Structural Characterization of Self-Assembling Hybrid Nanoparticles for Bisphosphonate Delivery in Tumors.

Hybrid self-assembling nanoparticles (hsaNPs) encapsulating bisphosphonates (BPs) recently showed very promising results in preclinic experiments for the treatment of brain tumor. However, the poor knowledge on the architecture of hybrid nanovectors is certainly one of the main reasons hampering further clinical and industrial development of these technologies. Here we propose to combine different techniques, that is, small angle neutron scattering (SANS) and X-ray Sscattering (SAXS), with cryo-electron transmission microscopy (cryo-TEM) to study the architecture of the final hsaNPs as well as of the four components before the assembling process. Data analysis based on SANS and SAXS experiments suggested a multiple compartment architecture of the final product, consisting of two bilayers sourrounding a core. Structures consisting of two shells surrounding an internal core were also observed in the cryo-TEM analysis. Such high resolution insight, also combined with size distribution and zeta potential of the NPs, provides exhaustive characterization of hsaNPs encapsulating BPs, and it is aimed at supporting further their clinical and industrial development.

Micro-spherical cochleate composites: method development for monodispersed cochleate system.

Cochleates have been of increasing interest in pharmaceutical research due to their extraordinary stability. However the existing techniques used in the production of cochleates still need significant improvements to achieve sufficiently monodispersed formulations. In this study, we report a simple method for the production of spherical composite microparticles (3-5 µm in diameter) made up of nanocochleates from phosphatidylserine and calcium (as binding agent). Formulations obtained from the proposed method were evaluated using electron microscopy and small angle X-ray scattering and were compared with conventional cochleate preparation techniques. In this new method, an ethanolic lipid solution and aqueous solution of a binding agent is subjected to rapid and uniform mixing with a microfluidic device. The presence of high concentration of organic solvent promotes the formation of composite microparticles made of nanocochleates. This simple methodology eliminates elaborate preparation methods, while providing a monodisperse cochleate system with analogous quality.

Understanding cochleate formation: insights into structural development.

Understanding the structure and the self-assembly process of cochleates has become increasingly necessary considering the advances of this drug delivery system towards the pharmaceutical industry. It is well known that the addition of cations like calcium to a dispersion of anionic lipids such as phosphatidylserines results in stable, multilamellar cochleates through a spontaneous assembly. In the current investigation we have studied the intermediate structures generated during this self-assembly of cochleates. To achieve this, we have varied the process temperature for altering the rate of cochleate formation. Our findings from electron microscopy studies showed the formation of ribbonlike structures, which with proceeding interaction associate to form lipid stacks, networks and eventually cochleates. We also observed that the variation in lipid acyl chains did not make a remarkable difference to the type of structure evolved during the formation of cochleates. More generally, our observations provide a new insight into the self-assembly process of cochleates based on which we have proposed a pathway for cochleate formation from phosphatidylserine and calcium. This knowledge could be employed in using cochleates for a variety of possible biomedical applications in the future.

Electron microscopy and theoretical modeling of cochleates.

Cochleates are self-assembled cylindrical condensates that consist of large rolled-up lipid bilayer sheets and represent a novel platform for oral and systemic delivery of therapeutically active medicinal agents. With few preceding investigations, the physical basis of cochleate formation has remained largely unexplored. We address the structure and stability of cochleates in a combined experimental/theoretical approach. Employing different electron microscopy methods, we provide evidence for cochleates consisting of phosphatidylserine and calcium to be hollow tubelike structures with a well-defined constant lamellar repeat distance and statistically varying inner and outer radii. To rationalize the relation between inner and outer radii, we propose a theoretical model. Based on the minimization of a phenomenological free energy expression containing a bending, adhesion, and frustration contribution, we predict the optimal tube dimensions of a cochleate and estimate ratios of material constants for cochleates consisting of phosphatidylserines with varied hydrocarbon chain structures. Knowing and understanding these ratios will ultimately benefit the successful formulation of cochleates for drug delivery applications.

