Kislay Roy

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets (“apatopes”) with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer–aptamer, aptamer–nonaptamer biomacromolecules (siRNAs, proteins) and aptamer–nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.

Multifunctional Iron Bound Lactoferrin and Nanomedicinal Approaches to Enhance Its Bioactive Functions

Lactoferrin (Lf), an iron-binding protein from the transferrin family has been reported to have numerous functions. Even though Lf was first isolated from milk, it is also found in most exocrine secretions and in the secondary granules of neutrophils. Antimicrobial and anti-inflammatory activity reports on lactoferrin identified its significance in host defense against infection and extreme inflammation. Anticarcinogenic reports on lactoferrin make this protein even more valuable. This review is focused on the structural configuration of iron-containing and iron-free forms of lactoferrin obtained from different sources such as goat, camel and bovine. Apart for emphasizing on the specific beneficial properties of lactoferrin from each of these sources, the general antimicrobial, immunomodulatory and anticancer activities of lactoferrin are discussed here. Implementation of nanomedicinial strategies that enhance the bioactive function of lactoferrin are also discussed, along with information on lactoferrin in clinical trials.

Applications of aptamers in nanodelivery systems in cancer, eye and inflammatory diseases.

Aptamers are an interesting class of molecules that have potential in many facets of human health. They are characterized by high affinity and specificity to their targets, are small in size, have similar properties to antibodies, but are made synthetically. All of these properties, among others, give aptamers the potential to diagnose, image and treat like no other molecules. By combining the unique properties of aptamers with the ever expanding field of nanotechnology and all it has to offer, we are entering a very promising new area of targeted nanodelivery treatments. These treatments have found success in the complex disease processes of cancer, eye and inflammatory diseases.

LNA aptamer based multi-modal, Fe3O4-saturated lactoferrin (Fe3O4-bLf) nanocarriers for triple positive (EpCAM, CD133, CD44) colon tumor targeting and NIR, MRI and CT imaging

This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe3O4 nanoparticles (NPs) together, as a saturated Fe3O4-bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe3O4-bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe3O4-bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe3O4-bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin, survivin splice variant (survivin 2B) and other anti-apoptotic proteins Bad, Bcl-2 and XIAP. Apart from the promising anti-cancer efficacy and the exceptional tumor targeting ability observed by multimodal imaging using near-infrared (NIR) imaging, magnetic resonance imaging (MRI) and computerized tomographic (CT) techniques, these NCs also maintained the immunomodulatory benefits of bLf as they were able to increase the RBC, hemoglobin, iron calcium and zinc levels in mice.

Fe-bLf nanoformulation targets survivin to kill colon cancer stem cells and maintains absorption of iron, calcium and zinc

AIM To validate the anticancer efficacy of alginate-enclosed, chitosan-conjugated, calcium phosphate, iron-saturated bovine lactoferrin (Fe-bLf) nanocarriers/nanocapsules (NCs) with improved sustained release and ability to induce apoptosis by downregulating survivin, as well as cancer stem cells. MATERIALS & METHODS The stability, nanotoxicity of the modified nanoformulation was evaluated and their anticancer efficacy was re-examined. Their mechanism of internalization was studied and we identified the role of various miRNAs in absorption of these NCs/iron in various body parts of mice. We determined the effect of these NCs on survivin, stem cell markers, red blood cell count, iron, calcium and zinc concentration in mice, determined the antiangiogenic properties of these NCs and studied their effect on cancer stem-like cells. RESULTS Spherical NCs (396.1 ± 27.2 nm) exceedingly reduced viability of Caco-2 cells (32 ± 2.83%). The NCs also showed effective internalization and reduction of cancer stem cell markers in triple-positive CD133, survivin and CD44 cancer stem-like cells. Mice treated with the NCs showed no nanotoxicity and did not develop any tumors in xenograft colon cancer models. We found that the serum iron, zinc and calcium absorption were increased. DMT1, LRP, transferrin and lactoferrin receptors were responsible for internalization of the NCs. Different miRNAs were responsible for iron regulation in different organs. Interestingly, NCs inhibited survivin and its different isoforms. CONCLUSION Our results confirmed that NCs internalized and changed the expression of selected miRNAs that further enhanced their uptake. The NCs activated both extrinsic, as well as intrinsic apoptotic pathways to induce apoptosis by targeting survivin in cancer cells and cancer stem cells, without inducing any nonspecific nanotoxicity. Apart from inhibiting angiogenesis and stem cell markers, NCs also maintained iron and calcium levels.

