Physico-chemistry and Cytotoxicity of Tenofovir-Loaded Acid Phosphatase-Responsive Chitosan Nanoparticles.

This study assesses the in vitro release of tenofovir (TFV)-loaded triphosphate (TPP) cross-linked chitosan nanoparticles (NPs) catalyzed by human prostatic acid phosphatase (hPAP) for 24 h. The physico-chemical characterization of the NPs included particle mean diameter (PMD), zeta potential (ζ), percent drug encapsulation efficiency (% EE), Fourier transform infra-red (FTIR) spectroscopy, powder X-ray diffractometry analysis (PXRD), and drug release kinetics. The first-order rate constant (k) and activation energy (Ea) of the cross-link (TPP) are determined by the integrated rate law and Arrhenius's equations. The hPAP Michaelis-Menten constant (Km) is determined by the Lineweaver-Burk's equation. The NP's safety profile is evaluated on vaginal epithelial cells (VK2/E6E7). The lyophilized drug-loaded NPs' PMD, ζ, and PDI are 149.97 nm, 4.4 mV, and 0.3, respectively. The % EE after lyophilization is 93.7 ± 4.4%. These NPs released drug at faster rate (63% of TFV within 6 h) under the enzyme's influence. The similarity and difference factors of drug release profiles (absence vs presence of hPAP) are 56.5 and 40.3, respectively. The hPAP's Km value of 0.019 mM suggests it has a good affinity for TPP at physiological pH ~ 7.4. The enhanced hydrolysis of TPP or degradation of chitosan NPs is fundamentally due to a decrease of TPP's activation energy by hPAP. In fact, the Ea value is 22.50 ± 3.06 kJ/mol or 16.33 ± 0.62 kJ/mol in the absence or presence of hPAP, respectively. The NPs are non-cytotoxic to the treated vaginal cell line. These hPAP-responsive NPs are promising topical nanomicrobicides for HIV/AIDS prevention.

Engineering fast dissolving sodium acetate mediated crystalline solid dispersion of docetaxel.

It is hypothesized that a novel crystalline solid dispersion (CSD) of docetaxel (C-DXT) can be engineered by dispersing native docetaxel (DXT, a BCS class II drug) in sodium acetate crystal (SA). DXT is dissolved in glacial acetic/SA solution and freeze-dried. The resulting C-DXT is characterized by differential scanning calorimetry (DSC), powder X-ray analysis (PXRD), LC-MS/MS, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Quartz crystal microbalance with dissipation monitoring (QCM-D) and dynamic light scattering (DLS). Its cytotoxicity on model cancerous (MCF-7, MDA-MB-468) and normal breast cells (MCF-10A) is assessed by MTS assay. SEM/TEM data and the absence of the characteristics peaks of DXT on the DSC curve (at 193.4 °C) and the XRD scan (at 2θ = 15.31 °C and 23.04 °C) confirm the presence of C-DXT in SA. The LC-MS/MS data indicates the chemical stability of DXT. The yield and C-DXT loading are 95.2% and 6.52% w/w, respectively. The C-DXT rapidly forms an aqueous non-rigid nanosuspension with a faster drug dissolution rate compared to native DXT. Unlike, control Tween 80/ethanol, SA is noncytotoxic to normal cells. However, C-DXT's cytotoxicity is time and dose dependent for all diseased cells. This unique CSD process might be applicable to other hydrophobic bioactive agents to enhance their safety and efficacy.

Label-Free Ferrocene-Loaded Nanocarrier Engineering for In Vivo Cochlear Drug Delivery and Imaging.

