Introduction

Cancer is a burgeoning problem related to public health and a global threat to the human race. According to the Globocan2018 study, 18.1 million new cancer cases and 9.6 million cancer deaths were reported in 2018 [1]. Cancer-related deaths increased by about 17% in comparison to data available in 2012 [2]. Irrespective of sex, people are mostly diagnosed with lung cancer, which is also the major cause of cancer death among males. However, breast cancer is most prevalent in females and is also life threatening [2].

All types of cancer therapies which have evolved to date are depicted in Figure 1. The most common and conventional therapies against this deadly disease include surgery, radiation, and chemotherapy. These conventional therapeutics have several side effects, which causes a lot of physical as well as psychological stress among patients [5]. Chemotherapeutic drugs induce certain toxicity in our body, including hematotoxicity, cardiotoxicity, gastrointestinal toxicity, neurotoxicity, nephrotoxicity, and hair follicle toxicity, etc. [6]. These drugs target rapidly multiplying cells, which leads to inefficiency in differentiating between cancerous and normal cells [7]. This restricts the maximum allowable dose of drugs. On the other hand, these chemotherapeutic drugs get rapidly eliminated from the body through renal or other metabolic processes. Therefore, administration of a high dosage of drug is required to avoid rapid elimination and for widespread distribution of the drug to the targeted area, which is not economical and causes undesirable toxicity [8].

Figure 1. Evolution of cancer therapy techniques to date [3,4]. (Cliparts are adapted and modified from clipartlibrary.com).

Subsequent advances and development have led to various alternative therapies to overcome such problems. These include liposomal therapy, targeted therapy, immunotherapy, hormone therapy, and stem cell therapy. In liposomal therapy, drugs are encapsulated inside vesicles made up of a phospholipid bilayer, known as liposomes. This therapy emerged in the 1990s and minimizes some of the side effects of chemotherapy. The first drug-encapsulated liposome approved by the FDA was DaunoXome (daunorubicin liposomal) in 1996, after which came Doxil (doxorubicin liposomal) (2004) and DepoCyt (cytarabine liposomal), etc. Simultaneously, with the discovery of developing antibodies or other targeted molecules artificially, inhibitor and targeted therapy emerged. Rituximab (RituxanTM) (1997) was the first approved monoclonal chimeric antibody which was targeted against CD20 of non-Hodgkin’s lymphomas and Sorafenib (2005) was the first FDA-approved kinase inhibitor drug which was developed for the treatment of renal cell cancer (RCC) [3]. However, one of the main disadvantages of targeted therapy is that antibodies are proteins which undergo enzymatic digestion inside the body and are converted into lower molecular weight fragments. These fragments are usually cleared through the renal system. Therefore, conjugation of proteins with nanocarriers such as polymer enhances their solubility, stability, and immunological profile [9].

Furthermore, with the advancement in nanostructure-based therapeutics and diagnostic agents, certain conjugated nanoformulations and nanoparticles have been developed for treating cancer. Some examples are Genexol-PM (paclitaxel loaded in a polyethylene glycol-polylactic acid copolymer (PEG-PLA)), Zinostatin Stimalmer (polymer protein conjugate), Abraxane (paclitaxel conjugated with albumin), NanoTherm nanoparticles (aminosilane-coated iron oxide), Gliadel (biodegradable polymeric wafer loaded with carmustine), and Feridex/Endorem (superparamagnetic iron oxide nanoparticles (SPIONs) coated with dextrane), etc. [3]. Drug dosages used in developing these nanomedicines are lower than individual chemotherapy. This reduces the side effects related to large doses of drugs and enhances their therapeutic effect and safety profiles. These nano-delivery systems contain drugs, therapeutic agents, and imaging agents which are either conjugated, encapsulated, dispersed, or adsorbed [10]. These nanoformulations have a high surface area to volume ratio, good stability, and enhanced permeability and retention (EPR) [11]. This technique is a traditional method for delivering drugs via nanocarriers in which the drug associated with the carrier is aggregated into the cancer tissues. The structural abnormality in blood vessels near cancer cells leads to increased tissue permeability, delivery, and retention of drug molecules inside the cancer cells. This passive delivery of drugs is not very efficient and eventually, the drug is released back towards the high concentration area into the blood [10]. Hence, the bioconjugation technique can be used to deliver drugs selectively to the targeted cancer site. Bioconjugation consists of linking two molecules, usually via a covalent bond. Here, at least one molecule should be of biological origin or a biomolecule [12]. It is a tool that bridges chemistry and biology [13]. In the case of cancer therapeutic agents, these biologically originated molecules used for conjugation are primarily the ligands that target tumor-specific antigens [14,15]. Alternatively, they can be peptides [16,17], glycoproteins [18], aptamers [19,20], or interferons [21], etc.; these all have anticancerous properties. The unique advantage of bioconjugates is their ability to selectively deliver therapeutics to pathological sites and to increase the retention of the molecule in the blood circulation system. Their delivery mechanism is based on active delivery of drugs [22].