Nucleic Acid-Based Aptamers: Applications, Development and Clinical Trials

Short single-stranded oligonucleotides called aptamers, often termed as chemical antibodies, have been developed as powerful alternatives to traditional antibodies with respect to their obvious advantages like high specificity and affinity, longer shelf-life, easier manufacturing protocol, freedom to introduce chemical modifications for further improvement, etc. Reiterative selection process of aptamers over 10-15 cycles starting from a large initial pool of random nucleotide sequences renders them with high binding affinity, thereby making them extremely specific for their targets. Aptamer-based detection systems are well investigated and likely to displace primitive detection systems. Aptamer chimeras (combination of aptamers with another aptamer or biomacromolecule or chemical moiety) have the potential activity of both the parent molecules, and thus hold the capability to perform diverse functions at the same time. Owing to their extremely high specificity and lack of immunogenicity or pathogenicity, a number of other aptamers have recently entered clinical trials and have garnered favorable attention from pharmaceutical companies. Promising results from the clinical trials provide new hope to change the conventional style of therapy. Aptamers have attained high therapeutic relevance in a short time as compared to synthetic drugs and/or other modes of therapy. This review follows the various trends in aptamer technology including production, selection, modifications and success in clinical fields. It focusses largely on the various applications of aptamers which mainly depend upon their selection procedures. The review also sheds light on various modifications and chimerizations that have been implemented in order to improve the stability and functioning of the aptamers, including introduction of locked nucleic acids (LNAs). The application of various aptamers in detection systems has been discussed elaborately in order to stress on their role as efficient diagnostic agents. The key aspect of this review is focused on success of aptamers on the basis of their performance in clinical trials for various diseases.

Targeting VEGF with LNA-stabilized G-rich oligonucleotide for efficient breast cancer inhibition

In this study, we investigated the efficacy of an LNA (locked nucleic acid)-modified DNA aptamer named RNV66 targeting VEGF against various breast cancer cell lines. Our results demonstrate that RNV66 efficiently inhibits breast cancer cell proliferation both in vitro and in vivo. Introduction of LNA nucleotides were crucial for higher efficacy. Furthermore, the binding interaction of RNV66 with VEGF was investigated using molecular dynamic simulations leading to the first computational model of the LNA aptamer-VEGF complex blocking its interaction with VEGF-receptor.

Oral administration of iron-saturated bovine lactoferrin–loaded ceramic nanocapsules for breast cancer therapy and influence on iron and calcium metabolism

