International Journal of Pharmaceutics (v.502, #1-2)

Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line by Elena Fernández Fernández; Beatriz Santos-Carballal; Wolf-Michael Weber; Francisco M. Goycoolea (1-9).
Display OmittedSuccessful gene therapy requires the development of suitable vehicles for the selective and efficient delivery of genes to specific target cells at the expense of minimal toxicity. In this work, we investigated a non-viral gene delivery system based on chitosan (CS) to specifically address cystic fibrosis (CF). Thus, electrostatic self-assembled CS-pEGFP and CS-pEGFP-siRNA complexes were prepared from high-pure fully characterized CS (Mw ∼20 kDa and degree of acetylation ∼30%). The average diameter of positively-charged complexes (i.e. ζ ∼ +25 mV) was ∼200 nm. The complexes were found relatively stable over 14 h in Opti-MEM. Cell viability study did not show any significant cytotoxic effect of the CS-based complexes in a human bronchial cystic fibrosis cell line (CFBE41o-). We evaluated the transfection efficiency of this cell line with both CS-pEGFP and co-transfected with CS-pEGFP-siRNA complexes at (N/P) charge ratio of 12. We reported an increase in the fluorescence intensity of CS-pEGFP and a reduction in the cells co-transfected with CS-pEGFP-siRNA. This study shows proof-of-principle that co-transfection with chitosan might be an effective delivery system in a human CF cell line. It also offers a potential alternative to further develop therapeutic strategies for inherited disease treatments, such as CF.
Keywords: Cystic fibrosis; Chitosan; Gene delivery; Nanoparticles; Transfection;

Display OmittedUnderstanding the molecular aggregation of therapeutic agents is particularly important when applying low doses of a drug to the surface of the skin. The aim of this study was to understand how the concentration of a drug influenced its molecular aggregation and its subsequent percutaneous penetration after topical application. A model drug tetracaine was shown to form a series of different aggregates across the μM (fluorescence spectroscopy) to mM (light scattering analysis) concentration range. The aggregate formation process was sensitive to the pH of the vehicle in which the drug was dissolved (pH 4, critical aggregation concentration (CAC) – 11 μM; pH 8, CAC – 7 μM) and it appeared to have an impact upon the drug’s percutaneous penetration. At pH 4, increasing the concentration of the drug in the donor solution decreased the skin permeability coefficient (Kp) of tetracaine (13.7 ± 4.3 × 10−3  cm/h to0.06 ± 0.02 × 10−3  cm/h), whilst at pH 8, it increased the Kp (29.9 ± 9.9 × 10−3  cm/h to 75.1 ± 41.7 × 10−3  cm/h). These data trends were reproduced in a silicone membrane and this supported the notion that the more polar aggregates formed at pH 4 acted to decrease the proportion of species available to pass through the skin, whilst the more hydrophobic aggregates formed in pH 8 increased the membrane diffusing species.
Keywords: Tetracaine; Transport; Aggregation; Skin; Penetration enhancer; Pain; Drug delivery;

A simple approach to predict the stability of phospholipid vesicles to nebulization without performing aerosolization studies by Sneha Subramanian; Iftikhar Khan; Oshadie Korale; Mohamed Albed Alhnan; Waqar Ahmed; Mohammad Najlah; Kevin M.G. Taylor; Abdelbary Elhissi (18-27).
Display OmittedMembrane extrusion was investigated for predicting the stability of soya phosphatidylcholine liposomes and surfactosomes (Tween 80-enriched liposomes) to nebulization. Formulations were prepared with or without cholesterol, and salbutamol sulfate (SBS) or beclometasone dipropionate (BDP) were incorporated as model hydrophilic or hydrophobic drugs respectively. Formulations were extruded through 5, 2, 1 and 0.4 μm polycarbonate membrane filters to study the influence of membrane pore size on drug retention by the vesicles. Surfactosomes were found to be very leaky to SBS, such that even without extrusion greater than 50% of the originally entrapped drug was lost; these losses were minimized by the inclusion of cholesterol. The smaller the membrane pore size, the greater the leakage of SBS; hence only around 10% were retained in cholesterol-free surfactosomes extruded through 0.4 μm filters. To study the influence of vesicle size on SBS retained entrapment, an excessive extrusion protocol was proposed (51 extrusion cycles through 1 μm filters) to compare the stability of freshly prepared vesicles (i.e. unextruded; median size approx. 4.5–6.5 μm) with those previously extruded through 1 μm pores. Cholesterol was essential for minimizing losses from liposomes, whilst for surfactosomes size reduction prior to extrusion was the only way to minimize SBS losses which reached up to 93.40% of the originally entrapped drug when no cholesterol was included. When extrusion was applied to BDP-loaded vesicles, greater proportions of the drug were retained in the vesicles compared to SBS. Even with extrusion through 0.4 μm, BDP retention was around 50–60% with little effect of formulation. Excessive extrusion showed BDP retention using small liposomes (1 μm) to be as high as 71–87%, compared to 50–66% for freshly prepared vesicles. The findings, based on extrusion, were compared to studies of vesicle stability to nebulization, published by a range of investigators. It was concluded that extrusion is a valid method for predicting the stability of liposomes to nebulization.
Keywords: Aerosol; Drug delivery; Drug development; Extruder; Nebulizer;

