Evaluating the photodynamic efficacy of nebulized curcumin-loaded liposomes prepared by thin-film hydration and dual centrifugation: In vitro and in ovo studies.

Lung cancer, one of the most common causes of high mortality worldwide, still lacks appropriate and convenient treatment options. Photodynamic therapy (PDT) has shown promising results against cancer, especially in recent years. However, pulmonary drug delivery of the predominantly hydrophobic photosensitizers still represents a significant obstacle. Nebulizing DPPC/Cholesterol liposomes loaded with the photosensitizer curcumin via a vibrating mesh nebulizer might overcome current restrictions. In this study, the liposomes were prepared by conventional thin-film hydration and two other methods based on dual centrifugation. The liposomes' physicochemical properties were determined before and after nebulization, showing that liposomes do not undergo any changes. However, morphological characterization of the differently prepared liposomes revealed structural differences between the methods in terms of lamellarity. Internalization of curcumin in lung adenocarcinoma (A549) cells was visualized and quantified. The generation of reactive oxygen species because of the photoreaction was also proven. The photodynamic efficacy of the liposomal formulations was tested against A549 cells. They revealed different phototoxic responses at different radiant exposures. Furthermore, the photodynamic efficacy was investigated after nebulizing curcumin-loaded liposomes onto xenografted tumors on the CAM, followed by irradiation, and evaluated using positron emission tomography/computed tomography and histological analysis. A decrease in tumor metabolism could be observed. Based on the efficacy of curcumin-loaded liposomes in 2D and 3D models, liposomes, especially with prior film formation, can be considered a promising approach for PDT against lung cancer.

Lipid-Coated Polymeric Nanoparticles for the Photodynamic Therapy of Head and Neck Squamous Cell Carcinomas.

Next to alcohol and tobacco abuse, infection with human papillomaviruses (HPVs) is a major risk factor for developing head and neck squamous cell carcinomas (HNSCCs), leading to 350,000 casualties worldwide each year. Limited therapy options and drug resistance raise the urge for alternative methods such as photodynamic therapy (PDT), a minimally invasive procedure used to treat HNSCC and other cancers. We prepared lipid-coated polymeric nanoparticles encapsulating curcumin as the photosensitizer (CUR-LCNPs). The prepared CUR-LCNPs were in the nanometer range (153.37 ± 1.58 nm) and showed an encapsulation efficiency of 92.69 ± 0.03%. Proper lipid coating was visualized using atomic force microscopy (AFM). The CUR-LCNPs were tested in three HPVpos and three HPVneg HNSCC lines regarding their uptake capabilities and in vitro cell killing capacity, revealing a variable but highly significant tumor cell inhibiting effect in all tested HNSCC cell lines. No significant differences were detected between the HPVpos and HPVneg HNSCC groups (mean IC50: (9.34 ± 4.73 µmol/L vs. 6.88 ± 1.03 µmol/L), suggesting CUR-LCNPs/PDT to be a promising therapeutic option for HNSCC patients independent of their HPV status.

Photodynamic and antiangiogenic activities of parietin liposomes in triple negative breast cancer.

Parietin (PTN) is an anthraquinone with promising efficacy in the inhibition of cancer cell proliferation and tumor growth. Due to its hydrophobicity, PTN is sparingly soluble under physiological conditions and has a low bioavailability. Hence, we presented PTN in liposomes to overcome these drawbacks. The prepared liposomes were characterized and their stability was also assessed in serum. Singlet oxygen quantum yield of PTN loaded liposomes was indirectly quantified using uric acid. The intracellular uptake of liposomes was studied by CLSM which indicated the perinuclear localization of PTN liposomes. Cellular viability assay and live/dead staining demonstrated both light and dose-dependent phototoxicity of PTN on the human breast cancer cell line. The mechanism of cellular uptake was investigated using different pathway inhibitors and the results showed that clathrin-mediated endocytosis is predominant. The colocalization experiment indicated that PTN is localized in both mitochondria and lysosomes. These findings together with flow cytometry analysis elucidated that apoptosis is the main mechanism underlying cell death post-PDT. Finally, the antiangiogenic effect of PTN liposomes was further evaluated in the chorioallantoic membrane (CAM) model and the results indicated that PDT induced vascular response was confined to the irradiated area leaving the non-irradiated unscathed.

