Development of a Spray-Dried Formulation of Peptide-DNA Nanoparticles into a Dry Powder for Pulmonary Delivery Using Factorial Design.

BACKGROUND: Gene therapy via pulmonary delivery holds the potential to treat various lung pathologies. To date, spray drying has been the most promising method to produce inhalable powders. The present study determined the parameters required to spray dry nanoparticles (NPs) that contain the delivery peptide, termed RALA (N-WEARLARALARALARHLARALARALRACEA-C), complexed with plasmid DNA into a dry powder form designed for inhalation. METHODS: The spray drying process was optimised using full factorial design with 19 randomly ordered experiments based on the combination of four parameters and three centre points per block. Specifically, mannitol concentration, inlet temperature, spray rate, and spray frequency were varied to observe their effects on process yield, moisture content, a median of particle size distribution, Z-average, zeta potential, encapsulation efficiency of DNA NPs, and DNA recovery. The impact of mannitol concentration was also examined on the spray-dried NPs and evaluated via biological functionality in vitro. RESULTS: The results demonstrated that mannitol concentration was the strongest variable impacting all responses apart from encapsulation efficiency. All measured responses demonstrated a strong dependency on the experimental variables. Furthermore, spray drying with the optimal variables in combination with a low mannitol concentration (1% and 3%, w/v) produced functional RALA/pDNA NPs. CONCLUSION: The optimal parameters have been determined to spray dry RALA/pDNA NPs into an dry powder with excellent biological functionality, which have the potential to be used for gene therapy applications via pulmonary delivery.

Exploiting the anticancer effects of a nitrogen bisphosphonate nanomedicine for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an incurable aggressive brain cancer in which current treatment strategies have demonstrated limited survival benefit. In recent years, nitrogen-containing bisphosphonates (N-BPs) have demonstrated direct anticancer effects in a number of tumour types including GBM. In this study, a nano-formulation with the RALA peptide was used to complex the N-BP, alendronate (ALN) into nanoparticles (NPs) < 200 nm for optimal endocytic uptake. Fluorescently labelled AlexaFluor®647 Risedronate was used as a fluorescent analogue to visualise the intracellular delivery of N-BPs in both LN229 and T98G GBM cells. RALA NPs were effectively taken up by GBM where a dose-dependent response was evidenced with potentiation factors of 14.96 and 13.4 relative to ALN alone after 72 h in LN229 and T98G cells, respectively. Furthermore, RALA/ALN NPs at the IC50, significantly decreased colony formation, induced apoptosis and slowed spheroid growth in vitro. In addition, H-Ras membrane localisation was significantly reduced in the RALA/ALN groups compared to ALN or controls, indicative of prenylation inhibition. The RALA/ALN NPs were lyophilised to enhance stability without compromising the physiochemical properties necessary for functionality, highlighting the suitability of the NPs for scale-up and in vivo application. Collectively, these data show the significant potential of RALA/ALN NPs as novel therapeutics in the treatment of GBM.

Pharmacokinetics of the Protein Microbicide 5P12-RANTES in Sheep following Single-Dose Vaginal Gel Administration.