It is hypothesized that ferrocene (FC)-loaded nanocarriers (FC-NCs) are safe label-free contrast agents for cochlear biodistribution study by transmission electron microscopy (TEM). To test this hypothesis, after engineering, the poly(epsilon-caprolactone)/polyglycolide NCs are tested for stability with various types and ratios of sugar cryoprotectants during freeze-drying. Their physicochemical properties are characterized by UV-visible spectroscopy, dynamic light scattering, Fourier transform infrared spectroscopy, and scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDS). The biodistribution of the FC-NCs in the cochlear tissue after intratympanic injection in guinea pigs is visualized by TEM. Auditory brainstem responses are measured before and after 4-day treatments. These FC-NCs have 153.4 ± 8.7 nm, 85.5 ± 11.2%, and -22.1 ± 1.1 mV as mean diameters, percent drug association efficiency, and zeta potential, respectively (n = 3). The incorporation of FC into the NCs is confirmed by Fourier transform infrared spectroscopy and SEM/EDS spectra. Lactose (3:1 ratio, v/v) is the most effective stabilizer after a 12-day study. The administered NCs are visible by TEM in the scala media cells of the cochlea. Based on auditory brainstem response data, FC-NCs do not adversely affect hearing. Considering the electrondense, radioactive, and magnetic properties of iron inside FC, FC-NCs are promising nanotemplate for future inner ear theranostics.

Sodium Acetate Coated Tenofovir-Loaded Chitosan Nanoparticles for Improved Physico-Chemical Properties.

PURPOSE: It is hypothesized that sodium acetate (SA) can be used for in situ coating of drug loaded chitosan NPs for improved physico-chemical properties. METHODS: Tenofovir (TFV) is used as a model drug. Uncoated chitosan NPs are prepared by ionic gelation. SA is generated in situ from half neutralization of acetic acid with sodium hydroxide, and coats chitosan NPs during freeze-drying. The NPs' physico-chemical properties [e.g., particle mean diameters (PMD) zeta potential (ζ), EE%, drug release profile, morphology] are characterized by dynamic light scattering, spectrophotometry, Korsmeyer-Peppas model, transmission electron microscopy (TEM), respectively. Melting point (MP), non-aqueous titration, Fourier transform infrared (FTIR) analysis, and powder X-ray diffractometry (XRD) pattern evaluate the SA coated chitosan NPs. The NPs' cytotoxicity on macrophages Raw 264.7 is assessed by neutral red, resazurin, nitrite oxide (NO) and cytokines assays. RESULTS: Collectively, FTIR, ζ, XRD, MP, and TEM data confirm that SA coats chitosan NPs. The PMD range is 136-348 nm (uncoated) and 171-379 nm (coated NPs). The ζ values range is +24.3-28.5 mV (uncoated) and 0.1-3.1 mV (coated NPs). The EE% ranges from 5.5 to 11.7% (uncoated NPs) and increased up to 86.3-92.7%(8-17-fold) after coating. The SA also prevents NPs aggregation during the freeze-drying and aqueous dispersion. The core-shell NPs exhibited a sustain release of TFV following anomalous transport mechanism (R(2) ~ 0.99). The coated NPs are non-cytotoxic (cell viability ~100%) and without any proinflammatory response. CONCLUSIONS: This SA coating chitosan NPs mechanism may be useful for (i) efficient encapsulation, (ii) stabilizing colloidal dispersions, (iii) controlling the release and solubility of bioactive agents.

Strong microwave absorption of hydrogenated wide bandgap semiconductor nanoparticles.

Electromagnetic interactions in the microelectronvolt (µeV) or microwave region have numerous important applications in both civil and military fields, such as electronic communications, signal protection, and antireflective coatings on airplanes against microwave detection. Traditionally, nonmagnetic wide-bandgap metal oxide semiconductors lack these µeV electronic transitions and applications. Here, we demonstrate that these metal oxides can be fabricated as good microwave absorbers using a 2D electron gas plasma resonance at the disorder/order interface generated by a hydrogenation process. Using ZnO and TiO2 nanoparticles as examples, we show that large absorption with reflection loss values as large as -49.0 dB (99.99999%) is obtained in the microwave region. The frequency of absorption can be tuned with the particle size and hydrogenation condition. These results may pave the way for new applications for wide bandgap semiconductors, especially in the µeV regime.

Evaluation of degradation kinetics and physicochemical stability of tenofovir.