Parietin Cyclodextrin-Inclusion Complex as an Effective Formulation for Bacterial Photoinactivation.

Multidrug resistance in pathogenic bacteria has become a significant public health concern. As an alternative therapeutic option, antimicrobial photodynamic therapy (aPDT) can successfully eradicate antibiotic-resistant bacteria with a lower probability of developing resistance or systemic toxicity commonly associated with the standard antibiotic treatment. Parietin (PTN), also termed physcion, a natural anthraquinone, is a promising photosensitizer somewhat underrepresented in aPDT because of its poor water solubility and potential to aggregate in the biological environment. This study investigated whether the complexation of PTN with (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD) could increase its solubility, enhance its photophysical properties, and improve its phototoxicity against bacteria. At first, the solubilization behavior and complexation constant of the PTN/HP-ß-CD inclusion complexes were evaluated by the phase solubility method. Then, the formation and physicochemical properties of PTN/HP-ß-CD complexes were analyzed and confirmed in various ways. At the same time, the photodynamic activity was assessed by the uric acid method. The blue light-mediated photodegradation of PTN in its free and complexed forms were compared. Complexation of PTN increased the aqueous solubility 28-fold and the photostability compared to free PTN. PTN/HP-ß-CD complexes reduce the bacterial viability of Staphylococcus saprophyticus and Escherichia coli by > 4.8 log and > 1.0 log after irradiation, respectively. Overall, the low solubility, aggregation potential, and photoinstability of PTN were overcome by its complexation in HP-ß-CD, potentially opening up new opportunities for treating infections caused by multidrug-resistant bacteria.

Lipoparticles for Synergistic Chemo-Photodynamic Therapy to Ovarian Carcinoma Cells: In vitro and in vivo Assessments.

PURPOSE: Lipoparticles are the core-shell type lipid-polymer hybrid systems comprising polymeric nanoparticle core enveloped by single or multiple pegylated lipid layers (shell), thereby melding the biomimetic properties of long-circulating vesicles as well as the mechanical advantages of the nanoparticles. The present study was aimed at the development of such an integrated system, combining the photodynamic and chemotherapeutic approaches for the treatment of multidrug-resistant cancers. METHODS: For this rationale, two different sized Pirarubicin (THP) loaded poly lactic-co-glycolic acid (PLGA) nanoparticles were prepared by emulsion solvent evaporation technique, whereas liposomes containing Temoporfin (mTHPC) were prepared by lipid film hydration method. Physicochemical and morphological characterizations were done using dynamic light scattering, laser doppler anemometry, atomic force microscopy, and transmission electron microscopy. The quantitative assessment of cell damage was determined using MTT and reactive oxygen species (ROS) assay. The biocompatibility of the nanoformulations was evaluated with serum stability testing, haemocompatibility as well as acute in vivo toxicity using female albino (BALB/c) mice. RESULTS AND CONCLUSION: The mean hydrodynamic diameter of the formulations was found between 108.80 ± 2.10 to 405.70 ± 10.00 nm with the zeta (ζ) potential ranging from -12.70 ± 1.20 to 5.90 ± 1.10 mV. Based on the physicochemical evaluations, the selected THP nanoparticles were coated with mTHPC liposomes to produce lipid-coated nanoparticles (LCNPs). A significant (p< 0.001) cytotoxicity synergism was evident in LCNPs when irradiated at 652 nm, using an LED device. No incidence of genotoxicity was observed as seen with the comet assay. The LCNPs decreased the generalized in vivo toxicity as compared to the free drugs and was evident from the serum biochemical profile, visceral body index, liver function tests as well as renal function tests. The histopathological examinations of the vital organs revealed no significant evidence of toxicity suggesting the safety and efficacy of our lipid-polymer hybrid system.

Lipodendriplexes mediated enhanced gene delivery: a cellular to pre-clinical investigation.

