International Journal of Pharmaceutics (v.504, #1-2)

Ultra-small lipid-dendrimer hybrid nanoparticles as a promising strategy for antibiotic delivery: In vitro and in silico studies by Sandeep J. Sonawane; Rahul S. Kalhapure; Sanjeev Rambharose; Chunderika Mocktar; Suresh B. Vepuri; Mahmoud Soliman; Thirumala Govender (1-10).
Display OmittedThe purpose of this study was to explore the preparation of a new lipid-dendrimer hybrid nanoparticle (LDHN) system to effectively deliver vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) infections. Spherical LDHNs with particle size, polydispersity index and zeta potential of 52.21 ± 0.22 nm, 0.105 ± 0.01, and −14.2 ± 1.49 mV respectively were prepared by hot stirring and ultrasonication using Compritol 888 ATO, G4 PAMAM- succinamic acid dendrimer, and Kolliphor RH-40. Vancomycin encapsulation efficiency (%) in LDHNs was almost 4.5-fold greater than in lipid-polymer hybrid nanoparticles formulated using Eudragit RS 100. Differential scanning calorimetry and Fourier transform-infrared studies confirmed the formation of LDHNs. The interactions between the drug-dendrimer complex and lipid molecules using in silico modeling revealed the molecular mechanism behind the enhanced encapsulation and stability. Vancomycin was released from LDHNs over the period of 72 h with zero order kinetics and super case II transport mechanism. The minimum inhibitory concentration (MIC) against S. aureus and MRSA were 15.62 μg/ml and 7.81 μg/ml respectively. Formulation showed sustained activity with MIC of 62.5 μg/ml against S. aureus and 500 μg/ml against MRSA at the end of 72 and 54 h period respectively. The results suggest that the LDHN system can be an effective strategy to combat resistant infections.
Keywords: Lipid; Dendrimer; Hybrid nanoparticles; Antibiotic; Sustained release; Bacterial resistance; MRSA;

Magnetic solid lipid nanoparticles in hyperthermia against colon cancer by María Muñoz de Escalona; Eva Sáez-Fernández; José C. Prados; Consolación Melguizo; José L. Arias (11-19).
Display OmittedA reproducible double emulsion/solvent evaporation procedure is developed to formulate magnetic solid lipid nanoparticles (average size ≈ 180 nm) made of iron oxide cores embedded within a glyceryl trimyristate solid matrix. The physicochemical characterization of the nanocomposites ascertained the efficacy of the preparation conditions in their production, i.e. surface properties (electrokinetic and thermodynamic data) were almost indistinguishable from those of the solid lipid nanomatrix, while electron microscopy characterizations and X-ray diffraction patterns confirmed the satisfactory coverage of the magnetite nuclei. Hemocompatibility of the particles was established in vitro. Hysteresis cycle determinations defined the appropriate magnetic responsiveness of the nanocomposites, and their heating characteristics were investigated in a high frequency alternating gradient of magnetic field: a constant maximum temperature of 46 °C was obtained within 40 min. Finally, in vitro tests performed on human HT29 colon adenocarcinoma cells demonstrated a promising decrease in cell viability after treatment with the nanocomposites and exposure to that alternating electromagnetic field. To the best of our knowledge, this is the first time that such type of nanoformulation with very promising hyperthermia characteristics has been developed for therapeutic aims.
Keywords: Cancer therapy; Colon cancer; Hemocompatibility; Magnetic colloid; Magnetic fluid hyperthermia; Magnetic solid lipid nanoparticle; Nanocomposite;

The influence of co-formers on the dissolution rates of co-amorphous sulfamerazine/excipient systems by Katarzyna Gniado; Korbinian Löbmann; Thomas Rades; Andrea Erxleben (20-26).
Display OmittedA comprehensive study on the dissolution properties of three co-amorphous sulfamerazine/excipient systems, namely sulfamerazine/deoxycholic acid, sulfamerazine/citric acid and sulfamerazine/sodium taurocholate (SMZ/DA, SMZ/CA and SMZ/NaTC; 1:1 molar ratio), is reported. While all three co-formers stabilize the amorphous state during storage, only co-amorphization with NaTC provides a dissolution advantage over crystalline SMZ and the reasons for this were analyzed. In the case of SMZ/DA extensive gelation of DA protects the amorphous phase from crystallization upon contact with buffer, but at the same time prevents the release of SMZ into solution. Disk dissolution studies showed an improved dissolution behavior of SMZ/CA compared to crystalline SMZ. However, enhanced dissolution properties were not seen in powder dissolution testing due to poor dispersibility. Co-amorphization of SMZ and NaTC resulted in a significant increase in dissolution rate, both in powder and disk dissolution studies.
Keywords: Co-amorphization; Dissolution studies; Dispersibility; Sulfamerazine;

