International Journal of Pharmaceutics (v.516, #1-2)

Style and precision in scientific papers by Alexander T. Florence (1-2).

Display OmittedThe objective was to investigate poly vinyl acetate (Kollicoat® SR 30 D) and ammonio methacrylate copolymer (Eudragit® RL 30 D) blends as coatings to increase the mechanical robustness of hydroxypropyl methylcellulose (HPMC) matrix tablets. Poly vinyl acetate (Kollicoat® SR 30 D – KSR) was selected for its flexibility and ammonio methacrylate copolymer (Eudragit® RL 30 D – ERL) because of its high permeability. Films based on KSR:ERL blends were prepared by casting or spraying aqueous dispersions of these polymers and were characterized by water uptake, dry mass loss and mechanical properties. KSR:ERL blends were investigated as coating materials to improve the robustness, mechanical strength and drug release from the HPMC matrix tablets containing propranolol HCl, caffeine and carbamazepine as model drugs. Both HPMC and the polymer coating affected the propranolol release. The release and the mechanical properties could be easily adjusted by varying the polymer blend ratio. The flexibility increased with increasing KSR content. At an 8% w/w coating level, a force of 3.2 N was required to rupture the coating of the swollen tablet after 16 h in the release medium; the coated tablets were thus robust to withstand gastrointestinal forces. The coating level (6%–10%, w/w) and dissolution agitation rate (50 rpm to 150 rpm) had no effect on the drug release. The water-insoluble carbamazepine was not released from the coated tablets as HPMC erosion, which is necessary for the release of a poorly water-soluble drug was hindered by the coating. The release of the water-soluble propranolol increased with increasing drug content and decreased with increasing HPMC content.Poly vinyl acetate and ammonio methacrylate copolymer could be a proper polymer blend for coating HPMC matrix tablets to increase mechanical robustness, which characterized by its flexibility and permeability.
Keywords: Poly vinyl acetate; Ammonio methacrylate copolymer; HPMC; Matrix tablet; Mechanical properties; Oral controlled release;

The effect of material attributes and process parameters on the powder bed uniformity during a low-dose dosator capsule filling process by S. Stranzinger; E. Faulhammer; V. Calzolari; S. Biserni; R. Dreu; R. Šibanc; A. Paudel; J.G. Khinast (9-20).
Display OmittedThe objective of this work was to assess the effect of process parameters of a dosator nozzle machine on the powder bed uniformity of inhalation powders with various characteristics during a low-dose dosator capsule filling process. Three grades of lactose excipients were extensively characterized and filled into size 3 capsules using different dosing chamber lengths (2.5, 5 mm), nozzle diameters (1.9, 3.4 mm), powder bed heights (5, 10 mm) and filling speeds (500, 3000 capsules/h).The fill weight and the weight variability of Lactohale 100 (large particles, good flowability, low cohesion) remained almost the same, regardless of the process parameters throughout the capsule filling run time. Moreover, for this powder an increase in the fill weight at a higher filling speed was observed in all cases. Fill weight variability was significantly higher for lower dosing chamber volumes at a filling speed of 3000 capsules per hour. Lactohale 220 (small particles, poor flowability, high cohesion) delivered entirely different results. After a certain run time, depending on instrumental settings, a ‘steady-state’ with constant fill weights and low weight variability was achieved. For this highly cohesive powder, a high dosing chamber volume requires a low filling speed in order for the powder to completely fill the dosator nozzle. Moreover, it was established that a dosing chamber length of 2.5 mm and a powder bed height of 10 mm were required due to the powder’s high fill weight variability over time, while the dosator size had no effect on it.In summary, the layer uniformity, the fill weight and the weight variability strongly depend on the powder characteristics and the instrumental settings. The results indicate that Lactohale 220 requires special attention during low-dose capsule filling. The study presents excellent insights into the effect of material attributes and process parameters on the layer uniformity and the quality of end product.
Keywords: Low-dose capsule filling; Dosator nozzle machine; Inhalation powders; Powder bed uniformity;

Display OmittedpH-sensitive nanoparticles have a great potential for dermal and transfollicular drug delivery. In this study, pH-sensitive, dexamethasone-loaded Eudragit® L 100, Eudragit® L 100-55, Eudragit® S 100, HPMCP-50, HPMCP-55 and cellulose acetate phthalate nanoparticles were prepared by nanoprecipitation and characterized. The pH-dependent swelling, erosion, dissolution and drug release kinetics were investigated in vitro using dynamic light scattering and Franz diffusion cells, respectively. Their toxicity potential was assessed by the ROS and MTT assays. 100–700 nm nanoparticles with high drug loading and entrapment efficiency were obtained. The nanoparticles bear no toxicity potential. Cellulose phthalates nanoparticles were more sensitive to pH than acrylates nanoparticles. They dissolved in 10 mM pH 7.5 buffer and released > 80% of the drug within 7 h. The acrylate nanoparticles dissolved in 40 mM pH 7.5 buffer and released 65–70% of the drug within 7 h. The nanoparticles remained intact in 10 and 40 mM pH 6.0 buffers (HPMCP nanoparticles dissolved in 40 mM pH 6.0 buffer) and released slowly. The nanoparticles properties could be modulated by blending the different polymers. In conclusion, various pH-sensitive nanoparticles that could release differently on the skin surface and dissolve and release in the hair follicles were obtained.
Keywords: Cellulose acetate phthalate; Dexamethasone; Eudragit®; HPMCP; pH-sensitive nanoparticle; Skin nanocarrier;

Development of near zero-order release PLGA-based microspheres of a novel antipsychotic by Jinlong Zhao; Lexi Wang; Chunyu Fan; Kongtong Yu; Ximing Liu; Xiaolei Zhao; Dan Wang; Wenhua Liu; Zhengxing Su; Fengying Sun; Youxin Li (32-38).
Display OmittedThe novel antipsychotic isoperidone, a prodrug of paliperidone, was designed to improve liposolubility for the development of poly(D,L-lactide-co-glycolide) (PLGA)-based microspheres to achieve near zero-order release behaviour in vivo. Microspheres with a smooth surface were obtained using the oil-in-water emulsion solvent evaporation method and yielded a high encapsulation efficiency of 92%. Pharmacokinetic studies in beagle dogs showed a one-week plateau phase followed by a two-week quasi-zero-order release with no burst release. The in vitro release method with a good in vitro-in vivo correlation was also established. Pharmacodynamic evaluation was performed using the MK-801-induced schizophrenic behavioural mouse model, and the sustained suppressive effect lasted two weeks. The pharmacokinetic-pharmacodynamic (PK-PD) relationship of isoperidone microspheres was compared to that of oral administration of free drug. The results revealed a strong correlation between the plasma drug level and the antipsychotic effect. A stable drug plasma concentration was detected in mice both intraday and interday from 8 to 22 d after a single injection of isoperidone microspheres, and a sustained suppressive effect on the schizophrenic model was also observed. In comparison, the mouse group receiving oral daily administration exhibited more dose-dependent effects, and the pharmacological effect diminished rapidly in conjunction with a reduction of the plasma drug levels 8 h after the last administration of isoperidone on day 3. The above results confirm the superiority of long-acting release over oral administration and indicate a valuable alternative for the clinical treatment of schizophrenia.
Keywords: Antipsychotic; Microsphere; Zero-order release; PK-PD relationship;