5P12-RANTES, a chemokine analogue that potently blocks the HIV CCR5 coreceptor, is being developed as both a vaginal and rectal microbicide for prevention of sexual transmission of HIV. Here, we report the first pharmacokinetic data for 5P12-RANTES following single-dose vaginal gel administration in sheep. Aqueous gel formulations containing low (1.24-mg/ml), intermediate (6.18-mg/ml), and high (32.0-mg/ml; suspension-type gel) concentrations of 5P12-RANTES were assessed via rheology, syringeability, and in vitro release testing. Following vaginal gel administration to sheep, 5P12-RANTES concentrations were measured in vaginal fluid, vaginal tissue, and serum over a 96-h period. All gels showed non-Newtonian pseudoplastic behavior, with the high-concentration gels exhibiting a greater viscosity and cohesive structure than the intermediate- and low-concentration gels. In in vitro release testing, >90% 5P12-RANTES was released from the low- and intermediate-concentration gels after 72 h. For the high-concentration gel, ∼50% 5P12-RANTES was detected, attributed to protein denaturation during lyophilization and/or subsequent solvation of the protein within the gel matrix. In sheep, 5P12-RANTES concentrations in vaginal fluid, vaginal tissue, and serum increased in a dose-dependent manner. The highest concentrations were measured in vaginal fluid (105 to 107 ng/ml), followed by vaginal tissue (104 to 106 ng/ml). Both of these concentration ranges are several orders of magnitude above the reported half-maximal inhibitory concentrations. The lowest concentration was measured in serum (<102 ng/ml). The 5P12-RANTES pharmacokinetic data are similar to those reported previously for other candidate microbicides. These data, coupled with 5P12-RANTES's potency at picomolar concentrations, its strong barrier to resistance, and the full protection that it was observed to provide in a rhesus macaque vaginal challenge model, support the continued development of 5P12-RANTES as a microbicide.

Inhaled dry powder liposomal azithromycin for treatment of chronic lower respiratory tract infection.

A dry powder inhaled liposomal azithromycin formulation was developed for the treatment of chronic respiratory diseases such as cystic fibrosis and bronchiectasis. Key properties including liposome size, charge and encapsulation efficiency powder size, shape, glass transition temperature (Tg), water content and in vitro respiratory deposition were determined. Antimicrobial activity against cystic fibrosis (CF) respiratory pathogens was determined by MIC, MBC and biofilm assays. Cytotoxicity and cellular uptake studies were performed using A549 cells. The average liposome size was 105 nm, charge was 55 mV and encapsulation efficiency was 75 %. The mean powder particle size d[v,50] of 4.54 µm and Mass Median Aerodynamic Diameter (MMAD) was 5.23 µm with a mean Tg of 76˚C and water content of 2.1 %. These excellent physicochemical characteristics were maintained over one year. Liposomal loaded azithromycin demonstrated enhanced activity against P. aeruginosa clinical isolates grown in biofilm. The formulation was rapidly delivered into bacterial cells with > 75 % uptake in 1 h. Rapid uptake into A549 cells via a cholesterol-dependent endocytosis pathway with no cytotoxic effects apparent. These data demonstrate that this formulation could offer benefits over current treatment regimens for people with chronic respiratory infection.

Aerosolised micro and nanoparticle: formulation and delivery method for lung imaging.

Purpose: The application of contrast and tracing agents is essential for lung imaging, as indicated by the wide use in recent decades and the discovery of various new contrast and tracing agents. Different aerosol production and pulmonary administration methods have been developed to improve lung imaging quality. This review details and discusses the ideal characteristics of aerosol administered via pulmonary delivery for lung imaging and the methods for the production and pulmonary administration of dry or liquid aerosol. Methods: We explored several databases, including PubMed, Scopus, and Google Scholar, while preparing this review to discover and obtain the abstracts, reports, review articles, and research papers related to aerosol delivery for lung imaging and the formulation and pulmonary delivery method of dry and liquid aerosol. The search terms used were "dry aerosol delivery", "liquid aerosol delivery", "MRI for lung imaging", "CT scan for lung imaging", "SPECT for lung imaging", "PET for lung imaging", "magnetic particle imaging", "dry powder inhalation", "nebuliser", and "pressurised metered-dose inhaler". Results: Through the literature review, we found that the critical considerations in aerosol delivery for lung imaging are appropriate lung deposition of inhaled aerosol and avoiding toxicity. The important tracing agent was also found to be Technetium-99m (99mTc), Gallium-68 (68Ga) and superparamagnetic iron oxide nanoparticle (SPION), while the essential contrast agents are gold, iodine, silver gadolinium, iron and manganese-based particles. The pulmonary delivery of such tracing and contrast agents can be performed using dry formulation (graphite ablation, spark ignition and spray dried powder) and liquid aerosol (nebulisation, pressurised metered-dose inhalation and air spray). Conclusion: A dual-imaging modality with the combination of different tracing or contrast agents is a future development of aerosolised micro and nanoparticles for lung imaging to improve diagnosis success.

