A comprehensive review on anticancer evaluation techniques.

The development of effective anticancer strategies and the improvement of our understanding of cancer need analytical tools. Utilizing a variety of analytical approaches while investigating anti-cancer medicines gives us a thorough understanding of the traits and mechanisms concerned to cancer cells, which enables us to develop potent treatments to combat them. The importance of anticancer research may be attributed to various analytical techniques that contributes to the identification of therapeutic targets and the assessment of medication efficacy, which are crucial things in expanding our understanding of cancer biology. The study looks at methods that are often used in cancer research, including cell viability assays, clonogenic assay, flow cytometry, 2D electrophoresis, microarray, immunofluorescence, western blot caspase activation assay, bioinformatics, etc. The fundamentals, applications, and how each technique analytical advances our understanding of cancer are briefly reviewed.

Synthesis, Molecular Docking and Evaluation of 1,3,4-Oxadiazole-Isobenzofuran Hybrids as Antimicrobial and Anticancer Agents.

In drug discovery, the hybridization of bioactive pharmacophores is a powerful tool for targeting enzymes involved in cancer and microbial cell growth. A combination of 1,3,4-oxadiazole and isobenzofuran may improve the antitumor and antimicrobial properties of the hybrid molecules. A series of hybrid molecules having 1,3,4-oxadiazole and isobenzofuran were synthesized and structural characterization was done by FT-IR, 1 H-NMR, 13 C-NMR, and mass spectrometry. Molecular docking studies were performed to investigate binding interactions of compounds with proteins (PDB NO: 2R3J and 1GII), and the results were consistent with in vitro anticancer data. All the synthesized compounds were tested for antimicrobial activity against S. aureus, E. faecalis (Gram-positive) and E. coli and P. aeruginosa (Gram-negative) bacterial strains. Among the synthesized compounds, 7a and 7b displayed good activity against the tested bacterial strains. Also, compounds were tested for their anti-tumor activity against breast cancer (MCF-7) and colon cancer (HCT-116) cell lines via SRB assay. In comparison to doxorubicin (1.14 µM), hybrids 7e (4.32 µM), 7f (4.15 µM), 7g (4.66 µM), and 7h (4.83 µM) demonstrated comparable IC50 value against the HCT 116 cell line.

A nanocomposite hydrogel delivery system for mesenchymal stromal cell secretome.

BACKGROUND: Mesenchymal stromal cell conditioned medium (MSC-CM) contains a cocktail of bioactive factors that act synergistically to induce therapeutic effects. This has been clearly demonstrated by in vivo applications of MSC-CM, but the establishment of controlled delivery systems is an unmet requirement for clinical translation. METHODS: We developed a nanocomposite-hydrogel (NP-H) comprised of poly-L-lactide nanoparticles (NPs) embedded in gelatin/hyaluronic acid (Gel/HA) hydrogel as a delivery vehicle for MSC-CM. First, we optimized the culture conditions for bone marrow-derived MSCs using serum-containing medium (SCM) and serum-free medium (SFM) and characterized the corresponding CM (serum-containing conditioned medium (ScCM) and serum-free conditioned medium (SfCM), respectively) for its potency and xeno markers. Then we prepared a composite matrix followed by physiochemical characterization and functional assays were performed. RESULTS: Nanocomposite hydrogel displayed an even distribution of NPs along with high porosity (> 60%) and swelling ratios > 1500%, while its protein release pattern corresponded to a mix of degradation and diffusion kinetics. Functional evaluation of the composites was determined using MSCs and human fibroblasts (HFFs). The cells seeded directly onto the composites displayed increasing metabolic activities over time, with ScCM-NP-H groups having maximum activity. The cells treated in vitro with 5% and 10% extracts of ScCM-NP-H and SfCM-NP-H exhibited a dose- and duration-dependent response. Cell activities reduced considerably for all groups, except 10% ScCM-NP-H, which displayed a significant increase over time. CONCLUSION: We observed that sustained release of MSC-CM is required to prevent dose-dependent cytotoxicity. The proposed nanocomposite hydrogel for MSC-CM delivery can open up a new array for its clinical application.

Enhancing the efficacy of fluocinolone acetonide by encapsulating with PLGA nanoparticles and conjugating with linear PEG polymer.