Improved formulation of cationic solid lipid nanoparticles displays cellular uptake and biological activity of nucleic acids by Anna Fàbregas; Silvia Prieto-Sánchez; Marc Suñé-Pou; Sofía Boyero-Corral; Josep Ramón Ticó; Encarna García-Montoya; Pilar Pérez-Lozano; Montserrat Miñarro; Josep Mª Suñé-Negre; Cristina Hernández-Munain; Carlos Suñé (39-44).
Display OmittedNon-viral delivery using cationic solid lipid nanoparticles (SLNs) represents a useful strategy to introduce large DNA and RNA molecules to target cells. A careful selection of components and their amounts is critical to improve transfection efficiency. In this work, a selected and optimized formulation of SLNs was used to efficiently transfect circular DNA and linear RNA molecules into cells. We characterized the main physicochemical characteristics and binding capabilities of these SLNs and show that they deliver DNA and RNA molecules into cells where they display full bioactivity at nontoxic concentrations using fluorescence- and luminescence-based methodologies. Hence, we established a novel and simple SLN formulation as a powerful tool for future therapeutic use.
Keywords: Cationic solid lipid nanoparticles (SLNs); Plasmid DNA; siRNA; Binding efficiency; Transfection; Luciferase;

Ionic liquids as potential enhancers for transdermal drug delivery by Daniela Monti; Emanuele Egiziano; Susi Burgalassi; Patrizia Chetoni; Cinzia Chiappe; Angelo Sanzone; Silvia Tampucci (45-51).
Display OmittedThe aim of this study was to verify the effect of several cyclic onium based ionic liquids (ILs), including mono- and dicationic derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO), a dialkyl morpholinium salt and a Brønsted acidic IL, as enhancers of the in vitro transdermal permeation and skin retention of diltiazem through and into hairless rat skin. The drug was used as both the hydrochloride salt (DZHCl) and the free base (DZB) to highlight the relationship between the enhancement effect and the physico-chemical characteristics of the active agent. Permeation tests were carried out using Gummer-type diffusion cells and excised rat skin with a 0.005 M aqueous solution of diltiazem hydrochloride or diltiazem free base with and without the addition of 1% w/w ionic liquids. At the end of the permeation experiments with diltiazem hydrochloride, a suitable extraction procedure allowed for the determination of the drug content retained in the skin. Depending on the ionic liquid structure, a significant enhancement in diltiazem hydrochloride levels in the receiving phase was observed, and the transdermal permeation of the diltiazem free base was markedly increased by treatment with all of the ionic liquids. N-dodecyldabco bromide was the best enhancer for both salified and free base drug forms, even though it showed a certain toxicity. On the other hand, N-methyl-N-decylmorpholinium bromide showed a good balance between enhancer activity and cytotoxicity.
Keywords: ionic liquids; hairless rat skin; cutaneous permeation and penetration; diltiazem; enhancers;

The effects of hydration media on the characteristics of non-ionic surfactant vesicles (NISV) prepared by microfluidics by Mohammad A. Obeid; Ibrahim Khadra; Alexander B. Mullen; Rothwelle J. Tate; Valerie A. Ferro (52-60).
Display OmittedNon-ionic surfactant vesicles (NISV) are colloidal particles that provide a useful delivery system for drugs and vaccines. One of the methods that is used for NISV preparation is microfluidics in which the lipid components dissolved in organic phase are mixed with an aqueous medium to prepare the particles through self-assembly of the lipids. In this work, we examined the effect of using different types of aqueous media on the characteristics of the NISV prepared by microfluidics. Five aqueous media were tested: phosphate buffered saline, HEPES buffer, Tris buffer, normal saline and distilled water. The resulting particles were tested for their physical characteristics and cytotoxicity. The aqueous media were found to have significant effects on the physical characteristics of the particles, as well as their overall stability under different conditions and their cytotoxicity to different human cell lines. Careful consideration should be taken when choosing the aqueous media for preparing NISV through microfluidics. This is an important factor that will also have implications with respect to the entrapped material, but which in addition may help to design vesicles for different uses based on changing the preparation medium.
Keywords: Non-ionic surfactant vesicles; Microfluidics; Hydration media; Drug delivery;

Folic acid-decorated and PEGylated PLGA nanoparticles for improving the antitumour activity of 5-fluorouracil by Mazen M. El-Hammadi; Ángel V. Delgado; Consolación Melguizo; José C. Prados; José L. Arias (61-70).
Display Omitted5-Fluorouracil (5-FU) is a broad spectrum cytotoxic agent being used in chemotherapy of malignancies. However, 5-FU shows a number of limitations like short half-life, non-selective biodistribution, and the development of drug resistances by tumour cells. It was investigated the potential use of folic acid-decorated and PEGylated poly(D,L-lactide-co-glycolide) nanoparticles (FOL-PEG-PLGA NPs) for the targeted delivery of 5-FU to colon and breast cancers. PEG-PLGA and FOL-PEG-PLGA conjugates were synthesized and characterized. NPs of PLGA, PEG-PLGA, and FOL-PEG-PLGA were prepared by nanoprecipitation under optimal formulation conditions. They were found to be haemocompatible, and exhibited negligible cytotoxicity in normal (CCD-18 and MCF-10A) and tumour (HT-29 and MCF-7) human cell lines. 5-FU loading capabilities were also defined, and the NPs exhibited an initial burst drug release followed by a sustained 5-FU release. In vitro cytotoxicity studies in folate-overexpressed HT-29 colon cancer cells and MCF-7 breast cancer cells demonstrated that the half maximal inhibitory concentration (IC50) of 5-FU-loaded FOL-PEG-PLGA NPs was approximately 4-fold less than that of the 5-FU-loaded PLGA NPs (p  < 0.05). Consequently, FOL-PEG-PLGA NPs could have great potential as a targeted 5-FU delivery system for colon and breast cancer treatment.
Keywords: Cancer chemotherapy; 5-Fluorouracil; FOL-PEG-PLGA conjugate; Haemocompatibility; Ligand-receptor mediated drug delivery; Nanoprecipitation.;

Polymeric particle-mediated molecular therapies to treat spinal cord injury by Farideh Ordikhani; Saahil Sheth; Silviya Petrova Zustiak (71-81).
Display OmittedSpinal cord injury (SCI) is a physically and psychologically debilitating condition that mainly affects young, healthy males who are at the peak of their personal and professional development. SCI damages axons and disrupts myelination, which interrupts sensory and motor neuronal function. Current treatments are mostly palliative, aimed at reducing further damage and pain, but do not provide a cure. Polymeric particles have shown tremendous promise to provide patients with effective treatments that can bring partial or full functional recovery. Their unique properties can facilitate delivery of therapeutic agents to the injury site, provide protection from the host immunity or provide platforms to stimulate the regeneration of damaged axons. This review highlights the current benefits and challenges of the use of polymeric particles to control the release of molecular therapeutics as potential strategies for SCI treatment.
Keywords: Spinal cord injury; Polymeric particle; Molecular therapy; Neurotrophic factors; Hydrogels;

Impact of additives on the formation of protein aggregates and viscosity in concentrated protein solutions by Katharina Christin Bauer; Susanna Suhm; Anna Katharina Wöll; Jürgen Hubbuch (82-90).
Display OmittedIn concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregation.
Keywords: Microrheology; Phase behavior; Protein conformation; Glycine; NaCl; ArgHCl; Protein interactions;