Rational design and characterisation of an amphipathic cell penetrating peptide for non-viral gene delivery.

RALA is a cationic amphipathic peptide which has shown great promise as an efficient, multifunctional delivery system for the delivery of nucleic acids. Rational peptide design was utilised in this study to understand the essential amino acids required for delivery and if any improvements to the RALA peptide could be made. Six amphipathic peptides were synthesised with strategic sequences and amino acid substitutions to reduce peptide sequence, while maintaining the functional characteristics of RALA including amphipathicity, alpha-helicity and pH responsiveness for endosomal escape. Data demonstrated that all six peptides complexed pEGFP-N1 to produce cationic nanoparticles <200 nm in diameter, but not all peptides resulted in successful transfection; indicating the influence of peptide design for cellular uptake and endosomal escape. Pep2, produced nanoparticles with similar characteristics and transfection efficiency to the parent peptide, RALA. However, Pep2 had issues with toxicity and a lack of pH-responsive alpha-helcity. Therefore, RALA remains the superior sequence for non-toxic gene delivery.

Vaginal rings with exposed cores for sustained delivery of the HIV CCR5 inhibitor 5P12-RANTES.

Antiretroviral-releasing vaginal rings are at the forefront of ongoing efforts to develop microbicide-based strategies for prevention of heterosexual transmission of the human immunodeficiency virus (HIV). However, traditional ring designs are generally only useful for vaginal administration of relatively potent, lipophilic, and small molecular weight drug molecules that have sufficient permeability in the non-biodegradable silicone elastomer or thermoplastic polymers. Here, we report a novel, easy-to-manufacture 'exposed-core' vaginal ring that provides sustained release of the protein microbicide candidate 5P12-RANTES, an experimental chemokine analogue that potently blocks the HIV CCR5 coreceptor. In vitro release, mechanical, and stability testing demonstrated the utility and practicality of this novel ring design. In a sheep pharmacokinetic model, a ring containing two »-length excipient-modified silicone elastomer cores - each containing lyophilised 5P12-RANTES and exposed to the external environment by two large windows - provided sustained concentrations of 5P12-RANTES in vaginal fluid and vaginal tissue between 10 and 10,000 ng/g over 28days, at least 50 and up to 50,000 times the reported in vitro IC50 value.

Development and pharmacokinetics of a combination vaginal ring for sustained release of dapivirine and the protein microbicide 5P12-RANTES.

The past fifteen years have witnessed a resurgence of interest in vaginal ring technologies for drug delivery applications, mostly driven by the impetus for development of vaginally-administered antiretroviral microbicides to help reduce the high acquisition rates for human immunodeficiency virus (HIV) among Sub-Saharan African women. Currently, the lead candidate microbicide is a 28-day silicone elastomer vaginal ring releasing dapivirine (Ring-004), an experimental non-nucleoside reverse transcriptase inhibitor. The ring was tested in two pivotal Phase III clinical studies in 2016 and is currently undergoing review by the European Medicines Agency. Recently, we described a new type of silicone elastomer vaginal ring offering sustained release of the protein molecule 5P12-RANTES, a potent experimental chemokine analogue that potently blocks the HIV CCR5 coreceptor. Building on our previous work, here we report the preclinical development of a new combination vaginal ring that offers sustained release of both 5P12-RANTES and dapivirine, in which the 5P12-RANTES is incorporated into an exposed core within the ring body and the dapivirine in the sheath. In this way, in vitro release of dapivirine matches closely that for Ring-004. Also, we report the pharmacokinetic testing of this combination ring formulation in sheep, where vaginal concentrations of both drugs are maintained over 28 days at levels potentially useful for preventing HIV infection in women.

DNA vaccination via RALA nanoparticles in a microneedle delivery system induces a potent immune response against the endogenous prostate cancer stem cell antigen.

