Darunavir-Loaded Lipid Nanoparticles for Targeting to HIV Reservoirs

Darunavir has a low oral bioavailability (37%) due to its lipophilic nature, metabolism by cytochrome P450 enzymes and P-gp efflux. Lipid nanoparticles were prepared in order to overcome its low bioavailability and to increase the binding efficacy of delivery system to the lymphoid system. Darunavir-loaded lipid nanoparticles were prepared using high-pressure homogenization technique. Hydrogenated castor oil was used as lipid. Peptide, having affinity for CD4 receptors, was grafted onto the surface of nanoparticles. The nanoparticles were evaluated for various parameters. The nanoparticles showed size of less than 200 nm, zeta potential of ? 35.45 mV, and a high drug entrapment efficiency (90%). 73.12% peptide was found conjugated to nanoparticles as studied using standard BSA calibration plot. Permeability of nanoparticles in Caco-2 cells was increased by 4-fold in comparison to plain drug suspension. Confocal microscopic study revealed that the nanoparticles showed higher uptake in HIV host cells (Molt-4 cells were taken as model containing CD4 receptors) as compared to non-CD4 receptor bearing Caco-2 cells. In vivo pharmacokinetic in rats showed 569% relative increase in bioavailability of darunavir as compared to plain drug suspension. The biodistribution study revealed that peptide-grafted nanoparticles showed higher uptake in various organs (also in HIV reservoir organs namely the spleen and brain) except the liver compared to non-peptide-grafted nanoparticles. The prepared nanoparticles resulted in increased binding with the HIV host cells and thus could be promising carrier in active targeting of the drugs to the HIV reservoir.     

Preparation of carbon dot‐based ratiometric fluorescent probes for cellular imaging from Curcuma longa

This work derived biocompatible and stable probes based on fluorescent nanoparticles (FNPs) from a natural source, Curcuma longa. The multi‐color fluorescence emissions from carbonized Curcuma longa (C‐FNPs) obtained through defined dehydration conditions are soluble in water and have a small particle size (~17 nm). The surface passivation with polyethylene glycol (PEG) capped with amine groups in FNPs (P‐FNPs) generated a probe with a higher quantum yield and longer fluorescence lifetime than obtained with C‐FNPs. The X‐ray photoelectron spectroscopy and X‐ray diffraction spectra confirmed the associated chemical moieties of C‐FNPs and P‐FNPs. Furthermore, the prepared material showed non‐toxic effects with almost 100% cell viability, even at high concentrations. In conclusion, fluorescence sensors from natural sources may be useful for numerous biomedical research applications.     

A model predicting delivery of saquinavir in nanoparticles to human monocyte/macrophage (Mo/Mac) cells

Modeling the influence of a technology such as nanoparticle systems on drug delivery is beneficial in rational formulation design. While there are many studies showing drug delivery enhancement by nanoparticles, the literature provides little guidance regarding when nanoparticles are useful for delivery of a given drug. A model was developed predicting intracellular drug concentration in cultured cells dosed with nanoparticles. The model considered drug release from nanoparticles as well as drug and nanoparticle uptake by the cells as the key system processes. Mathematical expressions for these key processes were determined using experiments in which each process occurred in isolation. In these experiments, intracellular delivery of saquinavir, a low solubility drug dosed as a formulation of poly(ethylene oxide)-modified poly(epsilon- caprolactone) (PEO-PCL) nanoparticles, was studied in THP-1 human monocyte/macrophage (Mo/Mac) cells. The model accurately predicted the enhancement in intracellular concentration when drug was administered in nanoparticles compared to aqueous solution. This simple model highlights the importance of relative kinetics of nanoparticle uptake and drug release in determining overall enhancement of intracellular drug concentration when dosing with nanoparticles.     