Clinical success of effective gene therapy is mainly hampered by the insufficiency of safe and efficient internalization of a transgene to the targeted cellular site. Therefore, the development of a safe and efficient nanocarrier system is one of the fundamental challenges to transfer the therapeutic genes to the diseased cells. Polyamidoamine (PAMAM) dendrimer has been used as an efficient non-viral gene vector (dendriplexes) but the toxicity and unusual biodistribution induced by the terminal amino groups (-NH2) limit its in vivo applications. Hence, a state of the art lipid modification with PAMAM based gene carrier (lipodendriplexes) was planned to investigate theirs in vitro (2D and 3D cell culture) and in vivo behaviour. In vitro pDNA transfection, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, cellular protein contents, live/dead staining and apoptosis were studied in 2D cell culture of HEK-293 cells while GFP transfection, 3D cell viability and live/dead staining of spheroids were performed in its 3D cell culture. Acute toxicity studies including organ to body index ratio, hematological parameters, serum biochemistry, histopathological profiles and in vivo transgene expression were assessed in female BALB/c mice. The results suggested that, in comparison to dendriplexes the lipodendriplexes exhibited significant improvement of pDNA transfection (p < 0.001) with lower LDH release (p < 0.01) and ROS generation (p < 0.05). A substantially higher cellular protein content (p < 0.01) and cell viability were also observed in 2D culture. A strong GFP expression with an improved cell viability profile (p < 0.05) was indicated in lipodendriplexes treated 3D spheroids. In vivo archives showed the superiority of lipid-modified nanocarrier system, depicted a significant increase in green fluorescent protein (GFP) expression in the lungs (p < 0.01), heart (p < 0.001), liver (p < 0.001) and kidneys (p < 0.001) with improved serum biochemistry and hematological profile as compared to unmodified dendriplexes. No tissue necrosis was evident in the animal groups treated with lipid-shielded molecules. Therefore, a non-covalent conjugation of lipids with PAMAM based carrier system could be considered as a promising approach for an efficient and biocompatible gene delivery system.

Downregulation of MDR 1 gene contributes to tyrosine kinase inhibitor induce apoptosis and reduction in tumor metastasis: A gravity to space investigation.

P-glycoprotein (P-gp) associated multidrug resistance (MDR) represents a major failure in cancer treatment. The overexpression of P-gp is responsible for ATP-dependent efflux of drugs that decrease their intracellular accumulation. An effective downregulation of MDR1 gene using small interfering RNA (siRNA) is one of the safe and effective tools to overcome the P-gp triggered MDR. Therefore, the development of an efficient and non-toxic carrier system for siRNA delivery is a fundamental challenge for effective cancer treatment. Polyamidoamine (PAMAM) dendrimer has been used for efficient delivery of siRNA (dendriplexes) to the tumor cells but the associated toxicity problems render its use in biological applications. A non-covalent lipid modification (lipodendriplexes) is supposed to offer a promising strategy to overcome the demerits linked to the naked dendriplexes system. In the current study, we deliver siRNA, designed against MDR1 gene (si-MDR1), in colorectal carcinoma cells (Caco-2), having overexpression of P-gp, to check the role of MDR1 gene in tumor progression and multidrug resistance using two dimensional (2D) and three dimensional (3D) environment. Imatinib mesylate (IM), a P-gp substrate, was used as model drug. Our results revealed that the effective knockdown by lipodendriplexes system can significantly reduce the tumor cell migration in 2D (p < 0.001) and 3D (p < 0.001) cell cultures as compared to unmodified dendriplexes and si-Control groups. It was also observed that lipodendriplexes aided downregulation of MDR1 gene effectively, re-sensitized the Caco-2 cells for IM uptake and showed a significantly (p < 0.001) higher apoptosis. Our findings imply that our lipodendriplexes system has a great potential for siRNA delivery, however, further in vivo application using a suitable targeted system can play a major role for better cancer therapeutics.

Wavelength dependent photo-cytotoxicity to ovarian carcinoma cells using temoporfin loaded tetraether liposomes as efficient drug delivery system.