In situ Formation of Polymer Microparticles in Bacterial Nanocellulose Using Alternative and Sustainable Solvents to Incorporate Lipophilic Drugs.

Bacterial nanocellulose has been widely investigated in drug delivery, but the incorporation of lipophilic drugs and controlling release kinetics still remain a challenge. The inclusion of polymer particles to encapsulate drugs could address both problems but is reported sparely. In the present study, a formulation approach based on in situ precipitation of poly(lactic-co-glycolic acid) within bacterial nanocellulose was developed using and comparing the conventional solvent N-methyl-2-pyrrolidone and the alternative solvents poly(ethylene glycol), CyreneTM and ethyl lactate. Using the best-performing solvents N-methyl-2-pyrrolidone and ethyl lactate, their fast diffusion during phase inversion led to the formation of homogenously distributed polymer microparticles with average diameters between 2.0 and 6.6 µm within the cellulose matrix. Despite polymer inclusion, the water absorption value of the material still remained at ~50% of the original value and the material was able to release 32 g/100 cm2 of the bound water. Mechanical characteristics were not impaired compared to the native material. The process was suitable for encapsulating the highly lipophilic drugs cannabidiol and 3-O-acetyl-11-keto-ß-boswellic acid and enabled their sustained release with zero order kinetics over up to 10 days. Conclusively, controlled drug release for highly lipophilic compounds within bacterial nanocellulose could be achieved using sustainable solvents for preparation.

Encapsulation of the Anti-inflammatory Dual FLAP/sEH Inhibitor Diflapolin Improves the Efficiency in Human Whole Blood.

Diflapolin is a dual FLAP/sEH inhibitor with potent anti-inflammatory efficiency in cellular assays and experimental in vivo studies. Despite these outstanding characteristics, its high lipophilicity and plasma protein binding hamper the bioactivity in blood. To overcome these limitations, diflapolin was encapsulated in poly(lactic-co-glycolic acid) nanoparticles to develop an efficient and biocompatible drug delivery system. Two different cosolvent approaches were tested showing the possibility to exchange dimethyl sulfoxide in the organic phase by the sustainable 400 g/mol poly(ethylene glycol). A particle size of 220 nm and the amorphous encapsulation of diflapolin in high amounts rendered the nanoparticles appropriate for the intended application. Excellent biocompatibility of the nanoparticles was demonstrated in an ex ovo hen's egg model. The potent suppression of FLAP-dependent 5-lipoxygenase product formation by the nanoparticles in human whole blood, superior to the free drug, makes them to a promising drug delivery system to improve the bioactivity of diflapolin.

Lecithin-based novel cationic nanocarriers (Leciplex) II: improving therapeutic efficacy of quercetin on oral administration.

The objective of the present investigation was to evaluate ability of the novel self-assembled phospholipid- based cationic nanocarriers (LeciPlex) in improving the therapeutic efficacy of a poorly water-soluble natural polyphenolic agent, quercetin (QR), on oral administration. Quercetin loaded LeciPlex (QR-LeciPlex) were successfully fabricated using a biocompatible solvent Transcutol HP. The QR-LeciPlex were characterized for particle size, encapsulation efficiency, zeta potential, and particle morphology by cryo-TEM. UV and fluorescence spectral characterization was carried out to find out the association of QR with LeciPlex. Small angle neutron scattering studies (SANS) were carried out to understand the internal structure of Leciplex and to evaluate the influence of the incorporation of QR in the LeciPlex. Anti-inflammatory and antitumorigenic activity of QR-LeciPlex was determined in comparison to QR suspension to evaluate the potential of LeciPlex in improving oral delivery of QR. QR-LeciPlex exhibited a particle size of ∼400 nm and had excellent colloidal stability. The QR-LeciPlex had a zeta potential greater than +30 mV and exhibited very high encapsulation efficiency of QR (>90%). UV and fluorescence spectral characterization indicated the interaction/association of QR with LeciPlex components. Cryo-TEM studies showed that LeciPlex and QR-LeciPlex have a unilamellar structure. SANS confirmed the unilamellar structure of LeciPlex and indicated that the incorporation of QR does not have any effect on the internal structure of the LeciPlex. QR-LeciPlex exhibited significantly higher anti-inflammatory and antitumorigenic activity (p < 0.01) as compared to that of QR suspension on oral administration.

