Specific Ligands Research Articles

Identification and validation of Sertoli cell homing peptides as molecular steering for testis targeted drug delivery

The testicle, an organ privileged with immunity because of Blood-Testis Barrier (BTB), poses a major impediment to developing and delivering drugs to the testes. These problems can be prevented by targeting testicular cells using specific ligands, such as homing peptides. This is the first study to demonstrate the successful selection of Sertoli cell homing peptides using a phage display peptide library. The identification of peptides is performed with Sanger sequencing and high-throughput NGS. The Sertoli cell and testis targeting potential of the SCHP1 and SCHP2 was confirmed using confocal microscopy and flow cytometry of the FITC-labelled peptides and in vivo bio-distribution of the corresponding Cy5.5-tagged peptides. Secondary structures were predicted in the setting of different polarity by circular dichroism. The results suggest that SCHP1 and SCHP2 can effectively target Sertoli cells. In vivo bio-distribution in mouse models indicated significantly higher uptake of SCHP1 and SCHP2 by testes compared with the heart, brain, and spleen. SCHP1 and SCHP2 can be adopted as molecular steering for targeted male contraceptive delivery, treatment of testicular cancer, and male infertility. Further development of the peptides into peptidomimetics may increase their stability, and information on the molecular targets of these peptides may reveal their therapeutic potential.

Construction of mouse cochlin mutants with different GAG-binding specificities and their use for immunohistochemistry.

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

The development of immunosorbents for the treatment of systemic lupus erythematosus via hemoperfusion.

Systemic lupus erythematosus (SLE) is an autoimmune disease (AID) that involves multiple organ systems and is characterized by elevated levels of autoantibodies (ANA) and immune complexes. The immunoadsorption technique uses an extracorporeal clearance process to remove pathogenic toxins from patients' blood and alleviate disease symptoms. An immunosorbent is a key component of the immunoadsorption system that determines therapeutic efficacy and safety. Immunosorbents are prepared by immobilizing antibodies, antigens, or ligands with specific physicochemical affinities on a supporting matrix. Immunosorbents and pathogenic toxins bind via affinity adsorption, which involves electrostatic interactions, hydrogen bonds, hydrophobic interactions, and van der Waals forces. Immunosorbents are classified on the basis of their interaction mechanism with toxins into three categories: non-selective, semi-selective, and highly selective. This review aimed to summarize the current status of various commercial immunosorbents that are used to treat SLE. Moreover, recent developments in immunosorbents have heightened the need for a brief discussion about specific ligands and a supporting matrix.

Current state of NK cell-mediated immunotherapy in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has become one of the most common hematological diseases in western countries, with an annual incidence of 42/100,000. Conventional chemotherapy and targeted therapeutic drugs showed limitations in prognosis or in efficiency in high-risk patients. Immunotherapy represented is one of the most effective therapeutic approaches with the potential of better effect and prognosis. Natural killer (NK) cells are good options for immunotherapy as they can effectively mediate anti-tumor activity of immune system by expressing activating and inhibiting receptors and recognizing specific ligands on various tumor cells. NK cells are critical in the immunotherapy of CLL by enhancing self-mediated antibody-dependent cytotoxicity (ADCC), allogeneic NK cell therapy and chimeric antigen receptor-natural killer (CAR-NK) cell therapy. In this article, we reviewed the features, working mechanisms, and receptors of NK cells, and the available evidence of the advantages and disadvantages of NK cell-based immunotherapies, and put forward future study directions in this field.

Improved detection and phylogenetic analysis of plant proteins containing LysM domains.

Plants perceive N-acetyl-d-glucosamine-containing oligosaccharides that play a role in the interaction with bacteria and fungi, through cell-surface receptors containing a tight bundle of three LysM domains in their extracellular region. However, the identification of LysM domains of receptor-like kinases (RLK)/receptor-like proteins (RLP) using sequence based methods has led to some ambiguity, as some proteins have been annotated with only one or two LysM domains. This missing annotation was likely produced by the failure of the LysM hidden Markov model (HMM) from the Pfam database to correctly identify some LysM domains in proteins of plant origin. In this work, we provide improved HMMs for LysM domain detection in plants, that were built from the structural alignment of manually curated LysM domain structures from the Protein Data Bank and AlphaFold Protein Structure Database. Furthermore, we evaluated different sets of ligand-specific HMMs that were able to correctly classify a limited set of fully characterised RLK/Ps by their ligand specificity. In contrast, the phylogenetic analysis of the extracellular region of RLK/Ps, or of their individual LysM domains, was unable to discriminate these proteins by their ligand specificity. The HMMs reported here will allow a more sensitive detection of plant proteins containing LysM domains and help improve their characterisation.

