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.

pH-Sensitive Nanodrug Carriers for Codelivery of ERK Inhibitor and Gemcitabine Enhance the Inhibition of Tumor Growth in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), a metabolic disorder, remains one of the leading cancer mortality sources worldwide. An initial response to treatments, such as gemcitabine (GEM), is often followed by emergent resistance reflecting an urgent need for alternate therapies. The PDAC resistance to GEM could be due to ERK1/2 activity. However, successful ERKi therapy is hindered due to low ligand efficiency, poor drug delivery, and toxicity. In this study, to overcome these limitations, we have designed pH-responsive nanoparticles (pHNPs) with a size range of 100-150 nm for the simultaneous delivery of ERKi (SCH 772984) and GEM with tolerable doses. These pHNPs are polyethylene glycol (PEG)-containing amphiphilic polycarbonate block copolymers with tertiary amine side chains. They are systemically stable and capable of improving in vitro and in vivo drug delivery at the cellular environment's acidic pH. The functional analysis indicates that the nanomolar doses of ERKi or GEM significantly decreased the 50% growth inhibition (IC50) of PDAC cells when encapsulated in pHNPs compared to free drugs. The combination of ERKi with GEM displayed a synergistic inhibitory effect. Unexpectedly, we uncover that the minimum effective dose of ERKi significantly promotes GEM activities on PDAC cells. Furthermore, we found that pHNP-encapsulated combination therapy of ERKi with GEM was superior to unencapsulated combination drug therapy. Our findings, thus, reveal a simple, yet efficient, drug delivery approach to overcome the limitations of ERKi for clinical applications and present a new model of sensitization of GEM by ERKi with no or minimal toxicity.

Metabolic disorder in Alzheimer's disease.

Alzheimer's disease (AD), a well known aging-induced neurodegenerative disease is related to amyloid proteinopathy. This proteinopathy occurs due to abnormalities in protein folding, structure and thereby its function in cells. The root cause of such kind of proteinopathy and its related neurodegeneration is a disorder in metabolism, rather metabolomics of the major as well as minor nutrients. Metabolomics is the most relevant "omics" platform that offers a great potential for the diagnosis and prognosis of neurodegenerative diseases as an individual's metabolome. In recent years, the research on such kinds of neurodegenerative diseases, especially aging-related disorders is broadened its scope towards metabolic function. Different neurotransmitter metabolisms are also involved with AD and its associated neurodegeneration. The genetic and epigenetic backgrounds are also noteworthy. In this review, the physiological changes of AD in relation to its corresponding biochemical, genetic and epigenetic involvements including its (AD) therapeutic aspects are discussed.

Chemical Architecture of Block Copolymers Differentially Abrogate Cardiotoxicity and Maintain the Anticancer Efficacy of Doxorubicin.

The molecular architecture of pH-responsive amphiphilic block copolymers, their self-assembly behavior to form nanoparticles (NPs), and doxorubicin (DOX)-loading technique govern the extent of DOX-induced cardiotoxicity. We observed that the choice of pH-sensitive tertiary amines, surface charge, and DOX-loading techniques within the self-assembled NPs strongly influence the release and stimulation of DOX-induced cardiotoxicity in primary cardiomyocytes. However, covalent conjugation of DOX to a pH-sensitive nanocarrier through a "conditionally unstable amide" linkage (PCPY-cDOX; PC = polycarbonate and PY = 2-pyrrolidine-1-yl-ethyl-amine) significantly reduced the cardiotoxicity of DOX in cardiomyocytes as compared to noncovalently encapsulated DOX NPs (PCPY-eDOX). When these formulations were tested for drug release in serum-containing media, the PCPY-cDOX systems showed prolonged control over drug release (for ∼72 h) at acidic pH compared to DOX-encapsulated nanocarriers, as expected. We found that DOX-encapsulated nanoformulations triggered cardiotoxicity in primary cardiomyocytes more acutely, while conjugated systems such as PCPY-cDOX prevented cardiotoxicity by disabling the nuclear entry of the drug. Using 2D and 3D (spheroid) cultures of an ER + breast cancer cell line (MCF-7) and a triple-negative breast cancer cell line (MDA-MB-231), we unravel that, similar to encapsulated systems (PCPY-eDOX-type) as reported earlier, the PCPY-cDOX system suppresses cellular proliferation in both cell lines and enhances trafficking through 3D spheroids of MDA-MB-231 cells. Collectively, our studies indicate that PCPY-cDOX is less cardiotoxic as compared to noncovalently encapsulated variants without compromising the chemotherapeutic properties of the drug. Thus, our studies suggest that the appropriate selection of the nanocarrier for DOX delivery may prove fruitful in shifting the balance between low cardiotoxicity and triggering the chemotherapeutic potency of DOX.