Cyrene™ as an Alternative Sustainable Solvent for the Preparation of Poly(lactic-co-glycolic acid) Nanoparticles.

Toxic and environmental harmful organic solvents are widely applied to prepare poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NP) in standard preparation methods. Alternative non-toxic solvents suffer from disadvantages like high viscosity and plasticizing effects. To overcome these hurdles, Cyrene™ as a new sustainable, non-toxic and low viscous solvent was used to formulate PLGA NPs. A new preparation method was developed and optimized. Small sized blank NPs around 220 nm with a narrow size distribution and highly negative charge (<-23 mV) were obtained. To test the application for drug delivery, the lipophilic model drug atorvastatin was encapsulated in high drug loads with comparable physicochemical characteristics as the blank NPs, and a total drug release within 24 h. No changes of the crystallinity or plasticizing effects could be observed. Highly purified NPs were obtained with a residual Cyrene™ content <2.5%. Finally, the biocompatibility of Cyrene™ itself and of the NPs formed in the presence of Cyrene™ was demonstrated in a hen's egg test. Conclusively, the use of Cyrene™ as solvent offers a simple, fast and non-toxic procedure for preparation of PLGA NPs as drug delivery systems circumventing the downsides of standard methods.

Overcoming the hydrophilicity of bacterial nanocellulose: Incorporation of the lipophilic coenzyme Q10 using lipid nanocarriers for dermal applications.

Although used in a wide range of medical and pharmaceutical applications, the potential of the natural biopolymer bacterial nanocellulose (BNC) as drug delivery system is by far not fully exploited. Particularly, the incorporation of lipophilic drugs is still considered as an unsolved task. In the present study, the homogeneous incorporation of the lipophilic coenzyme Q10 (CoQ10) into BNC was accomplished by several post-synthesis techniques utilizing different nanoemulsions and liposomes. All colloidal carriers were in the range of about 90-120 nm with negative zeta potentials and storage stabilities up to 30 days. The biphasic drug release profiles of loaded BNC were found to be dependent on the type of colloidal carrier and the loading technique. Favorable characteristics such as high mechanical stability and high loading capacity were retained after the incorporation of the lipophilic components. Penetration studies using excised porcine skin revealed CoQ10 distributions also in deeper skin layers dependent on the type of the colloidal carrier system. In conclusion, hydrophilic BNC could be loaded with water-insoluble drugs as shown for the model drug CoQ10 by the use of lipidic colloidal carriers which offers new possibilities of application in pharmacy and medicine.

Sustainable preparation of anti-inflammatory atorvastatin PLGA nanoparticles.

In the present study, the anti-inflammatory lipophilic drug atorvastatin was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) using a sustainable method in comparison to the standard emulsion-diffusion-evaporation technique. For the sustainable method the organic solvent ethyl acetate was fully replaced by 400 g/mol poly(ethylene glycol) (PEG 400). Both techniques led to the formation of nanoparticles with comparable sizes of about 170 to 247 nm depending on the polymer type, with monomodal size distribution and negative zeta potential. All nanoparticles demonstrated a high biocompatibility in a shell-less hen's egg model and displayed an anti-inflammatory effect in human monocytes. The use of PEG 400 resulted in plasticizing effects and a lower crystallinity of the PLGA nanoparticles as determined by differential scanning calorimetry and Raman spectroscopy, which correlated with a faster drug release. Interestingly, the particles prepared by the sustainable method showed a crystallinity and drug release kinetics similar to nanoparticles made of PEG-PLGA using the standard method. Conclusively, the sustainable method is a fast and easy to perform technique suitable to prepare atorvastatin-loaded PLGA nanoparticles avoiding toxic and environmentally damaging drawbacks frequently associated with classical organic solvents.