Tenofovir (TFV) has been proven to prevent the transmission of the Human Immunodeficiency Virus (HIV) through the vagina. But, there is little information available about its stability under various storage and stress conditions. Hence, this study aimed to investigate the degradation behavior and physicochemical stability of TFV using liquid chromatography coupled mass spectrometry (LC-MS) and solid state X-ray diffraction (XRD) analyses. The LC-MS analysis was performed on a QTrap mass spectrometer with an enhanced mass spectrum (EMS) scan in positive mode. A reversed phase C18 column was used as the stationary phase. TFV exhibited degradation under acidic and alkaline hydrolytic conditions. The degradation products with m/z 289.2 and 170 amu have been proposed as 6-Hydroxy adenine derivative of TFV, and (2-hydroxypropan-2-yloxy) methylphosphonic acid, respectively. A pseudo-first-order degradation kinetic allowed for estimating the shelf-life, half-life, and time required for 90% degradation of 3.84, 25.34, and 84.22 h in acidic conditions, and 58.26, 384.49, and 1277.75 h in alkaline conditions, respectively. No significant degradation was observed at pH 4.5 (normal cervicovaginal pH) and oxidative stress conditions of 3% and 30% v/v hydrogen peroxide solutions. The shelf life of TFV powder at room temperature was 23 months as calculated by using an Arrhenius plot. The XRD pattern showed that the drug was stable and maintained its original crystallinity under the accelerated and thermal stress conditions applied. Stability analyses revealed that the TFV was stable in various stress conditions; however, formulation strategies should be implemented to protect it in strong acidic and alkaline environments.

Resveratrol-loaded nanocarriers: formulation, optimization, characterization and in vitro toxicity on cochlear cells.

The present work aimed to investigate the suitability of polymeric nanoparticles (NPs) loaded with resveratrol (RES) for drug delivery to cochlear cells. RES-loaded NPs were prepared by a solvent-diffusion method without surfactant. The Box-Behnken design was used to study the effect of the formulation variables on the particle mean diameter (PMD), polydispersity index (PDI), zeta-potential (ζ), percent drug encapsulation efficiency (EE%), and ratio between NP size before and after freeze-drying (Sf/Si). The physicochemical stability of the RES-loaded NPs during freeze-drying was investigated using four well-known cryoprotectants (i.e., lactose, mannitol, sucrose, and trehalose) at different concentrations. The RES-loaded NPs were also characterized by powder X-ray diffraction (PXRD) and in vitro drug release studies. Finally, the in vitro toxicity of the synthesized NPs was evaluated on two cochlear cell lines: HEI-OC1 and SVK-1 cells. The optimal formulation (desirability: 0.86) had 135.5±37.3nm as PMD, 0.126±0.080 as PDI, -26.84±3.31mV as ζ, 99.83±17.59% as EE%, and 3.30±0.92 as Sf/Si ratio. The PMD and PDI of the RES-loaded NPs were maintained within the model space only when trehalose was used at concentrations higher than 15% (w/v). Results from the in vitro cytotoxicity studies showed that blank NPs did not alter the viability of both cells lines, except for concentrations higher than 600µg/mL. However, the cell viability was significantly decreased at high concentrations of native RES (>50µM, p<0.05) in both cell lines. Overall, the results suggested that the RES-loaded polymeric NPs could be a suitable template for cochlea antioxidant delivery and otoproctection.

Influence of surface chemistry on cytotoxicity and cellular uptake of nanocapsules in breast cancer and phagocytic cells.

The present work tests the hypothesis that stabilizers have a critical role on nanocarrier stealthiness and anticancer drug efficacy. Two different types of docetaxel (Doc)-loaded nanocapsules (NCs) stabilized with polysorbate 80 (NC(T80)) and polyvinyl alcohol (NC(PVA)) were synthesized using the emulsion solvent diffusion method. These NCs were characterized for particle mean diameter (PMD), drug content, morphology, surface composition, and degree of crystallinity. Furthermore, the cytotoxicity and cellular uptake of the NCs were investigated in MDA-MB 231 cells, THP-1 monocytes, and THP-1-derived macrophages. The optimized spherical NC(T80) had 123.02 ± 14.6 nm, 0.27 ± 0.1, and 101 ± 37.0% for PMD, polydispersity index, and drug encapsulation efficiency, respectively. Doc release kinetics from NC(T80) and NC(PVA) mostly provided better fit to zero-order and Higuchi models, respectively. Powder X-ray diffraction (PXRD) and X-ray photoelectron spectroscopy (XPS) results revealed the presence of amorphous stabilizers on the surface of the NCs. At high drug concentration, the cytotoxicity of NC(T80) was substantially improved (1.3-1.6-fold) compared with that of NC(PVA) in MDA-MB 231 cells. The uptake of both NCs was inhibited by latrunculin A and dynasore, indicating an actin- and dynamin-dependent endocytosis in MDA-MB 231 cells. This occurred via a multifaceted mechanism involving clathrin, caveolin, cytoskeleton, and macropinocytosis. Interestingly, the uptake of NC(PVA) was 2.7-fold greater than that of NC(T80) and occurred through phagocytosis in monocytes and macrophages. This study demonstrates the potential impact of the surface chemistry on the cytotoxicity and phagocytic clearance of nanocarriers for a subsequent improvement of the efficacy of Doc intended for breast cancer chemotherapy.

