3.3.1. Albumin based prodrugs and albumin-drug conjugates

Albumin based prodrugs and drug conjugates are widely used in cancer therapy. The unique properties of albumin like biodegradability, solid tumor accumulation ability, non-toxicity etc. makes it suitable drug carrier. Albumin also has different binding sites which can bind drugs by covalent or non-covalent bonding. Binding of drugs with albumin alters the properties of the drug molecule like toxicity profile, circulation half life and other pharmacokinetic properties [54]. Albumin-drug conjugate enhances the circulation half of drug due to long plasma half life (~ 19 days) of albumin in human body. Apart from this, it also overcomes the multi drug resistance of anticancer drugs [55]. On the basis of above mentioned properties of albumin-drug conjugates, lots of research work is carried out in last few years for the treatment of cancer and is also in clinical trials.

Many anticancer drugs like methotrexate (MTX), doxorubicin (DOX), cisplatin, oxaliplatin, carboplatin, docetaxel etc. were used to prepare albumin-drug conjugates. Stehle et al. prepared MTX-HSA conjugate for the treatment of cancer and this was the first albumin-drug conjugate that was under gone clinical trials. MTX-HSA conjugate was prepared by the direct coupling of MTX with the lysine residue of HSA and found to be more effective in targeting tumor [56], [57]. 7-Ethyl-10-hydroxycamptothecin (SN38) - HSA conjugate was prepared by Sepeheri et al. to improve the solubility as well as tumor tissue targeting as compared to active form of SN38 by chemical coupling using carbodiimide chemistry (EDC/NHS) [55]. The results revealed better solubility and stability of SN38-HSA conjugate along with prolonged circulation time in blood as compared to free SN38. Esmaeili et al. developed docetaxel-albumin conjugate for enhancing the solubility and tumor targeting [58]. Apart from drugs, different dyes and photosensitizing agents have also been conjugated to albumin for cancer diagnosis and therapy. Jeong et al. prepared chlorin e6 (Ce6) conjugated HSA nanoparticles for photodynamic therapy. Ce6-HSA conjugate was prepared by the carbodiimide chemistry using EDC/NHS. This conjugate was able to form self-assembled nanoparticles which upon illumination with specific wavelength light produced singlet oxygen which damaged the target tumor cells in cell culture. The in vivo results also revealed superior biodistribution of Ce6-HSA conjugate at the tumor site as compared to free Ce6 [59].

Different prodrugs have been designed to target the cancer cells that utilized endogenous albumin as drug carrier. These prodrugs are designed in such a way that binds rapidly and selectively to the cysteine-34 position (Cys-34) of circulating serum albumin after administration because approximately 70% of blood circulating albumin is mercaptalbumin containing an accessible Cys-34 [34]. Many platinum based prodrugs have been designed to selectively bind with the serum albumin for cancer treatment. Zheng et al. designed Pt (IV) prodrugs to bind non-covalently to HSA for drug delivery. In this study, axial ligand of cisplatin was asymmetrically functionalized by two different groups i.e. succinate and an unbranched aliphatic carbamate. Optimized compound of this study (compound 4e) bound with serum albumin non-covalently and after conversion of active form demonstrated 9 to 70 time better anticancer activity than parent compound cisplatin in lung and ovarian cancer cell lines along with prolonged half life (6.8 h) in blood as compared to cisplatin (t1/2 ∼ 20 min) or satraplatin (t1/2 ∼ 6 min). This prodrug compound (compound 4e) was interacted with the biological reductants, present in the cancer cells, which convert it into Pt(II) form. This Pt(II) form further interacted with DNA and causes DNA damage, cell cycle arrest and apoptosis [60]. In another study, Mayr et al. prepared oxaliplatin prodrugs to bind with HSA and improve the anticancer activity. Prodrugs were designed by the functionalization of the maleimide group in the parent compound which converted the Pt (II) form of oxaliplatin to Pt (IV) prodrug. Apart from oxaliplatin, cisplatin prodrug was also prepared with the same functional moiety and results were compared. The results indicated the faster reduction of cisplatin analogue as compared to oxaliplatin analogues. Apart from the reducibility, tumor-targeting potential of the albumin-bound drugs 24 h after i.v. administration also varied which may be due to different reducibility of the drugs. All these results revealed the potential of prodrug of oxaliplatin in enhancing plasma half life of drug by avoiding fast renal clearance and better tumor accumulation of the drug due to EPR effect [61]. Apart from the platinum based prodrugs, ferric prodrugs have also been designed to interact with serum albumin and targeting cancer. Qi et al. designed the ferric prodrug based on N-donor residues (Lys199 or/and His242) of human serum albumin (HSA) carrier IIA subdomain for cancer treatment. They synthesized six Fe (III) compounds derived from 2-hydroxy-1-naphthaldehyde thiosemicarbazone and compared their potential alone as well as after binding with the HSA. Among these compounds, compound 12 and compound 12-HSA complex demonstrated better in vivo performance. As compared to compound 12, compound 12-HAS complex exhibited better targeting ability and activity in liver cancer [62]. All these results suggest that HSA carrier prodrug strategy for intravenous administration of novel anticancer compounds may be a promising approach for targeted cancer therapy.

