Improving Cancer Targeting: A Study on the Effect of Dual-Ligand Density on Targeting of Cells Having Differential Expression of Target Biomarkers.

Silica nanoparticles with hyaluronic acid (HA) and folic acid (FA) were developed to study dual-ligand targeting of CD44 and folate receptors, respectively, in colon cancer. Characterization of particles with dynamic light scattering showed them to have hydrodynamic diameters of 147-271 nm with moderate polydispersity index (PDI) values. Surface modification of the particles was achieved by simultaneous reaction with HA and FA and results showed that ligand density on the surface increased with increasing concentrations in the reaction mixture. The nanoparticles showed minimal to no cytotoxicity with all formulations showing ≥ 90% cell viability at concentrations up to 100 µg/mL. Based on flow cytometry results, SW480 cell lines were positive for both receptors, the WI38 cell line was positive for CD44 receptor, and Caco2 was positive for the folate receptor. Cellular targeting studies demonstrated the potential of the targeted nanoparticles as promising candidates for delivery of therapeutic agents. The highest cellular targeting was achieved with particles synthesized using folate:surface amine (F:A) ratio of 9 for SW480 and Caco2 cells and at F:A = 0 for WI38 cells. The highest selectivity was achieved at F:A = 9 for both SW480:WI38 and SW480:Caco2 cells. Based on HA conjugation, the highest cellular targeting was achieved at H:A = 0.5-0.75 for SW480 cell, at H:A = 0.75 for WI38 cell and at H:A = 0.5 for Caco2 cells. The highest selectivity was achieved at H:A = 0 for both SW480:WI38 and SW480:Caco2 cells. These results demonstrated that the optimum ligand density on the nanoparticle for targeting is dependent on the levels of biomarker expression on the target cells. Ongoing studies will evaluate the therapeutic efficacy of these targeted nanoparticles using in vitro and in vivo cancer models.

Cell Membrane Coated Nanoparticles: An Emerging Biomimetic Nanoplatform for Targeted Bioimaging and Therapy.

Biomimetic nanoplatform being a recent and emerging strategy plays an important role in a wide variety of applications. The different types of membranes used for coating include membranes from red blood cells, platelets, leucocytes, neutrophils, cancer cells, stem cells, etc. The as obtained membrane vesicles are fused onto the core nanoparticles through extrusion, sonication, electroporation. Biomimetic nanoparticles attain special functions which include ligand recognition and targeting, long blood circulation, immune escaping, tumor targeting depending on the core-shell interactions. The membrane coated nanoparticles indeed mimic the source cells and improves the therapeutic efficacy of drugs other cargos through specific delivery and enhanced accumulation in the tumor.

Engineered liposomes mediated approach for targeted colorectal cancer drug Delivery: A review.

Colorectal cancer (CRC) continues to be one of the most prevalent and deadliest forms of cancer worldwide, despite notable advancements in its management. The prognosis for metastatic CRC remains discouraging, with a relative 5-year survival rate for stage IV CRC patients. Conventional treatments for advanced malignancies such as chemotherapy, often face limitations in effectively targeting cancer cells resulting in off-target distribution and significant side effects. In the quest for better strategies, researchers have explored numerous alternatives. Among these, nanoparticles (NPs) specifically liposomes have emerged as one of the most promising candidates in developing targeted delivery systems for cancer therapeutics. This review discusses the current approaches employing functionalised liposomes to overcome major biological barriers in therapeutics delivery for CRC treatment. We have also shared our perspectives on the technological development of liposomes for future clinical use and highlighted a few useful insights on the material choices for future research work in CRC.

Aptamers and nanobodies as alternatives to antibodies for ligand-targeted drug delivery in cancer.

Targeted drug delivery (TDD) is the selective delivery of a therapeutic agent specifically to the site of action to avoid adverse effects and systemic toxicity and to reduce the dose required. Ligand TDD or active TDD involves using a ligand-drug conjugate comprising a targeting ligand linked to an active drug moiety that can either be free or encapsulated within a nanocarrier (NC). Aptamers are single-stranded oligonucleotides that bind to specific biomacromolecules because of their 3D conformation. Nanobodies are the variable domains of unique heavy chain-only antibodies (HcAbs) produced by animals of the Camelidae family. Both these types of ligand are smaller than antibodies and have been used to efficiently target drugs to particular tissues or cells. In this review, we describe the applications of aptamers and nanobodies as ligands for TDD, their advantages and disadvantages compared with antibodies, and the various modalities for targeting cancers using these ligands. Teaser: Aptamers and nanobodies are macromolecular ligands that can actively chaperone drug molecules to particular cancerous cells or tissues in the body to target their pharmacological effects and improve their therapeutic index and safety.