Castrate resistant prostate cancer (CRPC) remains a major challenge for healthcare professionals. Immunotherapeutic approaches, including DNA vaccination, hold the potential to harness the host's own immune system to mount a cell-mediated, anti-tumour response, capable of clearing disseminated tumour deposits. These anti-cancer vaccines represent a promising strategy for patients with advanced disease, however, to date DNA vaccines have demonstrated limited efficacy in clinical trials, owing to the lack of a suitable DNA delivery system. This study was designed to evaluate the efficacy of a two-tier delivery system incorporating cationic RALA/pDNA nanoparticles (NPs) into a dissolvable microneedle (MN) patch for the purposes of DNA vaccination against prostate cancer. Application of NP-loaded MN patches successfully resulted in endogenous production of the encoded Prostate Stem Cell Antigen (PSCA). Furthermore, immunisation with RALA/pPSCA loaded MNs elicited a tumour-specific immune response against TRAMP-C1 tumours ex vivo. Finally, vaccination with RALA/pPSCA loaded MNs demonstrated anti-tumour activity in both prophylactic and therapeutic prostate cancer models in vivo. This is further evidence that this two-tier MN delivery system is a robust platform for prostate cancer DNA vaccination. STATEMENT OF SIGNIFICANCE: This research describes the development and utilisation of our unique microneedle (MN) DNA delivery system, which enables penetration through the stratum corneum and deposition of the DNA within the highly immunogenic skin layers via a dissolvable MN matrix, and facilitates cellular uptake via complexation of pDNA cargo into nanoparticles (NPs) with the RALA delivery peptide. We report for the first time on using the NP-MN platform to immunise mice with encoded Prostate Stem Cell Antigen (mPSCA) for prostate cancer DNA vaccination. Application of the NP-MN system resulted in local mPSCA expression in vivo. Furthermore, immunisation with the NP-MN system induced a tumour-specific cellular immune response, and inhibited the growth of TRAMP-C1 prostate tumours in both prophylactic and therapeutic challenge models in vivo.

Calorimetric and spatial characterization of polymorphic transitions in caffeine using quasi-isothermal MTDSC and localized thermomechanical analysis.

We describe a novel integrated approach to the study of polymorphic transformation that includes quasi-isothermal modulated temperature differential scanning calorimetry (QI-MTDSC) and microthermal analysis (MTA), with a view to studying the thermal, kinetic and spatial characteristics of the process. Form II and I caffeine was prepared and conventional DSC and hot stage microscopy performed. The Form II to I transition at circa 413 K was associated with a change in crystal habit to needle shaped crystals. QI-MTDSC was used to measure the heat capacity of the system as a function of temperature, while MTA was able to spatially differentiate between the two polymorphs in compressed systems. We present a novel extension of the reduced temperature method whereby we apply it for the first time to linear rising temperature data corresponding to the transition; the analysis suggests a close approximation to Arrhenius behavior. We also describe a heat transfer model that allows calculation of the thermal gradients within a hermetically sealed pan for the first time. The combined approach has therefore allowed the characterization of the thermodynamics and kinetics of the transformation process as well as spatial identification of the distribution of the transformation in compressed systems.

DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system.

Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 µg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer.

Gene therapy with RALA/iNOS composite nanoparticles significantly enhances survival in a model of metastatic prostate cancer.