We determined the anticancer efficacy and internalization mechanism of our polymeric–ceramic nanoparticle system (calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate nanocapsules/nanocarriers [ACSC NCs]) loaded with iron-saturated bovine lactoferrin (Fe-bLf) in a breast cancer xenograft model. ACSC-Fe-bLf NCs with an overall size of 322±27.2 nm were synthesized. In vitro internalization and anticancer efficacy were evaluated in the MDA-MB-231 cells using multicellular tumor spheroids, CyQUANT and MTT assays. These NCs were orally delivered in a breast cancer xenograft mice model, and their internalization, cytotoxicity, biodistribution, and anticancer efficacy were evaluated. Chitosan-coated calcium phosphate Fe-bLf NCs effectively (59%, P≤0.005) internalized in a 1-hour period using clathrin-mediated endocytosis (P≤0.05) and energy-mediated pathways (P≤0.05) for internalization; 3.3 mg/mL of ACSC-Fe-bLf NCs completely disintegrated (~130-fold reduction, P≤0.0005) the tumor spheroids in 72 hours and 96 hours. The IC50 values determined for ACSC-Fe-bLf NCs were 1.69 mg/mL at 10 hours and 1.62 mg/mL after 20 hours. We found that Fe-bLf-NCs effectively (P≤0.05) decreased the tumor size (4.8-fold) compared to the void NCs diet and prevented tumor recurrence when compared to intraperitoneal injection of Taxol and Doxorubicin. Receptor gene expression and micro-RNA analysis confirmed upregulation of low-density lipoprotein receptor and transferrin receptor (liver, intestine, and brain). Several micro-RNAs responsible for iron metabolism upregulated with NCs were identified. Taken together, orally delivered Fe-bLf NCs offer enhanced antitumor activity in breast cancer by internalizing via low-density lipoprotein receptor and transferrin receptor and regulating the micro-RNA expression. These NCs also restored the body iron and calcium levels and increased the hematologic counts.

Locked nucleic acid modified bi-specific aptamer-targeted nanoparticles carrying survivin antagonist towards effective colon cancer therapy

We investigated the anti-cancer activity of alginate coated chitosan nanoparticles (CHNP) encapsulating cell-permeable dominant negative survivin (SR9) with locked nucleic acid (LNA) aptamers targeting EpCAM and nucleolin (termed as “nanobullets”) in vitro (2D and 3D cell culture models) and in vivo (colon cancer mouse xenograft model). We incorporated three LNA modifications in each sequence in order to enhance the stability of these aptamers. Confocal microscopy revealed binding of the LNA-aptamers to their specific markers with high affinity. The muco-adhesive nanobullets showed 6-fold higher internalization in cancer cells when compared to non-cancerous cells, suggesting a tumour specific uptake. A higher intensity of nanobullets was observed in both the periphery and the core of the multicellular tumour spheroids compared to non-targeted CHNP-SR9. The nanobullets were found to be the highly effective as they led to a 2.26 fold (p < 0.05) reduction at 24 h and a 4.95 fold reduction (p ≤ 0.001) in the spheroid size at 72 h. The tumour regression was 4 fold higher in mice fed on a nanobullet diet when compared to a control diet. The nanobullets were able to show a significantly high apoptotic (p ≤ 0.0005) and necrotic index in the tumour cell population (p ≤ 0.005) when compared to void NPs. Therefore, our nanoparticles have shown highly promising results and therefore deliver a new conduit towards the approach of cancer-targeted nanodelivery.

Molecular targets in arthritis and recent trends in nanotherapy.

Due to its severity and increasing epidemiology, arthritis needs no description. There are various forms of arthritis most of which are disabling, very painful, and common. In spite of breakthroughs in the field of drug discovery, there is no cure for arthritis that can eliminate the disease permanently and ease the pain. The present review focuses on some of the most successful drugs in arthritis therapy and their side effects. Potential new targets in arthritis therapy such as interleukin-1β, interleukin-17A, tumor necrosis factor alpha, osteopontin, and several others have been discussed here, which can lead to refinement of current therapeutic modalities. Mechanisms for different forms of arthritis have been discussed along with the molecules that act as potential biomarkers for arthritis. Due to the difficulty in monitoring the disease progression to detect the advanced manifestations of the diseases, drug-induced cytotoxicity, and problems with drug delivery; nanoparticle therapy has gained the attention of the researchers. The unique properties of nanoparticles make them highly attractive for the design of novel therapeutics or diagnostic agents for arthritis. The review also focuses on the recent trends in nanoformulation development used for arthritis therapy. This review is, therefore, important because it describes the relevance and need for more arthritis research, it brings forth a critical discussion of successful drugs in arthritis and analyses the key molecular targets. The review also identifies several knowledge gaps in the published research so far along with the proposal of new ideas and future directions in arthritis therapy.