Fluocinolone acetonide (FA), a glucocorticoid is used to treat inflammation in the posterior segment of the eye. Due to short half-life and body clearance, it will not be able to give therapeutic effect for long time with a single injection. Formulating FA nanoparticles (NPs) or PEG conjugates can be an effective way to overcome these disadvantages. We prepared two formulations, FA loaded in PLGA nanoparticles (NPs-FA) and FA conjugated to linear PEG (PEG-FA). The NPs-FA were characterised for size and zeta potential using particle size analyser and shape and morphology by using scanning electron microscope (SEM). The amount of drug loaded per mg of NPs and in-vitro release of FA from NPs were calculated using reverse phase high pressure liquid chromatography (RP-HPLC). NPs synthesis was optimized with factorial and Response Surface Methodology (RSM). Chemically synthesized PEG-FA conjugates were characterized using H-NMR and purity of the conjugate was analysed using RP-HPLC. Visualization of cellular uptake of NPs was done by coumarin-6 loaded NPs under fluorescent microscope. RAW 264.7 macrophages were treated with NPs-FA and PEG-FA conjugates to study their effectiveness in inhibiting TNF-α levels compared to free FA treatment. Stability test confirmed that FA is more stable within NPs than in free form. Particle size and zeta potential were found to be 183.6 ± 12.47nm and -25.6 ± 4.4mV, respectively. 149.58 ± 11.3µg of FA was encapsulated per mg of NPs and 61 µg of FA was present per mg of PEG-FA conjugate. In vitro drug release study showed a sustained release of FA from the NPs for a period of 30 days. Fluorescent microscope images showed uptake of NPs by RAW 264.7 cells. TNF-α assay confirmed that substantial inhibition of TNF-α levels from both formulations compared to free FA. From the results, we conclude that new formulations will greatly reduce drug dosage and frequency of administration for long term treatment of inflammation in posterior part of the eye.

Antimycobacterial susceptibility evaluation of rifampicin and isoniazid benz-hydrazone in biodegradable polymeric nanoparticles against Mycobacterium tuberculosis H37Rv strain.

INTRODUCTION: Tuberculosis (TB) is the single largest infectious disease which requires a prolonged treatment regime with multiple drugs. The present treatment for TB includes frequent administration of a combination of four drugs for a duration of 6 months. This leads to patient's noncompliance, in addition to developing drug-resistant strains which makes treatment more difficult. The formulation of drugs with biodegradable polymeric nanoparticles (NPs) promises to overcome this problem. MATERIALS AND METHODS: In this study, we focus on two important drugs used for TB treatment - rifampicin (RIF) and isoniazid (INH) - and report a detailed study of RIF-loaded poly lactic-co-glycolic acid (PLGA) NPs and INH modified as INH benz-hydrazone (IH2) which gives the same therapeutic effect as INH but is more stable and enhances the drug loading in PLGA NPs by 15-fold compared to INH. The optimized formulation was characterized using particle size analyzer, scanning electron microscopy and transmission electron microscopy. The drug release from NPs and stability of drug were tested in different pH conditions. RESULTS: It was found that RIF and IH2 loaded in NPs release in a slow and sustained manner over a period of 1 month and they are more stable in NPs formulation compared to the free form. RIF- and IH2-loaded NPs were tested for antimicrobial susceptibility against Mycobacterium tuberculosis H37Rv strain. RIF loaded in PLGA NPs consistently inhibited the growth at 70% of the minimum inhibitory concentration (MIC) of pure RIF (MIC level 1 µg/mL), and pure IH2 and IH2-loaded NPs showed inhibition at MIC equivalent to the MIC of INH (0.1 µg/mL). CONCLUSION: These results show that NP formulations will improve the efficacy of drug delivery for TB treatment.

Design of aqueous two-phase systems for purification of hyaluronic acid produced by metabolically engineered Lactococcus lactis.

Hyaluronic acid has a wide range of biomedical applications and its commercial value is highly dependent on its purity and molecular weight. This study highlights the utility of aqueous two-phase separation as a primary recovery step for hyaluronic acid and for removal of major protein impurities from fermentation broths. Metabolically engineered cultures of a lactate dehydrogenase mutant strain of Lactococcus lactis (L. lactis NZ9020) were used to produce high-molecular-weight hyaluronic acid. The cell-free fermentation broth was partially purified using a polyethylene glycol/potassium phosphate system, resulting in nearly 100% recovery of hyaluronic acid in the salt-rich bottom phase in all the aqueous two-phase separation experiments. These experiments were optimized for maximum removal of protein impurities in the polyethylene glycol rich top phase. The removal of protein impurities resulted in substantial reduction of membrane fouling in the subsequent diafiltration process, carried out with a 300 kDa polyether sulfone membrane. This step resulted in considerable purification of hyaluronic acid, without any loss in recovery and molecular weight. Diafiltration was followed by an adsorption step to remove minor impurities and achieve nearly 100% purity. The final hyaluronic acid product was characterized by Fourier-transform IR and NMR spectroscopy, confirming its purity.

Reformulating Tylocrebrine in Epidermal Growth Factor Receptor Targeted Polymeric Nanoparticles Improves Its Therapeutic Index.