In the development of albumin based prodrugs and albumin-drug conjugates, several factors may affect the activity of the compound. The major factors are: molar ratio of drug and albumin, structure and stereochemistry of the drug, nature of the drug and substitution moiety etc. For example, presence of hydrophobic moiety in the drug may enhance the affinity of drug to bind with the serum albumin which leads to stabilization of the drug-albumin conjugate [47], [59].

3.3.2. Albumin-polymer conjugates

Apart from tremendous advantageous properties, some draw backs are also associated with albumin that restrict its use in drug delivery such as lack of intrinsic targeting group, limited proteolytic stability and lesser suitability for hydrophilic and charged drugs [63], [64]. These drawbacks can be overcome by conjugating albumin with another polymer. Similar to albumin-drug conjugates and other protein polymer hybrids, albumin-polymer conjugates can be prepared either by chemical conjugation using covalent or non-covalent conjugation strategies or simply by the physical attachment of different polymers with albumin. Interactions between albumin and polymers are possible because of the presence of various active groups present on the surface of albumin like amino (Lys residues), carboxyl (Asp and Glu residues) and thiol (Cys residue). As compared to non-covalent conjugation strategy, covalent conjugates of albumin-polymer are more stable under physiological conditions. But on the basis of preparation method, non-covalent conjugation methods are relatively simple and flexible [47], [64]. There are two basic techniques for preparing the albumin-polymer conjugates: 1) conjugation of polymer to albumin by various coupling reactions like maleimide coupling, carbodiimide chemistry etc. [65] and 2) in-situ polymerization method (atom transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer polymerization (RAFT), ring opening polymerization (ROP) etc.) [65], [66], [67], [68]. Albumin-polymer conjugates have been prepared by conjugating several polymers like polyethylene glycol (PEG) and its derivatives [69], poly(methyl methacrylate) (PMMA) [66], [70], poly (lactide co glycolide) (PLGA) [71], poly(N-isopropylacrylamide) (PNIPAAm) [68], poly(ε-caprolactone) (PCL) [65], hydroxyethylacrylate (HEA) [67] etc. Albumin-polymer conjugates based nanoparticles can be prepared by different methods like self assembly, coating/conjugation of polymer over the albumin nanoparticles and vice-versa [65], [70], [71] and have been extensively explored in the treatment of cancer. Dag et al. developed polymer-albumin conjugate for the delivery of macromolecular platinum drugs and its effectiveness was tested against the ovarian cancer. In their study, two monomers N-(2-hydroxypropyl)methacrylamide (HPMA) and Boc protected 1,3-diaminopropan-2-yl acrylate (Ac-DAP-Boc) were copolymerized using RAFT polymerization to form macromolecular ligand and this was further conjugated with the platinum drug. This polymer‑platinum conjugate was further conjugated with albumin to form self assembled core (polymer‑platinum)-shell (albumin) nanoparticles having the size of 80 nm. Albumin coated polymer‑platinum conjugate was readily taken up by the ovarian cancer cells and demonstrated superior toxicity as compared to albumin coating free polymer‑platinum conjugate [72]. In another study, Liu et al. prepared DOX-encapsulated cetuximab-functionalized BSA–PCL nano vesicle as a tumor-targeted nanocarrier. BSA-PCL conjugates were synthesized by maleimide–sulfhydryl coupling reaction. DOX-encapsulated cetuximab-functionalized BSA–PCL nano vesicle showed enhanced antitumor activity as compared to free DOX [65]. Camptothecin (CPT) encapsulated BSA-PMMA nanoparticles were prepared by the simple nanoprecipitation method and showed enhanced anti-tumor activity both in vitro and in animals [70].