Uptake and cytotoxicity of docetaxel-loaded hyaluronic acid-grafted oily core nanocapsules in MDA-MB 231 cancer cells.

PURPOSE: It is hypothesized that docetaxel (Doc)-loaded hyaluronic acid (HA)-polyethylene glycol/poly(ε-caprolactone)-grafted oily core nanocapsules (NCs) can enhance the drug cytotoxicity and uptake in CD44 expressing breast cancer (BC) cells (MDA-MB 231). METHODS: NCs were prepared, optimized and characterized by dynamic light scattering, transmission electron microscopy (TEM), and powder X-ray diffraction (PXRD). In vitro cytotoxicity tests [MTS, level of reactive oxygen species (ROS) and level of reduced glutathione (GSH)] were performed in BC cells. The contribution of CD44 to the NCs cellular uptake was elucidated using an anti CD44 antibody blockage and a CD44 negative NIH3T3 cell line. RESULTS: The optimum formulation of Doc-loaded HA oily core NCs had respective mean diameter, polydispersity, and drug encapsulation efficiency of 224.18 nm, 0.32, and 60.38%. The NCs appeared spherical with low drug crystallinity, while the drug release data fitted to first order equation. Compared to that of ungrafted NCs, the cytotoxicity of Doc-loaded HA-grafted NCs was significantly enhanced (p<0.05). A decrease of the intracellular level of ROS was reversely correlated with that of GSH. Interestingly, the cellular internalization of HA-grafted NCs mediated CD44 was dramatically enhanced (3 to 4-fold) with respect to the absence of specific biomarker or targeting ligand. CONCLUSIONS: The use of HA-grafted NCs enhanced the selective drug payload, cytotoxicity and uptake in MDA-MB 231 cells. Therefore, it could be a promising template for safe and effective delivery of Doc and similar chemotherapeutic agents in cancer cells.

Directional heat dissipation across the interface in anatase-rutile nanocomposites.

Understanding the structures and properties of interfaces in (nano-)composites helps to reveal their important influence on reactivity and overall performance. TiO2 is a technologically important material, and anatase/rutile TiO2 composites have been shown to display enhanced photocatalytic performance over pure anatase or rutile TiO2. This has been attributed to a synergistic effect between the two phases, but the origin of this effect as well as the structure of the interface has not been established. Using Raman spectroscopy, here we provide evidence of distinct differences in the thermal properties of the anatase and rutile moieties in the composite, with anatase becoming effectively much warmer than the rutile phase under laser irradiation. With the help of first-principles calculations, we analyze the atomic structure and unique electronic properties of the composite and infer possible reasons for the directional heat dissipation across the interface.

Built-in electric field-assisted surface-amorphized nanocrystals for high-rate lithium-ion battery.

High-power batteries require fast charge/discharge rates and high capacity besides safe operation. TiO2 has been investigated as a safer alternative candidate to the current graphite or incoming silicon anodes due to higher redox potentials in effectively preventing lithium deposition. However, its charge/discharge rates are reluctant to improve due to poor ion diffusion coefficients, and its capacity fades quickly with rate as only thinner surface layers can be effectively used in faster charge/discharge processes. Here, we demonstrate that surface-amorphized TiO2 nanocrystals greatly improve lithium-ion rechargeable battery performance: 20 times rate and 340% capacity improvement over crystalline TiO2 nanocrystals. This improvement is benefited from the built-in electric field within the nanocrystals that induces much lower lithium-ion diffusion resistance and facilitates its transport in both insertion and extraction processes. This concept thus offers an innovative and general approach toward designing battery materials with better performance.