5,10,15,20-Tetrakis(3-hydroxyphenyl)chlorin (mTHPC; temoporfin) is one of the most potent second-generation photosensitizers available today for the treatment of a variety of clinical disorders and has a unique capability of being activated at different wavelengths. However, due to its highly lipophilic nature, poor solubility in the aqueous media and poor bioavailability limits its application in anticancer therapies. To overcome these potential issues, we developed three different liposomal formulations with mTHPC encapsulated in hydrophobic milieu thus increasing the bioavailability of the drug. The prepared formulations were characterized in terms of hydrodynamic diameter, surface charge, encapsulation efficiency, and stability studies. The mean size of the liposomes was found to be in the nanoscale range (about 100 nm) with zeta potential ranging from -6.0 to -13.7 mV. mTHPC loaded liposomes were also evaluated for morphology using atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM). Data obtained from the hemocompatibility experiments showed that these formulations were compatible with blood showing less than 10% hemolysis and coagulation time lower than 40 s. The results obtained from the single-cell gel electrophoresis assay also demonstrated no incidence of genotoxicity. Photodynamic destruction of SK-OV-3 cells using mTHPC loaded liposomes showed a dose-response relationship upon irradiation with two different wavelength lights (blue λ = 457 nm & red λ = 652 nm). A 10-fold pronounced effect was produced when liposomal formulations were irradiated at 652 nm as compared to 457 nm. This was also evaluated by the quantitative assessment of reactive oxygen production (ROS) using fluorescence microscopy. The qualitative assessment of PDT pre- and post-irradiation was visualized using confocal laser scanning microscopy (CLSM) which demonstrated an intense localization of mTHPC liposomes in the perinuclear region. Chick chorioallantoic membrane assay (CAM) was used as an alternative in-ovo model to demonstrate the localized destruction of tumor microvasculature. Overall, the prepared nanoformulation is a biocompatible, efficient and well characterized delivery system for mTHPC for the safe and effective PDT.

Targeted ErbB3 cancer therapy: A synergistic approach to effectively combat cancer.

Surface modification of nanoparticles with aptamer is gaining popularity lately due to its selective targeting and low immunogenicity. In this study, sorafenib tosylate (SFB) was loaded in biodegradable PLGA nanoparticles prepared by solvent evaporation method. The surfaces of drug deprived and drug-loaded particles (PN and PNS, respectively) were coupled with aptamer to target ErbB3 using EDC/NHS chemical modification. Nanoparticles were characterized with regard to their size, shape and chemical composition by dynamic light scattering, atomic force microscopy, FTIR and elemental analysis respectively. To evaluate the particles in vitro cell culture studies were performed. Cell viability assay, pathway analysis and apoptosis assay showed cellular toxicity in the presence of aptamer in PNS-Apt (p < 0.001). Metastatic progression assay showed decreased cell migration in the presence of aptamer and SFB. Confocal laser scanning microscopy was used to visualize the receptor-mediated time-dependent intracellular uptake and distribution of the nanoparticles throughout the cytoplasm. The findings of the current study demonstrated the potential efficacy of the surface modified SFB-loaded particles against ErbB3.

Selective anti-ErbB3 aptamer modified sorafenib microparticles: In vitro and in vivo toxicity assessment.

The delivery of aptamer modified therapeutic moieties to specific tissue sites has become one of the major therapeutic choices to reduce the toxicity of inhibitory drugs. Bearing this in mind, the current study was designed using sorafenib (SFB) encapsulated microparticles (MP) prepared with biodegradable poly (D, L-lactic-co-glycolic acid) (PLGA) copolymer. The surfaces of these microparticles were modified with RNA aptamer having a binding affinity towards ErbB3 receptors. SFB-loaded MP (MPS) were prepared by o/w solvent evaporation method and the surface was coupled with the amino group of aptamer by EDC/NHS chemistry. Physiochemical investigations were done by dynamic light scattering, scanning electron microscopy and FTIR. In vitro apoptosis assay, cell viability assay and metastatic progression showed a significant decrease (p < 0.001) in vitro cell viability for MPS and MPS-Apt as compared to MP. The synergistic combination of SFB and aptamer also decreased the metastatic progression of cells for an extended period. Microparticles were also evaluated for in vivo toxicity in female BALB/c mice. It was evident that the presence of aptamer decreased the generalized toxicity of MPS-Apt, as measured by mean body weight loss and blood profiles, keeping all the blood formed elements level within acceptable limits. The histopathological investigations showed some necrotic and pyknotic bodies. In a similar fashion, liver function test and renal function tests showed pronounced effects of formulations on vital organs.

Photodynamic Therapy of Ovarian Carcinoma Cells with Curcumin-Loaded Biodegradable Polymeric Nanoparticles.