Several promising anticancer drug candidates have been sidelined owing to their poor physicochemical properties or unfavorable pharmacokinetics, resulting in high overall cost of drug discovery and development. Use of alternative formulation strategies that alleviate these issues can help advance new molecules to the clinic at a significantly lower cost. Tylocrebrine is a natural product with potent anticancer activity. Its clinical trial was discontinued following the discovery of severe central nervous system toxicities. To improve the safety and potency of tylocrebrine, we formulated the drug in polymeric nanoparticles targeted to the epidermal growth factor receptor (EGFR) overexpressed on several types of tumors. Through in vitro studies in different cancer cell lines, we found that EGFR targeted nanoparticles were significantly more effective in killing tumor cells than the free drug. In vivo pharmacokinetic studies revealed that encapsulation in nanoparticles resulted in lower brain penetration and enhanced tumor accumulation of the drug. Further, targeted nanoparticles were characterized by significantly enhanced tumor growth inhibitory activity in a mouse xenograft model of epidermoid cancer. These results suggest that the therapeutic index of drugs that were previously considered unusable could be significantly improved by reformulation. Application of novel formulation strategies to previously abandoned drugs provides an opportunity to advance new molecules to the clinic at a lower cost. This can significantly increase the repertoire of treatment options available to cancer patients.

Folic acid functionalized nanoparticles for enhanced oral drug delivery.

The oral absorption of drugs that have poor bioavailability can be enhanced by encapsulation in polymeric nanoparticles. Transcellular transport of nanoparticle-encapsulated drug, possibly through transcytosis, is likely the major mechanism through which nanoparticles improve drug absorption. We hypothesized that the cellular uptake and transport of nanoparticles can be further increased by targeting the folate receptors expressed on the intestinal epithelial cells. The objective of this research was to study the effect of folic acid functionalization on transcellular transport of nanoparticle-encapsulated paclitaxel, a chemotherapeutic with poor oral bioavailability. Surface-functionalized poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles loaded with paclitaxel were prepared by the interfacial activity assisted surface functionalization technique. Transport of paclitaxel-loaded nanoparticles was investigated using Caco-2 cell monolayers as an in vitro model. Caco-2 cells were found to express folate receptor and the drug efflux protein, p-glycoprotein, to high levels. Encapsulation of paclitaxel in PLGA nanoparticles resulted in a 5-fold increase in apparent permeability (Papp) across Caco-2 cells. Functionalization of nanoparticles with folic acid further increased the transport (8-fold higher transport compared to free paclitaxel). Confocal microscopic studies showed that folic acid functionalized nanoparticles were internalized by the cells and that nanoparticles did not have any gross effects on tight junction integrity. In conclusion, our studies indicate that folic acid functionalized nanoparticles have the potential to enhance the oral absorption of drugs with poor oral bioavailability.

Intrinsic targeting of inflammatory cells in the brain by polyamidoamine dendrimers upon subarachnoid administration.

AIM: Understanding the interactions between nanomaterials and disease processes is crucial for designing effective therapeutic approaches. This article explores the unusual neuroinflammation targeting of dendrimers (with no targeting ligands) in the brain, with significant consequences for nanoscale materials in medicine. METHOD: The in vivo biodistribution of fluorescent-labeled neutral generation-4- polyamidoamine dendrimers (∼4 nm) in a rabbit model of cerebral palsy was explored following subarachnoid administration. RESULTS: These dendrimers, with no targeting ligands, were localizing in activated microglia and astrocytes (cells responsible for neuroinflammation), even in regions far moved from the site of injection, in newborn rabbits with maternal inflammation-induced cerebral palsy. CONCLUSION: This intrinsic ability of dendrimers to localize inactivated microglia and astrocytes can enable targeted delivery of therapeutics in disorders such as cerebral palsy, Alzheimer's and multiple sclerosis.

Interfacial activity assisted surface functionalization: a novel approach to incorporate maleimide functional groups and cRGD peptide on polymeric nanoparticles for targeted drug delivery.

Nanoparticles formulated using poly(d,l-lactide-co-glycolide) (PLGA) copolymer have emerged as promising carriers for targeted delivery of a wide variety of payloads. However, an important drawback with PLGA nanoparticles is the limited types of functional groups available on the surface for conjugation to targeting ligands. In the current report, we demonstrate that the interfacial activity assisted surface functionalization (IAASF) technique can be used to incorporate reactive functional groups such as maleimide onto the surface of PLGA nanoparticles. The surface maleimide groups were used to conjugate cRGD peptide to nanoparticles. The cRGD peptide targets alpha(v)beta(3) integrins overexpressed on tumor vasculature and some tumor cells, and was used as model targeting ligand in this study. Incorporation of biologically active cRGD peptide on the surface of nanoparticles was confirmed by in vitro cell uptake studies and in vivo tumor accumulation studies. Functionalization of nanoparticles with cRGD peptide increased the cellular uptake of nanoparticles 2-3-fold, and this enhancement in uptake was substantially reduced by the presence of excess cRGD molecules. In a syngeneic mouse 4T1 tumor model, cRGD functionalization resulted in increased accumulation and retention of nanoparticles in the tumor tissue (nearly 2-fold greater area under the curve), confirming the in vivo activity of cRGD functionalized nanoparticles. In conclusion, the IAASF technique enabled the incorporation of reactive maleimide groups on PLGA nanoparticles, which in turn permitted efficient conjugation of biologically active cRGD peptide to the surface of PLGA nanoparticles.