pH-sensitive micelles based on acid-labile pluronic F68–curcumin conjugates for improved tumor intracellular drug delivery by Xiao-Bin Fang; Jin-Ming Zhang; Xi Xie; Di Liu; Cheng-Wei He; Jian-Bo Wan; Mei-Wan Chen (28-37).
Display OmittedCurcumin (Cur) is a highly pleiotropic anticancer agent that inhibits cell proliferation and induces apoptosis in cancer cells. A variety of nano-systems constituted by polymer–drug conjugates have been designed to overcome its shortages on water solubility, chemical instability, and poor bioavailability. However, most of them suffer from ineffective release of Cur in cancer cells in vivo. This work developed a novel flexible acid-responsive micelle formulation by covalently conjugating Cur on the hydrophilic terminals of pluronic F68 chains via cis-aconitic anhydride linkers. The synthesized F68-Cis–Cur conjugates can readily precipitate to form homogeneous micelles with average size about 100 nm in aqueous solution. In acid environments, F68-Cis–Cur conjugates would break down and subsequently release Cur rapidly, for the reason of pH-sensitive cleavage of cis-aconitic anhydride linkers. In vitro anticancer activity tests demonstrated that F68-Cis–Cur micelles induced higher cytotoxicity against both A2780 and SMMC 7721 cells than free Cur. It provided a larger decrease of mitochondrion membrane potential and induced cellular apoptosis. F68-Cis–Cur micelles remarkably increased cellular uptake of Cur than free Cur through caveolae-mediated endocytosis in an energy-dependent manner. This study demonstrates F68-Cis–Cur conjugation as a promising tool for improving intracellular drug delivery in cancer therapy.
Keywords: Polymer–drug conjugate; Curcumin; pH-sensitive; Pluronic F68; Cancer;

Preparation and characterization of fast dissolving flurbiprofen and esomeprazole solid dispersion using spray drying technique by Roshan Pradhan; Tuan Hiep Tran; Sung Yub Kim; Kyu Bong Woo; Yong Joo Choi; Han-Gon Choi; Chul Soon Yong; Jong Oh Kim (38-46).
Display OmittedWe aimed to develop an immediate-release flurbiprofen (FLU) and esomeprazole (ESO) combination formulation with enhanced gastric aqueous solubility and dissolution rate. Aqueous solubility can be enhanced by formulating solid dispersions (SDs) with a polyvinylpyrrolidone (PVP)-K30 hydrophilic carrier, using spray-drying technique. Aqueous and gastric pH dissolution can be achieved by macro-environmental pH modulation using sodium bicarbonate (NaHCO3) and magnesium hydroxide (Mg(OH)2) as the alkaline buffer. FLU/ESO-loaded SDs (FLU/ESO-SDs) significantly improved aqueous solubility of both drugs, compared to each drug powder. Dissolution studies in gastric pH and water were compared with the microenvironmental pH modulated formulations. The optimized FLU/ESO-SD powder formulation consisted of FLU/ESO/PVP-K30/sodium carbonate (Na2CO3) in a weight ratio 1:0.22:1.5:0.3, filled in the inner capsule. The outer capsule consisted of NaHCO3 and Mg(OH)2, which created the macro-environmental pH modulation. Increased aqueous and gastric pH dissolution of FLU and ESO from the SD was attributed to the alkaline buffer effects and most importantly, to drug transformation from crystalline to amorphous SD powder, clearly revealed by scanning electron microscopy, differential scanning calorimetry, and powder X-ray diffraction studies. Thus, the combined FLU and ESO SD powder can be effectively delivered as an immediate-release formulation using the macro-environmental pH modulation concept.
Keywords: Flurbiprofen; Esomeprazole; Solubility; Solid dispersion; Spray-drying; Dissolution;

Novel galactosylated biodegradable nanoparticles for hepatocyte-delivery of oridonin by Ying Wang; Xinquan Liu; Guangpu Liu; Hejian Guo; Caiyun Li; Yongchun Zhang; Fang Zhang; Zhongxi Zhao; Huiling Cheng (47-60).
Display OmittedNanoparticles based on the newly synthesized copolymers of linear PLGA blocked with two TPGS ends and galactosylated TPGS were successfully constructed as carriers of oridonin for liver-targeting. The novel copolymers were characterized by 1H-NMR and TGA. The drug-loaded nanoparticles were prepared by a nanoprecipitation technique and characterized in terms of physicochemical properties, such as particle size, zeta potential, morphology, encapsulation efficiency, in vitro drug release behavior and physical state of the entrapped drug. The ORI-Gal-PT NPs were found to have the highest antitumor efficacy in comparison with the oridonin solution and non-galactosylated nanoparticles and induced a higher apoptotic rate of tumor cells. The targeting nanoparticles could enhance the therapeutic effect of oridonin by increasing uptake of the nanoparticles through asialoglycoprotein receptor-mediated endocytosis. The ORI-Gal-PT NPs system could be a highly promising drug delivery system to be used in liver cancer therapy.
Keywords: Oridonin; Nanoparticles; Liver targeting; Biodegradable polymer; Drug delivery;

