Characterization and cytotoxicty of nanostructured lipid carriers formulated with olive oil, hydrogenated palm oil and polysorbate 80.

Nanostructured lipid carriers (NLC) composed of solid and liquid lipids, and surfactants are potentially good colloidal drug carriers. Before NLC can be used as drug carriers, the cytotoxicity of their components must be ascertained. The cytotoxicity of solid lipids (trilaurin, palmitin, docosanoid acid, and hydrogenated palm oil [HPO]) and surfactants (Polysorbate 20, 80, and 85) were determined on BALB/c 3T3 cells. The HPO and Polysorbate 80 were least cytotoxic and used with olive oil in the formulation of NLC. The particle size, polydispersity index, zeta potential, specific surface area, and crystallinity index of the NLC were 61.14 nm, 0.461, -25.4 mV, and 49.07 m2 and 27.12% respectively, while the melting point was 4.3°C lower than of HPO. Unlike in serum-free, NLC incubated in fetal bovine serum-supplemented medium did not show particle growth, suggesting that serum proteins in medium inhibit nanoparticles aggregation. The study also showed that NLC was less toxic to BALB/c 3T3 cells than Polysorbate 80. Thus, NLC with olive oil, HPO, and Polysorbate 80 as components are potentially good drug carriers with minimal cytotoxicity on normal cells

Tamoxifen-loaded nanostructured lipid carrier as a drug delivery system: characterization, stability assessment and cytotoxicity

Cancer nanotherapeutics is beginning to overwhelm the global research and viewed to be the revolutionary treatment regime in the medical field. This investigation describes the development of a stable nanostructured lipid carrier (NLC) system as carrier for Tamoxifen (TAM). The TAM-loaded NLC (TAM-NLC) developed with 200 mg of TAM showed a spherical particle with the size of 46.6 nm, polydispersity index of 0.267, entrapment efficiency of 99.74% and with the zeta potential of -23.78 mV. Besides, the equivalent cytotoxicity of TAM and TAM-NLC to human (MCF-7) and mice (4T1) mammary breast cancer cell lines were observed. Incubating the formulation at the physiological pH resulted into reduced Ostwald ripening rate but without any significant change in the absorptivity. When coupled with the measurements of zeta potential and Ostwald ripening rate, the absorbance assay may be used to predict the long-term stability of drug-loaded nanoparticle formulations. The results of the study also suggest that TAM-NLC is a promising drug delivery system for breast cancer therapy. This is the first encouraging report on the in vitro effect of TAM-NLC against human and mouse mammary adenocarcinoma cell lines

PAMAM dendrimer roles in gene delivery methods and stem cell research

Nanotechnology has provided new technological opportunities, which could help in challenges confronting stem cell research. Polyamidoamine (PAMAM) dendrimers, a new class of macromolecular polymers with high molecular uniformity, narrow molecular distribution specific size and shape and highly functionalised terminal surface have been extensively explored for biomedical application. PAMAM dendrimers are also nanospherical, hyperbranched and monodispersive molecules exhibiting exclusive properties which make them potential carriers for drug and gene delivery

Zerumbone-loaded nanostructured lipid carrier induces apoptosis in human colorectal adenocarcinoma (Caco2) cell line

The incorporation of zerumbone (Zer) into nanostructured lipid carrier (NLC) is hypothesized to increase the efficacy of the drug. Nanostructured lipid carrier has sustained-drug release characteristics and is able to improve the solubility and bioavailability of the lipophilic drug. In this study, the anti-cancer effect of Zer was tested on human colorectal adenocarcinoma (Caco-2) cell line. The effect of Zer, zerumbone-loaded nanostructured lipid carrier (Zer-NLC) and NLC on the Caco-2 cell viability were determined using the MTT assay. The treatment concentration ranges from 0 to 120 μM at four different time intervals (i.e., 0 h, 24 hrs, 48 hrs and 72 hrs) were evaluated. At 24 hrs, the half-growth inhibitory concentration (GI50) of Zer-NLC (i.e., 4.25 μM) is lower than that of Zer (i.e., 23.75 μM). However, Zer outperformed the Zer-NLC at the subsequent time points. Similar trend was observed in other parameters including the cytostatic concentration (CC) and half-lethal concentration 50 (LC50). Phase contrast imaging and AO/PI fluorescence staining were performed at the CC and LC50 values. The morphological changes and the apoptosis features could be seen in cells treated with Zer and Zer-NLC while cells treated with NLC showed minor morphological changes. The cells treated with Zer-NLC demonstrated a slightly slower progression of apoptosis, which could be due to the controlled release of Zer from the NLC matrix. It was concluded that the incorporation of Zer into NLC did not compromise the potency and efficacy of the drug