A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells.

BACKGROUND: Betulinic acid (BA), a member of pentacyclic triterpenes has shown important biological activities like anti-bacterial, anti-malarial, anti-inflammatory and most interestingly anticancer property. To overcome its poor aqueous solubility and low bioavailability, structural modifications of its functional groups are made to generate novel lead(s) having better efficacy and less toxicity than the parent compound. BA analogue, 2c was found most potent inhibitor of colon cancer cell line, HT-29 cells with IC50 value 14.9 µM which is significantly lower than standard drug 5-fluorouracil as well as parent compound, Betulinic acid. We have studied another mode of PCD, autophagy which is one of the important constituent of cellular catabolic system as well as we also studied proteasomal degradation pathway to investigate whole catabolic pathway after exploration of 2c on HT-29 cells. METHODS: Mechanism of autophagic cell death was studied using fluorescent dye like acridine orange (AO) and monodansylcadaverin (MDC) staining by using fluorescence microscopy. Various autophagic protein expression levels were determined by Western Blotting, qRT-PCR and Immunostaining. Confocal Laser Scanning Microscopy (CLSM) was used to study the colocalization of various autophagic proteins. These were accompanied by formation of autophagic vacuoles as revealed by FACS and transmission electron microscopy (TEM). Proteasomal degradation pathway was studied by proteasome-Glo™ assay systems using luminometer. RESULTS: The formation of autophagic vacuoles in HT-29 cells after 2c treatment was determined by fluorescence staining--confirming the occurrence of autophagy. In addition, 2c was found to alter expression levels of different autophagic proteins like Beclin-1, Atg 5, Atg 7, Atg 5-Atg 12, LC3B and autophagic adapter protein, p62. Furthermore we found the formation of autophagolysosome by colocalization of LAMP-1 with LC3B, LC3B with Lysosome, p62 with lysosome. Finally, as proteasomal degradation pathway downregulated after 2c treatment colocalization of ubiquitin with lysosome and LC3B with p62 was studied to confirm that protein degradation in autophagy induced HT-29 cells follows autolysosomal pathway. CONCLUSIONS: In summary, betulinic acid analogue, 2c was able to induce autophagy in HT-29 cells and as proteasomal degradation pathway downregulated after 2c treatment so protein degradation in autophagy induced HT-29 cells follows autolysosomal pathway.

pH-responsive targeted nanoparticles release ERK-inhibitor in the hypoxic zone and sensitize free gemcitabine in mutant K-Ras-addicted pancreatic cancer cells and mouse model.