Accumulation of photosensitisers in photodynamic therapy in healthy tissues is often the cause of unwanted side effects. Using nanoparticles, improved bioavailability and site-specific drug uptake can be achieved. In this study, curcumin, a natural product with anticancer properties, albeit with poor aqueous solubility, was encapsulated in biodegradable polymeric poly(lactic-co-glycolic acid) (PLGA) nanoparticles (CUR-NP). Dynamic light scattering, laser Doppler anemometry and atomic force microscopy were used to characterise the formulations. Using haemolysis, serum stability and activated partial thromboplastin time tests, the biocompatibility of CUR-NP was assessed. Particle uptake and accumulation were determined by confocal laser scanning microscopy. Therapeutic efficacy of the formulation was tested in SK-OV-3 human ovarian adenocarcinoma cells post low level LED irradiation by determining the generation of reactive oxygen species and cytotoxicity. Pharmacologic inhibitors of cellular uptake pathways were used to identify the particle uptake mechanism. CUR-NP exhibited better physicochemical properties such as stability in the presence of light and improved serum stability compared to free curcumin. In addition, the novel nanoformulation facilitated the use of higher amounts of curcumin and showed strong apoptotic effects on tumour cells.

Nano spray dried antibacterial coatings for dental implants.

Nanostructured coatings of dental implants have shown great potential in overcoming many challenges responsible for implant failure. In this study, nano spray drying technology was utilized to produce novel biocompatible nanocoatings with antibacterial activity. The experiments were applied on titanium discs, which were used as a model material for dental implants. The produced nanocoatings consisted of poly(lactic-co-glycolic acid) as a biodegradable polymer and norfloxacin as a model antibiotic. Scanning electron microscopy results revealed an average particle size ranging between 400 and 600 nm. In vitro release studies showed a biphasic drug release profile with a burst release within the first 48 h, followed by a sustained release phase until the end of the experiment. The antibacterial activity of the nanocoatings was evaluated against Escherichia coli where the norfloxacin loaded nanocoatings achieved up to 99.83% reduction in the number of viable bacterial colonies. Finally, in vitro biocompatibility of the nanocoatings was investigated using mouse fibroblasts (L929) as a standard sensitive cell line for cytotoxicity assessment. Cell proliferation on the surface of the titanium discs was studied using fluorescence microscopy followed by cell counting assay. Both methods confirmed the biocompatibility of the examined nanocoatings. In conclusion, nano spray drying is a promising technique for preparing tailor-made nanocoatings, thereby representing an innovative approach for the surface modification of dental implants.

Curcumin loaded nanoparticles as efficient photoactive formulations against gram-positive and gram-negative bacteria.

The constant increase in multi-resistant bacterial strains and the decline in the number of newly approved antibiotics necessitate the development of alternative approaches to antibiotic treatment. In this study, a modern alternative approach to antibiotic therapy using photosensitiser encapsulated polymeric nanoparticles is presented. Cationic nanoparticles were prepared using a biodegradable and biocompatible polymer poly (lactic-co-glycolic acid), a stabiliser poly (vinyl alcohol) and chitosan. Dynamic light scattering and laser Doppler anemometry were used to determine particle size distribution and ζ-potential respectively. To quantify the antibacterial photodynamic effect of the nanoparticles, in vitro studies were performed using Staphylococcus saprophyticus subsp. bovis and Escherichia coli DH5 alpha to represent both a gram-positive as well as a gram-negative strain. It was demonstrated that the particle ζ-potential significantly influenced the antibacterial phototoxicity, gaining up to 3 log10 higher efficacy for chitosan coated nanoparticles. Furthermore, neither irradiation alone nor curcumin in absence of light led to a significant growth reduction, confirming the photodynamic effect of curcumin. Electron microscopy has been used to study the morphological characteristics of the nanoparticles as well as their interaction with bacteria and the changes of bacterial morphology and ultrastructure upon photodynamic treatment. An increased adherence of the chitosan modified nanoparticles to the bacteria and structural damage upon photodynamic treatment was clearly evident and confirmed the results from in vitro studies.

Development of inhalable curcumin loaded Nano-in-Microparticles for bronchoscopic photodynamic therapy.