Spray drying tenofovir loaded mucoadhesive and pH-sensitive microspheres intended for HIV prevention.

PURPOSE: To develop spray dried mucoadhesive and pH-sensitive microspheres (MS) based on polymethacrylate salt intended for vaginal delivery of tenofovir (a model HIV microbicide) and assess their critical biological responses. METHODS: The formulation variables and process parameters are screened and optimized using a 2(4-1) fractional factorial design. The MS are characterized for size, zeta potential, yield, encapsulation efficiency, Carr's index, drug loading, in vitro release, cytotoxicity, inflammatory responses and mucoadhesion. RESULTS: The optimal MS formulation has an average size of 4.73µm, zeta potential of -26.3mV, 68.9% yield, encapsulation efficiency of 88.7%, Carr's index of 28.3 and drug loading of 2% (w/w). The MS formulation release 91.7% of its payload in the presence of simulated human semen. At a concentration of 1mg/ml, the MS are noncytotoxic to vaginal endocervical/epithelial cells and Lactobacillus crispatus when compared to control media. There is also no statistically significant level of inflammatory cytokine (IL1-α, IL-1ß, IL-6, IL-8, and IP-10) release triggered by these MS. Their percent mucoadhesion is 2-fold higher than that of 1% HEC gel formulation. CONCLUSION: These data suggest the promise of using such MS as an alternative controlled microbicide delivery template by intravaginal route for HIV prevention.

Entrapment and release kinetics of furosemide from pegylated nanocarriers.

This study was designed to test the hypothesis that furosemide (Fur) can be entrapped into surfactant free pegylated nanocarriers (NCs) for controlled drug release. To test this hypothesis, Fur-loaded NCs were prepared by emulsion solvent diffusion method. A 2(3) factorial design was used to optimize the effect of three formulation variables [amounts of Fur (X(1)), poly(lactic-co-glycolic acid) (X(2)) and poly-ε-caprolactone-polyethylene glycol (X(3))] on particle mean diameter (Y(1)), polydispersity index (PDI, Y(2)), and percent drug encapsulation efficiency (EE%, Y(3)). The NCs were characterized for morphology, thermal behavior, optical properties, crystallinity, and drug release kinetics using electron microscopy (EM), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, powder X-ray diffraction (PXRD), and high performance liquid chromatography, respectively. The optimum formula produced with 6 mg of Fur, 7 mg of PLGA, and 1mg of PCL-PEG corresponded to 183.26 nm, 0.26, and 88.29% as Y(1), Y(2) and Y(3) values, respectively. DSC thermograms, FTIR spectra and PXRD diffractograms indicated that Fur was encapsulated in its polymorphic crystalline form I within the NCs polymeric matrix. This was further confirmed by a comparative study between native Fur, Fur nanocrystal and Fur loaded NCs using scanning EM, PXRD and drug release kinetics. The release kinetics of the optimized formula fit the Higuchi model indicating that the drug was released by diffusion in 12h. These results indicate that pegylated Fur-loaded NCs could be successfully prepared with high EE% and sustained release profile intended for the inner ear drug delivery.

Encapsulation of docetaxel in oily core polyester nanocapsules intended for breast cancer therapy.

This study is designed to test the hypothesis that docetaxel [Doc] containing oily core nanocapsules [NCs] could be successfully prepared with a high percentage encapsulation efficiency [EE%] and high drug loading. The oily core NCs were generated according to the emulsion solvent diffusion method using neutral Labrafac CC and poly(d, l-lactide) [PLA] as oily core and shell, respectively. The engineered NCs were characterized for particle mean diameter, zeta potential, EE%, drug release kinetics, morphology, crystallinity, and cytotoxicity on the SUM 225 breast cancer cell line by dynamic light scattering, high performance liquid chromatography, electron microscopies, powder X-ray diffraction, and lactate dehydrogenase bioassay. Typically, the formation of Doc-loaded, oily core, polyester-based NCs was evidenced by spherical nanometric particles (115 to 582 nm) with a low polydispersity index (< 0.05), high EE% (65% to 93%), high drug loading (up to 68.3%), and a smooth surface. Powder X-ray diffraction analysis revealed that Doc was not present in a crystalline state because it was dissolved within the NCs' oily core and the PLA shell. The drug/polymer interaction has been indeed thermodynamically explained using the Flory-Huggins interaction parameters. Doc release kinetic data over 144 h fitted very well with the Higuchi model (R2 > 0.93), indicating that drug release occurred mainly by controlled diffusion. At the highest drug concentration (5 µM), the Doc-loaded oily core NCs (as a reservoir nanosystem) enhanced the native drug cytotoxicity. These data suggest that the oily core NCs are promising templates for controlled delivery of poorly water soluble chemotherapeutic agents, such as Doc.