Display OmittedP-glycoprotein (P-gp), an ATP-binding cassette (ABC) multidrug transporter, can actively transport a broad spectrum of chemically diverse substrates out of cells and is heavily involved in multidrug resistance (MDR) in tumors. So far, the multiple specific binding sites remain a major obstacle in developing an efficient prediction method for P-gp substrates. Herein, emerging chemical pattern (ECP) combined by hierarchical cluster analysis was utilized to predict P-gp substrates as well as their potential binding sites. An optimal ECP model using only 3 descriptors was established with prediction accuracies of 0.80, 0.81 and 0.74 for 803 training samples, 120 test samples, and 179 independent validation samples, respectively. Hierarchical cluster analysis (HCA) of the ECPs of P-gp substrates derived 2 distinct ECP groups (ECPGs). Interestingly, HCA of the P-gp substrates based on ECP similarities also showed 2 distinct classes, which happened to be dominated by the 2 ECPGs, respectively. In the light of available experimental proofs and molecular docking results, the 2 distinct ECPGs were proved to be closely related to the binding profiles of R- and H-site substrates, respectively. The present study demonstrates, for the first time, a successful ECP model, which can not only accurately predict P-gp substrates, but also identify their potential substrate-binding sites.
Keywords: P-glycoprotein; Emerging chemical pattern; Substrat e-binding site; Prediction; Hierarchical cluster analysis; Molecular docking;

In situ gelling systems based on Pluronic F127/Pluronic F68 formulations for ocular drug delivery by Kosai Al Khateb; Elvira K. Ozhmukhametova; Marat N. Mussin; Serzhan K. Seilkhanov; Tolebai K. Rakhypbekov; Wing Man Lau; Vitaliy V. Khutoryanskiy (70-79).
Display OmittedThis study evaluated the use of Pluronic F127 and Pluronic F68 as excipients for formulating in situ gelling systems for ocular drug delivery. Thermal transitions have been studied in aqueous solutions of Pluronic F127, Pluronic F68 as well as their binary mixtures using differential scanning calorimetry, rheological measurements, and dynamic light scattering. It was established that the formation of transparent gels at physiologically relevant temperatures is observed only in the case of 20 wt% of Pluronic F127. The addition of Pluronic F68 to Pluronic F127 solutions increases the gelation temperature of binary formulation to above physiological range of temperatures. The biocompatibility evaluation of these formulations using slug mucosa irritation assay and bovine corneal erosion studies revealed that these polymers and their combinations do not cause significant irritation. In vitro drug retention study on glass surfaces and freshly excised bovine cornea showed superior performance of 20 wt% Pluronic F127 compared to other formulations. In addition, in vivo studies in rabbits demonstrated better retention performance of 20 wt% Pluronic F127 compared to Pluronic F68. These results confirmed that 20 wt% Pluronic F127 offers an attractive ocular formulation that can form a transparent gel in situ under physiological conditions with minimal irritation.
Keywords: In situ gelling systems; Ocular drug delivery; Pluronics; Gelation; Mucoadhesion;

Lipid-based nanoformulations for peptide delivery by Nada Matougui; Lukas Boge; Anne-Claire Groo; Anita Umerska; Lovisa Ringstad; Helena Bysell; Patrick Saulnier (80-97).
Possible peptide localization in LNFs.Display OmittedNanoformulations have attracted a lot of attention because of their size-dependent properties. Among the array of nanoformulations, lipid nanoformulations (LNFs) have evoked increasing interest because of the advantages of their high degree of biocompatibility and versatility. The performance of lipid nanoformulations is greatly influenced by their composition and structure. Therapeutic peptides represent a growing share of the pharmaceutical market. However, the main challenge for their development into commercial products is their inherent physicochemical and biological instability. Important peptides such as insulin, calcitonin and cyclosporin A have been incorporated into LNFs. The association or encapsulation of peptides within lipid-based carriers has shown to protect the labile molecules against enzymatic degradation. This review describes strategies used for the formulation of peptides and some methods used for the assessment of association efficiency. The advantages and drawbacks of such carriers are also described.
Keywords: Nanoformulations; Lipids; Peptides; Drug delivery;