Modeling of the In Vitro Release Kinetics of Sonosensitive Targeted Liposomes.

Targeted liposomes triggered by ultrasound are a promising drug delivery system as they potentially improve the clinical outcomes of chemotherapy while reducing associated side effects. In this work, a comprehensive model fitting was performed for a large dataset of liposomal release profiles with seven targeting moieties (albumin, cRGD, estrone, hyaluronic acid, Herceptin, lactobionic acid, and transferrin) in addition to the control liposomes under ultrasound release protocols. Two levels of ultrasound frequencies were tested: low frequency (20 kHz) at 6.2, 9, and 10 mW/cm2 as well as high frequencies (1.07 MHz and 3 MHz) at 10.5 and 173 W/cm2. At a low frequency, Hixson-Crowell, Korsmeyer-Peppas, Gompertz, Weibull, and Lu-Hagen showed good fits to our release profiles at all three power densities. At high frequencies, the former three models reflected the best fit. These models will aid in predicting drug release profiles for future in vitro studies.

Drug discovery and development scheme for liver-targeting bridged nucleic acid antisense oligonucleotides.

Antisense oligonucleotides (ASOs) containing bridged nucleic acids (BNAs) have been proven to be very powerful. However, ensuring a reliable discovery and translational development scheme for this class of ASOs with wider therapeutic windows remains a fundamental challenge. We here demonstrate the robustness of our scheme in the context of the selection of ASOs having two different BNA chemistries (2,'4'-BNA/locked nucleic acid [LNA] and amido-bridged nucleic acid [AmNA]) targeting human proprotein convertase subtilisin/kexin type 9 (PCSK9). The scheme features a two-step process, including (1) a unique and sensitive in vitro screening approach, called Ca2+ enrichment of medium (CEM) transfection, and (2) a ligand-targeted drug delivery approach to better reach target tissues, averting unintended accumulation of ASOs. Using CEM screening, we identified a candidate ASO that shows >70% cholesterol-lowering action in monkeys. An N-acetylgalactosamine (GalNAc) ligand then was appended to the candidate ASO to further broaden the therapeutic margin by altering the molecule's pharmacokinetics. The GalNAc conjugate, HsPCSK9-1811-LNA, was found to be at least ten times more potent in non-human primates (compared with the unconjugated counterpart), with reduced nephrotoxicity in rats. Overall, we successfully showed that our drug development scheme is better suited for selecting clinically relevant BNA-based ASOs, especially for the treatment of liver-associated diseases.

Octreotide-Targeted Lcn2 siRNA PEGylated Liposomes as a Treatment for Metastatic Breast Cancer.

Lcn2 overexpression in metastatic breast cancer (MBC) can lead to cancer progression by inducing the epithelial-to-mesenchymal transition and enhancing tumor angiogenesis. In this study, we engineered a PEGylated liposomal system encapsulating lipocalin 2 (Lcn2) small interfering RNA (Lcn2 siRNA) for selective targeting MBC cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231. The PEGylated liposomes were decorated with octreotide (OCT) peptide. OCT is an octapeptide analog of somatostatin growth hormone, having affinity for somatostatin receptors, overexpressed on breast cancer cells. Optimized OCT-targeted Lcn2 siRNA encapsulated PEGylated liposomes (OCT-Lcn2-Lipo) had a mean size of 152.00 nm, PDI, 0.13, zeta potential 4.10 mV and entrapment and loading efficiencies of 69.5% and 7.8%, respectively. In vitro uptake and intracellular distribution of OCT-Lcn2-Lipo in MCF-7 and MDA-MB-231 and MCF-12A cells demonstrated higher uptake for the OCT-targeted liposomes at 6 h by flow cytometry and confocal microscopy. OCT-Lcn2-lipo could achieve approximately 55-60% silencing of Lcn2 mRNA in MCF-7 and MDA-MB-231 cells. OCT-Lcn2-Lipo also demonstrated in vitro anti-angiogenic effects in MCF-7 and MDA-MB-231 cells by reducing VEGF-A and reducing the endothelial cells (HUVEC) migration levels. This approach may be useful in inhibiting angiogenesis in MBC.