Optimization of formulation variables affecting spray-dried oily core nanocapsules by response surface methodology.

The formulation variables required for the production of spray-dried oily core nanocapsules (NCs) with targeted size and drug payload were optimized using a Box-Behnken experimental design. These NCs were characterized for size, morphology, encapsulation efficiency (EE%) and drug release kinetics, crystallinity, and density, by dynamic light scattering, electron microscopy, ultraviolet spectrometry, powder X-ray diffraction, and density-gradient centrifugation, respectively. The size of the NCs ranged from 208.6 to 504.4 nm, with EE% from 64.7% to 94.6%. The amounts of oil and surfactant (Pluronic F127) significantly affected size. The amounts of polymer [polylactide (PLA)], oil, and surfactant significantly affected EE%. The optimum formulation parameters were set to be 300 mg of PLA, 0.56 mL of oil, and 239.57 mg of Pluronic F127, which corresponded to size of 284.1 nm and EE% of 95.7%. Morphological analysis and density-gradient centrifugation showed the existence of an oily core and spherical nanostructure with no detectable drug crystals. The NCs had longer sustained drug release than nanosphere control, with a good fit to the Ritger-Peppas model (R(2) > 0.930). Spray-dried oily core NCs were successfully produced, and the Box-Behnken design appears to be an effective tool to predict the size and encapsulation of NCs.

Thermodynamics of drug nanoencapsulation: case study of phenytoin-poly (D, L-lactide) nanocarrier.

In order to rationalize the nanoencapsulation process, a thermodynamic analysis allowed to predict the limit of a model drug (phenytoin, PHT) loading into poly (d, l-lactic acid) nanocarrier (PLA NC) prepared by solvent displacement method. The NC were characterized for size, morphology, thermal behavior and crystallography by dynamic light scattering, electron microscopies, differential scanning calorimetry, and powder X-ray diffraction, respectively. The drug loading, encapsulation efficiency (EE) and in vitro drug release profile were determined using high performance liquid chromatography. There was a logarithmic correlation between the partial molar free energy change based on the molecular descriptors and the drug / polymer weight (D/P) ratio predicting an equilibrium state at ratio= 0.29. This was consistent with experimental data as PHT appeared to be loaded either in crystalline or molecular/amorphous state within the PLA matrix with an average EE coefficient of 91.5%. Overall, the smallest PHT NC had a mean diameter of ≈231 nm and was produced at the limiting D/P ratio of 0.33, using PHT: 18 mg, PLA: 60 mg and surfactant: 0.3%. The drug release occurred by diffusion following Higuchi model within 24 hours. These data suggested that thermodynamic analysis allows elucidating PHT encapsulation within PLA NC and may be useful in the rationale design of other macromolecular based-nanocarriers.

Formulation of dacarbazine-loaded cubosomes. Part III. Physicochemical characterization.

The purpose of this study was to investigate the physicochemical properties of dacarbazine-loaded cubosomes. The drug-loaded cubosome nanocarriers were prepared by a fragmentation method and then freeze dried. They were then characterized for size, morphology, thermal behavior, and crystallography using dynamic light scattering, transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD), respectively. The drug loading and encapsulation efficiency were determined by UV spectrophotometry. The results showed that the prepared dacarbazine-loaded cubosomes had mean diameters ranging from 86 to 106 nm. In addition to the TEM, the characteristic peaks from PXRD data suggested that the freeze-dried nanoformulations were indeed cubic in nature. DSC and PXRD analysis suggested the 0.06 or 0.28% w/w actual drug loaded inside cubosomes was in the amorphous or molecular state. These physicochemical characteristics would affect the nanoformulation shelf-life, efficacy, and safety.