Process control and scale-up of modified bacterial cellulose production for tailor-made anti-inflammatory drug delivery systems.

Bacterial cellulose (BC) has proven its high potential as active wound dressing and drug delivery system in many scientific studies, but the transferability of the methods to efficient manufacturing still needs to be demonstrated. This study presents a technically feasible, straightforward and efficient approach to modify BC according to specific medical requirements, to scale-up the cultivation and to load the active pharmaceutical ingredient of interest. By means of in situ-modification of the network structure using water-soluble poly(ethylene glycol) 400 and 4000 on pilot-scale, up to 41.5 ±â€¯3.0 % higher transparency of the dressing, 40.6 ±â€¯3.8 % increased loading capacity and 9% increased total release of the anti-inflammatory model drug diclofenac sodium could be obtained. Spray loading was investigated as material efficient alternative to absorption loading allowing a significant reduction in loading time.

Development and characterization of bacterial nanocellulose loaded with Boswellia serrata extract containing nanoemulsions as natural dressing for skin diseases.

The combination of the anti-inflammatory lipophilic Boswellia serrata extract with the natural hydropolymer bacterial nanocellulose (BNC) for the treatment of skin diseases is counteracted by their different hydro/lipophilicity. To overcome the hydrophilicity of the BNC, the water in its network was exchanged by single and double nanoemulsions. Incorporation of the Boswellia serrata extract in the nanoemulsions formed particles of about 115 to 150 nm with negative zeta potential and storage stability over 30 days at temperatures between 4 and 32 °C. Their loading into the BNC did not change the preferential characteristics of the nanocellulose like water absorption and retention, softness, and pressure stability in a relevant way. Loaded BNC could be sterilized by an electron-beam procedure. A biphasic drug release profile of lead compounds was observed by Franz cell diffusion test. The biocompatibility of the loaded BNC was confirmed ex ovo by a shell-less hen's egg test. Tape stripping experiments using porcine skin determined a dependency of the drug penetration into skin on the type of nanoemulsion, single vs. repeated applications and the incubation time. In conclusion, the hydrophilicity of BNC could be overcome using nanoemulsions which offers the possibility for the anti-inflammatory skin treatment with Boswellia serrata extract.

Tailor-made material characteristics of bacterial cellulose for drug delivery applications in dentistry.

Bacterial cellulose (BC) has shown high potential as innovative wound dressing and drug delivery system. Bringing both together, drug-loaded BC was investigated for applications in dental therapies such as dental extraction or mucosal transplantation. Both applications would benefit from a material which degrades under physiological conditions, and from an antibiotic environment. Consequently, periodate-oxidation of BC was investigated to facilitate modified degradation behaviour. A periodate concentration of 0.14 mol/L at ϑ = 25 °C and t = 8 h resulted in a material loss of <10%, but at the same time a sufficient degree of degradation. Additionally, native and oxidised BC loaded with doxycycline was tested for prophylaxis against infection. An in vitro-toxicity test (MTT assay) provided a first confirmation of biocompatibility, whereas agar diffusion tests proved antibiotic efficiency against pathogenic oral bacteria. Release studies of the drug from native and oxidised BC confirmed a comparative biphasic release behaviour.

Simulation of the long-term fate of superparamagnetic iron oxide-based nanoparticles using simulated biological fluids.

Aim: To simulate the stability and degradation of superparamagnetic iron oxide nanoparticles (MNP) in vitro as part of their life cycle using complex simulated biological fluids. Materials & methods: A set of 13 MNP with different polymeric or inorganic shell materials was synthesized and characterized regarding stability and degradation of core and shell in simulated biological fluids. Results: All MNP formulations showed excellent stability during storage and in simulated body fluid. In endosomal/lysosomal media the degradation behavior depended on shell characteristics (e.g., charge, acid-base character) and temperature enabling the development of an accelerated stress test protocol. Conclusion: Kinetics of transformations depending on the MNP type could be established to define structure-activity relationships as prediction model for rational particle design.