BACKGROUND: Recent approvals of gene therapies by the FDA and the EMA for treatment of inherited disorders have further opened the door for assessment of nucleic acid pharmaceuticals for clinical usage. Arising from the presence of damaged or inappropriate DNA, cancer is a condition particularly suitable for genetic intervention. The RALA peptide has been shown to be a potent non-viral delivery platform for nucleic acids. This study examines the use of RALA to deliver a plasmid encoding inducible nitric oxide synthase (iNOS) as an anti-cancer treatment. METHODS: The physiochemical properties of the RALA/DNA nanoparticles were characterized via dynamic light scattering and transmission electron microscopy. The nanoparticles were labelled with fluorophores and tracked over time using confocal microscopy with orthogonal sections to determine cellular location. In vitro studies were employed to determine functionality of the nanoparticles both for pEGFP-N1 and CMV-iNOS. Nanoparticles were injected intravenously into C57/BL6 mice with blood and serum samples analysed for immune response. PC3-luc2M cells were injected into the left ventricle of SCID mice followed by treatment with RALA/CMV-iNOS nanoparticles to evaluate the tumour response in a metastatic model of prostate cancer. RESULTS: Functional cationic nanoparticles were produced with gene expression in PC-3 prostate cancer cells. Furthermore, repeated administrations of RALA/DNA nanoparticles into immunocompetent mice did not produce any immunological response: neutralization of the vector or release of inflammatory mediators. RALA/CMV-iNOS reduced the clonogenicity of PC-3 cells in vitro, and in an in vivo model of prostate cancer metastasis, systemically delivered RALA/CMV-iNOS significantly improved the survival of mice. CONCLUSION: These studies further validate RALA as a genetic cargo delivery vehicle and iNOS as a potent therapy for the treatment of cancer.

Investigation into the subambient behavior of aqueous mannitol solutions using temperature-controlled Raman microscopy.

The aim was twofold; to demonstrate the ability of temperature-controlled Raman microscopy (TRM) to locate mannitol within a frozen system and determine its form; to investigate the annealing behavior of mannitol solutions at -30 degrees C. The different polymorphic forms of anhydrous mannitol as well as the hemihydrate and amorphous form were prepared and characterized using crystal or powder X-ray diffractometry (XRD) as appropriate and Raman microscopy. Mannitol solutions (3% w/v) were cooled before annealing at -30 degrees C. TRM was used to map the frozen systems during annealing and was able to differentiate between the different forms of mannitol and revealed the location of both beta and delta polymorphic forms within the structure of the frozen material for the first time. TRM also confirmed that the crystalline mannitol is preferentially deposited at the edge of the frozen drop, forming a rim that thickens upon annealing. While there is no preference for one form initially, the study has revealed that the mannitol preferentially transforms to the beta form with time. TRM has enabled observation of spatially resolved behavior of mannitol during the annealing process for the first time. The technique has clear potential for studying other crystallization processes, with particular advantage for frozen systems.

Novel freeze-dried DDA and TPGS liposomes are suitable for nasal delivery of vaccine.

There is a pressing need for effective needle-free vaccines that are stable enough for use in the developing world and stockpiling. The inclusion of the cationic lipid DDA and the PEG-containing moiety TPGS into liposomes has the potential to improve mucosal delivery. The aim of this study was to develop stable lyophilized cationic liposomes based on these materials suitable for nasal antigen delivery. Liposomes containing DDA and TPGS were developed. Size and zeta potential measurements, ex vivo, CLSM cell penetration study and cell viability investigations were made. Preliminary immunisation and stability studies using ovalbumin were performed. The liposomes exhibited suitable size and charge for permeation across nasal mucosa. DDA and TPGS increased tissue permeation in ex vivo studies and cell uptake with good cell viability. The liposomes improved immune response both locally and vaginally when compared to i.m administration or control liposomes delivered nasally. Additionally, the lyophilized products demonstrated good stability in terms of Tg, size and antigen retention. This study has shown that the novel liposomes have potential for development as a mucosal vaccine delivery system. Furthermore, the stability of the lyophilized liposomes offers potential additional benefits in terms of thermal stability over liquid formats.

Systemic RALA/iNOS Nanoparticles: A Potent Gene Therapy for Metastatic Breast Cancer Coupled as a Biomarker of Treatment.