Tumor targeted delivery of octreotide-periplogenin conjugate: Synthesis, in vitro and in vivo evaluation by Hui-Yun Zhang; Wen-Qian Xu; Yuan-Wen Wang; Emmanuel Omari-Siaw; Yan Wang; Yuan-yuan Zheng; Xia Cao; Shan-Shan Tong; Jiang-nan Yu; Xi-ming Xu (98-106).
Display OmittedPeriplogenin (PPG), a cardiac glycoside prepared from Cortex periplocae, with similar structure to bufalin, has been found to induce apoptosis in many tumor cells. However, lots of cardiac glycosides possessing strong antitumor activity in vitro have still not passed phase I clinical trials, mostly due to poor tumor selectivity and systemic toxicity. To overcome this drawback, we designed octreotide-periplogenin (OCT-PPG) conjugate by coupling PPG–succinate to the amino-terminal end of octreotide. In comparison with free PPG, the conjugate exhibited significantly stronger cytotoxicity on HepG2 cells (SSTRs overexpression) but much less toxicity in L-02 cells. After intravenous injection of OCT-PPG conjugate into H22 tumor-bearing mice, its total accumulation in tumor was 2.3 fold higher than that of free PPG, but was 0.71- and 0.84-fold lower in heart and liver, respectively, suggesting somatostatin-mediated target delivery of PPG into the tumor tissue and reduced distribution in heart and liver. In vivo studies using H22 tumor model in mice confirmed the remarkable therapeutic effect of this conjugate. These results suggested that OCT-PPG conjugate could provide a new approach for clinical application of cardiac glycosides and as a targeting agent for cancer therapy.
Keywords: Cardiac glycosides; Periplogenin; Octreotide; Target delivery; Octreotide-periplogenin conjugate;

Multivariate calibration of the degree of crystallinity in intact pellets by X-ray powder diffraction by Krisztina Nikowitz; Attila Domján; Klára Pintye-Hódi; Géza Regdon (107-116).
Display OmittedXRPD is the method of choice to determine crystalline content in an amorphous environment. While several studies describe its use on powders, little information is available on its performance on finished products. The method’s use may be limited not only by the need of sample pretreatment and its validation but also by the propensity of some materials to recrystallize when exposed to heat or mechanical stress. In this work the authors describe an attempt at constructing a model based on the XRPD measurement of intact layered pellets using univariate methods based on peak heights and PLS regression. Results indicate that neither the goodness-of-fit (below 0.9 for all tested variables), nor the RMSEC values (above 5 for all tested variables) of any model based on peak height were good enough to consider them for everyday use. PLS regression however provided a model with improved characteristics (R2  = 0.9581, RMSEC = 3.04) despite the low API content and individual loading characteristics also reflected the validity of the model. PLS analysis also indicated that a specific sample may be different in some formulation characteristic that did not register on other examinations. This further indicates the method’s usefulness in the analysis of intact dosage forms.
Keywords: Crystallinity in intact pellets; XRPD; NMR; PLS regression; RMSEC values;

Formulation, physicochemical characterization and stability study of lithium-loaded microemulsion system by Abdelkader Mouri; Philippe Legrand; Abdeslam El Ghzaoui; Christophe Dorandeu; Jean Claude Maurel; Jean-Marie Devoisselle (117-124).
Display OmittedLithium biocompatible microemulsion based on Peceol®, lecithin, ethanol and water was studied in attempt to identify the optimal compositions in term of drug content, physicochemical properties and stability. Lithium solubilization in microemulsion was found to be compatible with a drug-surfactant binding model. Lithium ions were predominantly solubilized within lecithin head group altering significantly the interfacial properties of the system. Pseudo-ternary phase diagrams of drug free and drug loaded microemulsions were built at constant ethanol/lecithin weight ratio (40/60). Lithium loaded microemulsion has totally disappeared in the Peceol® rich part of phase diagram; critical fractions of lecithin and ethanol were required for the formation of stable microemulsion. The effect of lithium concentration on the properties and physical stability of microemulsions were studied using microscopy, Karl Fischer titrations, rheology analyses, conductivity measurements and centrifugation tests. The investigated microemulsions were found to be stable under accelerated storage conditions. The systems exhibited low viscosity and behaved as Newtonian fluid and no structural transition was shown.
Keywords: Lithium; Phase diagram; W/O microemulsion; Lecithin; Binding process;

A facile Friedel–Crafts acylation for the synthesis of polyethylenimine-grafted multi-walled carbon nanotubes as efficient gene delivery vectors by Azadeh Hashem Nia; Abbas Amini; Sahar Taghavi; Hossein Eshghi; Khalil Abnous; Mohammad Ramezani (125-137).
Display OmittedLow chemical reactivity of carbon nanotubes is one of the major obstacles in their functionalization via chemical reactions. As a non-destructive method, Friedel-Crafts acylation was suggested among the explored reactions for which only a few methods have been reported under harsh reaction conditions, e.g., high temperature all leading to low yields. In this study, we propose a novel method for the acylation of multi-walled carbon nanotubes (MWCNTs) at a low temperature (i.e., 42 °C), using SiO2-Al2O3 as a catalyst and 6-bromohexanoic acid as the acylating agent to produce high yield functionalized MWCNTs. After acylation, MWCNTs are conjugated with polyethylenimines (PEIs) with three molecular weights (1.8, 10 and 25 kDa). Three different MWCNT-PEI conjugates are synthesized and evaluated for their condensation ability, viability, size and zeta potential properties. The transfection efficiency of the functionalized MWCNTs is evaluated using luciferase assay and flow cytometry in a Neuroblastoma cell line. MWCNT-PEI (10 kDa) conjugate shows the highest transfection efficacy compared to others. For this carrier transfection efficacy exceeds the amount of PEI 25 kDa at similar carrier to plasmid weight ratio (C/P) and is around 3 times higher compared to PEI 25 kDa at C/P = 0.8 as positive control regarding its high transfection efficiency and low cytotoxicity.
Keywords: Multi-walled carbon nanotube; Friedel-Crafts acylation; Functionalization; Polyethylenimine; Gene delivery;

