EpCAM-targeted betulinic acid analogue nanotherapy improves therapeutic efficacy and induces anti-tumorigenic immune response in colorectal cancer tumor microenvironment.

BACKGROUND: Betulinic acid (BA) has been well investigated for its antiproliferative and mitochondrial pathway-mediated apoptosis-inducing effects on various cancers. However, its poor solubility and off-target activity have limited its utility in clinical trials. Additionally, the immune modulatory role of betulinic acid analogue in the tumor microenvironment (TME) is largely unknown. Here, we designed a potential nanotherapy for colorectal cancer (CRC) with a lead betulinic acid analogue, named as 2c, carrying a 1,2,3-triazole-moiety attached to BA through a linker, found more effective than BA for inhibiting CRC cell lines, and was chosen here for this investigation. Epithelial cell adhesion molecule (EpCAM) is highly overexpressed on the CRC cell membrane. A single-stranded short oligonucleotide sequence, aptamer (Apt), that folds into a 3D-defined architecture can be used as a targeting ligand for its specific binding to a target protein. EpCAM targeting aptamer was designed for site-specific homing of aptamer-conjugated-2c-loaded nanoparticles (Apt-2cNP) at the CRC tumor site to enhance therapeutic potential and reduce off-target toxicity in normal cells. We investigated the in vitro and in vivo therapeutic efficacy and anti-tumorigenic immune response of aptamer conjugated nanotherapy in CRC-TME. METHODS: After the characterization of nanoengineered aptamer conjugated betulinic acid nanotherapy, we evaluated therapeutic efficacy, tumor targeting efficiency, and anti-tumorigenic immune response using cell-based assays and mouse and rat models. RESULTS: We found that Apt-2cNP improved drug bioavailability, enhanced its biological half-life, improved antiproliferative activity, and minimized off-target cytotoxicity. Importantly, in an in vivo TME, Apt-2cNP showed promising signs of anti-tumorigenic immune response (increased mDC/pDC ratio, enhanced M1 macrophage population, and CD8 T-cells). Furthermore, in vivo upregulation of pro-apoptotic while downregulation of anti-apoptotic genes and significant healing efficacy on cancer tissue histopathology suggest that Apt-2cNP had predominantly greater therapeutic potential than the non-aptamer-conjugated nanoparticles and free drug. Moreover, we observed greater tumor accumulation of the radiolabeled Apt-2cNP by live imaging in the CRC rat model. CONCLUSIONS: Enhanced therapeutic efficacy and robust anti-tumorigenic immune response of Apt-2cNP in the CRC-TME are promising indicators of its potential as a prospective therapeutic agent for managing CRC. However, further studies are warranted.

H-ras-targeted genetic therapy remarkably surpassed docetaxel treatment in inhibiting chemically induced hepatic tumors in rats.

AIMS: Hepatocellular carcinoma (HCC) is still a leading cause of cancer-related death worldwide. But its chemotherapeutic options are far from expectation. We here compared H-ras targeted genetic therapy to a commercial docetaxel formulation (DXT) in inhibiting HCC in rats. MAIN METHODS: After the physicochemical characterization of phosphorothioate-antisense oligomer (PS-ASO) against H-ras mutated gene, the PS-ASO-mediated in vitro hemolysis, in vivo hepatic uptake, its pharmacokinetic profile, tissue distribution in some highly perfused organs, its effect in normal rats, antineoplastic efficacy in carcinogen-induced HCC in rats were evaluated and compared against DXT treatment. Mutated H-ras expression by in situ hybridization, hep-par-I, CK-7, CD-15, p53 expression patterns by immunohistochemical methods, scanning electron microscopic evaluation of hepatic architecture, various hepatic marker enzyme levels and caspase-3/9 apoptotic enzyme activities were also carried out in the experimental rats. KEY FINDINGS: PS-ASO showed low in vitro hemolysis (<3 %), and had a sustained PS-ASO blood residence time in vivo compared to DTX, with a time-dependent hepatic uptake. It showed no toxic manifestations in normal rats. PS-ASO distribution was although initially less in the lung than liver and kidney, but at 8 h it accumulated more in lung than kidney. Antineoplastic potential of PS-ASO (treated for 6 weeks) excelled in inhibiting chemically induced tumorigenesis compared to DTX in rats, by inhibiting H-ras gene expression, some immonohistochemical modulations, and inducing caspase-3/9-mediated apoptosis. It prevented HCC-mediated lung metastatic tumor in the experimental rats. SIGNIFICANCE: PS-ASO genetic therapy showed potential to inhibit HCC far more effectively than DXT in rats.