Study on the in situ aggregation of liposomes with negatively charged phospholipids for use as injectable depot formulation.

Compared to conventional parenteral formulations injectable depot formulations, owing to a sustained drug release, offer several advantages, such as a reduced dosing frequency - and consequent improved compliance - or a predictable release profile. Additionally, fluctuations in the drug blood level may be smoothened and consequently side effects reduced. Because of their capability to encapsulate water soluble drugs and their very low toxicity profile liposomes comprising phospholipids, most certainly represent a vehicle of choice for subcutaneous (s.c.) or intramuscular (i.m.) administration typical for depot injections too. In the past, especially liposomes containing negatively charged phosphatidylserines were investigated regarding their aggregation and fusion behavior upon addition of calcium ions. Liposomes need to have a large size to prevent fast removal from the s.c. or i.m. injection site to make them useful as depot vehicle. In order to obtain such large liposomes, aggregation of smaller liposomes may be considered. Aim of the present study was to induce and investigate controlled aggregation of vesicles containing other negatively charged phospholipids besides phosphatidylserines upon mixing with a solution of divalent cations. L-α-phosphatidylcholine from egg (EPC) liposomes formulated with 25 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) or 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG) proved to be possible lipid-based depot candidates due to their strong aggregation induced by calcium and magnesium cations. Different aggregation profiles with both cations could be observed. Morphological investigations of the aggregates showed that individual liposomes remain stable in the aggregates and no fusion occurred. A fluorescence-based fusion assay confirmed these results. Differential scanning calorimetry measurements supported the findings of the diverse aggregation profiles with calcium or magnesium owing to different binding sites of the cations to the lipid molecules.

Polyester-based particles to overcome the obstacles of mucus and biofilms in the lung for tobramycin application under static and dynamic fluidic conditions.

Pulmonary infections with Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) are difficult to treat and related with high mortality in some diseases like cystic fibrosis due to the recurrent formation of biofilms. The biofilm formation hinders efficient treatment with inhaled antibiotics due to a low penetration of the antibiotics through the polyanionic biofilm matrix and increased antimicrobial resistance of the biofilm-embedded bacteria. In this study, tobramycin (Tb) was encapsulated in particles based on poly(d,l,-lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-co-poly(d,l,-lactide-co-glycolide) diblock (PEG-PLGA) to overcome the biofilm barrier with particle sizes of 225-231 nm (nanoparticles) and 896-902 nm (microparticles), spherical shape and negative zeta potentials. The effectiveness against biofilms of P. aeruginosa and B. cepacia was strongly enhanced by the encapsulation under fluidic experimental condition as well as under static conditions in artificial mucus. The biofilm-embedded bacteria were killed by less than 0.77 mg/l encapsulated Tb, whereas 1,000 mg/l of free Tb or the bulk mixtures of Tb and the particles were ineffective against the biofilms. Moreover, encapsulated Tb was even effective against biofilms of the intrinsically aminoglycoside-resistant B. cepacia, indicating a supportive effect of PEG and PLGA on Tb. No cytotoxicity was detected in vitro in human lung epithelial cells with any formulation.

Preparation of PEGylated liposomes incorporating lipophilic lomeguatrib derivatives for the sensitization of chemo-resistant gliomas.