Controlled release of all-trans-retinoic acid from PEGylated gelatin nanopaticles by enzymatic degradation

A new targeting drug carrier for anticancer drug, all-trans-retinoic acid (atRA), was proposed by using angiogenesis which is one of the specific physiological properties of cancer cells. The proposed drug carrier was prepared as PEGylated gelatin nanoparticle (176 nm size). The gelatin molecules were aggregated by coupled deoxycholic acid and the surface of the nanoparticles was covered by polyethylene glycol to reduce reticuloendothelial system (RES) uptake. To prove the feasibility of the nanoparticles as a targeting drug carrier, the degradation of the nanoparicles by collagenase IV and the release pattern of atRA from the nanoparticles by enzymatic degradation were evaluated. The PEGylated gelatin nanoparticles were significantly degraded by collagenase IV within 10 seconds, with most of them degraded within 1 min. When atRA loaded in the PEGylated gelatin nanoparticles was released in phosphate buffered saline (PBS), only twelve percent of atRA were released for one hour. However, when the nanoparticles were put into PBS with collagenase IV of 0.1 μM, a burst effect of atRA was about 40% for the initial 10 min, followed by a continuous release of atRA upto 75% for 5 hr. Therefore, the PEGylated gelatin nanoparticles released anticancer drug very sensitively by collagenase IV, which is one of major matrix metalloproteases involved in angiogenesis. These results showed a feasibility that PEGylated gelatin nanoparticles could be used as a new targeting anticancer drug carrier using angiogenesis as a specific physiological property of cancer cells.     

Effect of cross-linking on the performance of micelles as drug delivery carriers: a cell uptake study

Poly(polyethylene glycol methyl ether methacrylate-co-methacrylic acid)-block-poly(methyl methacrylate) P(PEGMEMA-co-MAA)-b-PMMA block copolymer were prepared via RAFT (reversible addition-fragmentation chain transfer) polymerization and subsequently self-assembled into micelles as a drug delivery carrier for albendazole (ABZ). For comparison, the micelles were additionally cross-linked to study the effect of shell-cross-linking on the biological activity. The hydrodynamic diameter of cross-linked and un-cross-linked micelles was approximately 40 nm in both cases. While the cross-linked micelle was stable even in good solvents for both blocks, the un-cross-linked micelle was found to lose its integrity in cell growth media. Crosslinking had a major effect on the rate of drug release reducing it dramatically from 50% (uncrosslinked) to around 20% (crosslinked) over a 30 h incubation period. Both drug delivery systems were tested on human prostate cancer cells (PC-3, DU-145) and human ovarian cancer cells (OVCAR-3, A-2780). No toxic effects were measured with the unloaded micelle while the ABZ loaded un-cross-linked micelle lead to IC(50) values between 0.2 and 0.9 μM depending on the cell line. The IC(50) dropped to values between 0.006 and 0.06 μM, depending on cell line, once the micelles were stabilized by cross-linking. Three treatment cycles with ABZ for one day, followed by two days incubation in media using ABZ-loaded drug carriers led to complete cell death even at low concentrations in the case of the cross-linked micelle only. Cellular uptake has been studied using fluorescently labeled micelles and Nile red as model drug, showing cell uptake above the CMC but no micelle uptake below the CMC. Additional biological studies, such as colony formation assay and tubulin disorganization tests, were also performed to gain more insight into the effect of cross-linking of the shell of the micelle. In conclusion, shell-cross-linking is highly recommended, even for glassy micelles, for an efficient cellular uptake at low concentrations.     

Size- and coating-dependent uptake of polymer-coated gold nanoparticles in primary human dermal microvascular endothelial cells

A library-orientated approach is used to gain understanding of the interactions of well-defined nanoparticles with primary human endothelial cells, which are a key component of the vasculature. Fifteen sequentially modified gold nanoparticles (AuNPs) based on three different core sizes (18, 35, 65 nm) and five polymeric coatings were prepared. The synthetic methodology ensured homogeneity across each series of particles to allow sequential investigation of the chemical features on cellular interactions. The toxicity of these nanoparticles, their uptake behavior in primary human dermal microvascular endothelial cells (HDMECs), and quantification of uptake were all investigated. The results of our studies indicated that high concentrations of gold nanoparticles (250 μg/mL) were nontoxic and that the number of internalized nanoparticles was related to nanoparticle size and surface chemistry. In summary, the positive-charged ethanediamine-coated AuNPs were internalized to a greater extent than the negative- or neutral-charged AuNPs. Moreover, differences in the amounts of internalized AuNPs could be shown for the three neutral-charged AuNPs, whereas the uptake of hydroxypropylamine-coated particles was preferred compared with glucosamine-coated or PEGylated AuNPs. Hydroxypropylamine-coated AuNPs were found to be the most efficient neutral-charged particles in overcoming the endothelial cell barrier and entering the cell.     