Microfluidic manufacturing of phospholipid nanoparticles: Stability, encapsulation efficacy, and drug release by Mariana Guimarães Sá Correia; Maria L. Briuglia; Fabio Niosi; Dimitrios A. Lamprou (91-99).
Display OmittedLiposomes have been the centre of attention in research due to their potential to act as drug delivery systems. Although its versatility and manufacturing processes are still not scalable and reproducible. In this study, the microfluidic method for liposomes preparation is presented. DMPC and DSPC liposomes containing two different lipid/cholesterol ratios (1:1 and 2:1) are prepared. Results from this preparation process were compared with the film hydration method in order to understand benefits and drawbacks of microfluidics. Liposomes characterisation was evaluated through stability studies, encapsulation efficacy and drug release profiles of hydrophilic and lipophilic compounds. Stability tests were performed during 3 weeks and the liposomes properties of the most stable formulations were determined using Infrared Microscopy and Atomic Force Microscopy. Microfluidic allows loading of drugs and assembly in a quick single step and the chosen flow ratio for liposomes formulation plays a fundamental role for particle sizes. One hydrophilic and one lipophilic compounds were incorporated showing how formulation and physic-chemical characteristics can influence the drug release profile.
Keywords: Liposomes; Microfluidics; Encapsulation efficacy; Controlled release;

Microfluidics platform for glass capillaries and its application in droplet and nanoparticle fabrication by Bárbara Herranz-Blanco; Eloy Ginestar; Hongbo Zhang; Jouni Hirvonen; Hélder A. Santos (100-105).
Display OmittedThe accessibility to microfluidics of a broader scientific community is often limited by the costly and complex manufacture of the chips. In this respect, we present a simple and reusable platform for the flexible and easy assembly of glass capillaries to create a microfluidics chip within minutes, with excellent chemical compatibility and durability, and without the need of using specialized infrastructure. To demonstrate the application of the proposed platform, we have used it to produce microparticles by the double emulsion approach, nanoparticles by nanoprecipitation, and screened the nanoparticles’ size and polydispersity obtained upon modification of various parameters.
Keywords: Microfluidics; Glass capilaries; Microparticles; Double emulsion; Nanoprecipitation; Drug delivery;

Polysaccharide-coated liposomal formulations for dental targeting by Sara Pistone; Morten Rykke; Gro Smistad; Marianne Hiorth (106-115).
Display OmittedThe efficacy of treatments of oral ailments is often challenged by a low residence time of the conventional pharmaceutical formulations in the oral cavity, which could be improved by using bioadhesive formulations. This in vitro study investigated charged liposomes, both uncoated and coated through electrostatic deposition with polysaccharides (chitosan, alginate and pectin), as bioadhesive systems for the oral cavity. First, formulations that provided liposomes fully coated with polysaccharide were selected. Thereafter, the stability of both the uncoated and the polysaccharide-coated liposomes was investigated in artificial saliva simulating pH, ionic strength, and ionic content of natural saliva. Additionally, adsorption to hydroxyapatite (model for tooth enamel) was tested. The surface charge was of high importance for both the stability in salivary environment and bioadhesion. In artificial saliva, the negatively charged liposomes were the most stable, and the stability of the positively charged liposomes was improved through coating with a negatively charged polysaccharide. On the contrary, the positively charged liposomes were the most bioadhesive, although a moderate adsorption was recorded for the negatively charged liposomes. Based on the present results, the negatively charged liposomes seem to be the most promising formulations used as a tooth adhesive nanosystem and could as such provide improved treatment of tooth ailments.
Keywords: Dental drug delivery; Polymer-coated liposome; Oral cavity; Hydroxyapatite; Adsorption; Artificial saliva;

New nano-matrix oral formulation of nanoprecipitated cyclosporine A prepared with multi-inlet vortex mixer by Hiroki Suzuki; Shun Hamao; Yoshiki Seto; Hideyuki Sato; Jennifer Wong; Robert K. Prud’homme; Hak-Kim Chan; Satomi Onoue (116-119).
Display OmittedWe present a combined nanoparticle (NP) formation process and spray drying to create a nano-matrix formulation of cyclosporine A (CsA) and mannitol (nCsA/MAN) to increase the bioavailability of CsA. CsA NPs were prepared by flash nano precipitation (FNP) using a multi-inlet vortex mixer, and spray-dried with or without mannitol to prepare nCsA/MAN or nanoprecipitated CsA powder (nCsA), respectively. Pre-forming the NPs by FNP uncouples the sizes of the CsA inclusions from the ultimate micron-sized powders produced by spray drying. Both CsA formulations were physicochemically characterized, and a pharmacokinetic study in rats was conducted after oral administration of CsA samples (10 mg-CsA/kg). In the nCsA/MAN, CsA NPs dispersed in the mannitol matrix. In water, both the nCsA and nCsA/MAN reconstituted into NP form with average sizes of 317 and 298 nm, respectively. In dissolution testing, nCsA and nCsA/MAN exhibited marked improvement in the dissolution with 31- and 41-fold higher drug release at 60 min, compared with amorphous CsA, suggesting the significant impact of CsA NP size on dissolution. The higher dissolution rate with the mannitol matrix indicates the role of mannitol on wetting and dispersing the NP aggregates. Both nCsA formulations provided enhancement in CsA exposure in rats with a ca. 3-fold increase of the oral bioavailability compared with amorphous CsA. The nCsA/MAN showed faster absorption in vivo than nCsA, possibly due to the improved dissolution rate. From these findings, the nano-matrix formulation appears to be an efficacious oral dosage form to enhance the biopharmaceutical properties of CsA.
Keywords: Cyclosporine A; Dissolution; Multi-inlet vortex mixer; Nano-matrix formulation; Oral absorption;

Display OmittedThe present investigation aims to develop lumefantrine loaded binary solid lipid nanoparticles (LF-SLNs) to improve its poor and variable oral bioavailability. The oral bioavailability of LF is poor and variable due to its limited aqueous solubility and P-gp mediated efflux occurring in small intestine. LF-SLNs were prepared using binary lipid mixture of stearic acid and caprylic acid stabilized with TPGS (D-alpha tocopheryl polyethylene glycol 1000 succinate) and Poloxamer 188. Developed LF-SLNs were characterized for particle size distribution, zeta potential, entrapment efficiency, solid state properties and biopharmaceutical properties including in situ intestinal permeability and oral bioavailability. The particle size distribution, zeta potential and entrapment efficiency of optimized batch (LF-SLN7) was found to be 357.7 ± 43.27 nm, 25.29 ± 1.15 mV and 97.35 ± 0.30%, respectively. DSC thermographs showed loss of crystalline nature of lumefantrine in LF-SLNs. In situ single pass intestinal permeability study (SPIP) study indicated significant enhancement in the effective intestinal permeability of LF from LF-SLN7 as compared to that of control. Pharmacokinetic study also showed significant increase in Cmax and area under curve (AUC0-) from LF-SLN7 (3860 ± 521 ng/mL and 43181 ± 2557 h × ng/mL, respectively) as compared to that of LF-control suspension (1425 ± 563 ng/mL and 19586 ± 1537 h × ng/mL, respectively). Thus, developed LF-SLNs can be promising to overcome P-gp efflux pump and enhance the oral bioavailability of lumefantrine.
Keywords: Lumefantrine; Solid lipid nanoparticle; P-glycoprotein pump efflux; Intestinal permeability; Oral bioavailability; In situ single pass intestinal permeability study (SPIP);