Photodynamic therapy is amongst the most rapidly developing therapeutic strategies against cancer. However, most photosensitizers are administered intravenously with very few reports about pulmonary applications. To address this issue, an inhalable formulation consisting of nanoparticles loaded with photosensitizer (i.e. curcumin) was developed. The nanoparticles were prepared using nanoprecipitation method. Dynamic light scattering measurements of the curcumin loaded nanoparticles revealed a hydrodynamic diameter of 181.20 ±â€¯11.52 nm. In vitro irradiation experiments with human lung epithelial carcinoma cells (A549) showed a selective cellular toxicity of the nanoparticles upon activation using LED irradiating device. Moreover, curcumin nanoparticles exhibited a dose-dependent photocytotoxicity and the IC50 values of curcumin were directly dependent on the radiation fluence used. The nanoparticles were subsequently spray dried using mannitol as a stabilizer to produce Nano-in-Microparticles with appropriate aerodynamic properties for a sufficient deposition in the lungs. This was confirmed using the next generation impactor, which revealed a large fine particle fraction (64.94 ±â€¯3.47%) and a mass median aerodynamic diameter of 3.02 ±â€¯0.07 µm. Nano-in-Microparticles exhibited a good redispersibility and disintegrated into the original nanoparticles upon redispersion in aqueous medium. The Langmuir monolayer experiments revealed an excellent compatibility of the nanoparticles with the lung surfactant. Results from this study showed that the Nano-in-Microparticles are promising drug carriers for the photodynamic therapy of lung cancer.

In situ intravenous photodynamic therapy for the systemic eradication of blood stream infections.

Photodynamic therapy is one of the most promising non-invasive strategies employed for the treatment of several kinds of bacterial infections. Though the vast majority of clinically approved photosensitisers are administered intravenously, most of the in vitro experiments are performed under static conditions which do not represent the physiological environment of the venous bloodstream. To address this issue, a dynamic circulation model was developed to facilitate in situ antibacterial photodynamic therapy under flow conditions to mimic blood stream infections.

Lipodendriplexes: A promising nanocarrier for enhanced gene delivery with minimal cytotoxicity.

Non-viral vectors are a safe, efficient and non-toxic alternative to viral vectors for gene therapy against many diseases ranging from genetic disorders to cancers. Polyamidoamine (PAMAM), a positively charged dendrimer has a tendency to complex with nucleic acids (to form dendriplexes) like plasmid DNA (pDNA) and small interfering RNA (siRNA) and can shield them from enzymatic degradation, thereby facilitating endocytosis and endosomal release. In this study, we developed an advanced variant of the dendriplexes by encapsulating them within liposomes to enhance their gene delivery efficiency. This liposome encapsulated dendriplex system can further reduce unwanted cytotoxicity and enhance cellular uptake of nucleic acids. A broad range of lipid combinations were used to optimize the lipodendriplexes in terms of their physicochemical characteristics including size, shape and zeta potential. The optimized lipodendriplexes were tested for pDNA transfection, in vitro cell viability, cellular uptake, siRNA mediated knockdown, hemocompatibility, metastatic progression and in ovo in chorioallantoic membrane model (CAM). The optimized system has shown significant improvement in pDNA transfection (p < 0.01) with higher GFP expression and gene silencing and has shown improved cell viability (p < 0.05) compared to the parent dendriplex system. The hemocompatibility and CAM analysis, revealed an efficient yet biocompatible gene delivery system in the form of lipodendriplexes.

Lipid coated chitosan-DNA nanoparticles for enhanced gene delivery.

Chitosan as a polycationic non-viral vector for gene delivery has the advantage of being a biocompatible and biodegradable polymer. However, without laborious chemical modifications to its structure, it is of limited use as a gene delivery vehicle due to its low ability to efficiently transfect under physiological conditions. To address this problem, we developed novel liposome encapsulated chitosan nanoparticles; lipochitoplexes (LCPs). Chitosan nanoparticles (CsNPs) were obtained using the ionic gelation technique. For this purpose, an ultrapure low molecular weight chitosan with a high degree of deacetylation was cross-linked using polyanionic tripolyphosphate resulting in efficient entrapment of plasmid DNA (pDNA) inside the nanoparticles. LCPs were prepared by incubating chitosan nanoparticles together with anionic liposomes (DPPC/Cholesterol). The LCPs offered better pDNA protection, reduced cytotoxicity and at least twofold increase in the transfection efficiency under physiological conditions. The efficiency of our delivery vehicle was also proved in vivo in the chorioallantoic membrane model (CAM). LCPs were able to transfect the CAM without traumatising the surrounding blood vessels. This new biocompatible composite system devoid of chemical modifications, organic solvents and harsh production conditions makes it an optimal gene delivery vehicle for in vivo applications offering new insights into the field of non-viral gene therapy.