Therapeutic options for managing Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest types of aggressive malignancies, are limited and disappointing. Therefore, despite suboptimal clinical effects, gemcitabine (GEM) remains the first-line chemotherapeutic drug in the clinic for PDAC treatment. The therapeutic limitations of GEM are primarily due to poor bioavailability and the development of chemoresistance resulting from the addiction of mutant-K-RAS/AKT/ERK signaling-mediated desmoplastic barriers with a hypoxic microenvironment. Several new therapeutic approaches, including nanoparticle-assisted drug delivery, are being investigated by us and others. This study used pH-responsive nanoparticles encapsulated ERK inhibitor (SCH772984) and surface functionalized with tumor-penetrating peptide, iRGD, to target PDAC tumors. We used a small molecule, SCH772984, to target ERK1 and ERK2 in PDAC and other cancer cells. This nanocarrier efficiently released ERKi in hypoxic and low-pH environments. We also found that the free-GEM, which is functionally weak when combined with nanoencapsulated ERKi, led to significant synergistic treatment outcomes in vitro and in vivo. In particular, the combination approaches significantly enhanced the GEM effect in PDAC growth inhibition and prolonged survival of the animals in a genetically engineered KPC (LSL-KrasG12D/+/LSL-Trp53R172H/+/Pdx-1-Cre) pancreatic cancer mouse model, which is not observed in a single therapy. Mechanistically, we anticipate that the GEM efficacy was increased as ERKi blocks desmoplasia by impairing the production of desmoplastic regulatory factors in PDAC cells and KPC mouse tumors. Therefore, 2nd generation ERKi (SCH 772984)-iRGD-pHNPs are vital for the cellular response to GEM and denote a promising therapeutic target in PDAC with mutant K-RAS.

CD24 negativity reprograms mitochondrial metabolism to PPARα and NF-κB-driven fatty acid ß-oxidation in triple-negative breast cancer.

CD24 is a well-characterized breast cancer (BC) stem cell (BCSC) marker. Primary breast tumor cells having CD24-negativity together with CD44-positivity is known to maintain high metastatic potential. However, the functional role of CD24 gene in triple-negative BC (TNBC), an aggressive subtype of BC, is not well understood. While the significance of CD24 in regulating immune pathways is well recognized in previous studies, the significance of CD24 low expression in onco-signaling and metabolic rewiring is largely unknown. Using CD24 knock-down and over-expression TNBC models, our in vitro and in vivo analysis suggest that CD24 is a tumor suppressor in metastatic TNBC. Comprehensive in silico gene expression analysis of breast tumors followed by lipidomic and metabolomic analyses of CD24-modulated cells revealed that CD24 negativity induces mitochondrial oxidative phosphorylation and reprograms TNBC metabolism toward the fatty acid beta-oxidation (FAO) pathway. CD24 silencing activates PPARα-mediated regulation of FAO in TNBC cells. Further analysis using reverse-phase protein array and its validation using CD24-modulated TNBC cells and xenograft models nominated CD24-NF-κB-CPT1A signaling pathway as the central regulatory mechanism of CD24-mediated FAO activity. Overall, our study proposes a novel role of CD24 in metabolic reprogramming that can open new avenues for the treatment strategies for patients with metastatic TNBC.

BCMA-targeted bortezomib nanotherapy improves therapeutic efficacy, overcomes resistance, and modulates the immune microenvironment in multiple myeloma.

Bortezomib (BTZ) is a standard-of-care treatment in multiple myeloma (MM); however, adverse side effects and development of resistance limit its long term benefit. To improve target specificity, therapeutic efficacy, and overcome resistance, we designed nanoparticles that encapsulate BTZ and are surface-functionalized with BCMA antibodies (BCMA-BTZ-NPs). We confirmed efficient cellular internalization of the BCMA-BTZ-NPs only in BCMA-expressing MM cells, but not in BCMA-knockout (KO) cells. In addition, BCMA-BTZ-NPs showed target-specific cytotoxicity against MM cell lines and primary tumor cells from MM patients. The BCMA-BTZ-NPs entered the cell through receptor-mediated uptake, which escapes a mechanism of BTZ resistance based on upregulating P-glycoprotein. Furthermore, BCMA-BTZ-NPs induced cell death more efficiently than non-targeted nanoparticles or free BTZ, triggering potent mitochondrial depolarization followed by apoptosis. In BTZ-resistant cells, BCMA-BTZ-NPs inhibited proteasome activity more effectively than free BTZ or non-targeted nanoparticles. Additionally, BCMA-BTZ-NPs enhanced immunogenic cell death and activated the autophagic pathway more than free BTZ. Finally, we found that BCMA-BTZ-NPs selectively accumulated at the tumor site in a murine xenograft model, enhanced tumor reduction, and prolonged host survival. These results suggest BCMA-BTZ-NPs provide a promising therapeutic strategy for enhancing the efficacy of BTZ and establish a framework for their evaluation in a clinical setting.