This report examines the energetics of aggregate formation between hydroxypropyl methylcellulose (HPMC) and model ionic surfactants including sodium dodecyl sulfate (SDS) at pharmaceutically relevant concentrations using the isothermal titration calorimetry (ITC) technique and a novel treatment of calorimetric data that accounts for the various species formed. The influence of molecular weight of HPMC, temperature and ionic strength of solution on the aggregate formation process was explored. The interaction between SDS and HPMC was determined to be an endothermic process and initiated at a critical aggregation concentration (CAC). The SDS-HPMC interactions were observed to be cooperative in nature and dependent on temperature and ionic strength of the solution. Molecular weight of HPMC significantly shifted the interaction parameters between HPMC and SDS such that at the highest molecular weight (HPMC K–100 M; >240 kDa), although the general shape of the titration curve (enthalpogram) was observed to remain similar, the critical concentration parameters (CAC, polymer saturation concentration (Csat) and critical micelle concentration (CMC)) were significantly altered and shifted to lower concentrations of SDS. Ionic strength was also observed to influence the critical concentration parameters for the SDS-HPMC aggregation and decreased to lower SDS concentrations with increasing ionic strength for both anionic and cationic surfactant-HPMC systems. From these data, other thermodynamic parameters of aggregation such as Δ H a g g ° , Δ G a g g ° , H a g g ° , Δ S a g g ° , and Δ C p were calculated and utilized to postulate the hydrophobic nature of SDS-HPMC aggregate formation. The type of ionic surfactant head group (anionic vs. cationic i.e., dodecyltrimethylammonium bromide (DTAB)) was found to influence the strength of HPMC-surfactant interactions wherein a distinct CAC signifying the strength of HPMC-DTAB interactions was not observed. The interpretation of the microcalorimetric data at different temperatures and ionic strengths while varying properties of polymer and surfactant was a very effective tool in investigating the nature and energetics of HPMC and ionic surfactant interactions.
Keywords: HPMC; Ionic surfactants; Isothermal titration calorimetry (ITC); Polymer-surfactant interactions; Nanosuspensions;

The strategic relevance of manufacturing technology: An overall quality concept to promote innovation preventing drug shortage by Michele Panzitta; Mauro Ponti; Giorgio Bruno; Giancarlo Cois; Alessandro D’Arpino; Paola Minghetti; Francesca Romana Mendicino; Luana Perioli; Maurizio Ricci (144-157).
Display OmittedManufacturing is the bridge between research and patient: without product, there is no clinical outcome. Shortage has a variety of causes, in this paper we analyse only causes related to manufacturing technology and we use shortage as a paradigm highliting the relevance of Pharmaceutical Technology. Product and process complexity and capacity issues are the main challenge for the Pharmaceutical Industry Supply chain. Manufacturing Technology should be acknowledged as a R&D step and as a very important matter during University degree in Pharmacy and related disciplines, promoting collaboration between Academia and Industry, measured during HTA step and rewarded in terms of price and reimbursement. The above elements are not yet properly recognised, and manufacturing technology is taken in to consideration only when a shortage is in place. In a previous work, Panzitta et al. proposed to perform a full technology assessment at the Health Technological Assessment stage, evaluating three main technical aspects of a medicine: manufacturing process, physicochemical properties, and formulation characteristics. In this paper, we develop the concept of manufacturing appraisal, providing a technical overview of upcoming challenges, a risk based approach and an economic picture of shortage costs. We develop also an overall quality concept, not limited to GMP factors but broaden to all elements leading to a robust supply and promoting technical innovation.
Keywords: Pharmaceutical technology; Manufacturing technology; GMP; HTA; Formulation; Manufacturing process; Shortage; Quality;

Display OmittedCurcumin (CUR) has a wide spectrum of biological and pharmacological activities, yet problems of its bioavailability remained a major challenge in preclinical studies. Thus, the design of the delivery systems with CUR as a model drug featuring dual release process – an initial burst followed by sustained release – to provide the optimal drug pharmacokinetics in the therapeutic region has been actively pursued. In this study, the 3-aminopropyltriethoxysilane (APTES)-functionalized electrospun poly(N-vinyl-2-pyrrolidone) fibers (NH2-PVP) were utilized as a free-standing substrate for the immobilization of CUR-PVP capped gold nanoparticles (CUR-PGNPs) conjugates. The conjugate was synthesized by sonication and the drug entrapment percentage was determined to be 54.2  ± 1.8. CUR-PGNPs immobilized on NH2-PVP fibers showed a moderate burst release during the first few hours, followed by a sustained release lasting for 2 days. The drug release was found pH-dependent (pH 5.0 > 6.0 > 7.4). The two-stage release profiles of CUR-PGNPs@NH2-PVP fibers were fitted well to Korsmeyer-Peppas model, indicating a non-Fickian diffusion mechanism for initial burst release and Fickian diffusion-controlled mechanism for the sustained release. Initial biocompatibility assessments based on lactate dehydrogenase (LDH) assay and morphological examination by SEM with L-929 mouse fibroblasts revealed that CUR-PGNPs@NH2-PVP nanofibrous scaffold was capable of supporting cell growth over a culture period of 3 days.
Keywords: Curcumin; Gold nanoparticles; Aminosilane functionalization; Sustained release; Biocompatibility;

Display OmittedThe use of nanoparticles for drug delivery is still restricted by their limited stability when stored in an aqueous medium. Freeze drying is the standard method for long-term storage of colloidal nanoparticles; however the method needs to be elaborated for each formulation. Spray freeze drying (SFD) is proposed here as a promising alternative for lyophilizing colloidal nanoparticles. Different types of polymeric and lipid nanoparticles were prepared and characterized. Afterwards, samples were spray freeze dried by spraying into a column of cold air with a constant concentration of different cryoprotectants, and the frozen spherules were collected for further freeze drying. Similar samples were prepared using the commonly used technique, freeze drying, as controls. Using SFD, fast-dissolving, spherical and porous nanocomposite microparticles with remarkably high flowability (CI ≤ 10) were produced. On the contrary to similar samples prepared using the freeze drying technique, the investigated polymeric and lipid nanoparticles were completely reconstituted (Sf/Si ratio <1.5) after SFD. SFD proved to be an effective platform for improving the long-term stability of colloidal nanoparticles.
Keywords: Spray freeze drying; Stability; Freeze drying; Polymeric nanoparticles; Lipid nanocapsules; Solid lipid nanoparticles; Liposomes;

Microbial biosensors to monitor the encapsulation effectiveness of Doxorubicin in chimeric advanced Drug Delivery Nano Systems: A calorimetric approach by Konstantinos Gardikis; Marco Signorelli; Chiara Ferrario; Alberto Schiraldi; Maria Grazia Fortina; Sophia Hatziantoniou; Costas Demetzos; Dimitrios Fessas (178-184).
Display OmittedThe release of the anticancer drug doxorubicin (DOX) incorporated in a new drug carrier, namely a chimeric nanosystem formed by liposomes and dendrimers, was studied following the influence of the drug on the growth kinetics of the Lactobacillus helveticus bacterium, that would mimic the intestinal microflora.The bacterial growth was followed at 37 °C by means of Isothermal Titration Calorimetry (ITC) and the method was assessed to monitor the overall effect of the delivered drug obtaining simple objective parameters to define the encapsulation effectiveness of the system, discriminating dose effects even in cases of very low release. Traditional microbiological investigations and in vitro release tests were also performed in parallel for validation.The achieved results suggest that L. helveticus is an excellent candidate as biosensor to assess the sealing effectiveness of these DOX drug carriers through ITC investigations. This approach can be extended for quantitative comparison of drug delivery systems with the same drug inserted in other supramolecular bodies for quantitative comparison.The peculiar results for the DOX drug carrier system investigated, indicate also that, the use of hydrophilic dendrimers in this case, produce a high sealing effect that seems promising in terms of the intestinal flora protection.
Keywords: Doxorubicin; Drug delivery; Liposomes; Dendrimers; Isothermal titration calorimetry (ITC); Lactobacillus helveticus;

