner for the FA located onto the cell surfaces.
H-binding	Hydrogen bond interactions
Levodopa	(l-3,4-dihydroxyphenylalanine), is an amino acid that is made and used as part of the normal biology of humans.
LSPR	An exclusive optical property of the AuNPs through which the particles are heated upon the exposure to a specific wavelength of the light in accordance to the size of the particles and their SPR preference.
LED	Light-emitting diode
MRI	Magnetic resonance imaging
MTD	Maximum-tolerated dose
PCN	Porous coordination network
Photodynamic therapy	Is a form of phototherapy involving light and a photosensitizing chemical substance.
Photosensitizer	Is a molecule that produces a chemical change in another molecule in a photochemical process.
(RG)5	A cell-penetrating peptide (CPP), arginine-glycine repeats.
TAT	Trans-activator of transcription is a protein consists of between 86 and 101 amino acids depending on the subtype.
4T1	A mouse mammary gland cancerous cell line
Zeta potential	Is the electrical potential at the slipping plane. This plane is the interface which separates mobile fluid from fluid that remains attached to the surface.

Declarations

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article.

Funding statement

This work was supported by the Sichuan Province Science and Technology Program (2022YFH0047).

Data availability statement

No data was used for the research described in the article.

Declaration of interests statement

The author MRH declares the following potential conflicts of interest: Scientific Advisory Boards: Transdermal Cap Inc, Cleveland, OH; BeWell Global Inc, Wan Chai, Hong Kong; Hologenix Inc. Santa Monica, CA; LumiThera Inc, Poulsbo, WA; Vielight, Toronto, Canada; Bright Photomedicine, Sao Paulo, Brazil; Quantum Dynamics LLC, Cambridge, MA; Global Photon Inc, Bee Cave, TX; Medical Coherence, Boston MA; NeuroThera, Newark DE; JOOVV Inc, Minneapolis-St. Paul MN; AIRx Medical, Pleasanton CA; FIR Industries, Inc. Ramsey, NJ; UVLRx Therapeutics, Oldsmar, FL; Ultralux UV Inc, Lansing MI; Illumiheal & Petthera, Shoreline, WA; MB Lasertherapy, Houston, TX; ARRC LED, San Clemente, CA; Varuna Biomedical Corp. Incline Village, NV; Niraxx Light Therapeutics, Inc, Boston, MA. Consulting; Lexington Int, Boca Raton, FL; USHIO Corp, Japan; Merck KGaA, Darmstadt, Germany; Philips Electronics Nederland B.V. Eindhoven, Netherlands; Johnson & Johnson Inc, Philadelphia, PA; SanofiAventis Deutschland GmbH, Frankfurt am Main, Germany. Stockholdings: Global Photon Inc, Bee Cave, TX; Mitonix, Newark, DE.

Additional information

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