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.

Polymeric Composite Matrix with High Biobased Content as Pharmaceutically Relevant Molecular Encapsulation and Release Platform.

Drug delivery systems (DDS) that can temporally control the rate and extent of release of therapeutically active molecules find applications in many clinical settings, ranging from infection control to cancer therapy. With an aim to design a locally implantable, controlled-release DDS, we demonstrated the feasibility of using cellulose nanocrystal (CNC)-reinforced poly (l-lactic acid) (PLA) composite beads. The performance of the platform was evaluated using doxorubicin (DOX) as a model drug for applications in triple-negative breast cancer. A facile, nonsolvent-induced phase separation (NIPS) method was adopted to form composite beads. We observed that CNC loading within these beads played a critical role in the mechanical stability, porosity, water uptake, diffusion, release, and pharmacological activity of the drug from the delivery system. When loaded with DOX, composite beads significantly controlled the release of the drug in a pH-dependent pattern. For example, PLA/CNC beads containing 37.5 wt % of CNCs showed a biphasic release of DOX, where 41 and 82% of the loaded drug were released at pH 7.4 and pH 5.5, respectively, over 7 days. Drug release followed Korsmeyer's kinetics, indicating that the release mechanism was mostly diffusion and swelling-controlled. We showed that DOX released from drug-loaded PLA/CNC composite beads locally suppressed the growth and proliferation of triple-negative breast cancer cells, MBA-MB-231, via the apoptotic pathway. The efficacy of the DDS was evaluated in human tissue explants. We envision that such systems will find applications for designing biobased platforms with programmed stability and drug delivery functions.

CCN5 activation by free or encapsulated EGCG is required to render triple-negative breast cancer cell viability and tumor progression.

Epigallocatechin-3-gallate (EGCG) has been considered an anticancer agent despite conflicting and discrepant bioavailability views. EGCG impairs the viability and self-renewal capacity of triple-negative breast cancer (TNBC) cells and makes them sensitive to estrogen via activating ER-α. Surprisingly, the mechanism of EGCG's action on TNBC cells remains unclear. CCN5/WISP-2 is a gatekeeper gene that regulates viability, ER-α, and stemness in TNBC and other types of cancers. This study aimed to investigate whether EGCG (free or encapsulated in nanoparticles) interacts with the CCN5 protein by emphasizing its bioavailability and enhancing its anticancer effect. We demonstrate that EGCG activates CCN5 to inhibit in vitro cell viability through apoptosis, the sphere-forming ability via reversing TNBC cells' stemness, and suppressing tumor growth in vivo. Moreover, we found EGCG-loaded nanoparticles to be functionally more active and superior in their tumor-suppressing ability than free-EGCG. Together, these studies identify EGCG (free or encapsulated) as a novel activator of CCN5 in TNBC cells and hold promise as a future therapeutic option for TNBC with upregulated CCN5 expression.

Functional Applications of Polyarginine-Hyaluronic Acid-Based Electrostatic Complexes.