Development of hydroxyapatite nanoparticles loaded with folic acid to induce osteoblastic differentiation by Catarina Santos; Pedro Gomes; José A. Duarte; Margarida M. Almeida; Maria E.V. Costa; Maria H. Fernandes (185-195).
Display OmittedRecently it has been shown that folic acid can have an important role in bone regeneration. For this reason, combining a classic bone regeneration system as, hydroxyapatite, loaded with folic acid, may be an important issue to be developed. To address this issue, hydroxyapatite nanoparticles loaded with folic acid were designed as an effective bone regenerative system, to induce osteoblast differentiation and improve the bone regeneration. HapNP were prepared by a hydrothermal method that used citric acid as a tailoring agent of particles morphology and, simultaneously, had the particularly to let carboxylic pendant groups in the particle surface, which provided a platform for the immobilization of folic acid (FA), producing HapNP-FA. A comparative study among hydroxyapatite nanoparticles loaded and unloaded with folic acid in presence of human mesenchymal stem cells was performed. The results demonstrate, that nanoparticles were able to be internalized by human mesenchymal stem cells. In addition, cell proliferation and viability were not affected in a wide concentration range. Both particles induced the expression of Runx2 and the expression and activity of alkaline phosphatase. However, HapNP-FA caused a significantly higher overexpression of Runx2. The osteoblastic differentiation confirms the potential applicability of HapNP-FA in the local bone regeneration.
Keywords: Folic acid; Nanohydroxyapatite; Osteoblastic induction; Bone regeneration;

Polymeric nanospheres for topical delivery of vitamin D3 by Tannaz Ramezanli; Brian E. Kilfoyle; Zheng Zhang; Bozena B. Michniak-Kohn (196-203).
Display OmittedThis study investigates the potential application of polymeric nanospheres (known as TyroSpheres) as a formulation carrier for topical delivery of cholecalciferol (i.e., Vitamin D3, VD3) with the goal to improve the skin delivery and stability of VD3. High drug loading and binding efficiencies were obtained for VD3 when loaded in TyroSpheres. VD3 was released from TyroSpheres in a sustained manner and was delivered across the stratum corneum, which occurred independent of the initial drug loading. An ex vivo skin distribution study showed that TyroSphere formulations delivered 3–10 μg of active into the epidermis which was significantly higher than that delivered from Transcutol® (the control vehicle). In addition, an in vitro cytotoxicity assay using keratinocytes confirmed that VD3 encapsulation in the nanoparticles did not alter the drug activity. Photodegradation of VD3 followed zero-order kinetics. TyroSpheres were able to protect the active against hydrolysis and photodegradation, significantly enhancing the stability of VD3 in the topical formulation.
Keywords: Vitamin D3; TyroSpheres; Nanoparticles; Skin permeation; Photostability;

The effect of pulse duration, power and energy of fractional Er:YAG laser for transdermal delivery of differently sized FITC dextrans by Barbara Zorec; Dejan Škrabelj; Marko Marinček; Damijan Miklavčič; Nataša Pavšelj (204-213).
Display OmittedWe studied fractional Er:YAG laser to enhance transdermal drug delivery of compounds possessing different molecular weights: FITC-dextrans (or FD) with average molecular weights of 4, 10 and 20 kDa. Vertical glass Franz diffusion cells were used to study molecular transport through dermatomed porcine skin and histological analysis of laser-treated skin was performed after treatment with different laser pulse protocols. We were comparing different pulse durations at constant or varying pulse energies. We found that the energy of the delivered pulses mostly dictates the size/depth of laser-created microchannels, while the duration of the pulses dictates the extent of thermally altered tissue. That is, tissue ablation threshold is lowered at shorter pulse durations with higher power, which is preferred as it lowers thermal effects on viable skin layers. Especially for smaller molecules, transdermal delivery is increased by increasing laser-created microchannel size, but also by making partitioning into tissue easier when less thermal damage is caused on tissue. For large molecules, molecular transport through the remainder of skin tissue becomes increasingly difficult regardless of laser pulse parameters.
Keywords: Transdermal drug delivery; Fractional laser; Er:YAG; Pulse duration; Energy modulation;

Co-delivery of zinc and 5-aminosalicylic acid from alginate/N-succinyl-chitosan blend microspheres for synergistic therapy of colitis by Haogang Duan; Shaoyu Lü; Hongyan Qin; Chunmei Gao; Xiao Bai; Yuhui Wei; Xin’an Wu; Mingzhu Liu; Xinyu Zhang; Zhen Liu (214-224).
Display OmittedThe present study developed novel zinc ion cross-linked alginate/N-succinyl-chitosan (NSC) blend microspheres (MS) for co-delivery of zinc and 5-aminosalicylic acid (5-ASA) for synergistic therapy of colitis. Physicochemical characterization of blend MS was assessed using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and energy dispersive X-ray spectrometer (EDS). In vitro release studies demonstrated that blend MS has a pH-dependent release property. Both 5-ASA and zinc have lower release in acid medium and higher release in colonic environment. The therapeutic efficacy of zinc cross-linked blend MS was evaluated using induced-colitis rat models, and showed a superior treatment effect in alleviating inflammation of colitis rats. No systemic toxicity was observed after oral administration of blend MS. Therefore, zinc ion cross-linked alginate/N-succinyl-chitosan blend MS appeared to be a good candidate for co-delivery of zinc and 5-ASA to colon, and had great potential application in inflammatory bowel diseases (IBD) treatment.
Keywords: Alginate; N-succinyl-chitosan; Blend microspheres; Co-delivery; Colon-specific;

Lipid vesicular nanocarrier: Quick encapsulation efficiency determination and transcutaneous application by Yibang Zhang; Weibeng Ng; Xue Feng; Fangying Cao; Huaxi Xu (225-230).
Display OmittedNanoscale delivery systems have been widely investigated to overcome the penetration barrier of stratum corneum for effective transcutaneous application. The aim of this study is the development of effective vesicular formulations of ovalbumin and saponin which are able to promote penetration through the skin layers. Three kinds of vesicular formulations have been investigated as carriers, including liposomes, transfersomes and ethosomes, in which cholesterol and/or cationic lipid stearylamine are incorporated. The impact of membrane composition variations on the protein entrapment has been evaluated for each vesicle type. Formulations were characterized for particle size, polydispersity and encapsulation efficiency. The best formulations for each type of vesicle were subjected to in vivo transdermal immunization in mice. Among the three kinds of vesicular carrier, ethosomal nano carrier not only showed the best stability over a two months’ storage, but also enabled the highest increase in the titer of serum antibody. In this regard, cationic nano-ethosomes can be considered as a promising vesicular carrier for transdermal vaccines. Meanwhile, we have developed a simple method to determine encapsulation efficiency of vesicular systems, which has potential application as a high throughput screening for vesicular formulations.
Keywords: Encapsulation efficiency; Liposome; Transfersome; Ethosome; Transdermal immunization;