Zerumbone-loaded nanostructured lipid carriers: preparation, characterization, and antileukemic effect

Zerumbone, a natural dietary lipophilic compound with low water solubility (1.296 mg/L at 25°C) was used in this investigation. The zerumbone was loaded into nanostructured lipid carriers using a hot, high-pressure homogenization technique. The physicochemical properties of the zerumbone-loaded nanostructured lipid carriers (ZER-NLC) were determined. The ZER-NLC particles had an average size of 52.68 ± 0.1 nm and a polydispersity index of 0.29 ± 0.004 μm. Transmission electron microscopy showed that the particles were spherical in shape. The zeta potential of the ZER-NLC was −25.03 ± 1.24 mV, entrapment efficiency was 99.03%, and drug loading was 7.92%. In vitro drug release of zerumbone from ZER-NLC was 46.7%, and for a pure zerumbone dispersion was 90.5% over 48 hours, following a zero equation. Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human T-cell acute lymphoblastic leukemia (Jurkat) cells, the half maximal inhibitory concentration (IC50) of ZER-NLC was 5.64 ± 0.38 μg/mL, and for free zerumbone was 5.39 ± 0.43 μg/mL after 72 hours of treatment. This study strongly suggests that ZER-NLC have potential as a sustained-release drug carrier system for the treatment of leukemia

Effect of recombinant human erythropoietin and bovine lactoferrin on canine mammary gland tumor cell

Adjuvant chemotherapy is recommended for metastatic canine mammary gland tumor. Erythropoietin receptors (EPOR) was once thought to be only expressed on the surfaces of the erythroid progenitor cells. Recently, EPOR have been identified in several neoplastic cell lines and solid tumors including human and canine mammary gland tumors. Bovine lactoferrin (bLF) has several biological activities, including anti-tumor effect on some human and animal tumors. Clinical trials have been carried out in human medicine based on these effects. In this in vitro study, doxorubicin, recombinant human erythropoietin (rHuEPO) and bLF were used separately and in combination in order to determine the effect of different drugs on canine mammary gland tumor. Recombinant human erythropoietin was found to have a non-significant effect on the canine mammary gland tumor cell line. Doxorubicin alone gave a more promising result in cytotoxic effect of the cells in a dose-dependent manner. Bovine lactoferrin however did not show a clear anti-proliferative pattern on the tumor cells. The drug combination treatment did not show better anti-proliferative or cytotoxic effect on the cells than doxorubicin alone. The combination of these drugs induced growth arrest at G2/M phase

Effect of hypoxia on the response of canine mammary gland tumor cells to bovine lactoferrin, doxorubucin and recombinant human erythropoietin

The exact role of hypoxia in tumor biology remains controversial because there is no conclusive evident on its effect on tumors. There is concern that tumor hypoxia is one of the causes of chemoresistance in cancer cells. Recently, erythropoietin receptors have been found in human breast cancer cells indicating that recombinant human erythropoietin (rHuEPO) treatment of cancer-related anemia can influence the functions of the cells. Bovine lactoferrin (bLF) was also shown to have antiproliferative effects on cancer cells. The aim of this study was to determine the in vitro effects of rHuEPO, bLF and Doxorubicin (DOX) on a canine mammary gland tumor cell line, CMT-stylo cells, under hypoxic condition. The cells were treated with bLF, rHuEPO, DOX rHuEPO and DOX and bLF, rHuEPO combinations. These treated cells were subjected to MTT assay. The results showed that hypoxia lowered the proliferation rate of the CMT-stylo cells while combination treatments showed improved killing. Flow cytometry analysis showed that DOX had cytotoxic while bLF had antiproliferative effects on the CMT-stylo cells