Cetuximab-conjugated PLGA nanoparticles as a prospective targeting therapeutics for non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most prevalent cancers diagnosed worldwide, yet managing it is still challenging. The epidermal growth factor receptor (EGFR) exhibits aberrant signalling in a wide range of human cancers, and it is reported to overexpress in most NSCLC cases. The monoclonal antibody [Cetuximab (Cet)] was conjugated onto the surface of the poly (lactide-co-glycolide) (PLGA) nanoparticles which were loaded with docetaxel (DTX) for the development of targeted therapy against lung cancer. This site-specific delivery system exhibited an enhanced cellular uptake in lung cancer cells which overexpress EGFR (A549 and NCI-H23). The nanoparticles also showed better therapeutic effectiveness against NSCLC cells, as evidenced by reduced IC50 values, cell cycle arrest at the G2/M phase, and increased apoptosis. The improved efficacy and in vivo tolerance of Cet-DTX NPs were demonstrated in benzo(a)pyrene (BaP)-induced lung cancer mice model. Histopathological analysis showed that intravenous injection of Cet-DTX NP to mice carrying lung cancer greatly reduced tumour development and proliferation. Comparing Cet-DTX NP to free drug and unconjugated nanoparticles, it also had negligible side effects and improved survival rates. Therefore, Cet-DTX NPs present a promising active targeting carrier for lung tumour-NSCLC-selective treatment.

Assessment of superiority of HSP70-targeting aptamer-functionalized drug-nanocarrier over non-targeted commercially available counterpart in HCC therapy: in vitro and in vivo investigations and molecular modeling.

AIMS: This research aims to compare the therapeutic potential of target-specific phosphorothioate backbone-modified aptamer L5 (TLS9a)-functionalized paclitaxel (PTX)-loaded nanocarrier (PTX-NPL5) that we formulated with that of non-targeted commercial formulation, protein albumin-bound nanoparticles of PTX, Abraxane® (CF) against hepatocellular carcinoma (HCC) through a myriad of preclinical investigations. MAIN METHODS: A variety of in vitro and in vivo assays have been executed to compare the therapeutic effects of the formulations under investigation, including the investigation of the degree of apoptosis induction and its mechanism, cell cycle analysis, the level of ROS production, and redox status, the morphological and histological characteristics of malignant livers, and in vivo imaging. The formulations were also compared concerning pharmacokinetic behaviors. Finally, in silico molecular docking has been performed to predict the possible interactions between aptamer and target(s). KEY FINDINGS: PTX-NPL5 exhibited therapeutic superiority over CF in terms of inducing apoptosis, cell cycle arrest, endorsing oxidative stress to neoplastic cells, and reducing hepatic cancerous lesions. Unlike CF, PTX-NPL5 did not exhibit any significant toxicity in healthy hepatocytes, proving enough impetus regarding the distinctive superiority of PTX-NPL5 over CF. The pharmacokinetic analysis further supported superior penetration and retention of PTX-NPL5 in neoplastic hepatocytes compared to CF. A molecular modeling study proposed possible interaction between aptamer L5 and heat shock protein 70 (HSP70). SIGNIFICANCE: The target-specificity of PTX-NPL5 towards neoplastic hepatocytes, probably achieved through HSP70 recognition, enhanced its therapeutic efficacy over CF, which may facilitate its real clinical deployment against HCC in the near future.

Phosphorothioated amino-AS1411 aptamer functionalized stealth nanoliposome accelerates bio-therapeutic threshold of apigenin in neoplastic rat liver: a mechanistic approach.