Measurement of O2 Binding by Sensory Hemeproteins.

The discovery of an increasing number of proteins that function in the detoxification and sensing of gaseous ligands has renewed interest in hemeproteins. It is critical to measure the affinities of these proteins for ligands like O2, CO, and NO, know with confidence when a protein is fully saturated with a specific ligand, and be able to estimate how well a ligand will compete against other ligands for a specific protein. Below we describe how to obtain an intact O2-binding hemeprotein with a full complement of heme, how to evaluate the factors that can impact its affinity for O2, and how to determine accurately the equilibrium and kinetic parameters Kd, kon, and koff for O2 binding.

Abstract P004: Targeting the RXR pathway for the prevention of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive cancer that lacks expression of the estrogen receptor (ER), progesterone receptor, and Erb-B2 receptor tyrosine kinase 2. TNBC patients exhibit a poor prognosis, even if treated with chemotherapy. Although studies using selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) have shown that breast cancer prevention is feasible, these drugs do not prevent ER-negative or TNBC tumors. It has been shown by our laboratory and others that retinoid X receptor (RXR)-specific ligands (rexinoids) can prevent breast cancers that are both ER-positive and ER-negative in mice. In our previous studies in MMTV-erbB2 mice, IRX-4204, a fourth generation rexinoid, prevented the development of most HER2/erbB2-positive tumors in these mice. For this study, we hypothesized that by targeting RXR pathway, we can prevent the development of triple negative, BRCA1-mutant mammary tumors in mice. To test the hypothesis, we treated MMTV-Cre/BRCA1co/co/p53+/- mice prior to their developing tumors with IRX-4204 to determine whether this RXR agonist effectively prevents triple-negative breast tumors. Methods: We tested the tumor preventative effect of IRX-4204 using the established MMTV-Cre/BRCA1co/co/p53+/- mouse model. These mice were produced by breeding MMTV-Cre/BRCA1co/co/p53+/- males and MMTV-Cre/BRCA1co/co/p53+/+ females, and female pups were PCR genotyped. All mice were separated into 4 groups: 1) sesame oil control, 2) IRX-4204 (10 mg/kg), 3) IRX-4204 (20 mg/kg) and 4) 9-cis-UAB-30 (5 mg/kg). All treatments were given by oral gavage, five days a week from 4 months of age. Mice were observed daily for tumor formation and toxicity. The percentage of tumor free mice were recorded, from which tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the Log-rank test. Results: In MMTV-Cre/BRCA1co/co/p53+/- mice, IRX-4204 reduced tumor incidence and was associated with an increase in median tumor free survival time from 209 days to 336 days at 10 mg/kg dose (p=0.005). At the higher dose (20 mg/kg) IRX-4204 also delayed tumor formation with median tumor free survival from 209 days to 260 days (p=0.039). The rexinoid 9-cis-UAB 30 also significantly delayed tumor formation in MMTV-Cre/BRCA1co/co/p53+/- mice with median survival from 209 days to 270 days (p=0.04). Long term treatment of IRX-4204 was not associated with any toxicity. Conclusion: RXR agonist IRX-4204 delayed ER-negative mammary tumor formation in BRCA1co/co; MMTV-Cre; p53+/- mice. Based on our results, IRX-4204 is an effective cancer preventive drug without observed toxicity. Our results suggest that studies with reduced IRX-4204 dose alone or in combination with other targeted therapies such as selective estrogen receptor modulators are warranted. In the future, clinical trials of the IRX-4204 should be considered for the prevention of breast cancer in high-risk patients. (Supported by NCI-PREVENT contract to P. Brown and A. Mazumdar HHSN26100008). Citation Format: Cassandra Moyer, Jamal Hill, Darian Coleman, Shizuko Sei, Altaf Mohammed, Martin Sanders, Powel Brown, Abhijit Mazumdar. Targeting the RXR pathway for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P004.

A Cross-Sectional Study to Evaluate the Alteration of Cytokine Expression and Activation of Inflammatory Pathway in Response to NOD1 and NOD2 Signal in Leprosy.