Background: Electrostatic complexes of poly (l-Arginine) (pArg) and hyaluronic acid (HA) have been investigated for their functional applications to supply free or polymeric form of l-Arginine (Arg) to target cells. As a vital amino acid, Arg plays significant role in multitude of pathophysiological processes ranging from wound healing to cancer. However, serum arginase expression and toxicity of Arg at cellular level renders exogenous delivery of this amino acid a challenging task. We showed that polyarginine-hyaluronic acid ionic nanocomplexes (pArg-HA iNCs) could be an effective way to deliver Arg to target cell populations. Materials and Methods: These electrostatic complexes were prepared by mixing HA (average m.w. of 200 kDa) with pArg (m.w. 5-15 kDa; Sigma) in aqueous solutions and purifying over glycerol. Nanocomplexes were characterized for their particle size, surface charge, capacity to release l-Arg, and intracellular uptake of complexes. Results: Synthesized nanocomplexes showed hydrodynamic diameter ranging from 140-306 nm depending on the content of pArg or HA within the formulation. With surface charge (ζ-potential) of -29 mV, the nanocomplexes showed pH-dependent release of Arg. At pH 7.4, pArg-HA iNCs released 30% of the total Arg-content, while at pH 5.0, 60% of Arg was released after 24 h. These electrostatically stabilized complexes were found to promote growth of human dermal fibroblasts (HDF) in wound-healing assay and increased nitric oxide (NO) activity in these cells in a time-dependent manner. Nanocomplexes also showed cellular uptake and enhanced dose-dependent toxicity against two pancreatic cancer cell lines, i.e. MIA PaCa-2 and Panc-1. Interestingly, the cytotoxic effect was synergized upon pre-treatment of the cells with a frontline chemotherapeutic agent, gemcitabine (GEM), and was not observed when the cells were treated with Arg alone. Conclusion: As such, this communication shows the prospect of pArg-HA iNC electrostatic nanocomplexes to interact and interfere with intracellular Arg metabolic machinery conducive to rescuing different pathological conditions.

Nanoencapsulated betulinic acid analogue distinctively improves colorectal carcinoma in vitro and in vivo.

Betulinic acid, a plant secondary metabolite, has gained significant attention due to its antiproliferative activity over a range of cancer cells. A promising betulinic acid analogue (2c) with better therapeutic efficacy than parent molecule to colon carcinoma cells has been reported. Despite impressive biological applications, low aqueous solubility and bioavailability create difficulties for its therapeutic applications. To overcome these lacunae and make it as a promising drug candidate we have encapsulated the lead betulinic acid derivative (2c) in a polymeric nanocarrier system (2c-NP) and evaluated its in vitro and in vivo therapeutic efficacy. Apoptosis that induces in vitro antiproliferative activity was significantly increased by 2c-NP compared to free-drug (2c), as assured by MTT assay, Annexin V positivity, JC1 analysis and cell cycle study. The therapeutic potential measured in vitro and in vivo reflects ability of 2c-NP as an effective therapeutic agent for treatment of colon carcinoma and future translation to clinical trials.

CD-340 functionalized doxorubicin-loaded nanoparticle induces apoptosis and reduces tumor volume along with drug-related cardiotoxicity in mice.