Hepatocellular carcinoma (HCC) is a leading cause of death globally. Even though the progressive invention of some very potent therapeutics has been seen, the success is limited due to the chemotherapeutic resistance and recurrence in HCC. Advanced targeted treatment options like immunotherapy, molecular therapy or surface-engineered nanotherapeutics could offer the benefits here owing to drug resistance over tumor heterogenicity. We have developed tumor-sensing phosphorothioate and amino-modified aptamer (AS1411)-conjugated stealth nanoliposomes, encapsulating with apigenin for precise and significant biodistribution of apigenin into the target tumor to exploit maximum bio-therapeutic assistances. The stable aptamer functionalized PEGylated nanoliposomes (Apt-NLCs) had an average vesicle size of 100-150 nm, a smooth surface, and an intact lamellarity, as ensured by DLS, FESEM, AFM, and Cryo-TEM. This study has specified in vitro process of optimum drug (apigenin) extrusion into the cancer cells by nucleolin receptor-mediated cellular internalization when delivered through modified AS1411 functionalized PEGylated nanoliposomes and ensured irreversible DNA damage in HCC. Significant improvement in cancer cell apoptosis in animal models, due to reduced clearance and higher intratumor drug accumulation along with almost nominal toxic effect in liver, strongly supports the therapeutic potential of aptamer-conjugated PEGylated nanoliposomes compared to the nonconjugated formulations in HCC. The study has established a robust superiority of modified AS1411 functionalized PEGylated nanoliposomes as an alternative drug delivery approach with momentous reduction of HCC tumor incidences.

Quantum dots: The cutting-edge nanotheranostics in brain cancer management.

Quantum dots (QDs) are semiconductor nanocrystals possessing unique optoelectrical properties in that they can emit light energy of specific tunable wavelengths when excited by photons. They are gaining attention nowadays owing to their all-around ability to allow high-quality bio-imaging along with targeted drug delivery. The most lethal central nervous system (CNS) disorders are brain cancers or malignant brain tumors. CNS is guarded by the blood-brain barrier which poses a selective blockade toward drug delivery into the brain. QDs have displayed strong potential to deliver therapeutic agents into the brain successfully. Their bio-imaging capability due to photoluminescence and specific targeting ability through the attachment of ligand biomolecules make them preferable clinical tools for coming times. Biocompatible QDs are emerging as nanotheranostic tools to identify/diagnose and selectively kill cancer cells. The current review focuses on QDs and associated nanoformulations as potential futuristic clinical aids in the continuous battle against brain cancer.

Inhibitory potential of iRGD peptide-conjugated garcinol-loaded biodegradable nanoparticles in rat colorectal carcinoma.

Targeted drug delivery has become attention in chemotherapy during the last decade. The principle of chemotherapy seeks maximum effect to the desired site and the minimum impact to other undesired sites of action. The nanoparticulated drug delivery system progressed a lot in this aspect in the last twenty years. Plant-derived natural products and their semisynthetic analogues boosted chemotherapy through their excellent mechanistic approach to killing cancer cells. Keeping in mind the available molecular targets in colorectal carcinoma (CRC), in this article, we proposed a peptide conjugated novel polymeric nanoparticle to deliver garcinol against colorectal carcinoma. Integrin binding peptide iRGD, sequence c(CRGDKGPDC), has been selected as a targeting moiety, as most CRC overexpress integrins. We encapsulated garcinol in biodegradable polymeric nanoparticle (PLGA)-conjugated with iRGD peptide on the particles' surface, and analyzed its (iRGD-GAR-NP's) in vitro and in vivo antineoplastic potential against CRC in a comparative way with gracinol (GAR) and garcinol-loaded PLGA nanoparticles (GAR-NP). In vitro cellular studies on human CRC cell lines, HCT116 and HT-29, revealed the superior cytotoxic potential of iRGD-GAR-NP over GAR and GAR-NP. The IC50 value on HCT116 cells was reduced by 2.3 times compared to GAR upon the application of iRGD-GAR-NP. At equivalent doses, iRGD-GAR-NP induced higher apoptosis in HCT116 cells and caused blockage of cell cycle at G0/G1 phase of the same. iRGD-GAR-NP increased the apoptotic population of HCT116 cells by 2.5 times compared to GAR. In vivo biodistribution study uncoiled the ability of GAR-NP and iRGD-GAR-NP to accumulate in the colons of dimethyl hydrazine-induced CRC-bearing Sprague-Dawely (SD) rats. In vivo antitumor efficacy study demonstrated the better effect of iRGD-GAR-NP to reduce CRC tumor progression in experimental animals. The survival rate of animals was also increased by 166% in the case of iRGD-GAR-NP compared to CRC-bearing animals received no treatment.