Leprae bacilli are identified as foreign by pattern recognition receptors (PRRs) present in the microbes but absent in the host. The Nucleotide oligomerization domain (NOD)-like receptor (NLR) family comprises the nucleotide-binding oligomerisation domain (NOD1 and NOD2) proteins, which are two well-known PRRs. The objectives of this study were to study the expression of cytoplasmic NOD1 and NOD2 in the pathogenesis of leprosy and the serum level of expressed cytokines and to measure the messenger Ribonucleic Acid (mRNA) expression. Clinically suspected Hansen's patients were analysed for 4 years. Newly diagnosed leprosy patients were considered leprosy disease control (LDC). The cases with active or new lesions and an increase in Bacteriological index (BI) by at least 2 + after 12 months of completion of Multidrug therapy (MDT) were considered leprosy disease relapse (LDR) cases. Age- and sex-matched healthy individuals served as our control group (healthy control (HC)). enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentration of five human cytokines in serum, including three pro-inflammatory cytokines (Tumor necrosis factor (TNF)-α, Interferon gamma (IFN-γ) and IL-6), one anti-inflammatory cytokine (IL-10) and one chemokine (IL-8). Quantitative expression of receptor genes (NOD1 and NOD2) and cytokine genes (TNF-α, IFN-γ, IL-6, IL-10 and IL-8) was evaluated by quantitative real-time polymerase chain reaction (PCR) (qRT-PCR). We studied NOD1 and NOD2 expression in the tissues through fluorescence immunohistochemistry. Differential NLR intracellular expression on peripheral blood monocytes (PBMs) and their response to stimulation with specific ligands (lipopolysaccharide (LPS) and muramyl dipeptide (MDP)) were studied. A significant difference in the expression of the NOD1 gene was observed in unstimulated monocytes of the LDC and LDR cases when compared to HC. The NOD2 transcript level was significantly higher in stimulated monocytes from LDC and LDR patients than in similarly stimulated cells from HC. The LDC patients had a significantly higher level of pro-inflammatory cytokines as compared to the HC. In conclusion, this study has demonstrated the expression of both cytokines and chemokines in response to NLR activation in the skin of leprosy patients.

Stable and Thermo-Responsive Dextran Sulfate-Graft-PNIPAm Amphiphilic Nanoparticles for Potential Target Methotrexate Delivery

Thermo-responsive copolymers grafted with N-isopropylacrylamide (NIPAm) are excellent candidates for drug release. Dextran sulfate (DS) acts as a specific ligand in inflamed regions, turning it highly useful as a target for drug delivery. DS was associated with NIPAm to produce amphiphilic graft copolymers prepared via free radicals. The molar ratio of feed reagents NIPAm/DS varied from 1 (DS-g-PNIPAm) to 4 (DS-g-4PNIPAm). The synthesis was confirmed by spectroscopic techniques (Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR)). All copolymers showed self-organization capacity in an aqueous medium in temperatures higher than 34 °C, and sizes less than 300 nm. DS-g-3PNIPAm exhibited stability in water and in phosphate buffer at pH 7.4. Scanning electron microscopy confirmed their spherical shape. This copolymer showed specificity to leukemic cells, and normal cells’ proliferation. Methotrexate (MTX) is a very low water-soluble drug used for rheumatoid arthritis and cancer. Unfortunately, MTX have severe collateral effects. MTX-loaded nanoparticles can overcome such issues as well as enhance bioactivity and stability. The MTX was encapsulated and delivered from the DS-g-3PNIPAm with potential target delivery due to the presence of DS. Comparison with MTX encapsulated in other nanoparticles reveals that the DS-g-PNIPam presents the best performance among the thermoresponsive and the second among the target MTX nanocarriers.

Single-Cell Quantification of the Mechanical Stability of Cell-Cell Adherens Junction Using Glass Micropipettes.

Micropipette-based methods have been widely used for the manipulation of cells and characterization of the mechanical properties at the cell or tissue level. Here, we introduce the glass micropipette-based mechanical assays for the stability of cell-cell adhesion. A probing microbead coated with specific adhesion ligands, captured by a glass micropipette, is manipulated to form the adhesion complexes with the corresponding receptors on a single cell. Once the cell is moving away from the micropipette, forces are generated from 20 pN to 100 nN to the adhesion complexes, which are quantified in real-time based on the bending of the glass micropipette. We specifically emphasize the principle and method to probe the rupturing forces of the adhesion complexes at controlled force loading rates, the ligand coating on the probe microbeads, the force calibration of the glass micropipette, and the applications of the method to probe the E-cadherin-based cell-cell adhesions. The principles can be broadly applied to other cell adhesions such as cell-matrix adhesions, neuronal synapses, and bacterial-cell adhesions.

Fast and selective reversed-phase high performance liquid chromatographic separation of UO2 2+ and Th4+ ions using surface modified C18 silica monolithic supports with target specific ionophoric ligands.