Assessing impact of formulation and process variables on in-vitro performance of directly compressed abuse deterrent formulations by Ziyaur Rahman; Yang Yang; Maxwell Korang-Yeboah; Akhtar Siddiqui; Xiaoming Xu; Muhammad Ashraf; Mansoor A. Khan (138-150).
Display OmittedPrescription drug products abuse/misuse is epidemic in United States. Opioids drug forms major portion of prescription drug product abuse. Abuse deterrence formulation (ADF) is one of the many approaches taken by sponsors to tackle this problem. It involves formulating opioids into dosage forms that will be difficult to abuse/misuse. Current investigation focused on evaluating the abuse deterrent properties (ADP) of ADF manufactured by direct compression method. Effect of process and formulation variables on ADP was investigated by statistical design of experiment (fractional factorial design). Independent factors studied were molecular weight of polyethylene oxide (Polyox™), curing time, temperature and method, and antioxidant type. Sotalol hydrochloride was selected as a model drug. ADP investigated were hardness/crush resistance, syringeability and injectability, physical manipulation (reduction into powder) and drug extraction in water and alcohol. Hardness and syringeability are evaluated by newly developed quantitative procedure. Other properties were also investigated such as morphology, crystallinity, assay and dissolution. The hardness and drug extraction was significantly (p < 0.05) affected by curing temperature. Formulations could be powdered in 3 min irrespective of their hardness. Syringeability and injectability were intrinsic properties of the polymer used in the formulation, and were not affected by the investigated factors. Crystallinity of the polymer and drug changed, and was dependent upon curing temperature and time. The dissolution and assay were independent of formulation and process parameters studied. In conclusion, the study indicated some advantages of ADF product compared to non-ADF prepared by direct compression. However, the ADF should not be viewed as abuse proof product rather as incrementally improved product.
Keywords: Abuse deterrence; Sotalol; Hardness; Crush resistance; Extraction; Syringeability and injectability;

Display OmittedThe study investigated the use of monoacyl phosphatidylcholine (MAPC) in self-nanoemulsifying drug delivery system (SNEDDS). A D-optimal design was used to generate two sets of formulations containing long-chain (LC) or medium-chain (MC) glycerides, caprylocaproyl macrogol-8 glycerides (Labrasol), Lipoid S LPC 80 (LPC) (80% MAPC) and ethanol. The formulations were characterized using dynamic light scattering, microscopy, in vitro lipolysis and viscometric measurements. All LC formulations within the investigated range were predicted to generate polydisperse emulsions while MC formulations generated nanoemulsions with droplet sizes from 23 to 167 nm. Using LPC in MC formulations reduced the nanoemulsion droplet sizes in simulated gastric and intestinal media. The nanoemulsion droplet size of MC SNEDDS containing LPC was not affected by gastrointestinal pH, while the zeta potentials increased at low pH. During in vitro lipolysis, less fatty acids were released when LPC was incorporated into the formulations (2.05 ± 0.02 mmol reduced to 1.76 ± 0.05 mmol when incorporating 30% LPC). Replacing Labrasol by LPC increased the formulation dynamic viscosity from 57 ± 1 mPa s (0% LPC) to 436 ± 8 mPa s (35% LPC) at 25 °C, however, this did not considerably prolong the formulation dispersion time. In conclusion, MC SNEDDS containing LPC are promising formulations when desiring to reduce the amount of synthetic surfactants and possibly modify the digestion rate.
Keywords: Monoacyl phosphatidylcholine; Self-nanoemulsifying drug delivery systems; Experimental design; Dynamic light scattering; Cryogenic transmission electron microscopy; In vitro lipolysis;

Biopharmaceutical evaluation of epigallocatechin gallate-loaded cationic lipid nanoparticles (EGCG-LNs): In vivo, in vitro and ex vivo studies by Joana F. Fangueiro; Ana C. Calpena; Beatriz Clares; Tatiana Andreani; Maria A. Egea; Francisco J. Veiga; Maria L. Garcia; Amélia M. Silva; Eliana B. Souto (161-169).
Transcorneal permeation profile of EGCG-CTAB LNs (■) and EGCG-DDAB LNs (♦), and respective stereomicrographs of the CAM after 5 min of exposure to lipid nanoparticles.Display OmittedCationic lipid nanoparticles (LNs) have been tested for sustained release and site-specific targeting of epigallocatechin gallate (EGCG), a potential polyphenol with improved pharmacological profile for the treatment of ocular pathologies, such as age-related macular edema, diabetic retinopathy, and inflammatory disorders. Cationic EGCG-LNs were produced by double-emulsion technique; the in vitro release study was performed in a dialysis bag, followed by the drug assay using a previously validated RP-HPLC method. In vitro HET-CAM study was carried out using chicken embryos to determine the potential risk of irritation of the developed formulations. Ex vivo permeation profile was assessed using rabbit cornea and sclera isolated and mounted in Franz diffusion cells. The results show that the use of cationic LNs provides a prolonged EGCG release, following a Boltzmann sigmoidal profile. In addition, EGCG was successfully quantified in both tested ocular tissues, demonstrating the ability of these formulations to reach both anterior and posterior segment of the eye. The pharmacokinetic study of the corneal permeation showed a first order kinetics for both cationic formulations, while EGCG-cetyltrimethylammonium bromide (CTAB) LNs followed a Boltzmann sigmoidal profile and EGCG-dimethyldioctadecylammonium bromide (DDAB) LNs a first order profile. Our studies also proved the safety and non-irritant nature of the developed LNs. Thus, loading EGCG in cationic LNs is recognised as a promising strategy for the treatment of ocular diseases related to anti-oxidant and anti-inflammatory pathways.
Keywords: Draize test; HET-CAM test; Epigallocatechin gallate; Lipid nanoparticles; Ocular delivery;