Microencapsulated SLN: An innovative strategy for pulmonary protein delivery by Diana P Gaspar; Carmen Serra; Paulo R Lino; Lídia Gonçalves; Pablo Taboada; Carmen Remuñán-López; António J Almeida (231-246).
Display OmittedAssociating protein with nanoparticles is an interesting strategy to improve their bioavailability and biological activity. Solid lipid nanoparticles (SLN) have been sought as carriers for therapeutic proteins transport to the lung epithelium. Nevertheless, because of their low inertia, nanoparticles intended for pulmonary application usually escape from lung deposition. To overcome this problem, the production of spray-dried powders containing nanoparticles has been recently reported. Herein we developed new hybrid microencapsulated SLN for pulmonary administration, containing a model protein (papain, PAP). PAP was adsorbed onto glyceryl dibehenate and glyceryl tristearate SLN. Physical characterization using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) confirmed the interaction between PAP and SLN corroborating that the protein was efficiently adsorbed at SLN’s surface. PAP adsorption onto SLN (PAP-SLN) slightly increased particle size, while decreasing the SLN negative surface charge. The adsorption process followed a Freundlich type of adsorption isotherm. Nanoformulations were then spray-dried, originating spherical microparticles with suitable aerodynamic characteristics. Full characterization of microparticles was performed using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and isothermal titration calorimetry (ITC). PAP was released from dry powders in a higher extent when compared with non spray-dried SLN. Nevertheless, protein stability was kept throughout microsphere production, as assessed by SDS-PAGE.
Keywords: Solid lipid nanoparticles; Adsorption; Proteins; Spray-drying; Microencapsulation; Pulmonary administration;

Spherical agglomerates of lactose with enhanced mechanical properties by Dejan Lamešić; Odon Planinšek; Zoran Lavrič; Ilija Ilić (247-257).
Display OmittedThe aim of this study was to prepare spherical agglomerates of lactose and to evaluate their physicochemical properties, flow properties, particle friability and compaction properties, and to compare them to commercially available types of lactose for direct compression (spray-dried, granulated and anhydrous β-lactose). Porous spherical agglomerates of α-lactose monohydrate with radially arranged prism-like primary particles were prepared exhibiting a high specific surface area. All types of lactose analysed had passable or better flow properties, except for anhydrous β-lactose, which had poor flowability. Particle friability was more pronounced in larger granulated lactose particles; however, particle structure was retained in all samples analysed. The mechanical properties of spherical agglomerates of lactose, in terms of compressibility, established with Walker analysis, and compactibility, established with a compactibility profile, were found to be superior to any commercially available types of lactose. Higher compactibility of spherical agglomerates of lactose is ascribed to significantly higher particle surface area due to a unique internal structure with higher susceptibility to fragmentation.
Keywords: Spherical crystallization; Spherical agglomerates; Lactose; Compactibility; Compressibility;

Development of flexible nanocarriers for siRNA delivery into tumor tissue by Hyunkyung Jung; Yuri Shimatani; Mahadi Hasan; Kohei Uno; Susumu Hama; Kentaro Kogure (258-265).
Display OmittedVarious non-viral delivery systems for small interfering RNAs (siRNA) have been developed. Such delivery systems generally exhibit tightly formed spherical structures. While such carriers have demonstrated good transfection activity in mono-layered cell systems, effects against solid tumors are often less apparent and difficult to demonstrate, likely due to the rigid structures of the carriers, which may prevent penetration to deeper regions within tumor tissue. Herein, we developed a flexible nanocarrier (FNC) system that is able to penetrate to deeper regions within tumor tissue. Specifically, we employed previously found flexible polyplexes comprised of siRNA and poly-l-lysine as wick structures for the preparation of FNCs. FNCs were constructed by coating the wick structures with lipids using a liposomal membrane fusion method. The diameters of the resulting FNCs were ca. 170 nm, and the shapes were non-spherical. Lipid coating was confirmed using a nuclease resistance assay. Furthermore, FNCs showed significant RNA interference effects, comparable to Lipofectamine 2000, in a mono-layered cell system. To accelerate tumor penetration, the FNC surface was modified with polyethylene glycol (PEG) and the tight junction opener peptide AT1002. Surface-modified FNCs demonstrated effective penetrability into a cancer spheroid. Thus, we developed a novel and unique tumor-penetrable siRNA FNC system.
Keywords: Flexible nanocarrier; Intercellular penetrability; siRNA/PLL polyplexes;

Application of DoE approach in the development of mini-capsules, based on biopolymers and manuka honey polar fraction, as powder formulation for the treatment of skin ulcers by Marika Tenci; Silvia Rossi; Maria Cristina Bonferoni; Giuseppina Sandri; Irene Mentori; Cinzia Boselli; Antonia Icaro Cornaglia; Maria Daglia; Anna Marchese; Carla Caramella; Franca Ferrari (266-277).
Display OmittedThe aim of the present work was the development of a powder formulation for the delivery of manuka honey (MH) bioactive components in the treatment of chronic skin ulcers. In particular pectin (PEC)/chitosan glutamate (CS)/hyaluronic acid (HA) mini-capsules were obtained by inverse ionotropic gelation in presence of calcium chloride and subsequently freeze-dried. Optimization of unloaded (blank) formulation was performed using DoE approach. In a screening phase, the following three factors were investigated at two levels: CS (0.5–1% w/w), PEC (0.5–1% w/w) and HA (0.3–0.5% w/w) concentrations. For the optimization phase a “central composite design” was used. The response variables considered were: particle size, buffer (PBS) absorption and mechanical resistance. In a previously work two different MH fractions were investigated, in particular MH fraction 1 (Fr1), rich in polar substances (sugars, methylglyoxal (MGO), dicarbonyl compounds, …), was able to enhance human fibroblasts in vitro proliferation. In the present work, the loading of MH Fr1 into mini-capsules of optimized composition determined a significant increase in cell proliferation in comparison with the unloaded ones. Loaded particles showed antimicrobial activity against Staphylococcus aureus and Streptococcus pyogenes; they were also able to improve wound healing in vivo on a rat wound model.
Keywords: DoE approach; Pectin; Chitosan glutamate; Hyaluronic acid; Wound healing;

γ-Cyclodextrin by Phennapha Saokham; Thorsteinn Loftsson (278-292).
Display Omittedγ-Cyclodextrin (γCD) is a cyclic oligosaccharide formed by bacterial digestion of starch and used as solubilizing agent and stabilizer in a variety of pharmaceutical and food products. γCD is a large (molecular weight 1297 Da) hydrophilic molecule that does not readily permeate biological membranes and is rapidly digested by bacteria in the gastrointestinal tract. In humans γCD is metabolized by α-amylase that is found in, for example, saliva, bile fluid and tears. Thus, bioavailability of γCD is negligible. Also, γCD is readily excreted unchanged in the urine after parenteral administration. Like other cyclodextrins, γCD can form water-soluble inclusion complexes with many poorly-soluble compounds. In comparison with the natural αCD and βCD, γCD has the largest hydrophobic cavity, highest water solubility and the most favorable toxicological profile. The focus of this review is production, physiochemical properties, pharmacokinetics, toxicity and applications of γCD and its derivatives. Also, the aggregation behavior of γCD in aqueous media is discussed.
Keywords: γ-Cyclodextrin; ADME; Complexing agent; Inclusion complex; Solubilization; Aggregation;

Needleless coaxial electrospinning: A novel approach to mass production of coaxial nanofibers by Lucie Vysloužilová; Matej Buzgo; Pavel Pokorný; Jiří Chvojka; Andrea Míčková; Michala Rampichová; Jiří Kula; Karel Pejchar; Martin Bílek; David Lukáš; Evžen Amler (293-300).
Display OmittedHerein, we describe a simple spinneret setup for needleless coaxial electrospinning that exceeds the limited production capacity of current approaches. The proposed weir spinneret enables coaxial electrospinning from free liquid surface. This approach leads to the formation of coaxial nanofibers with higher and uniform shell/core ratio, which results in the possibility of better tuning of the degradation rate. The throughput and quality increase favor the broader application of coaxial nanofibers from weir spinnerets as systems for controlled drug delivery in regenerative medicine and tissue engineering.
Keywords: Core-shell nanofibers; Coaxial electrospinning; Needleless electrospinning; Fluorescent spectroscopy;