Cellular and Molecular Targeted Drug Delivery in Central Nervous System Cancers: Advances in Targeting Strategies.

Central nervous system (CNS) cancers are among the most common and treatment-resistant diseases. The main reason for the low treatment efficiency of the disorders is the barriers against targeted delivery of anticancer agents to the site of interest, including the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB). BBB is a strong biological barrier separating circulating blood from brain extracellular fluid that selectively and actively prevents cytotoxic agents and majority of anticancer drugs from entering the brain. BBB and BBTB are the major impediments against targeted drug delivery into CNS tumors. Nanotechnology and its allied modalities offer interesting and effective delivery strategies to transport drugs across BBB to reach brain tissue. Integrating anticancer drugs into different nanocarriers improves the delivery performance of the resultant compounds across BBB. Surface engineering of nanovehicles using specific ligands, antibodies and proteins enhances the BBB crossing efficacy as well as selective and specific targeting to the target cancerous tissues in CNS tumors. Multifunctional nanoparticles (NPs) have brought revolutionary advances in targeted drug delivery to brain tumors. This study reviews the main anatomical, physiological and biological features of BBB and BBTB in drug delivery and the recent advances in targeting strategies in NPs-based drug delivery for CNS tumors. Moreover, we discuss advances in using specific ligands, antibodies, and surface proteins for designing and engineering of nanocarriers for targeted delivery of anticancer drugs to CNS tumors. Finally, the current clinical applications and the perspectives in the targeted delivery of therapeutic molecules and genes to CNS tumors are discussed.

Advances in Targeted Drug Delivery Approaches for the Central Nervous System Tumors: The Inspiration of Nanobiotechnology.

At present, brain tumor is among the most challenging diseases to treat and the therapy is limited by the lack of effective methods to deliver anticancer agents across the blood-brain barrier (BBB). BBB is a selective barrier that separates the circulating blood from the brain extracellular fluid. In its neuroprotective function, BBB prevents the entry of toxins, as well as most of anticancer agents and is the main impediment for brain targeted drug delivery approaches. Nanotechnology-based delivery systems provide an attractive strategy to cross the BBB and reach the central nervous system (CNS). The incorporation of anticancer agents in various nanovehicles facilitates their delivery across the BBB. Moreover, a more powerful tool in brain tumor therapy has relied surface modifications of nanovehicles with specific ligands that can promote their passage through the BBB and favor the accumulation of the drug in CNS tumors. This review describes the physiological and anatomical features of the brain tumor and the BBB, and summarizes the recent advanced approaches to deliver anticancer drugs into brain tumor using nanobiotechnology-based drug carrier systems. The role of specific ligands in the design of functionalized nanovehicles for targeted delivery to brain tumor is reviewed. The current trends and future approaches in the CNS delivery of therapeutic molecules to tumors are also discussed.

Dual-ligand α5ß1 and α6ß4 integrin targeting enhances gene delivery and selectivity to cancer cells.

Nanoparticles functionalized with cancer-targeting ligands have shown promise but are still limited by off-tumor binding to healthy tissues that express low levels of the molecular target. Targeting two cancer biomarkers using dual-targeted heteromultivalent nanoparticles presents a possible solution to this challenge by requiring overexpression of two separate ligands for localization. In order to guide experimental design, a kinetic model was built to explore how the affinity and valency of dual-ligand liposomes affect the binding and selectivity of delivery to cells with various receptor expression. α5ß1 and α6ß4 integrin expression levels were quantified on 20 different cell lines to identify appropriate model cells for in vitro investigation. Dual-targeting heteromultivalent liposomes covered with polyethylene glycol (PEG) were synthesized using the PR_b peptide that binds to the α5ß1 integrin and the AG86 peptide that binds to the α6ß4 integrin. PEGylated liposomes with varying ratios of the targeting peptides were delivered to cells with different integrin concentrations. Nanoparticle binding and internalization as well as integrin internalization as a function of time were evaluated to understand the effect of valency and avidity on delivery. Results showed that of all formulations and cells tested, dual-ligand liposomes with equal ligand valencies achieved enhanced binding and selectivity for cancer cells expressing equal and high levels of receptor expression. These trends were consistent between theoretical and experimental results. The optimized liposomes were further used to achieve efficient and selective transfection in dual-receptor expressing cancer cells. With a quantitative understanding of dual-ligand liposome binding, the insights gained from this study can inform rational design of modular heteromultivalent nanoparticles for enhanced specificity to target tissue for the creation of more effective cancer treatments.