Display OmittedTo crystallize diclofenac (DF) from diclofenac sodium (DFNa), to micronize DF and DFNa, and to evaluate in vitro aerodynamic performance of the jet-milled formulationsFrom the acidic titration of aqueous DFNa, DF crystals were formed and were identified using thermal analysis, spectroscopy, and X-ray powder diffraction. Following the micronization of the DF and DFNa powders, the recovered samples were imaged, and their particle size distributions were evaluated. Samples before and after jet millings were characterized, and in vitro aerodynamic performance testing was performed on the DF sample before jet milling and the DF and DFNa samples following jet milling.Hollow needles of DF were precipitated. With similar particle size distributions, the jet-milled DFNa sample from the collection bag, and the DF sample from the cyclone were used for further characterization. Despite different deposition patterns in the Next Generation Impactor, the DF hollow needles had a comparable respirable fraction percentage to the jet-milled DF and DFNa particles. However, the jet-milled DF formulation had the best in vitro aerodynamic performance.Hollow, crystalline needles of DF were formed and possessed promising aerosol performance in comparison with the jet-milled powders.
Keywords: Jet milling; Precipitation; Diclofenac sodium; Diclofenac; Next generation impactor; Dry powder inhaler;

Gastrointestinal stability of therapeutic anti-TNF α IgG1 monoclonal antibodies by Vipul Yadav; Felipe Varum; Roberto Bravo; Esther Furrer; Abdul W. Basit (181-187).
Display OmittedMonoclonal antibodies (mAbs) are highly effective therapeutic agents, administered exclusively by the parenteral route owing to their previously-documented instability in the gastrointestinal (GI) tract when delivered orally. To investigate the extent of the validity of this assumption, the stability of the tumor necrosis factor alpha (TNF-α) neutralizing IgG1 mAbs, infliximab and adalimumab, was studied in human GI conditions. In gastric fluid, infliximab and adalimumab degraded rapidly, with complete degradation occurring within 1 min. In small intestinal fluid, the molecules were shown to be more stable, but nonetheless degraded within a short time frame of 30 min. Investigations into the mechanisms responsible for infliximab and adalimumab instability in the small intestine revealed that the proteolytic enzyme elastase, and to a lesser extent the enzymes trypsin and chymotrypsin, was responsible for their degradation. By contrast, in the human colon, 75% and 50% of the dose of infliximab and adalimumab, respectively, were intact after 60 min, with conversion of mAbs into F(ab’)2 Fab and Fc fragments detected in colonic conditions. These data indicate that therapeutic IgG1 antibodies are more stable in the colon than in the upper GI tract, therefore highlighting the potential for oral delivery of anti-TNF-α mAbs targeted to the colon.
Keywords: Infliximab; Adalimumab; Monoclonal antibodies; Oral delivery; Gastrointestinal stability; Tumor necrosis factor alpha (TNF-α);

Enhanced pulmonary delivery of fluticasone propionate in rodents by mucus-penetrating nanoparticles by Alexey Popov; Lisa Schopf; James Bourassa; Hongming Chen (188-197).
Display OmittedMost attempts to achieve sustained drug delivery to pulmonary tissues using nanoparticles have focused on mucoadhesive particles (MAP). However, MAP become trapped in the luminal mucus layer and, as a result, are largely eliminated from the respiratory tract by mucociliary escalator and expiratory clearance, which undermines their sustained release potential. Recent studies have shown that mucus-penetrating particles (MPP) engineered to diffuse through mucus can avoid rapid mucociliary clearance in vivo and persist in the lung longer. Nonetheless, it has not been confirmed that MPP encapsulating small molecules can sustain drug release in the lung longer than MAP of similar size and core composition. As a proof of concept, we encapsulated fluticasone propionate (FP) into poly(lactide)-based MPP and MAP (both ∼200 nm diameter, ∼30–35% drug loading) and evaluated their pulmonary residence by measuring FP levels in mouse lungs over 24 h following intratracheal instillation. Furthermore, we evaluated the duration of action of FP MPP in a rat lung inflammation model compared to that of a non-encapsulated FP control. In rodents, pulmonary delivery of FP formulated as MPP provided a 60% higher local exposure compared to MAP and extended the single dose efficacy by at least 16 h compared to non-encapsulated FP.
Keywords: Drug delivery; Fluticasone; Inflammation; Mucoadhesive; Mucus-penetrating particles; Nanoparticles; Pulmonary;