Dispersibility of lactose fines as compared to API in dry powders for inhalation by Kyrre Thalberg; Simon Åslund; Marcus Skogevall; Patrik Andersson (27-38).
Display OmittedThis work investigates the dispersion performance of fine lactose particles as function of processing time, and compares it to the API, using Beclomethasone Dipropionate (BDP) as model API. The total load of fine particles is kept constant in the formulations while the proportions of API and lactose fines are varied.Fine particle assessment demonstrates that the lactose fines have higher dispersibility than the API. For standard formulations, processing time has a limited effect on the Fine Particle Fraction (FPF). For formulations containing magnesium stearate (MgSt), FPF of BDP is heavily influenced by processing time, with an initial increase, followed by a decrease at longer mixing times. An equation modeling the observed behavior is presented. Surprisingly, the dispersibility of the lactose fines present in the same formulation remains unaffected by mixing time.Magnesium analysis demonstrates that MgSt is transferred to the fine particles during the mixing process, thus lubrication both BDP and lactose fines, which leads to an increased FPF. Dry particle sizing of the formulations reveals a loss of fine particles at longer mixing times. Incorporation of fine particles into the carrier surfaces is believed to be behind this, and is hence a mechanism of importance as regards the dispersion performance of dry powders for inhalation.
Keywords: Dispersibility; Dry powder inhalation; Fine particle fraction; High shear mixing; Lactose carrier; Lactose fines;

Macroporous natural capsules extracted from Phoenix dactylifera L. spore and their application in oral drugs delivery by Saad M. Alshehri; Hamad A. Al-Lohedan; Eida Al-Farraj; Norah Alhokbany; Anis Ahmad Chaudhary; Tansir Ahamad (39-47).
Display OmittedMacroporous natural sporopollenin exine capsules (SEC) were extracted from date palm (Phoenix dactylifera L.) and coated by natural polymer composite (carboxymethyl cellulose with epichlorohydrin). The polymer coated exine capsules (PCEC) were used in in-vitro investigations for controlled delivery of paracetamol. SEC, PCEC, and drugs loaded capsules (PCEC-PAR) were characterized by scanning electron microscope (SEM), surface area (BET), Fourier-transform infrared (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The length of SEC was found to be 20–20.5 μm, and the pore sized was 50–135 nm, as measured using SEM. The studies revealed that maximum loading of the drug was at pH 6.0 (97.2%, with 50 mg mL−1). The results indicate that by increasing the pH from 1.4 to 7.4, the cumulative release rates of paracetamol in physiological buffer solution (PBS) is more than two times as in simulated gastric fluid (SGF). In addition, the in-vitro toxicity of PCEC against Caco-2 cells was tested by the 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay, and the results revealed that PCEC are biocompatible materials. The overall results encourage further studies on the clinical use of PCEC as drug carriers.
Keywords: Sporopollenin; FTIR; Drugs delivery; Paracetamol; X-ray diffraction;

Percutaneous absorption of benzophenone-3 loaded lipid nanoparticles and polymeric nanocapsules: A comparative study by E. Gilbert; L. Roussel; C. Serre; R. Sandouk; D. Salmon; P. Kirilov; M. Haftek; F. Falson; F. Pirot (48-58).
Display OmittedFor the last years, the increase of the number of skin cancer cases led to a growing awareness of the need of skin protection against ultraviolet (UV) radiations. Chemical UV filters are widely used into sunscreen formulations as benzophenone-3 (BP-3), a usually used broad spectrum chemical UV filter that has been shown to exercise undesirable effects after topical application. Innovative sunscreen formulations are thus necessary to provide more safety to users. Lipid carriers seem to be a good alternative to formulate chemical UV filters reducing their skin penetration while maintaining good photo-protective abilities. The aim of this work was to compare percutaneous absorption and cutaneous bioavailability of BP-3 loaded into solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), nanostructured polymeric lipid carriers (NPLC) and nanocapsules (NC). Particle size, zeta potential and in vitro sun protection factor (SPF) of nanoparticle suspensions were also investigated. Results showed that polymeric lipid carriers, comprising NPLC and NC, significantly reduced BP-3 skin permeation while exhibiting the highest SPF. This study confirms the interesting potential of NPLC and NC to formulate chemical UV filters.
Keywords: Percutaneous absorption; Cutaneous bioavailability; Chemical UV-filters; Lipid nanocarriers; In vitro sun protection factor;