Calorie restriction modulates neuro-immune system differently in young and aged rats.

Aging weakens and deregulates the immune system and plays an impact on the central nervous system (CNS). A crosstalk in between the CNS-mediated immune system and the body's overall innate immunity is often found to increase and subsequently accelerate neurodegeneration and behavioural impairment during aging. Dietary calorie restriction (CR) is found to be a beneficial non-invasive anti-aging therapy as it shows rejuvenation of stress response, brain functions and behaviour during aging. The present investigation deals with the consequence of CR diet supplementation for two different duration (one and two consecutive months) on aging-related alteration of the immune response in male albino Wistar rats at the level of (a) lymphocyte viability, proliferation, cytotoxicity, and DNA fragmentation in blood, spleen, and thymus and (b) cytokines (IL-6, IL-10, and TNF-α) in blood, spleen, thymus and different brain-regions to understand the effect of CR diet on neuroimmune system. The results depict that CR diet consumption for consecutive one and two months by the aged (18 and 24 months) rats significantly attenuated the aging-related (a) decrease of blood, splenic and thymic lymphocyte viability, proliferative activity, cytotoxicity, and IL-10 level and (b) increase of (i) blood, splenic and thymic DNA fragmentation and (ii) IL-6 and TNF-α level in those tissues and also in different brain regions. Unlike older rats, in young (4 months) rats, the consumption of CR diet under similar conditions affected those above-mentioned immune parameters reversibly and adversely. This study concludes that (a) aging significantly (p < 0.01) deregulates the above-mentioned immune parameters, (b) consecutive consumption of CR diet for one and two months is (i) beneficial (p < 0.05) to the aging-related immune system [lymphocyte viability, lymphocyte proliferation, cytotoxicity, pro (IL-6 and TNF-α)- and anti (IL-10)-inflammatory cytokines], but (ii) adverse (p < 0.05) to the immune parameters of the young rats, and (c) consumption of CR diet for consecutive two months is more potent (p < 0.05) than that due to one month.

Antimicrobial loaded gum odina - gelatin based biomimetic spongy scaffold for accelerated wound healing with complete cutaneous texture.

The main objective of this study was to assess the therapeutic activity of gum odina and gelatin based biomimetic scaffold which was previously established as an excellent wound dressing material. In the accelerated stability study, the changes in physicochemical properties were found to be negligible. The cytotoxicity studies were carried out in-vitro and the results showed that upto 90% of the cells remained viable in presence of the scaffold, confirming its biocompatibility. Moreover, results depicted the superior ability of the scaffold to promote cutaneous healing by increasing the rate of wound contraction (about 98%), granulation formation, collagen deposition and formation of an intact epidermis within 18 days. A satisfactory amount of hydroxyproline (240.2 ± 6.67 µg/100 mg tissue) in scaffold treated groups at 21 days ensured the significant deposition of collagen to re-epithelialization. Further it can be hypothesized that the controlled levels of antioxidant enzymes (SOD, CAT) to diminish the oxidative stress in the wounded sites were due to the innate antioxidant properties of both blank and drug loaded scaffold. These results strongly indicated that the prepared scaffolds have strong potential for biomedical applications and it may serve as promising candidate for the next generation of wound treatment systems.

Apigenin-Loaded PLGA-DMSA Nanoparticles: A Novel Strategy to Treat Melanoma Lung Metastasis.