Display OmittedIn this project, synergistic cancer cell death was achieved by a targeted delivery system comprising Bcl-xL-specific shRNA and a very low DOX content, which simultaneously activated an intrinsic apoptotic pathway. A modified branched polyethylenimine (PEI 10 kDa) was grafted through polyethylene glycol (PEG) linker to carboxylated single-walled carbon nanotubes (SWCNT) to serve as a vehicle for shRNA delivery. The SWNT-PEG-PEI conjugate was covalently attached to AS1411 aptamer as the nucleolin ligand to target the co-delivery system to the tumor cells overexpressing nucleolin receptors on their surface. The final vehicle was eventually obtained after intercalation of DOX with pBcl-xL shRNA-SWCNT-PEG-10-10%PEI-Apt. Cell viability assay, GFP expression and transfection experiment against L929 (-nucleolin) and AGS (+nucleolin) cells illustrated that the tested targeted delivery system inhibited the growth of nucleolin-abundant gastric cancer cells with strong cell selectivity. Subsequently, we illustrated that the combination treatment of the selected shRNAs and DOX had excellent tumoricidal efficacy as verified by MTT assay. Furthermore, very low concentration of DOX, approximately 58-fold lower than its IC50 concentration, was used which could mitigate toxic side effects of DOX. Overall, our work revealed that combination of shRNA-mediated gene-silencing strategy with chemotherapeutic agents constitutes a valuable and safe approach for antitumor activity.
Keywords: AS1411 aptamer; Nucleolin; pBcl-xL shRNA; SWCNT; Doxorubicin;

Paclitaxel-loaded polymeric nanoparticles combined with chronomodulated chemotherapy on lung cancer: In vitro and in vivo evaluation by Jie Hu; Shaozhi Fu; Qiuxia Peng; YunWei Han; Jie Xie; Ning Zan; Yue Chen; Juan Fan (313-322).
In this work, a passive targeting paclitaxel-loaded nanoparticles(PTX-NPs) was prepared and used to investigate its synergistic anti-tumor efficacy by combination with circadian chronomodulated chemotherapy in xenografted human lung cancer. The synergistic mechanism may be related to inhibit tumor cell proliferation through its action on Ki-67, and decreased micro-vessel density (MVD) associated with CD31 and promoted cell apoptosis.Display OmittedThe objective of our study was to examine the anti-tumor effect of paclitaxel (PTX)-loaded polymeric nanoparticles (PTX-NPs) combined with circadian chronomodulated chemotherapy. Our intention was to screen out the best time of the day for the drug to be administered. PTX-NPs with a diameter of approximately 168 nm were prepared through a thin film dispersion technique. The PTX in PTX-NPs showed an initial fast release subsequently a slower and sustained release. The cytotoxicity of chronomodulated administration of PTX-NPs in vitro confirmed that its cytotoxic effect was lower than that of PTX injection, and showed a time-dependent effect. In addition, anti-tumor effect was examined by analysing tumor growth inhibition rate, micro-vessel density (MVD), cell proliferation and cell apoptosis, following either injection with PTX or administration of PTX-NPs. Micro fluorine-18-deoxyglucose PET/computed tomography (18F-FDG PET/CT) was used to evaluate tumor reactivity to PTX-NPs combined with chronomodulated chemotherapy. Taken these results into consideraion, our experiment indicates that PTX-NPs exhibit greater anti-tumor activity against A549 cells, in comparison with PTX injection, and the anti-tumor effect at 15 h after light onset (HALO) administration is the best in all groups. Therefore, prepared PTX-NPs combined with chronomodulated chemotherapy could be a potential treatment for lung cancer.
Keywords: Polymeric nanoparticles; PCL-PEG-PCL; Paclitaxel; Chronomodulated chemotherapy; Anti-tumor activity;

Display OmittedThis paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0 ± 0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6–2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL.
Keywords: DOPE (CID 9546744); CHEMS (CID 65082); DSPC (CID 94190); (CID 406952); (CID 5997); (CID 44134771); pH-sensitive liposomes; Active loading; Gene-directed enzyme-prodrug therapy; Endosome escape;

Surfactant effect on the physicochemical characteristics of cationic solid lipid nanoparticles by Chiara Botto; Nicolò Mauro; Erika Amore; Elisabetta Martorana; Gaetano Giammona; Maria Luisa Bondì (334-341).
Display OmittedSolid lipid nanoparticles (SLNs) may be considered as a new approach for therapeutics for many diseases. In addition to drug delivery, their use as non-viral vectors for gene delivery can be obtained by including cationic lipids, which provide a positive surface potential that favors binding to the nucleic acids as DNA, siRNA, miRNA, etc. In fact, the addition of cationic surfactants is indispensable for obtaining nanoparticles with surface positive charge. In this study, three different cationic lipids (dioctadecyl dimethyl ammonium bromide, cetyltrimethyl ammonium bromide, cetylpyridinium chloride) and Brij 76 as nonionic surfactant were employed to formulate Precirol ATO 5 based cSLN using pEGFP-LUC as model plasmid. The physicochemical properties of cSLN were influenced by both type and amount of surfactants. Thermal analyses of bulk cSLN showed endothermic peaks significantly different from the ones of the single pure components, hinting a complete entanglement of the lipid matrix with the surfactants and justifying the different behavior of the cSLN in the ability to interact with the plasmid DNA. Finally, the biocompatibility of cSLN was demonstrated by hemolytic assays. These results may give an insight into the choice of surfactants in order to obtain non-toxic and highly effective delivery systems for gene therapy.
Keywords: Cationic solid lipid nanoparticles; Nanocarriers; Surfactant; Characterization; Gene delivery;

Pharmaceutical technology can turn a traditional drug, dexamethasone into a first-line ocular medicine. A global perspective and future trends by Javier Rodríguez Villanueva; Laura Rodríguez Villanueva; Manuel Guzmán Navarro (342-351).
Display OmittedDexamethasone is one of the most prescribed glucocorticoids. It is effective and safe in the treatment of a wide variety of ocular conditions, including anterior and posterior segment inflammation. However, its half-life in the vitreous humor is very short, which means that it typically requires frequent administrations, thus reducing patient adherence and causing therapeutic failure. Innovative dexamethasone delivery systems have been designed in an attempt to achieve sustained release and targeting. The FDA has approved dexamethasone implants for the treatment of macular edema secondary to retinal vein occlusion and posterior segment noninfectious uveitis. Lenses, micro- and nanoparticles, liposomes, micelles and dendrimers are also proving to be adequate systems for maintaining optimal dexamethasone levels in the site of action. Pharmaceutical technology is turning a classical drug, dexamethasone, into a fashionable medicine.
Keywords: Dexamethasone; Sustained release systems; Implants; Microparticles; Nanoparticles; Dendrimers; Ocular carriers;