Fibrin nanoparticles as Possible vehicles for drug delivery

Background

Several issues have been raised emphasizing the harmful toxic effects of metal nanoparticles towards biological systems. Search of biological nanoparticles with excellent biocompatibility and bioavailability could address this problem.

Methods

Fibrin nanoparticles (FNP) were prepared using a novel technique and characterized for their physico-chemical properties. In vitro studies were performed to examine cytotoxicity and cellular uptake of FNP. Innate immune response to FNP was studied by (i) estimating in vitro generation of complement split products, C3a and C4d and (ii) in vivo expression of pro-inflammatory cytokines, TNF-α, IL-1 and IL-6. In vivo biodistribution study was carried out by intravenous administration of FITC-labelled FNP in mice.

Results

FNP were spherical with size ranging from 25 to 28 nm. In vitro studies proved the biocompatibility of the nanoparticles, with their distribution across the cytoplasm and nucleus of treated cells. Complement activation studies showed insignificant increase in the level of C3a when compared with positive control. RT-PCR results revealed significant upregulation of TNF-α and downregulation of IL-6 cytokines after 6 h of FNP administration. In vivo biodistribution studies showed moderate blood circulation time, with predominant distribution of nanoparticles in the liver followed by the lungs, kidney and spleen. Haematology, serum biochemistry, and histopathology analyses demonstrated that FNP were non-toxic.

Conclusion

Owing to their small size, low cost, ease of preparation and excellent biocompatibility, FNP might be a promising novel material for drug delivery applications.

General significance

Our results demonstrate the safe and promising use of FNP for biomedical applications.     

Docetaxel-loaded nanostructured lipid carriers functionalized with trastuzumab (Herceptin) for HER2-positive breast cancer cells

Abstract

The aim of the present study was to prepare Herceptin targeted nanostructured lipid carriers (NLCs) of docetaxel (DTX). Herceptin was conjugated by chemical and physical methods to NLCs prepared by solvent extraction technique followed by probe sonication. Different types of fatty amines were used in construction of NLCs. The NLCs were characterized for their antibody coupling efficiency, particle size, zeta potential, polydispersity index, drug entrapment efficiency and drug release profiles. The toxicity of NLCs on MDA-MB-468 (HER2 negative receptor) and BT-474 (HER2 positive) breast cancer cell lines was evaluated by MTT assay. Also their cellular uptake was studied by flow-cytometry and fluorescent microscopy. The results showed the NLCs containing stearyl amine had the lowest particle size, the highest zeta potential and antibody coupling efficiency values. Herceptin binding to NLCs led to reduction in zeta potential and drug entrapment efficiency while, particle size increased. The NLCs containing spermine(SP) released DTX slower than other fatty amines. Non-conjugated nanoparticles containing DTX had more toxicity than the free DTX on both cell lines. Herceptin targeted NLCs caused more mortality on BT-474 cells than MDA-MB-468 cells. Flow-cytometry studies revealed enhanced cellular uptake of nanoparticles chemically conjugated by Herceptin on the BT-474 cells. DTX loaded in chemically conjugated NLCs to Herceptin showed more cytotoxic effects than the physically coated nanoparticles. The Herceptin conjugated NLCs seem promising in oriented delivery of DTX to HER2 positive breast cancer cells.     

Cellular uptake mechanism of an inorganic nanovehicle and its drug conjugates: Enhanced efficacy due to clathrin-mediated endocytosis

We present the mechanism for the cellular uptake of layered double hydroxide (LDH) nanoparticles that are internalized into MNNG/HOS cells principally via clathrin-mediated endocytosis. The intracellular LDHs are highly colocalized with not only typical endocytic proteins, such as clathrin heavy chain, dynamin, and eps15, but also transferrin, a marker of the clathrin-mediated process, suggesting their specific internalization pathway. LDHs loaded with an anticancer drug (MTX-LDH) were also prepared to confirm the efficacy of LDHs as drug delivery systems. The cellular uptake of MTX was higher in MTX-LDH-treated cells than in MTX-treated cells, giving a lower IC50 value for MTX-LDH than for MTX only. The inhibition of the cell cycle was greater for MTX-LDH than for MTX only. This result clearly shows that the internalization of LDH nanoparticles via clathrin-mediated endocytosis may allow the efficient delivery of MTX-LDH in cells and thus enhance drug efficacy.     