Reprocessing nuclear-spent fuels is highly demanded for enhanced resource efficacy and removal of the associated radiotoxicity. The present work elucidates the rapid separation of UO2 2+ and Th4+ ions using a reversed-phase high-performance liquid chromatographic (RP-HPLC) technique by dynamically modifying the surface of a C18 silica monolith column with target-specific ionophoric ligands. For the dynamic modification, four analogous aromatic amide ligands, N 1,N 1,N 3,N 3,N 5,N 5-hexa(alkyl)benzene-1,3,5-tricarboxamide (alkyl = butyl, hexyl, octyl, and decyl) as column modifiers were synthesized. The complexation properties and retention profiles of the amide-based column modifiers for the selective and sequential separation of UO2 2+ and Th4+ ions were investigated. In addition, the selective separation of UO2 2+ and Th4+ ions among the competitive ions of similar chemical properties were also studied. The ionophore immobilized C18 silica monolith columns demonstrated a varying degree of retention behavior for UO2 2+ and Th4+ ions (UO2 2+ is retained longer than Th4+ under all analytical conditions), eventually leading to rapid separations within a period of ≤5 min. A 0.1 M solution of 2-hydroxyisobutyric acid (HIBA, 1 mL min-1) served as the mobile phase, and the qualitative and quantitative assessment of the sequentially separated 5f metal ions was achieved through post-column derivatization reaction, using arsenazo(iii) as a post-column reagent (PCR; 1.5 mL min-1) prior to analysis using a UV-vis detector, at 665 nm (λ max). The developed technique was further evaluated by standardizing various analytical parameters, including modifier concentration, mobile phase pH, mobile phase flow rate, etc., to yield the best chromatographic separation. Also, the conceptual role of alkyl chain length (in the modifier) on the retention behavior of the studied metal ions was evaluated for cutting-edge future applications.

Investigation of PDGF-BB aptamer binding using growth factor-coated particles and flow cytometry

Platelet-derived growth factor-BB (PDGF-BB), a multifunctional growth factor secreted by platelets, endothelial and cancer cells, functions as an efficient and accurate biomarker. PDGF- BB has been employed in various therapeutic applications. Tuning its bioactivity via a specific binding ligand could enhance the PDGF-BB functionality. Aptamer, a single-stranded oligonucleotide that exhibits a selective binding to PDGF-BB, is the molecule of our interest. This research aims to evaluate the PDGF-BB aptamer binding by growth factor-coated particles. PDGF-BB-coated particles were confirmed by ELISA, and aptamer binding evaluation was investigated by flow cytometry. To optimize the condition for binding evaluation, the washing cycle and concentration of PDGF-BB, polystyrene particle and aptamer were studied using flow cytometer. Based on the flow cytometry results, the concentration of PDGF-BB showed an optimal level on the number of fluorescent particles. The percentage of fluorescent particles increased when the washing cycle was reduced and the particle concentration was decreased. The percentage of fluorescent particles also increased proportionally with the aptamer concentration. Using a flow cytometer to detect fluorescent signal from growth factor-coated particle is a promising strategy to study biomolecular binding.

Surface functionalization of extracellular vesicle nanoparticles with antibodies: a first study on the protein corona "variable".

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as extracellular vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, to date, studies dealing with the functionalization of the EV surface with proteins have never considered the protein corona "variable", namely the fact that extrinsic proteins may spontaneously adsorb on the EV surface, contributing to determine the surface, and in turn the biological identity of the EV. In this work, we explore and compare the two edge cases of EVs modified with the antibody Cetuximab (CTX) by chemisorption of CTX (through covalent binding via biorthogonal click-chemistry) and by formation of a physisorbed CTX corona. The results indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significant differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells, very likely due to the higher robustness of the CTX anchorage.

G4-DNA formation and chromatin remodelling are interdependent in human cells.

DNA G-quadruplexes (G4s) have been identified as important biological targets for transcriptional, translational, and epigenetic regulation. The stabilisation of G4s with small molecule ligands has emerged as a technique to regulate gene expression and as a potential therapeutic approach for human diseases. Here, we demonstrate that ligand stabilisation of G4s causes altered chromatin accessibility dependent on the targeting specificity of the molecule. In particular, stabilisation of a target G4 using the highly specific GTC365 ligand resulted in differential accessibility of 61 genomic regions, while the broad-targeting G4 ligand, GQC-05, stabilised many G4s and induced a global shift towards increased accessibility of gene promoter regions. Interestingly, while we observed distinct effects of each ligand on RNA expression levels and the induction of DNA double-stranded breaks, both ligands modified DNA damage response pathways. Our work represents the dual possibility of G4-stabilising ligands for specific or global chromatin modulation via unique targeting characteristics.