Nanomaterials as Carriers of Biomolecules in Conjugates

Researchers have used various inorganic and organic nanocarriers for bioconjugation [38]. Such nanocarriers include nanoparticles of different metals, carbon nanotubes, quantum dots, dendrimers, hydrogels, and other nanocarriers of biological origin. Some of these nanocarriers have been depicted in Figure 4. The multifunctional properties of these nanocarriers could be employed for targeting, tracking, and therapy [11]. A good number of these products are under preclinical and clinical trials [38].

Figure 4. Different types of nanocarriers used for the preparation of bioconjugates.

Combination Therapy Via Bioconjugates

Nucleic Acid or Aptamer-Based Therapeutic Agents

Nucleic acid bioconjugates have been extensively used for detecting and treating cancers. Aptamers, single-stranded nucleic acid molecules, can be conjugated with drugs, nanocarriers, or chemotherapeutic agents. They can be used as both targeting agents and therapeutic agents. Due to the uniqueness of their structure, they can be specifically bound to cells with a high affinity via electrostatic forces, hydrogen bonding, or van der Waal’s interactions [100,101,102]. Aptamers are the ultimate in targeted therapy with respect to specificity. They are strong and versatile molecules which have amazing biomedical applications. However, they have some downlines which include degradation by nuclease in vivo and ineffective immobilization on carrier surfaces, leading to untargeted delivery. Furthermore, proper methodologies are unavailable to convert highly specific aptamer-targeted molecular recognition into detectable signals. Chemical modification and bioconjugation of aptamers with nanostructures is one of the solutions to these drawbacks [100,102]. The first aptamer to go through clinical trials for cancer treatment was AS1411. This aptamer targets nucleolin, a protein expressed in the nuclei of all cells but in the case of cancer cells overexpressed in the cytoplasm and on the plasma membrane compared to normal cells [101]. Researchers have conjugated the AS1411 aptamer to various molecules such as 67Ga-citrate in cobalt-ferrite nanoparticles within a silica shell matrix for radionuclide imaging. This versatile bioconjugated cancer-targeted imaging system has been observed to be used for specific cancer diagnosis and to study cellular metabolism [103]. AS1411 was also conjugated with the chemotherapeutic agent doxorubicin to form a synthetic drug-DNA adduct (DDA) to target hepatocellular carcinoma cells. Authors evaluated the efficiency of this bioconjugate in vitro and in vivo. They concluded that the bioconjugated drug showed less anticancer efficacy than the free drug; however, side effects were observed less in the case of the bioconjugates [30]. Guo et al. conjugated AS1411 with PEG-PLGA nanoparticles encapsulated with paclitaxel to enhance the anti-glioma efficacy of the drug [33]. Malignant brain tumor is difficult to treat because of the nonspecificity of drugs, and hence, targeted delivery of drugs through aptamers increases their specificity [33]. Recently, Tao et al. conjugated the AS1411 aptamer with docetaxel-loaded copolymeric nanoparticles to target breast cancer cell lines in vivo [20]. Prostate-specific membrane antigen, a surface protein expressed in healthy prostates, prostate cancer, and the vasculature of various solid tumors, is also one of the best tumor markers for imaging and therapy [101]. Lupold et al. demonstrated that the aptamers A9 and A10 specifically bind to prostate cancer cells via PSMA [86]. Wang et al. developed superparamagnetic iron oxide nanoparticles (SPIONs)–A10 aptamer bioconjugates as a theranostic tool to deliver doxorubicin to prostate cancer cells [24]. Furthermore, Jalalian et al. extended this method to other cancer cell lines and conjugated the 5TR1 aptamer (Apt), which targets mucin-1 (MUC-1) glycoform, with epirubicin-loaded SPIONs to target murine colon cancer cells (C26 cells) [31]. Another aptamer molecule, DM1 (a maytansine-derived high-potential cytotoxic agent) was conjugated with mesoporous silica nanoparticles (MSNs) that bind to epithelial cell adhesion molecules (EpCAMs). This conjugate was found to target tumors of epithelial origin such as colorectal adenocarcinoma [104]. Recently, an electrochemical sandwich biosensor was developed by conjugating two aptamers which targeted the MUC1 biomarker of MCF7 cell lines. The first aptamer (MUC1) acted as a capture aptamer that specifically bound to MCF-7 cells which had been introduced as a sample to be detected. The second aptamer, labelled with silver nanoparticles, acted as a detection aptamer which bound to captured cancer cells, forming a sandwich. This biosensor was found to be useful to identify breast cancer in the initial stages [105].