Metformin-Loaded Hyaluronic Acid Nanostructure for Oral Delivery

The objective of this study was to develop a nanodelivery system containing a mucoadhesive polymer hyaluronic acid (HA) for oral delivery. Metformin was used as a model drug. Blank and drug-loaded HA nanostructures were prepared by precipitation method and characterized for particle size (PS), zeta potential (ZP), physical stability (over 65 days), surface morphology, moisture content, and physical state of the drug in the nanostructures. The cytotoxicity and hemolysis potential of the delivery system was assessed in Caco-2 cells and whole human blood, respectively. The in vitro release of metformin and its uptake in Caco-2 cells was evaluated using high-performance liquid chromatography. Ex vivo permeability of metformin was measured through goat intestinal membrane. The nanoparticles were physically stable and neutrally charged with an average PS of 114.53?±?12.01 nm. This nanodelivery system existed as nanofibers containing metformin in a crystalline state. This delivery system released the drug rapidly with >?50% of metformin released within 1 h. Cellular uptake studies on Caco-2 cells indicated higher uptake of metformin from nanoparticle as compared to metformin in solution, up to first 45 min. Ex vivo permeability studies on the other hand showed a higher metformin permeability from solution relative to that from nanoparticles through the goat intestinal membrane. Metformin nanoparticles were non-toxic at therapeutic concentrations in Caco-2 cells and showed no hemolytic effect to RBCs. This study indicates the preparation, characterization, as well as the potential use of HA nanostructures for oral delivery.     

Clear,Aqueous Topical Drop of Triamcinolone Acetonide

The objective of this study was to develop a clear aqueous mixed nanomicellar formulation (NMF) of triamcinolone acetonide (TA) with a combination of nonionic surfactant hydrogenated castor oil 60 (HCO-60) and octoxynol-40 (Oc-40). In order to delineate the effects of drug-polymer interactions on entrapment efficiency (EE), loading efficiency (LE), and critical micellar concentration (CMC), a design of experiment (DOE) was performed to optimize the formulation. In this study, full-factorial design has been used with HCO-60 and OC-40 as independent variables. All formulations were prepared following solvent evaporation and film rehydration method, characterized with size, polydispersity, shape, morphology, EE, LE, and CMC. A specific blend of HCO-60 and Oc-40 at a particular wt% ratio (5:1.5) produced highest drug EE, LE, and smallest CMC (0.0216 wt%). Solubility of TA in NMF improved 20 times relative to normal aqueous solubility. Qualitative ¹H NMR studies confirmed the absence of free drug in the outer aqueous NMF medium. Moreover, TA-loaded NMF appeared to be highly stable and well tolerated on human corneal epithelial cells (HCEC) and human retinal pigment epithelial cells (D407 cells). Overall, these studies suggest that TA in NMF is safe and suitable for human topical ocular drop application.     

Cellular uptake of Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles synthesized through solvent emulsion evaporation and nanoprecipitation method

Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles have been widely studied for drug delivery. The aim of this study is to determine how cellular uptake of these nanoparticles is influenced by different surface properties, incubation time, particle concentration and cell types. Spherical coumarin-6 loaded PLGA nanoparticles with a size of about 100 nm were synthesized through solvent emulsion evaporation and nanoprecipitation methods. In vitro cellular uptake efficiency was determined using human bronchial epithelial cells (BEAS-2B) and murine monocyte-derived macrophage (RAW264.7) cells. PLGA nanoparticles were incubated with these cells in a concentration range of 10-300 μg/ml for different time periods. The results show that cellular uptake decreased for nanoparticles surface coated with PVA surfactant and was especially limited for severely aggregated particles. At higher particle concentration, the total amount of particles taken up by cells increased while the uptake efficiency decreased. In addition, cells could take up more particles with longer incubation time, although the uptake rate decreased gradually with time. Finally, RAW264.7 cells show increased uptake compared to BEAS-2B cells. The information drawn from this study would provide important clues on how nanomaterials interact with cells and how these interactions can influence biocompatibility or toxicity.     