Immune response elicited by an intranasally delivered HBsAg low-dose adsorbed to poly-ε-caprolactone based nanoparticles by Sandra Jesus; Edna Soares; João Costa; Gerrit Borchard; Olga Borges (59-69).
Display OmittedAmong new strategies to increase hepatitis B virus (HBV) vaccination, especially in developing countries, the development of self-administered vaccines is considered one of the most valuable. Nasal vaccination using polymeric nanoparticles (NPs) constitutes a valid approach to this issue. In detail, poly-ε-caprolactone (PCL)/chitosan NPs present advantages as a mucosal vaccine delivery system: the high resistance of PCL against degradation in biological fluids and the mucoadhesive and immunostimulatory properties of chitosan. In vitro studies revealed these NPs were retained in a mucus-secreting pulmonary epithelial cell line and were capable of entering into differentiated epithelial cells. The intranasal (IN) administration of 3 different doses of HBsAg (1.5 μg, 5 μg and 10 μg) adsorbed on a fixed amount of PCL/chitosan NPs (1614 μg) generated identical titers of serum anti-HBsAg IgG and anti-HBsAg sIgA in mice nasal secretions. Besides other factors, the NP surface characteristics, particularly, zeta potential differences among the administered formulations are believed to be implicated in the outcome of the immune response generated.
Keywords: Mucosal immunization; Vaccine adjuvants; Antigen delivery; Poly-ε-caprolactone (PCL)/chitosan nanoparticles; Dose-response; Polymeric biomaterials;

PD-PK evaluation of freeze-dried atorvastatin calcium-loaded poly-ε-caprolactone nanoparticles by Iman S. Ahmed; Rania El-Hosary; Samia Shalaby; Marwa M. Abd-Rabo; Dalia G. Elkhateeb; Samia Nour (70-79).
Display OmittedIn this work lyophilized poly-ε-caprolactone nanoparticles (NPs) loaded with atorvastatin calcium (AC) were developed in an attempt to improve the in-vivo performance of AC following oral administration. The individual and combined effects of several formulation variables were previously investigated using step-wise full factorial designs in order to produce optimized AC-NPs with predetermined characteristics including particle size, drug loading capacity, drug release profile and physical stability. Four optimized formulations were further subjected in this work to lyophilization to promote their long-term physical stability and were fully characterized. The pharmacodynamics (PD)/pharmacokinetics (PK) properties of two optimized freeze-dried AC-NPs formulations showing acceptable long-term stability were determined and compared to a marketed AC immediate release tablet (Lipitor®) in albino rats. PD results revealed that the two tested formulations were equally effective in reducing low density lipoproteins (LDL) and triglycerides (TG) levels when given in reduced doses compared to Lipitor® and showed no adverse effects. PK results, on the other hand, revealed that the two freeze-dried AC-NPs formulations were of significantly lower bioavailability compared to Lipitor®. Taken together the PD and PK results demonstrate that the improved efficacy obtained at reduced doses from the freeze-dried AC-NPs could be due to increased concentration of AC in the liver rather than in the plasma.
Keywords: Atorvastatin calcium; Freeze-drying; Nanoparticles; Pharmacodynamics/pharmacokinetic studies;

Novel nanoemulsion based lipid nanosystems for favorable in vitro and in vivo characteristics of curcumin by Kun Wan; Lili Sun; Xueyuan Hu; Zijun Yan; Yonghong Zhang; Xue Zhang; Jingqing Zhang (80-88).
Display OmittedThe goal of this study was to assess the enhanced elementary characteristics, in vitro release, anti-cancer cytotoxicity, in situ absorption and in vivo bioavailability of a novel nanoemulsion based lipid nanosystems containing curcumin (CNELNs) when administered orally. The CNELNs were first fabricated by loading water-in-oil nanoemulsions into lipid nanosystems using a nanoemulsion-film dispersion-sonication method. The gastro-intestinal absorption, in vitro release and in vivo kinetic property of CNELNs were investigated using an in situ perfusion method, a dialysis method and a concentration-time curve based method, respectively. The inhibitory effects of CNELNs on human lung cancer A549 cell growth were determined using MTT assay. The absorption constants and effective permeabilities of CNELNs in different gastro-intestinal tracts increased 2.29–4.04 times and 4.06–8.27 times that of curcumin (CUR), respectively. The relative bioavailability of CNELNs to free CUR was 733.59%. CNELNs inhibited A549 growth in a dose- and time-dependent manner. CNELNs markedly improved the oral bioavailability of CUR which was probably due to the increased gastro-intestinal absorption. CNELNs had stronger inhibitory effects on the viabilities of A549 cells than that of free CUR. CNELNs might be promising nanosystems for oral delivery of CUR to satisfy clinical requirements.
Keywords: Nanoemulsion based lipid nanosystems; Characteristics; In situ absorption; In vivo kinetic property; Anti-cancer cytotoxicity;