DrugCam®—An intelligent video camera system to make safe cytotoxic drug preparations by Frédéric Benizri; Benoit Dalifard; Christophe Zemmour; Maxime Henriquet; Emmanuelle Fougereau; Benoit Le Franc (198-207).
Display OmittedDrugCam® is a new approach to control the chemotherapy preparations with an intelligent video system that enables automatic verification during the critical stages of preparations combined with an a posteriori control with partial or total visualization of the video recording of preparations. The assessment was about the recognizing of anticancer drug vials (qualitative analysis) and syringe volumes (quantitative analysis). The qualitative analysis was conducted for a total of 120 vials with sensitivity of 100% for 84.2% of the vials and at least 97% for all the vials tested. Accuracy was at least 98.5% for all vials. The quantitative analysis was assessed by detecting 10 measures of each graduation for syringes. The identification error rate was 2.1% (244/11,640) i.e. almost 94% to the next graduation. Only 3% (35/1164) of the graduations tested, i.e. 23/35 for volume <0.13 ml of 1 ml syringes, presented a volume error outside the admissible limit of ±5% of a confidence band constructed for the estimated linear regression line for each syringe. In addition to the vial detection model, barcodes can also read when they are present on vials. DrugCam® offers an innovative approach for controlling chemotherapy preparations and constitutes an optimized application of telepharmacy.
Keywords: Chemotherapies; Video camera; Qualitative control; Quantitative control; Telepharmacy;

Electrospun formulations of acyclovir, ciprofloxacin and cyanocobalamin for ocular drug delivery by Alexandra Baskakova; Sahar Awwad; Jennifer Quirós Jiménez; Hardyal Gill; Oleg Novikov; Peng T. Khaw; Steve Brocchini; Elena Zhilyakova; Gareth R. Williams (208-218).
Display OmittedTwo series of fibers containing the active ingredients acyclovir, ciprofloxacin and cyanocobalamin, and combinations of these drugs, were prepared by electrospinning. One set used the hydrophilic poly(vinylpyrrolidone) (PVP) as the filament-forming polymer, while the other used the slow-dissolving poly(ε-caprolactone) (PCL). The fibers were found to have cylindrical morphologies, although there was evidence for solvent occlusion with the PVP systems and for some drug particles in the PCL materials. The active ingredients were generally present in the amorphous physical form in the case of PVP, but evidence of crystallinity was observed with PCL. The existence of intermolecular interactions between the drugs and polymers was proven using simple molecular modeling calculations. Drug release from the various fibers was tested in a validated in vitro outflow model of the eye, and the fiber formulations found to be capable of extending drug release. We thus conclude that electrospun matrices such as those prepared in this work have potential for use as intravitreal implants.
Keywords: Electrospinning; Ocular drug delivery; In vitro; Half-life; Antivirals; Posterior segment; Fibers; Sustained release;

Small is beautiful: Surprising nanoparticles by Dominique Duchêne; Ruxandra Gref (219-231).
Display OmittedIn the preparation of nanoparticles for drug delivery, it is well known that their size as well as their surface decorations can play a major role in interaction with living media. It is less known that their shape and internal structure can interplay with cellular and in vivo fate. The scientific literature is full of a large variety of surprising terms referring to their shape and structure. The aim of this review is to present some examples of the most often encountered surprising nanoparticles prepared and usable in the pharmaceutical technology domain. They are presented in two main groups related to their physical aspects: 1) smooth surface particles, such as Janus particles, “snowmen”, “dumbbells”, “rattles”, and “onions” and 2) branched particles, such as “flowers”, “stars” and “urchins”. The mode of preparation and potential applications are briefly presented. The topic has a serious, wider importance, namely in opportunity these structures have to allow exploration of the role of shape and structure on the utility (and perhaps toxicity) of these nanostructures.
Keywords: Nanoparticles; Janus; Dumbbells; Rattles; Onions; Branched nanoparticles;