The flavone apigenin (APG), alone as well as in combination with other chemotherapeutic agents, is known to exhibit potential anticancer effects in various tumors and inhibit growth and metastasis of melanoma. However, the potential of apigenin nanoparticles (APG-NPs) to prevent lung colonization of malignant melanoma has not been well investigated. APG-loaded PLGA-NPs were surface-functionalized with meso-2,3-dimercaptosuccinic acid (DMSA) for the treatment of melanoma lung metastasis. DMSA-conjugated APG-loaded NPs (DMSA-APG-NPs) administered by an oral route exhibited sustained APG release and showed considerable enhancement of plasma half-life, Cmax value, and bioavailability compared to APG-NPs both in plasma and the lungs. DMSA-conjugated APG-NPs showed comparably higher cellular internalization in B16F10 and A549 cell lines compared to that of plain NPs. Increased cytotoxicity was observed for DMSA-APG-NPs compared to APG-NPs in A549 cells. This difference between the two formulations was lower in B16F10 cells. Significant depolarization of mitochondrial transmembrane potential and an enhanced level of caspase activity were observed in B16F10 cells treated with DMSA-APG-NPs compared to APG-NPs as well. Western blot analysis of various proteins was performed to understand the mechanism of apoptosis as well as prevention of melanoma cell migration and invasion. DMSA conjugation substantially increased accumulation of DMSA-APG-NPs given by an intravenous route in the lungs compared to APG-NPs at 6 and 8 h. This was also corroborated by scintigraphic imaging studies with radiolabeled formulations administered by an intravenous route. Conjugation also allowed comparatively higher penetration as evident from an in vitro three-dimensional tumor spheroid model study. Finally, the potential therapeutic efficacy of the formulation was established in experimental B16F10 lung metastases, which suggested an improved bioavailability with enhanced antitumor and antimetastasis efficacy of DMSA-conjugated APG-NPs following oral administration.

Carnosine improves aging-induced cognitive impairment and brain regional neurodegeneration in relation to the neuropathological alterations in the secondary structure of amyloid beta (Aß).

Aging-induced proteinopathies, including deterioration of amyloid beta (Aß) conformation, are associated with reductions in endogenous levels of carnosine and cognitive impairments. Carnosine is a well-known endogenous antioxidant, which counteracts aging-induced Aß plaque formation. The aim of this study was to investigate the effects of exogenous carnosine treatments on aging-induced changes (a) in the steady-state level of endogenous carnosine and conformation of Aß secondary structure in the different brain regions (cerebral cortex, hippocampus, hypothalamus, pons-medulla, and cerebellum) and (b) cognitive function. Young (4 months) and aged (18 and 24 months) male albino Wistar rats were treated with carnosine (2.0 µg kg-1  day-1 ; i.t.) or equivalent volumes of vehicle (saline) for 21 consecutive days and were tested for cognition using 8-arm radial maze test. Brains were processed to assess the conformational integrity of Aß plaques using Raman spectroscopy and endogenous levels of carnosine were measured in the brain regions using HPLC. Results indicated that carnosine treatments improved the aging-induced deficits in cognitive function and reduced the ß-sheets in the secondary structure of Aß protein, as well as mitigating the reduction in the steady-state levels of carnosine and spine density in the brain regions examined. These results thus, suggest that carnosine can attenuate the aging-induced: (a) conformational changes in Aß secondary structure by reducing the abundance of ß-sheets and reductions in carnosine content in the brain regions and (b) cognitive impairment.

A comprehensive calibrated phytolith based climatic index from the Himalaya and its application in palaeotemperature reconstruction.