Researchers have also delivered microRNAs (miRNAs) as a therapeutic agent. They are small-sized (~22 nucleotides) noncoding RNAs that can control post-transcriptional gene expression via RNA silencing. However, RNAs are prone to degradation by nucleases present in serum and can also activate immunogenic and inflammatory responses; hence, they need to be delivered in conjugation with other molecules. Perepelyuk et al. synthesized an aptamer-hybrid nanoparticle bioconjugate delivery system which consisted of miRNA-29b as a therapeutic agent (tumor-suppressant miRNA) and MUC1 aptamer as a targeting agent. They made this nanoformulation by encapsulating miRNA-29b in human IgG and coating the nanoparticle with poloxamer-188. Copolymer poloxamer-188 provided stealth behavior to the nanoparticles [19]. The MUC1 aptamer was found to bind to the transmembrane protein, mucin, which is expressed on the surface of cancerous cells. This nucleic acid bioconjugate was used to downregulate oncoproteins DNA methyltransferase 3B (DNMT3b) and myeloid cell leukemia sequence 1 (MCL1) in A549 cells and thereby inhibit cancer cell proliferation. This bioconjugate induces cell apoptosis and prevents methylation of cancer suppressor genes [19].

siRNA therapy is also a gene silencing therapy useful for cancer treatment. Scientists have linked antibodies and ligands chemically with siRNA nanoparticles; this approach is known as the siRNA-mediated silencing (RNAi) of genes. siRNAs also target oncogenes that lead to tumor proliferation, metastasis, angiogenesis, and multidrug resistance, and inhibit apoptosis [106]. Tietze et al. utilized dendrimer as a siRNA carrier linked with EGFRvIII antibody. This antibody is the ligand most frequently used to target EGFR. This bioconjugated system was found to be highly specific, with great stability [106]. Misra et al. used the ‘nuclein’ type nanoparticle “siNozyme” from the nano-assembly of pamitoyl-bioconjugated acetyl co-enzyme-A. This system stably incorporated chemotherapeutics and biologics to melanoma cancer sites for inhibiting their growth. They targeted transcriptional gene cMyc with siRNA and used siNozyme as a carrier [107]. Recently, Shah et al. conjugated siRNA with a series of saturated and unsaturated fatty acids (palmitic acid). This bioconjugation improved the cellular uptake of siRNA, which targets oncogenic glucose regulated proteins (GRPs) and downregulates them, thus improving cancer gene therapy [32].

mRNA-based therapeutic approaches have also emerged in the past few years. Earlier, mRNAs were not popular as therapeutic agents because of their instability, immunogenicity, poor delivery mechanism, and high production cost. However, within the past several years, researchers have gained knowledge about mRNA delivery systems and have reduced their production cost. Lipids, polymers, proteins, and gold nanoparticles, etc. are some of the examples of delivery agents which have been evolved over the years [108]. Oberli et al. developed lipid nanoparticles to deliver an mRNA vaccine for cancer immunotherapy. In this study, mRNA was delivered to the cytosol of antigen-presenting immune cells to induce a cytotoxic CD 8 T-cell response. This delivery system successfully protected the mRNA from endonucleases and delivered it to the targeted cells of a B16F10 tumor animal model without damaging normal cells [109]. In another study, a nanomicelle of a PEG block copolymer attached to cholesterol at one terminal was used as a delivery carrier for mRNA to impede pancreatic tumor tissue growth [110]. Gold nanoparticles were also used to deliver mRNA to cancer cells. Yeom et al. bioconjugated BAX mRNA on gold nanoparticle-DNA oligonucleotide conjugates to deliver mRNA into a xenograft tumor model. BAX mRNA was found to synthesize BAX protein, which inhibits tumor growth by apoptosis. This gold-nanoparticle-based delivery system was found to be stable, safe, and effective in vivo [111].