Drug permeation and cellular interaction of amino acid-coated drug combination powders for pulmonary delivery by Ville Vartiainen; Luis M. Bimbo; Jouni Hirvonen; Esko I. Kauppinen; Janne Raula (89-97).
Display OmittedThe effect of three amino acid coatings (l-leucine, l-valine and l-phenylalanine) on particle integrity, aerosolization properties, cellular interaction, cytocompatibility, and drug permeation properties of drug combination powder particles (beclomethasone dipropionate and salbutamol sulphate) for dry powder inhalation (DPI) was investigated. Particles with crystalline l-leucine coating resulted in intact separated particles, with crystalline l-valine coating in slightly sintered particles and with amorphous l-phenylalanine coating in strongly fused particles. The permeation of beclomethasone dipropionate across a Calu-3 differentiated cell monolayer was increased when compared with its physical mixture. Drug crystal formation was also observed on the Calu-3 cell monolayer. The l-leucine coated particles were further investigated for cytocompatibility in three human pulmonary (Calu-3, A549 and BEAS-2B) and one human macrophage (THP-1) cell lines, where they showed excellent tolerability. The l-leucine coated particles were also examined for their ability to elicit reactive oxygen species in pulmonary BEAS-2B and macrophage THP-1 cell lines. The study showed the influence of the amino acid coatings for particle formation and performance and their feasibility for combination therapy for pulmonary delivery.
Keywords: Amino acid; Drug delivery; Inhalation; Epithelial cell; Cell viability;

Polymeric microcontainers improve oral bioavailability of furosemide by Line Hagner Nielsen; Ana Melero; Stephan Sylvest Keller; Jette Jacobsen; Teresa Garrigues; Thomas Rades; Anette Müllertz; Anja Boisen (98-109).
Display OmittedMicrocontainers with an inner diameter of 223 μm are fabricated using the polymer SU-8, and evaluated in vitro, in situ and in vivo for their application as an advanced oral drug delivery system for the poorly water soluble drug furosemide. An amorphous sodium salt of furosemide (ASSF) is filled into the microcontainers followed by applying a lid using Eudragit L100. It is possible to control the drug release in vitro, and in vitro absorption studies show that the microcontainers are not a hindrance for absorption of ASSF. In situ perfusion studies in rats are performed with ASSF-filled microcontainers coated with Eudragit and compared to a furosemide solution. The absorption rate constant of ASSF confined in microcontainers is found to be significantly different from the solution, and by light microscopy, it is observed that the microcontainers are engulfed by the intestinal mucus. An oral bioavailability study in rats is performed with ASSF confined in microcontainers coated with Eudragit and a control group with ASSF in Eudragit-coated capsules. A relative bioavailability of 220% for the ASSF in microcontainers compared to ASSF in capsules is found. These studies indicate that the microcontainers could serve as a promising oral drug delivery system.
Keywords: Micro devices; Furosemide; Oral delivery; Delivery systems; Oral bioavailability; Intestinal perfusion;

Hydroxyl-modified magnetite nanoparticles as novel carrier for delivery of methotrexate by Fatemeh Farjadian; Sahar Ghasemi; Soliman Mohammadi-Samani (110-116).
Novel hydroxyl-modified magnetite nanocarriers are introduced as efficient host for methotrexate conjugation known as (MNP-TRIS-MTX). The chemical structure was fully characterized and the topography was studied by well-known techniques. The anti-cancer behavior of drug conjugated nanoparticles was evaluated in vitro in MCF-7 cell line which showed enhanced toxicity and the structure remarked as potent anti-cancer agent.Display OmittedIn this work, novel hydroxyl-modified magnetite nanocarriers are introduced as efficient host for methotrexate conjugation. The modification was based on the Micheal type addition reaction between tris(hydroxymethyl) aminomethane and acrylamidopropyl functionalized, silica-coated magnetite nanoparticle. The chemical structure characterization was carried out by FT-IR and the organic content was determined by CHN analysis. The topography was studied by SEM, TEM, AFM. DLS was performed to show particles’ mean diameter. Furthermore, the magnetite properties of modified particles were evaluated by VSM and the crystallinity was proved by XRD. To illustrate the efficiency of the modified particles, the anti-cancer drug methotrexate was conjugated to hydroxyl groups through estric bond formation. The controlled release activity of established nanoparticles was evaluated in simulated cellular fluid. Later, the anti-cancer behavior of drug conjugated nanoparticles was evaluated in vitro in MCF-7 cell line which showed enhanced toxicity after 48 h. Conclusively, the modified nanoparticles have remarked as powerful carrier to be applied as an anti-cancer agent.
Keywords: Magnetite nanoparticles; Magnetic nanoparticle (MNP); Methotrexate (MTX); Anti-cancer; Drug delivery; MCF-7;