Calcium phosphate nanoparticles functionalized with alendronate-conjugated polyethylene glycol (PEG) for the treatment of bone metastasis by Weijing Chu; Yanjuan Huang; Chanzhen Yang; Yunhui Liao; Xuefei Zhang; Mina Yan; Shengmiao Cui; Chunshun Zhao (352-363).
Display OmittedBecause of the peculiarity of the bone microstructure, the uptake of chemotherapeutics often happens at non-targeted sites, which induces side effects. In order to solve this problem, we designed a bone-targeting drug delivery system that can release drug exclusively in the nidus of the bone. Alendronate (ALN), which has a high ability to target to hydroxyapatite, was used to fabricate double ALN-conjugated poly (ethylene glycol) 2000 material (ALN-PEG2k-ALN). The ALN-PEG2k-ALN was characterized using 1H NMR and 31P NMR and FTIR. ALN-PEG2k-ALN-modified calcium phosphate nanoparticles (APA-CPNPs) with an ALN targeting moiety and hydrophilic poly (ethylene glycol) arms tiled on the surface was prepared for bone-targeted drug delivery. The distribution of ALN-PEG2k-ALN was tested by X-ray photoelectron spectroscopy. Isothermal titration calorimetry data indicated that similar to free ALN, both ALN-PEG2k-ALN and APA-CPNPs can bind to calcium ions. The bone-binding ability of APA-CPNPs was verified via ex vivo imaging of bone fragments. An in vitro release experiment demonstrated that APA-CPNPs can release drug faster in an acid environment than a neutral environment. Cell viability experiments indicated that blank APA-CPNPs possessed excellent biocompatibility with normal cells. Methotrexate (MTX) loaded APA-CPNPs have the same ability to inhibit cancer cells as free drug at high concentrations, while they are slightly weaker at low concentrations. All of these experiments verified the prospective application of APA-CPNPs as a bone-targeting drug delivery system.
Keywords: Bone targeting; Alendronate; Bone metastasis; Calcium phosphate nanoparticle;

Display OmittedThe objective of this study was to prepare solid lipid nanoparticles (SLNs) for sustained pulmonary delivery of Yuxingcao essential oil (YEO). Three YEO loaded SLNs (SLN-200, SLN-400 and SLN-800) with different particle size were prepared and separated following a high-shear homogenization technique using Compritol 888 ATO as lipid and polyvinyl alcohol as an emulsifier. The particle size, zeta potential, drug encapsulation efficiency and drug loading of the SLNs were determined to be between 171 and 812 nm, −17.1 and −19.3 mV, between 76.6 and 90.2% and between 2.34 and 3.12%, respectively whereas the in vitro release data showed that the SLNs led to sustained drug release up to 48 h. In addition, the SLN suspensions after nebulization conferred the fine particle fractions (<5.4 μm) of 67.4–75.8%. Following intratracheal administration to rats, YEO loaded SLNs not only prolonged pulmonary retention up to 24 h, but also increased AUC values (15.4, 18.2 and 26.3 μg/g h for SLN-200, SLN-400 and SLN-800, respectively) by 4.5-7.7 folds compared to the intratracheally dosed YEO solution and by 257–438 folds to the intravenously dosed YEO solution, respectively. The present results were the first to show that YEO loaded SLNs may sustain YEO inhalation delivery and improve local bioavailability, representing a promising inhalable carrier to attain once daily application.
Keywords: Solid lipid nanoparticles; Yuxingcao essential oil; Pulmonary delivery; Pulmonary retention; Sustained release; Traditional Chinese medicine;

Display OmittedFor a highly hydrophobic and drug, it is difficult to formulate and solidify its nanocrystals with high drug loading and good redispersity. In this study, Allisartan Isoproxil was used as a model drug, and SDS was tested in combination with sugar alcohols to improve the drug loading and redispersity for its spray-dried nanocrystals, simultaneously. These spray-dried nanocrystals had high drug loading of 61.7% and good redispersity, which was mainly attributed to the addition of SDS. In addition, the nanocrystals were characterized by scanning electron microscopy, differential scanning calorimetry, X-ray power diffraction analysis, Fourier transform infrared spectroscopy and Raman spectroscopy. The results showed that Allisartan Isoproxil was unchanged in chemical structure, but was partially amorphous. Regarding the in vitro dissolution, the optimism formulation shown an increased dissolution compared with the bulk drug and aggregated nanocrystals. Importantly, the optimum formulation increased the oral bioavailability of crude ALS-3 for 4.73 times. In conclusion, we developed a method to solidify aqueous nanocrystals with increased drug loading, good redispersity and improved bioavailability for high hydrophobic drugs.
Keywords: Spray-drying; Nanocrystals; Drug loading; Redispersity; Characterization; Oral bioavailability;

A novel approach for dry powder coating of pellets with Ethylcellulose. Part I: Evaluation of film formulation and process set up by Beatrice Albertini; Serena Bertoni; Cecilia Melegari; Luisa Stella Dolci; Nadia Passerini (380-391).
Display OmittedAn innovative dry powder coating technology was developed in a high-shear granulator using ethylcellulose (E10) as polymer. Several solid plasticizers were investigated with the aim of decreasing the polymer Tg at least to the highest possible working temperature (80 °C). DSC analysis of physical mixtures of E10 and plasticizers evidenced that lauric acid (LA) was the most effective plasticizer. In order to reach the target temperature a liquid plasticizer, oleic acid (OA), was introduced in the coating formulation. Free films were then prepared and the target minimum film forming temperature (MFFT) was established in the range 70–80 °C. Depending on the LA:OA weight ratio, Kollidon VA64 was included to decrease the LA recrystallization, while talc served as anti-sticking agent. Curing at the MFFT ensured the formation of homogeneous and stable films with good stability on storage. The dry powder coating process of placebo pellets was then developed, consisting of a combination of liquid assisted and thermal adhesion methods. The best coating formulations in terms of yields, coating efficiency (expressed as Relative Standard Deviation of the weight applied) and low pellets aggregation were based on E10:LA:OA in a weight ratio of 65:20:15 and 60:20:20. Moreover pellets remained stable after 1 year of storage (25 °C/60% R.H.).
Keywords: Ethylcellulose; Films; Plasticizers; Dry powder coating; Placebo pellets; High-shear granulator;

Improved sustained release of antigen from immunostimulatory DNA hydrogel by electrostatic interaction with chitosan by Yumiko Ishii-Mizuno; Yuka Umeki; Yoshinori Onuki; Hiroshi Watanabe; Yuki Takahashi; Yoshinobu Takakura; Makiya Nishikawa (392-400).
Display OmittedImmunostimulatory DNA hydrogel (sDNA hydrogel) containing unmethylated cytosine-phosphate-guanine (CpG) sequences has been demonstrated to be a useful antigen delivery system, which can effectively induce an antigen-specific immune response through stimulation of the innate immune system. However, relatively rapid release of antigens from the sDNA hydrogel limits its potential. To enhance the potency of the sDNA hydrogel via improvement of its sustained release property, we selected chitosan, a biocompatible cationic polymer which electrostatically interacts with DNA, and mixed it with the sDNA hydrogel. Compared to unmixed sDNA hydrogel, sDNA hydrogel mixed with chitosan (Chitosan-sDNA hydrogel) was more stable, tougher, had more bound water, released a model antigen ovalbumin (OVA) more slowly in vitro, and provided longer retention of OVA at the injection site after intradermal injection into mice. Intradermal immunization of mice with the OVA-loaded Chitosan-sDNA hydrogel resulted in the induction of a higher level of OVA-specific IgG in serum compared with OVA-loaded sDNA hydrogel with no chitosan. These results indicate that the Chitosan-sDNA hydrogel is an improved sustained release formulation for efficient induction of antigen-specific immune responses.
Keywords: Hydrogel; CpG motif; Controlled release; Electrostatic interaction; Chitosan; Antigen delivery;