3.3.3. Albumin-metal conjugates (albumin coated inorganic nanoparticles)

Inorganic nanoparticles are widely used in the drug delivery system not only for delivering the active moiety but also for diagnostic and imaging application because of their unique optical, magnetic and other properties. For target specificity, effective bioavailability and colloidal stability of these inorganic nanoparticles, surface modification with suitable targeting moiety or biocompatible moiety is necessary [47], [73]. Albumin coated inorganic nanoparticles have been explored extensively because of the unique properties of albumin. It provides the stability, enhanced circulation and better accumulation of inorganic nanoparticles to the desired target site and can also provide target specificity due to the presence of reactive surface functional groups for the attachment of targeting ligands [74]. Chen et al. developed pH-/H2O2-Responsive albumin-MnO2 Nanoparticles for combinational cancer therapy to modulate tumor hypoxia. To prevent the decomposition of MnO2nanoparticles in acidic environment, premodified HSA either with Ce6 or with c,t,c-[Pt(NH3)2-(O2CCH2CH2COOH)(OH)Cl2] (cis‑Pt(IV)SA), as pro-drug of cis‑platinum, were used which acted as a template and coating molecules to induce the formation of MnO2 nanoclusters through biomineralization in alkaline conditions, obtaining multicomponent HSA-MnO2-Ce6&Pt (HMCP) nanoparticles. These HMCP nanoparticles acted as a multimodal therapeutic system in which pH-/H2O2-responsive behaviors of MnO2 simultaneously generated O2 in situ by reaction with endogenous H2O2 inside the tumor and overcame the tumor hypoxia-associated resistance of photodynamic therapy. On the other side, HMCP nanoparticles within the acidic tumor microenvironment were gradually degraded into individual therapeutic albumin-drug complexes with small sizes (< 10 nm) and exhibited greatly enhanced intratumoral permeability for improved effectiveness in combined photodynamic and chemotherapy [75]. Attachment of albumin with the inorganic nanoparticles can alter the biological behavior like cellular uptake of nanoparticles, blood circulation time, higher penetration and retention of nanoparticles in the tumor cells etc. and these were also verified by different studies [47], [74], [76], [77], [78], [79].

4.1.1. Small molecules

Small molecules like folate and biotin are widely used as targeting ligand for cancer cell targeting because of their easy availability, inexpensiveness, ease in handling and flexibility in chemical modification and characterization [80]. Folic acid, FR binding ligand, is an inexpensive and non-immunogenic small molecule having molecular weight of 44 Da. Folate conjugated drugs or biomarkers retain their ability to bind specifically with FR because of the high stability of folate over a wide range of temperatures and pH values [92]. It significantly binds with the glycosylphosphatidylinositol-linked FR which is over expressed (100–300 times higher than those observed in normal tissues) in different cancerous cells [80]. Folate conjugated proteins like albumin are selectively internalized into cytoplasm by receptor mediated endocytosis and promotes cellular uptake of therapeutic moiety like drug into cancerous cells [93]. Biotin is another vitamin used to target the cancer cells. It is basically cell growth promoter and present in significantly higher concentration in the cancer cells as compared to normal cells. Biotin receptors are also over expressed on the cancer cell surface because rapid proliferation occurs in cancer cells which need a higher quantity of biotin so it can also be utilized as a cancer targeting ligand [94].

4.1.2. Carbohydrates

Carbohydrate molecules (galactose, lactose and mannose) specifically bind to asialoglycoprotein receptors (membrane lectin receptors) commonly found in liver cells and are used for targeting hepatic and cervical cancer cells [95]. Hyaluronic acid which is a polysaccharide is also used as ligand for CD44 receptor mediated cancer targeting [96], [97].

4.1.3. Peptides

Integrin receptors (αvβ3) (membrane receptor for extracellular matrix ligands) are over expressed on the cancer cells surface and in tumor proliferating neovascular endothelial cells while lesser expressed in normal quiescent endothelial cells. These integrin receptors are very well targeted by the use of peptides. Among the various peptides, cyclic arginine-glycine-aspartic acid (RGD) is mostly used to target cancer cells because of high affinity to αvβ3integrin which can enhance cellular uptake and prolong retention of drug in the cancer cells [80], [83]. Some examples are mentioned in Table 3. Apart from RGD, other peptides like cell penetrating peptide (i.e. TAT), tumor homing peptide like CREKA and LyP-1 are also used in the targeted delivery of albumin based carrier for cancer [98].

4.1.4. Proteins

Proteins like transferrin, lactoferrin and Apolipoprotein E (Apo E) bind with the transferrin receptor, lactoferrin receptor and low density lipoprotein receptor respectively. These ligands are used for brain targeting because of over expression in blood brain barrier also. Apo E and transferrin conjugated HSA nanoparticles loaded with loperamide were also prepared for brain targeting. The results indicated higher concentration of drug after the conjugation of these ligands with HSA nanoparticles in the brain [99], [100], [101], [102]. Zhigui et al. prepared lactoferrin-modified polyethylene glycol grafted BSA nanoparticles as a dual targeting system for gliomas. Results showed higher permeability and cellular uptake in both primary brain capillary endothelial cells (BCECs) and glioma cells (C6) [103].

4.1.5. Monoclonal antibody (MAb)

MAbs (Y-shaped proteins) have been also utilized as a ligand for tumor targeting because of their unique properties like higher affinity (Kd ~ 0.1 nM) even with a low density and ability to recognize a specific part of their target [104]. Different MAbs used as ligands for tumor targeting are: anti-CD3 MAb, Herceptin, EGFRMAb, DI17E6 MAb, anti FAS MAb, anti-antigen rich breast cancer cells MAb, AMB8LK MAb, anti-CD20 MAb, anti-CEA MAb etc. [80]. MAb functionalized albumin nanocarriers are also summarized in Table 3.