Zerumbone-loaded nanostructured lipid carrier induces apoptosis of canine mammary Adenocarcinoma cells

Canine mammary gland tumor (CMT) is the most common tumor in intact female dog. Zerumbone (ZER) has promising anticancer properties, but plagued with poor water solubility, poor absorption, bioavailability, and delivery to target tissues. To solubilize, ZER was loaded into nanostructured lipid carrier (NLC) to produce ZER-loaded NLC (ZER-NLC). Te objectives of this study were to determine the antiproliferative efect and the mode of cell death induced by ZER-NLC and ZER on a canine mammary gland tumor (CMT) adenocarcinoma primary cell line. Tere was no signifcant diference (p>0.05) between ZER-NLC and ZER treatments in the inhibition of CMT cell proliferation; thus, the loading of ZER into NLC did not compromise the cytotoxic efect of ZER. Microscopically, ZER-NLC- and ZER-treated CMT cells showed apoptotic cell morphology. ZER-NLC and ZER treatments signifcantly downregulated the antiapoptotic Bcl-2 and upregulated the proapoptotic Bax gene expressions in CMT cells. Both ZER-NLC and ZER-treated CMT cells showed signifcant (p<0.0001) increases in caspase-8, -9, and -3/7 protein activities. In conclusion, ZER-NLC induced CMT cell death via regulation of Bcl-2 and Bax gene expressions and caspase activations, indicating the involvement of both the intrinsic and extrinsic pathways of apoptosis. Tis study provided evidences for the potential of ZERNLC as an anticanine mammary gland adenocarcinoma chemotherapy

Induction of cell cycle arrest and apoptosis by copper complex Cu(SBCM)₂ towards oestrogen-receptor positive MCF-7 breast cancer cells

Copper complexes have the potential to be developed as targeted therapy for cancer because cancer cells take up larger amounts of copper than normal cells. Copper complex Cu(SBCM)2 has been reported to induce cell cycle arrest and apoptosis towards triple-negative breast cancer cells. Nevertheless, its effect towards other breast cancer subtypes has not been explored. Therefore, the present study was conducted to investigate the effect of Cu(SBCM)₂ towards oestrogen-receptor positive MCF-7 breast cancer cells. Growth inhibition of Cu(SBCM)₂ towards MCF-7 and human non-cancerous MCF-10A breast cells was determined by MTT assay. Morphological changes of Cu(SBCM)2-treated-MCF-7 cells were observed under an inverted microscope. Annexin V/PI apoptosis assay and cell cycle analysis were evaluated by flow cytometry. The expression of wild-type p53 protein was evaluated by Western blot analysis. The intracellular ROS levels of MCF-7 treated with Cu(SBCM)₂ were detected using DCFH-DA under a fluorescence microscope. The cells were then co-treated with Cu(SBCM)₂ and antioxidants to evaluate the involvement of ROS in the cytotoxicity of Cu(SBCM)2. Docking studies of Cu(SBCM)2 with DNA, DNA topoisomerase I, and human ribonucleotide reductase were also performed. The growth of MCF-7 cells was inhibited by Cu(SBCM)2 in a dose-dependent manner with less toxicity towards MCF-10A cells. It was found that Cu(SBCM)₂ induced G2/M cell cycle arrest and apoptosis in MCF-7 cells, possibly via a p53 pathway. Induction of intracellular ROS was not detected in MCF-7 cells. Interestingly, antioxidants enhance the cytotoxicity of Cu(SBCM)2 towards MCF-7 cells. DNA topoisomerase I may be the most likely target that accounts for the cytotoxicity of Cu(SBCM)₂

Lipid nanoparticles in anti-breast cancer drug delivery systems and drug-membrane interactions