Novel polymeric nanoparticles targeting the lipopolysaccharides of Pseudomonas aeruginosa by Y. Long; Z. Li; Q. Bi; C. Deng; Z. Chen; S. Bhattachayya; C. Li (232-241).
We used serotype specific LPS as a natural template and a process of inverse microemulsion polymerization to prepare surface molecularly imprinted NPs that exhibited highly specific targeting to P. aeruginosa strains and infection sites, both in vivo and in vitro. In combination with drug delivery, these “magic plastic bullets” could serve as a promising alternative to the ligand-modified nanocarriers currently used for targeted diagnosis and therapy.Display OmittedConsidering outburst of various infectious diseases globally, nanoparticle assisted targeted drug delivery has emerged as a promising strategy that can enhance the therapeutic efficacy and minimize the undesirable side effects of an antimicrobial agents. Molecular imprinting is a newly developed strategy that can synthesize a drug carrier with highly stable ligand-like ‘cavity’, may serve as a new platform of ligand-free targeted drug delivery systems. In this study, we use the amphiphilic lipopolysaccharides, derived from Pseudomonas aeruginosa as imprinting template and obtained an evenly distributed sub-40 nm polymeric nanoparticles by using inverse emulsion method. These molecularly imprinted nanoparticles (MIPNPs) showed specific binding to the lipopolysaccharide as determined by fluorescence polarization and microscale thermophoresis. MIPNPs showed selective recognition of target bacteria as detected by flow cytometry. Additionally, MIPNPs exhibited the in vivo targeting capabilities in both the keratitis model and meningitis model. Moreover, the photosensitizer methylene blue-loaded MIPNPs presented significantly strong inhibition of bacterial Growth, compared to non-imprinted controls for in vitro model of the photodynamic therapy. Our study shows an attempt to design a magic bullet by molecular imprinting that may provide a novel approach to generate synthetic carrier for targeting pathogen and treatment for a variety of infectious human diseases.
Keywords: Polymeric nanoparticles; Molecular imprinting; Lipopolysaccharide; Drug targeted delivery; Photodynamic therapy;

Combinations of colistin solutions and nebulisers for lung infection management in cystic fibrosis patients by Francesca Buttini; Irene Rossi; Marica Di Cuia; Alessandra Rossi; Gaia Colombo; Lisa Elviri; Fabio Sonvico; Anna Giulia Balducci (242-248).
Display OmittedIn this work different nebulisers were investigated in order to assess their efficiency in combination with colistimethate sodium (CMS) inhalation products. Four nebulisers, namely I-neb®, Aeroneb® Go, eFlow® rapid and PARI LC® Sprint were studied in terms of delivered dose (DD), drug delivery rate (DDR) and respirable dose (RD) of CMS. The goal was to provide scientific data to physicians for prescribing the most appropriate nebuliser for the CMS specific user.All the apparatuses nebulised ColiFin 1MIU/3 ml solution (80 mg of CMS) with delivered doses between 31% and 41% of the loaded amount. Aeroneb Go showed the longest nebulisation time (more than 20 min). When ColiFin 2MIU/4 ml was nebulised with eFlow rapid or PARI LC Sprint, the CMS respirable dose was 45.3 mg and 39.2 mg, in times of 5.6 and 10.8 min, respectively. I-neb, having a medication cup capacity limited to 0.4 ml, loaded with Promixin 0.4MIU/0.4 ml (32 mg of CMS), provided in a time of 9 min a RD of 21.5 mg, a value slightly higher than those obtained by nebulising ColiFin 1MIU/3 ml with the other nebulisers (range 15.9–17.6 mg).The results illustrate that the clinical outcome depends on the comparative analysis of nebulisation efficiency (respirable dose) and convenience (time), not disregarding the ratios between the amount loaded, delivered and deposited at lung level.
Keywords: Colistimethate sodium; Promixin; ColiFin; Membrane nebuliser; Jet nebuliser; Aerosol;

Multifunctional TK-VLPs nanocarrier for tumor-targeted delivery by Yachao Ren; Yu Mu; Lei Jiang; Hui Yu; Shuman Yang; Yu Zhang; Jianzhong Wang; Hua Zhang; Hunan Sun; Cuihong Xiao; Haisheng Peng; Yulong Zhou; Weiyue Lu (249-257).
Display OmittedVirus-like particles (VLPs) have been exploited for various biomedical applications, such as the monitoring, prevention, diagnosis and therapy of disease. In this study, a novel multifunctional VLPs nanocarrier (TK-VLPs) was prepared and used for tumor-targeted delivery. The SPR and cell uptake results indicated that the TK peptide is a “bi-functional ligand” with high affinity for Caco-2, HRT-18 and HUVEC cells through the integrin α6β1 and integrin αvβ3 receptors. The results of the direct immunofluorescence, SDS-PAGE and western blot assays demonstrated that the TK-VLPs were successfully prepared using the baculovirus expression system. Confocal laser scanning microscopy and the flow cytometry analysis validated that the TK-VLPs could target to Caco-2, HRT-18 and HUVEC cells. An in vivo study further confirmed that the TK-VLPs could target and efficiently deliver fluorescein to tumor cells and the tumor vasculature in mice bearing subcutaneous tumors. TK-VLPs-DOX displayed a uniform, spherical shape and an average size of approximately 28 nm. The results of the cell uptake and cytotoxicity assays indicated that TK-VLPs-DOX could enhance the selectivity for colorectal cancer cells. Together, our studies provide strong evidence that TK-VLPs could target colon tumor cells and tumor angiogenesis with enhanced permeability and retention effects, suggesting that the TK-VLPs are a multifunctional nanocarrier with potential applications in a colon tumor-targeted drug delivery system.
Keywords: TK peptide; Virus-like particles (VLPs); Nanocarrier; Parvovirus capsid protein VP2; Colonic drug delivery;