Binding symmetric porphyrins to the c-MYC promoter Pu24I G-quadruplex: toward more specific ligand recognition by flanking bases

The symmetric cationic porphyrin derivative TPyP4 exhibits higher selectivity toward flanking nucleotides of the c-MYC promoter G-quadruplex Pu24I than TMPyP4.

Cytotoxic and microbial studies of mixed metal complexes

Breast cancer and its treatment have become a prominent and challenging problem today. The increasing multidrug resistance to microbial pathogens is the root cause of breast cancer. Women suffering from cancer showed high levels of E. coli and S. aureus. In the last few decades, there has been a considerable need in the medical field for the discovery of new compounds endowed with antimicrobial activity, despite the fact that several antibiotics and chemotherapy drugs are currently accessible. Substantial research was conducted, particularly on transition complexes as metal-based drugs in pharmacological applications to provide therapeutic options. The synthesis, characterization, antibacterial activity, and cytotoxic activity of copper complexes with specific ligands of amino acids such as tyrosine and arginine are discussed in this work.

Sequential self-assembly of calix[4]resorcinarene-based heterobimetallic Cd8Pt8 nano-Saturn complexes.

A rational molecular design strategy is introduced for selective metal-ligand coordination, enabling the quantitative self-assembly of heterobimetallic nano-Saturn complexes. During the sequential multicomponent self-assembly, the CdII ions and organometallic trans-PtII motifs demonstrate preferential binding to specific ligands. The pre-designed directive interactions allow for precise control over the structural characteristics.

PH-tunable membrane-active polymers, NCMNP2a-x, and their potential membrane protein applications.

Accurate 3D structures of membrane proteins are essential for comprehending their mechanisms of action and designing specific ligands to modulate their activities. However, these structures are still uncommon due to the involvement of detergents in the sample preparation. Recently, membrane-active polymers have emerged as an alternative to detergents, but their incompatibility with low pH and divalent cations has hindered their efficacy. Herein, we describe the design, synthesis, characterization, and application of a new class of pH-tunable membrane-active polymers, NCMNP2a-x. The results demonstrated that NCMNP2a-x could be used for high-resolution single-particle cryo-EM structural analysis of AcrB in various pH conditions and can effectively solubilize BcTSPO with the function preserved. Molecular dynamic simulation is consistent with experimental data that shed great insights into the working mechanism of this class of polymers. These results demonstrated that NCMNP2a-x might have broad applications in membrane protein research.

Development of Multifunctional Nanomaterials and Devices for Biomedical Applications

The development of multifunctional nanomaterials and devices for biomedical applications has garnered significant attention in recent years due to their potential to revolutionize healthcare. In this study, we report the synthesis and characterization of novel nanomaterials with tailored properties for targeted drug delivery, imaging, and biosensing applications. We employed a bottom-up approach to design and fabricate nanocomposites comprising of biocompatible polymers, metallic nanoparticles, and quantum dots, which exhibit unique optical, magnetic, and electronic properties. The nanocomposites were functionalized with specific ligands to enable active targeting of cancer cells and pathogens. We also developed microfluidic devices for the efficient capture and analysis of circulating tumor cells (CTCs) using the synthesized nanomaterials. The performance of the nanomaterials and devices was evaluated in vitro and in vivo, demonstrating enhanced drug delivery efficiency, high-resolution imaging, and sensitive biosensing capabilities. Furthermore, we investigated the biocompatibility and long-term stability of the nanomaterials in physiological conditions. Our findings indicate that the developed multifunctional nanomaterials and devices hold great promise for advancing personalized medicine, early diagnosis, and targeted therapy. This study provides a comprehensive understanding of the design principles and potential applications of multifunctional nanomaterials in the biomedical field, paving the way for future research and clinical translation.

Phage for drug delivery vehicles.

Viruses being the natural carriers of gene have been widely used as drug delivery systems. However, the commonly used eukaryotic viruses such as adenoviruses, retroviruses, and lentiviruses, besides efficiently targeting the cells, can also stimulate immunological response or disrupt tumour suppressor genes leading to cancer. Consequently, there has been an increase interest in the scientific fraternity towards exploring other alternatives, which are safer and equally efficient for drug delivery. Bacteriophages, in this context have been at the forefront as an efficient, reliable, and safer choice. Novel phage dependent technologies led the foundation of peptide libraries and provides way to recognising abilities and targeting of specific ligands. Hybridisation of phage with inorganic complexes could be an appropriate strategy for the construction of carrying bioinorganic carriers. In this chapter, we have tried to cover major advances in the phage species that can be used as drug delivery vehicles.