Current anticancer drugs are plagued with lack of sustained effect and poor delivery. Many current studies focus on the use of drug carriers, particularly lipid nanoparticles as new drug delivery systems. In this study, a nanostructured lipid carrier (NLC) was formulated to serve as a carrier for tamoxifen (TAM). The study is also undertaken to determine the drugmembrane interaction through the use of liposomes as a membrane model. Hence, the main objectives of this study are to develop and determine the physicochemical and biological properties of NLC loaded with TAM (TAMNLC), and to determine the interaction between the trimethoxybenzoyl analogue of catechin gallate (TMCG) and lipid membrane. The NLC and TAM-NLC were prepared by high pressure homogenisation method. The lipid phase consisted of hydrogenated palm oil, olive oil and phosphatidylcholine as the lipid phase, while the aqueous phases are polysorbate 80, sorbitol, thimerosal and double-distilled water. The major components in the formulation were carefully chosen based on the absence of cytotoxicity towards a normal cell line (murine fibroblasts, BALB/c 3T3). The physicochemical characteristics of NLC, i.e. particle size, zeta potential (ZP), thermal profile, crystallinity, morphology and stability were assessed by photon correlation spectroscopy (PCS), laser doppler velocimetry, differential scanning calorimetry (DSC), wide-angle X-ray diffractometry (WAXD), transmission electron microscopy (TEM) and spectrophotometry, respectively. The release kinetics of TAM-NLC was determined by the Franz diffusion cell system while its cytotoxicity was determined in vitro on the human breast (MCF-7) and mouse mammary (4T1) cancer cell lines. To determine the drug-membrane interaction, a dried lipid film of 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) was used to form liposomes and to entrap TMCG and quinone methide (QM, metabolite of TMCG) separately. The interaction of drug with the model membrane was assessed by DSC, WAXD, smallangle X-ray diffractometry (SAXD) and Fourier-transform infrared (FTIR). The PyMOL software was used to construct the molecular model of drug and membrane interaction.The study showed that hydrogenated palm oil, trilaurin and docosanoic acid were significantly less cytotoxic than palmitin. Since surfactants influenced the physicochemical properties of the NLC, polysorbate 20 and 80 were assessed for use in the NLC formulation. The NLC formulated with polysorbate 80 showed good compatibility with the lipid phase, while polysorbate 20 caused destabilisation of the nanoparticles that resulted in phase separation during storage. With polysorbate 80 as surfactant, the NLCs are relatively spherical, with an average size of 102.8 nm, zeta potential of -30.57 mV, and possessed superior particle surface area to volume ratios. The transition temperature of NLC formulated with polysorbate 80 was 55.85 °C, which was lower than that formulated with polysorbate 20 or unprocessed lipid. The results indicated that NLC formulated with polysorbate 80 is of lower crystallinity and this was confirmed by WAXD. The NLC was also shown to be of low cytotoxicity to BALB/c 3T3 cell line. The NLC incubated with foetal bovine serum-supplemented media did not show increase in particle size, suggesting that its stability is good and practicality for use in intravenous administration. The stability of TAM-NLC was determined by storage at physiological pHs. The formulation is more stable at pH 7.4 (blood pH) even though its ZP was lower compared to pH 2.3 (stomach pH). The release of TAM from TAM-NLC followed first-order kinetics, while showing high cytotoxicity to MCF-7 and 4T1 cell lines with halfminimal inhibitory concentration of 5.56 and 5.19 μg mL-1, respectively. To determine the drug-membrane interaction, TMCG was used as the prodrug model and liposomes as the cell membrane model. The DSC analysis showed that TMCG was incorporated into DPPC membranes and had intercalated in-between the phospholipids molecules while reducing the cooperativity and lowering the transition temperature of the gel to liquidcrystalline phase. In addition, TMCG did not affect the macroscopic bilayer organisation of the liposomes; instead it decreased the thickness of the bilayer by forming an interdigitated gel phase. Quinone methide, the active form of TMCG however, showed limited interaction with the phospholipid bilayer indicating that a superficial interaction had occurred between QM and the phospholipid membrane with a weak gel stabilising effect and decreased hydrogen-bonding pattern of the interfacial region of the phospholipid. These results concur with the molecular dynamics simulation studies, which showed that TMCG was incorporated into the membrane phospholipid palisade while QM was excluded and interacted weakly with the polar portion of the lipid bilayer. In conclusion, the study showed that the optimised NLC formulation with low cytotoxicity is a superior vehicle for encapsulation and carriage of TAM. The TAM-NLC developed in this study showed controlled-released characteristics, good stability at physiological pH with potential for tumour targeting. The study also reinforced the reliability of liposomes as cell membrane models, and TMCG interacts very well with it