Past climate reconstructions from palaeoecological records require an understanding of relationships between modern vegetation and climate. Phytoliths are being used widely to reconstruct variations in C3/C4 grasses in the past vegetation and corresponding climate. However, little understanding is available on their relationships with the climate driver(s). Even though, the driver(s) regulating C3/C4 grass distributions vary regionally, while reconstructing the past distributions, a grass phytolith-based climatic index (Ic) has often been found to be used globally without assessing its regional consistency. In the Himalaya, the working potential of Ic has proven to be unsatisfactory when compared to other regions of the globe. To improve the efficacy of Ic, we have identified the redundant grass phytolith morphs and revised it by including four exclusive C3-grass indicator morphotypes (bilobate trapezoidal, bilobate scooped, saddle tall and saddle plateaued) to the existing Ic calculation. Thus, a new climatic index, revised Ic (rIc) is proposed in this article. We have compared the rIc with modern climate variables and a relationship with mean annual temperature (MAT) is established with statistical validation. To assess the working potential of the proposed calibration function in the past temperature reconstructions, we have estimated the late Holocene MAT variations in the Himalaya using rIc. We infer that in the mountainous regions like the Himalaya, even with irregular precipitation distribution, variability in C3/C4 grass distributions and their phytolith spectra seem to be a primary function of temperature. Further, we recommend that rIc can be satisfactorily used to reconstruct past temperature variations in the Himalaya and similar mountainous regions where soil water availability is not a limiting factor.

Anticancer potential of docetaxel-loaded cobalt ferrite nanocarrier: an in vitro study on MCF-7 and MDA-MB-231 cell lines.

AIM: To develop a biocompatible cobalt ferrite (CF-NP) nanodrug formulation using oleic acid and poly (d,l-lactide-co-glycolic) acid (PLGA) for the delivery of docetaxel (DTX) specifically to breast cancer cells. METHODS: The CF-NP were synthesised by hydrothermal method and conjugated with DTX in a PLGA matrix and were systematically characterised using XRD, FE-SEM, TEM, DLS, FTIR, TGA, SQUID etc. The drug loading, in vitro drug release, cellular uptake, cytotoxicity were evaluated and haemolytic effect was studied. RESULTS: The CF-NP showed good crystallinity with an average particle size of 21 nm and ferromagnetic nature. The DTX-loaded CF-NP (DCF-NP) showed 8.4% (w/w) drug loading with 81.8% loading efficiency with a sustained DTX release over time. An effective internalisation and anti-proliferative efficiency was observed in MCF-7 and MDA-MB-231 breast cancer cells and negligible haemolytic effect. CONCLUSION: The DCF-NP can have the potential for the effective delivery of DTX for breast cancer treatment.

Calorie restriction improves aging-induced impairment of cognitive function in relation to deregulation of corticosterone status and brain regional GABA system.

Aging is known to affect adversely the corticosterone status and the brain function including cognition. Calorie restricted (CR) diet has been found to improve brain aging. The objective of the present investigation is to study the effect of short-term CR diet without any food deprivation on aging-induced impairment of cognitive function in relation to the corticosterone status and the brain regional GABA system. The result showed that aging-induced deregulation of the brain regional GABA system, increase in plasma and adrenal corticosterone levels and cognitive impairment were attenuated with short-term CR diet supplementation for consecutive 1 and 2 months to the aged (18 and 24 months) rats. But in young rats (4 months) consumption of the same CR diet under similar conditions reversibly affected those above-mentioned parameters. These results, thus suggest that (a) aging down-regulates brain regional GABA system with an up-regulation of corticosterone status and impairment of cognitive function, (b) CR diet consumption improves this aging-induced deregulation of brain regional GABA system, corticosterone status, and cognitive function, (c) these attenuating effects of CR diet are greater with a longer period of consumption but (d) CR diet consumption is harmful to young rats as observed in those parameters.

Plant-Based Antidiabetic Nanoformulations: The Emerging Paradigm for Effective Therapy.

Diabetes mellitus is a life-threatening metabolic syndrome. Over the past few decades, the incidence of diabetes has climbed exponentially. Several therapeutic approaches have been undertaken, but the occurrence and risk still remain unabated. Several plant-derived small molecules have been proposed to be effective against diabetes and associated vascular complications via acting on several therapeutic targets. In addition, the biocompatibility of these phytochemicals increasingly enhances the interest of exploiting them as therapeutic negotiators. However, poor pharmacokinetic and biopharmaceutical attributes of these phytochemicals largely restrict their clinical usefulness as therapeutic agents. Several pharmaceutical attempts have been undertaken to enhance their compliance and therapeutic efficacy. In this regard, the application of nanotechnology has been proven to be the best approach to improve the compliance and clinical efficacy by overturning the pharmacokinetic and biopharmaceutical obstacles associated with the plant-derived antidiabetic agents. This review gives a comprehensive and up-to-date overview of the nanoformulations of phytochemicals in the management of diabetes and associated complications. The effects of nanosizing on pharmacokinetic, biopharmaceutical and therapeutic profiles of plant-derived small molecules, such as curcumin, resveratrol, naringenin, quercetin, apigenin, baicalin, luteolin, rosmarinic acid, berberine, gymnemic acid, emodin, scutellarin, catechins, thymoquinone, ferulic acid, stevioside, and others have been discussed comprehensively in this review.