Liposomal delivery is a well-established approach to increase the therapeutic index of drugs, mainly in the field of cancer chemotherapy. Here, we report the preparation and characterization of a new liposomal formulation of a derivative of lomeguatrib, a potent O6-methylguanine-DNA methyltransferase (MGMT) inactivator. The drug had been tested in clinical trials to revert chemoresistance, but was associated with a low therapeutic index. A series of lomeguatrib conjugates with distinct alkyl chain lengths - i.e. C12, C14, C16, and C18 - was synthesized, and the MGMT depleting activity as well as cytotoxicity were determined on relevant mouse and human glioma cell lines. Drug-containing liposomes were prepared and characterized in terms of loading and in vitro release kinetics. The lipophilic lomeguatrib conjugates did not exert cytotoxic effects at 5 µM in the mouse glioma cell line and exhibited a similar MGMT depleting activity pattern as lomeguatrib. Overall, drug loading could be improved by up to 50-fold with the lipophilic conjugates, and the slowest leakage was achieved with the C18 derivative. The present data show the applicability of lipophilic lomeguatrib derivatization for incorporation into liposomes, and identify the C18 derivative as the lead compound for in vivo studies.

Lipid colloidal carriers for improvement of anticancer activity of orally delivered quercetin: formulation, characterization and establishing in vitro-in vivo advantage.

Novel lipid nanocarriers, GeluPearl (GP) comprising of Precirol ATO 5 lipid nanoparticles with (GPNLC) or without oil (GPSLN), loaded with Quercetin (QR), were successfully fabricated to improve therapeutic efficacy. QR loaded GP nanoparticles were optimized to yield adequate colloidal stability, mean particle size in range of 350-380 nm and entrapment efficiency of more than 90%. GPSLN and GPNLC were characterized for morphological evaluation by virtue of cryo-TEM, surface charge, protection offered to QR against alkali mediated degradation and fluorescence studies to evaluate QR-lipid interaction. DSC analysis was performed to get insight into physical state of QR loaded in nanosystems. The in vitro release studies demonstrated sustained drug release potential of QR loaded GP. In vitro lipolysis studies confirmed that lipidic nanocarriers can improve QR solubilization. QR loaded GP nanosystems significantly (P < 0.05) reduced flank tumor volumes in C57BL/6 mice over a 22 day study period compared to QR suspension. GPSLN significantly reduced lung colonization and enhanced antimetastatic activity (P < 0.05) of drug against B16F10 melanoma cells in C57BL/6 mice as compared to QR suspension. QR loaded GPSLN and GPNLC could be effectively lyophilized without much change in particle size and drug content using 15% w/v mannitol as cryoprotectant.

Lecithin-based novel cationic nanocarriers (LeciPlex) I: fabrication, characterization and evaluation.

AIMS: In the present investigation, the feasibility of fabricating novel self-assembled cationic nanocarriers (LeciPlex) containing cetyltrimethylammonium bromide (CTAB) or didodecyldimethylammonium bromide (DDAB) and soybean lecithin using pharmaceutically acceptable biocompatible solvents such as 2-Pyrrolidone (Soluphor P) and diethyleneglycol monoethyl ether (Transcutol) was established. MATERIALS & METHODS: The interaction between DDAB/CTAB and soybean lecithin in the nanocarriers was confirmed by differential scanning calorimetry and in vitro antimicrobial studies. The positive charge on the nanocarriers was confirmed by zeta potential analysis. RESULTS: Transmission electron microscopy analysis could not reveal sufficient information regarding the internal structure of the nanocarriers, whereas cryotransmission electron microscopy studies indicated that these novel nanocarriers have unilamellar structure. Small-angle neutron scattering studies confirmed interaction of cationic surfactant (DDAB) and lecithin in the nanocarriers and confirmed the presence of unilamellar nanostructures. CONCLUSION: Various hydrophobic drugs could be encapsulated in the CTAB/DDAB-based lecithin nanocarriers (CTAB-LeciPlex or DDAB-LeciPlex) irrespective of their difference in log p-values. In vitro antimicrobial studies on triclosan-loaded LeciPlex confirmed entrapment of triclosan in the nanocarriers. The ability of CTAB-LeciPlex and DDAB-LeciPlex to condense plasmid DNA was established using agarose gel electrophoresis. DDAB-LeciPlex could successfully transfect pDNA in HEK-293 cells indicating potential in gene delivery.