Targeted delivery of paclitaxel using folate-conjugated heparin-poly(β-benzyl-l-aspartate) self-assembled nanoparticles

A self-assembled nanoparticulate system composed of a folate-conjugated heparin-poly(β-benzyl-l-aspartate) (HP) amphiphilic copolymer was proposed for targeted delivery of the antineoplastic drug paclitaxel (PTX). PTX was incorporated into three types of heparin-based nanoparticles, including HP, folate-conjugated HP (FHP), and folate-polyethylene glycol (PEG)-conjugated HP (FPHP), using a simple dialysis method. The PTX-loaded nanoparticles were then characterized according to particle size (140-190 nm) and size distribution, drug-loading content and efficiency, and in vitro release behavior. In the cellular uptake study using KB cells positive for the folate-receptor (FR), FHP and FPHP nanoparticles showed a much higher cellular uptake than did unconjugated HP nanoparticles. Specifically, when the PEG spacer was inserted between the folate ligand and heparin backbone, FPHP nanoparticles had a greater cellular uptake than did FHP nanoparticles. The in vitro cytotoxicity of PTX-loaded HP, FHP, and FPHP nanoparticles was studied in KB cells and FR-negative A549 cells. Compared with the cytotoxicity in A549 cells, PTX-loaded FHP and FPHP nanoparticles exhibited more potent cytotoxicity in KB cells than did PTX-loaded HP nanoparticles and free-PTX, suggesting that the presence of folate enhanced intracellular uptake via FR-mediated endocytosis. In addition, FPHP nanoparticles exhibited much greater cytotoxicity in KB cells than did FHP nanoparticles. These results suggest that PTX-loaded folate-conjugated HP nanoparticles are a potentially useful delivery system for cancer cells positive for the folate-receptor.     

Intracellular Delivery of Nanoparticles of an Antiasthmatic Drug

The aim of the investigation was to prepare and characterize wheat germ agglutinin(WGA)-conjugated poly(d,l-lactic-co-glycolic) acid nanoparticles encapsulating mometasone furoate (MF) as a model drug and assess changes in its fate in terms of cellular interactions. MF loaded nanoparticles were prepared using emulsion–solvent evaporation technique. WGA-conjugation was done by carbodiimide coupling method. The nanoparticles were characterized for size, zeta potential, entrapment efficiency and in-vitro drug release. The intracellular uptake of nanoparticles, drug cellular levels, and anti-proliferative activity studies of wheat germ agglutinin-conjugated and unconjugated nanoparticles were assessed on alveolar epithelial (A549) cells to establish cellular interactions. Prepared nanoparticles were spherical with 10–15 μg/mg of WGA conjugated on nanoparticles. The size of nanoparticles increased after conjugation and drug entrapment and zeta potential reduced from 78 ± 5.5% to 60 ± 2.5% and −15.3 ± 1.9 to −2.59 ± 2.1 mV respectively after conjugation. From the cellular drug concentration–time plot, AUC was found to be 0.4745, 0.6791 and 1.24 for MF, MF-nanoparticles and wheat germ agglutinin-MF-nanoparticles respectively. The in-vitro antiproliferative activity was improved and prolonged significantly after wheat germ agglutinin-conjugation. The results conclusively demonstrate improved availability and efficacy of antiasthmatic drug in alveolar epithelial cell lines. Hence, a drug once formulated as mucoadhesive nanoparticles and incorporated in dry powder inhaler formulation may be used for targeting any segment of lungs for more improved therapeutic response in other lung disorders as well.     

Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells

The study was to develop paclitaxel-loaded formulations using a novel type of self-assembled nanoparticles that was composed of block copolymers synthesized from poly(gamma-glutamic acid) and poly(lactide) via a simple coupling reaction. The nanoparticles (the NPs) were prepared with various feed weight ratios of paclitaxel to block copolymer (the P/BC ratio). The morphology of all prepared nanoparticles was spherical and the surfaces were smooth. Increasing the P/BC ratio significantly increased the drug loading content of the prepared nanoparticles, but remarkably reduced the drug loading efficiency. The release rate of paclitaxel from the NPs decreased significantly as the P/BC ratio increased. For the potential of targeting liver cancer cells, galactosamine was further conjugated on the prepared nanoparticles (the Gal-NPs) as a targeting moiety. It was found that the activity in inhibiting the growth of HepG2 cells (a liver cancer cell line) by the Gal-NPs was comparable to that of a clinically available paclitaxel formulation, while the NPs displayed a significantly less activity. This may be attributed to the fact that the Gal-NPs had a specific interaction with HepG2 cells via ligand-receptor recognition. Cells treated with distinct paclitaxel formulations resulted in arrest in the G2/M phase. The arrest of cells in the G2/M phase was highly suggestive of interference by paclitaxel with spindle formation and was consistent with the morphological findings presented herein. In conclusion, the active targeting nature of the Gal-NPs prepared in the study may be used as a potential drug delivery system for the targeted delivery to liver cancers.     

Development of multifunctional hyaluronan-coated nanoparticles for imaging and drug delivery to cancer cells

Currently, there is high interest in developing multifunctional theranostic platforms for cancer monitoring and chemotherapy. Herein, we report hyaluronan (HA)-coated superparamagnetic iron oxide nanoparticles (HA-SPION) as a promising system for targeted imaging and drug delivery. When incubated with cancer cells, HA-SPIONs were rapidly taken up and the internalization of HA-SPION by cancer cells was much higher than the NPs without HA coating. The high magnetic relaxivity of HA-SPION coupled with enhanced uptake enabled magnetic resonance imaging of cancer cells. Furthermore, doxorubicin (DOX) was attached onto the nanoparticles through an acid responsive linker. While HA-SPION was not toxic to cells, DOX-HA-SPION was much more potent than free DOX to kill not only drug-sensitive but also multi-drug-resistant cancer cells. This was attributed to differential uptake mechanisms and cellular distributions of free DOX and DOX-HA-SPION in cancer cells.     

Novel microbial route to synthesize silver nanoparticles using spore crystal mixture of Bacillus thuringiensis

Metallic nanoparticles are traditionally synthesized by wet chemical techniques, where the chemicals used are often toxic and flammable. In the present study, the spore crystal mixture of Bacillus thuringiensis was used for the synthesis of silver nanoparticles. Nanoparticles were characterized using UV-Vis absorption spectroscopy, XRD and TEM. X-ray diffraction and TEM analysis showed the average particle size of 15 nm and mixed (cubic and hexagonal) structure. This is for the first time that any bacterial spore crystal mixture was used for the synthesis of nanoparticles. Further, these biologically synthesized nanoparticles were found to be highly toxic against different multi drug resistant human pathogenic bacteria.     

Sodium alginate/poly(vinyl alcohol)/nano ZnO composite nanofibers for antibacterial wound dressings

Sodium alginate (SA)/poly (vinyl alcohol) (PVA) fibrous mats were prepared by electrospinning technique. ZnO nanoparticles of size ∼160 nm was synthesized and characterized by UV spectroscopy, dynamic light scattering (DLS), XRD and infrared spectroscopy (IR). SA/PVA electrospinning was further carried out with ZnO with different concentrations (0.5, 1, 2 and 5%) to get SA/PVA/ZnO composite nanofibers. The prepared composite nanofibers were characterized using FT-IR, XRD, TGA and SEM studies. Cytotoxicity studies performed to examine the cytocompatibility of bare and composite SA/PVA fibers indicate that those with 0.5 and 1% ZnO concentrations are less toxic where as those with higher concentrations of ZnO is toxic in nature. Cell adhesion potential of this mats were further proved by studying with L929 cells for different time intervals. Antibacterial activity of SA/PVA/ZnO mats were examined with two different bacteria strains; Staphylococcus aureus and Escherichia coli, and found that SA/PVA/ZnO mats shows antibacterial activity due to the presence of ZnO. Our results suggest that this could be an ideal biomaterial for wound dressing applications once the optimal concentration of ZnO which will give least toxicity while providing maximum antibacterial activity is identified.f