Composite liposome-PEI/nucleic acid lipopolyplexes for safe and efficient gene delivery and gene knockdown.

Cytotoxicity is a major drawback impeding the therapeutic use of gene delivery and gene down-regulation vehicles. Apart from cytotoxicity, rapid degradation and low cellular uptake are other major factors affecting therapeutic use. Considering the above factors, formulation and development of PEI (Polyethylenimine) based, liposome encapsulated delivery vehicles with improved transfection efficiency and low cytotoxicity which can be used for gene delivery and gene knockdown. DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (Dipalmitoylphosphatidylcholine) and cholesterol have been considered as lipids of choice bearing in mind various factors such as rigidness and surface charge which greatly influence the formation of liposomes, polyplex encapsulation and transfection efficiency. For the condensation of plasmid DNA (pDNA) and short interfering RNA (siRNA), branched PEI 25kDa (bPEI) and deacylated linear PEI 22kDa (lPEI) were employed. lPEI and siRNA polyplexes encapsulated within DOPE/DPPC/Cholesterol (DDC) liposomes exhibited higher luc (luciferase) gene knockdown in vitro compared to the controls. They also showed superior transfection efficiencies compared to polyplexes in experiments using pCMV-luc (luciferase reporter plasmid) and pEGFP-N1 (Green Fluorescence protein reporter plasmid). This can partly be attributed to the improved integrity imparted by the liposomal layer which was confirmed by complex stability and integrity assays. Cytotoxicity and coagulation time assays of DDC-lPEI based lipopolyplexes showed decreased cytotoxic potential and negligible influence on coagulation respectively for compared to polyplexes, thus rendering them suitable for gene therapy.

Hydrophilic Ionic Liquids as Ingredients of Gel-Based Dermal Formulations.

Ionic liquids (ILs) have several properties that offer many advantages in dermal drug delivery systems. Depending on the chemical structure, ILs can be used for protection against microorganisms, to enhance skin penetration, and as a solvent. In the present work, SEPINEO™ P 600 formulations and hydroxyethylcellulose gels containing the hydrophilic ILs hexylpyridinium chloride, choline dihydrogen phosphate, and 1-ethyl-3-methylimidazolium ethyl sulfate were prepared, and the influence of the ILs on the formulation properties was evaluated. ILs were successfully incorporated into the emulsion structure, resulting in stable formulations. The antimicrobial activity of the ILs was estimated. The minimal inhibitory concentration values for hexylpyridinium chloride are about 2.5 mg/mL. The other two ILs have no antimicrobial activity. Skin penetration enhancement of caffeine, a hydrophilic model substance, was observed in the presence of hexylpyridinium chloride.

Degradation and protection of DNAzymes on human skin.

DNAzymes are catalytic nucleic acid based molecules that have become a new class of active pharmaceutical ingredients (API). Until now, five DNAzymes have entered clinical trials. Two of them were tested for topical application, whereby dermally applied DNAzymes had been prone to enzymatic degradation. To protect the DNAzymes the enzymatic activity of human skin has to be examined. Therefore, the enzymatic activity of human skin was qualitatively and quantitatively analyzed. Activity similar to that of DNase II could be identified and the specific activity was determined to be 0.59Units/mg. These results were used to develop an in vitro degradation assay to screen different kinds of protective systems on human skin. The chosen protective systems consisted of biodegradable chitosans or polyethylenimine, which forms polyplexes when combined with DNAzymes. The polyplexes were characterized in terms of particle size, zeta potential, stability and degree of complexation. The screening revealed that the protective efficiency of the polyplexes depended on the polycation and the charge ratio (ξ). At a critical ξ ratio between 1.0 and 4.1 and at a maximal zeta potential, sufficient protection of the DNAzyme was achieved. The results of this study will be helpful for the development of a protective dermal drug delivery systems using polyplexes.