BACKGROUND AND OBJECTIVE: Targeted drug delivery of nanoparticles decorated with site-specific recognition ligands is of considerable interest to minimize cytotoxicity of chemotherapeutics in the normal cells. The study was designed to develop CD-340 antibody-conjugated polylactic-co-glycolic acid (PLGA) nanoparticles loaded with a highly water-soluble potent anticancer drug, doxorubicin (DOX), to specifically deliver entrapped DOX to breast cancer cells. METHODS: The study showed how to incorporate water-soluble drug in a hydrophobic PLGA (85:15) based matrix which otherwise shows poor drug loading due to leaching effect. The optimized formulation was covalently conjugated to anti-human epidermal growth factor receptor-2 (HER2) antibody (CD-340). Surface conjugation of the ligand was assessed by flow cytometry, confocal microscopy, and gel electrophoresis. Selectivity and cytotoxicity of the experimental nanoparticles were tested on human breast cancer cells SKBR-3, MCF-7, and MDA-MB-231. Both CD-340-conjugated and unconjugated nanoparticles were undergone in vitro and in vivo characterization. RESULT: Higher level of incorporation of DOX (8.5% W/W), which otherwise shows poor drug loading due to leaching effect of the highly water-soluble drug, was seen in this method. In HER2-overexpressing tumor xenograft model, radiolabeled antibody-conjugated nanoparticles showed preferentially more of the formulation accumulation in the tumor area when compared to the treatments with the unconjugated one or with the other control groups of mice. The ligand conjugated nanoparticles showed considerable potential in reduction of tumor growth and cardiac toxicity of DOX in mice, a prominent side-effect of the drug. CONCLUSION: In conclusion, CD-340-conjugated PLGA nanoparticles containing DOX preferentially delivered encapsulated drug to the breast cancer cells and in breast tumor and reduced the breast tumor cells by apoptosis. Site-specific delivery of the formulation to neoplastic cells did not affect normal cells and showed a drastic reduction of DOX-related cardiotoxicity.

Aptamer-Conjugated Apigenin Nanoparticles To Target Colorectal Carcinoma: A Promising Safe Alternative of Colorectal Cancer Chemotherapy.

Apigenin has gained interest recently among researchers as a potential chemotherapeutic agent in cancer, including colorectal cancer, due to its established antiproliferative activity in vitro. Despite its impressive anticancer activity in vitro, poor water solubility and nonspecific distribution in vivo make it difficult for its emergence as a drug candidate. To overcome these problems, we formulated an aptamer-conjugated apigenin-loaded nanoparticle (apt-ANP) to target against the overexpressed colorectal cancer cell surface biomarker epithelial cell adhesion molecule (EpCAM). Aptamer conjugation was conducted on the prepared nanoparticle, characterized (by SEM, TEM, and AFM) and evaluated for its antiproliferative activity toward in vitro colon carcinoma cells and in vivo colorectal cancer model. The aptamer-conjugated nanoformulation had an average size about 226 nm, smooth surface, satisfactory drug loading 17.5 ± 1.3%, and sustained drug-release pattern. The pharmacokinetic profile as well as the biodistribution study demonstrated a maximum retention of apt-ANP in the colon as compared to free drug and aptamer-free apigenin-loaded nanoparticle (ANP). Apt-ANP enhanced therapeutic efficacy to colorectal cancer cells, whereas it minimized off-target cytotoxicity to normal cells.

Synthesis and biological evaluation of a novel betulinic acid derivative as an inducer of apoptosis in human colon carcinoma cells (HT-29).

A novel family of betulinic acid analogues, carrying a triazole unit at C-3 attached through a linker, was synthesized by the application of azide-alkyne "Click reaction". These were screened for their anticancer activity against different cancer cells and normal human PBMC by MTT assay. Compound 2c [(3S)-3-{2-(4-(hydroxymethyl-1H-1,2,3-triazol-1-yl)acetyloxy}-lup-20(29)-en-28-oic acid] was found as the most potent inhibitor of cell line HT-29 with IC50 value 14.9 µM. Its role as an inducer of apoptosis was investigated in this cell line by Annexin-V/PI binding assay and by following its capability for ROS generation, depolarization of mitochondrial transmembrane potential, activation of caspases, PARP cleavage, nuclear degradation and expression of pro- and anti-apoptotic proteins. It exhibited much higher cytotoxicity than the standard drug 5-fluorouracil but showed negligible cytotoxicity towards normal PBMC. Elevated level of ROS generation, activation of caspase 3 and caspase 9, DNA fragmentation, higher expression of Bax and Bad, lower expression of Bcl2 and Bcl-xl, and increased level of Bax/Bcl-xl ratio identified 2c as a promising inducer of apoptosis that follows a mitochondria dependent pathway. Bio-physical studies indicate that compound 2c acts as a minor groove binder to the DNA.