Zeta potential changing phosphorylated nanocomplexes for pDNA delivery by Sonja Bonengel; Felix Prüfert; Max Jelkmann; Andreas Bernkop-Schnürch (117-124).
Display OmittedThe objective of this study was to evaluate the suitability of a zeta potential changing system as gene delivery system. The phosphate ester bearing ligand 6-phosphogluconic acid (6-PGA) was attached to linear and branched polyethyleneimine (PEI) via a carbodiimide-mediated reaction whereby 287 μmol and 413 μmol 6-PGA could be coupled per gram polymer. Nanocomplexes of these modified polymers with pDNA showed a zeta potential of +12 mV for nanocomplexes with the linear PEI-6PGA and +16 mV in case of the branched derivative. By the addition of carboxymethylcellulose (CMC), zeta potentials of the complexes were reduced to +2.86 and +3.25, respectively. Phosphate release studies on Caco 2 cells and HEK-293 cells demonstrated the ability to cleave the phosphate ester. Compared to HEK-293 cells, enzymatic degradation of the phosphate ester in Caco 2 cells was 2.3-fold higher from nanocomplexes comprising linear PEI and 4.3-fold higher from those with branched PEI. Furthermore, incubation with alkaline phosphatase led to an increase in the zeta potential of nanocomplexes based on linear PEI-6PGA to +6.96 mV and +8.26 mV in nanocomplexes comprising branched PEI-6PGA. Studying transfection efficiency in Caco 2 cells and HEK-293 cells, a higher expression of the green fluorescent protein (GFP) could be detected in HEK-293 cells. In presence of a phosphate inhibitor, transfection efficiencies were decreased in both cells lines, due to a lacking shift of the zeta potential of the tested pDNA complexes. According to these results, zeta potential changing systems seem to be a promising strategy for future gene delivery systems, as this concept allows the in situ generation of positive charges in close proximity to the cellular surface.
Keywords: Zeta potential changing systems; pDNA; Polyethyleneimine; Phosphatase; 6-; Phosphogluconic acid;

Modified gold nanoparticles for intracellular delivery of anti-liver cancer siRNA by Hanan Shaat; Amany Mostafa; Moustafa Moustafa; Amira Gamal-Eldeen; Ahmed Emam; Enas El-Hussieny; Mahmoud Elhefnawi (125-133).
Display OmittedTo overcome the rapid enzymatic degradation and low transfection efficiency of siRNA, the delivery carriers for siRNA is a therapeutic demand to increase its stability. Gold nanoparticles (AuNPs) modified by branched polyethyleneimine (bPEI) were developed as an efficient and safe intracellular delivery carriers for siRNA. The current study implied that siRNA designed against an oncogene c-Myc could be delivered by a modified AuNPs complex without significant cytotoxicity. The comparative semi-quantitative and quantitative real time PCR were used to measure the c-Myc gene expression after transfection with naked siRNA and siRNA/bPEI/AuNPs, but AuNPs interfered with PCR. However, the c-Myc protein translation was successfully detected in the transfected HuH7 cells with naked siRNA and siRNA/bPEI/AuNPs and it was found to be inhibited by siRNA/bPEI/AuNPs more than naked siRNA. The results validate the successful silencing of c-Myc gene. Accordingly, it may confirm the promising and effective delivery of siRNA by bPEI/AuNPs. The complex enhances the cellular uptake of siRNA without significant cytotoxicity and confirms that bPEI modified AuNPs could be used as a good candidate for safe cellular delivery of siRNA.
Keywords: Gold nanoparticles; Small interfering RNA (siRNA); Polyethyleneimine; Polyelectrolyte complex; c-Myc; Gene delivery;