GE11 peptide modified and reduction-responsive hyaluronic acid-based nanoparticles induced higher efficacy of doxorubicin for breast carcinoma therapy.

Novel breast carcinoma dual-targeted redox-responsive nanoparticles (NPs) based on cholesteryl-hyaluronic acid conjugates were designed for intracellular delivery of the antitumor drug doxorubicin (DOX). A series of reduction-responsive hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety (HA-ss-Chol) and GE11 peptide conjugated HA-ss-Chol (GE11-HA-ss-Chol) were synthesized. The obtained conjugates showed attractive self-assembly characteristics and high drug loading capacity. GE11-HA-ss-Chol NPs were highly stable under conditions mimicking normal physiological conditions, while showing a fast degradation of the vehicle's structure and accelerating the drug release dramatically in the presence of intracellular reductive environment. Furthermore, the cellular uptake assay confirmed GE11-HA-ss-Chol NPs were taken up by MDA-MB-231 cells through CD44- and epidermal growth factor receptor-mediated endocytosis. The internalization pathways of GE11-HA-ss-Chol NPs might involve clathrin-mediated endocytosis and macropinocytosis. The intracellular distribution of DOX in GE11-HA-ss-Chol NPs showed a faster release and more efficient nuclear delivery than the insensitive control. Enhanced in vitro cytotoxicity of GE11-HA-ss-Chol DOX-NPs further confirmed the superiority of their dual-targeting and redox-responsive capacity. Moreover, in vivo imaging investigation in MDA-MB-231 tumor-bearing mice confirmed that GE11-HA-ss-Chol NPs labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide, a near-infrared fluorescence dye, possessed a preferable tumor accumulation ability as compared to the single-targeting counterpart (HA-ss-Chol NPs). The antitumor efficacy showed an improved therapy efficacy and lower systemic side effect. These results suggest GE11-HA-ss-Chol NPs provide a good potential platform for antitumor drugs.

Hepatocellular carcinoma dually-targeted nanoparticles for reduction triggered intracellular delivery of doxorubicin.

Hepatocellular carcinoma (HCC) dual targeted stimuli responsive nanoparticles (NPs) for intracellular delivery of doxorubicin (DOX) were developed based on a reduction cleavable hyaluronic acid-glycyrrhetinic acid conjugate (HA-Cyst-GA). HA-Cyst-GA conjugate readily formed NPs in aqueous milieu and exhibited a high drug loading capacity (33.9%). The NPs redox responsiveness evaluation showed a tendency to lose their structural integrity in response to a reductive stimulus while remaining stable at physiological conditions, and that drug release was dramatically accelerated in presence of an intracellular level of glutathione. Moreover, cellular uptake studies highlighted the affinity of hepatoma cells (HepG2) toward the NPs as compared to breast cancer cells (MDA-MB-231). HA-Cyst-GA DOX-NPs displayed an increased cytotoxic potency over their non-responsive counterparts and free DOX with IC50 of 5.75, 9.33 and 10.23µg/mL, respectively. CLSM observations showed that HA-Cyst-GA DOX-NPs mediated a faster intracellular release and nuclear delivery of DOX as compared to the insensitive control. In vivo imaging study performed on H22 tumor bearing mice revealed a selective accumulation of DiR labeled NPs in the tumor and liver upon systemic administration. The antitumor efficacy was evaluated in HepG2 tumor xenograft model. Overall HA-Cyst-GA NPs appear as a potential HCC targeted intracellular delivery platform for DOX.