Aptamer-Functionalized Drug Nanocarrier Improves Hepatocellular Carcinoma toward Normal by Targeting Neoplastic Hepatocytes.

Site-specific delivery of chemotherapeutics specifically to neoplastic hepatocytes without affecting normal hepatocytes should be a focus for potential therapeutic management of hepatocellular carcinoma (HCC). The aptamer TLS 9a with phosphorothioate backbone modifications (L5) has not been explored so far for preferential delivery of therapeutics in neoplastic hepatocytes to induce apoptosis. Thus, the objective of the present investigation was to compare the therapeutic potential of L5-functionalized drug nanocarrier (PTX-NPL5) with those of the other experimental drug nanocarriers functionalized by previously reported HCC cell-targeting aptamers and non-aptamer ligands, such as galactosamine and apotransferrin. A myriad of well-defined investigations such as cell cycle analysis, TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) assay, and studies related to apoptosis, histopathology, and immunoblotting substantiated that PTX-NPL5 had the highest potency among the different ligand-attached experimental formulations in inducing selective apoptosis in neoplastic hepatocytes via a mitochondrial-dependent apoptotic pathway. PTX-NPL5 did not produce any notable toxic effects in healthy hepatocytes, thus unveiling a new and a safer option in targeted therapy for HCC. Molecular modeling study identified two cell-surface biomarker proteins (tumor-associated glycoprotein 72 [TAG-72] and heat shock protein 70 [HSP70]) responsible for ligand-receptor interaction of L5 and preferential internalization of PTX-NPL5 via clathrin-mediated endocytosis in neoplastic hepatocytes. The potential of PTX-NPL5 has provided enough impetus for its rapid translation from the pre-clinical to clinical domain to establish itself as a targeted therapeutic to significantly prolong survival in HCC.

A Comparative Investigation of the Ability of Various Aptamer-Functionalized Drug Nanocarriers to Induce Selective Apoptosis in Neoplastic Hepatocytes: In Vitro and In Vivo Outcome.

Aptamers offer a significant promise to target various cancers including hepatocellular carcinoma (HCC), for their high affinity and ability to reach the target site(s), non-immunogenicity, and low cost. The targeting ability to neoplastic hepatocytes by the aptamer, TLS 9a with phosphorothioate backbone modification (designated as L5), has not been explored yet. Hence, we investigated the comparative potential of L5 with some other previously reported liver cancer cell-specific aptamers, conjugated on the surface of drug-nanocarriers. Various in vitro studies such as cytotoxicity, in vitro cellular uptake, cell cycle analysis, and investigations related to apoptosis were performed. In vivo studies carried out here include macroscopic and microscopic hepatic alterations in chemically induced hepatocarcinogenesis in rats, upon experimental treatments. The outcome of the investigations revealed that L5-functionalized drug-nanocarrier (PTX-NPL5) had the highest apoptotic potential compared with the other aptamer-conjugated experimental formulations. Further, its maximum internalization by neoplastic hepatocytes and minimum internalization by normal hepatocytes indicate that it had the potential to preferentially target the neoplastic hepatocytes. Data of in vivo studies revealed that PTX-NPL5 reduced tumor incidences and tumor progress. Superior potency of PTX-NPL5 may be due to the maximum affinity of L5 towards neoplastic hepatocytes resulting in maximum permeation of drug-nanocarrier in them. An effective site-specific targeting of neoplastic hepatocytes can be achieved by L5 for preferential delivery of therapeutics. Further, investigations are needed to identify the target protein(s) on neoplastic hepatocytes responsible for ligand-receptor interaction of L5.