This study aimed to determine the therapeutic benefit of a nanoparticular formulation for the delivery of inducible nitric oxide synthase (iNOS) gene therapy in a model of breast cancer metastasis. Nanoparticles comprising a cationic peptide vector, RALA, and plasmid DNA were formulated and characterized using a range of physiochemical analyses. Nanoparticles complexed using iNOS plasmids and RALA approximated 60 nm in diameter with a charge of 25 mV. A vector neutralization assay, performed to determine the immunogenicity of nanoparticles in immunocompetent C57BL/6 mice, revealed that no vector neutralization was evident. Nanoparticles harboring iNOS plasmids (constitutively active cytomegalovirus [CMV]-driven or transcriptionally regulated human osteocalcin [hOC]-driven) evoked iNOS protein expression and nitrite accumulation and impaired clonogenicity in the highly aggressive MDA-MB-231 human breast cancer model. Micrometastases of MDA-MB-231-luc-D3H1 cells were established in female BALB/c SCID mice by intracardiac delivery. Nanoparticulate RALA/CMV-iNOS or RALA/hOC-iNOS increased median survival in mice bearing micrometastases by 27% compared with controls and also provoked elevated blood nitrite levels. Additionally, iNOS gene therapy sensitized MDA-MB-231-luc-D3H1 tumors to docetaxel treatment. Studies demonstrated that systemically delivered RALA-iNOS nanoparticles have therapeutic potential for the treatment of metastatic breast cancer. Furthermore, detection of nitrite levels in the blood serves as a reliable biomarker of treatment.

High speed DSC (hyper-DSC) as a tool to measure the solubility of a drug within a solid or semi-solid matrix.

Conventional differential scanning calorimetry (DSC) techniques are commonly used to quantify the solubility of drugs within polymeric-controlled delivery systems. However, the nature of the DSC experiment, and in particular the relatively slow heating rates employed, limit its use to the measurement of drug solubility at the drug's melting temperature. Here, we describe the application of hyper-DSC (HDSC), a variant of DSC involving extremely rapid heating rates, to the calculation of the solubility of a model drug, metronidazole, in silicone elastomer, and demonstrate that the faster heating rates permit the solubility to be calculated under non-equilibrium conditions such that the solubility better approximates that at the temperature of use. At a heating rate of 400 degrees C/min (HDSC), metronidazole solubility was calculated to be 2.16 mg/g compared with 6.16 mg/g at 20 degrees C/min.

Development of polymeric-cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery.

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid-polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

An investigation into the subambient behavior of aqueous mannitol solutions using differential scanning calorimetry, cold stage microscopy, and X-ray diffractometry.

The subambient behavior of aqueous mannitol solutions is of considerable relevance to the preparation of freeze dried formulations. In this investigation the properties of 3% w/v mannitol solutions were investigated using differential scanning calorimetry (DSC), cold stage microscopy (CSM), and X-ray diffraction (XRD) to identify the thermal transitions and structural transformations undergone by this system. It was found that on cooling from ambient the system formed ice at circa -20 degrees C while a further exotherm was seen at approximately -30 degrees C. Upon reheating an endotherm was seen at circa -30 degrees C followed immediately by an exotherm at circa -25 degrees C. Temperature cycling indicated that the thermal transitions observed upon reheating were not reversible. Modulated temperature DSC (MTDSC) indicated that the transitions observed upon reheating corresponded to a glass transition immediately followed by recrystallization, XRD data showed that recrystallization was into the beta form. Annealing at -35 degrees C for 40 min prior to cooling and reheating resulted in a maximum enthalpy being observed for the reheating exotherm. It is concluded that on cooling 3% w/v aqueous mannitol solutions an amorphous phase is formed that subsequently recrystallises into the beta form. The study has also shown that DSC, CSM, and XRD are useful complementary techniques for the study of frozen systems.

Development of a method to quantify the DNA content in cationic peptide-DNA nanoparticles.

Gene therapy has the potential to provide safe and targeted therapies for a variety of diseases. A range of intracellular gene delivery vehicles have been proposed for this purpose. Non-viral vectors are a particularly attractive option and among them cationic peptides have emerged as promising candidates. For the pharmaceutical formulation and application to clinical studies it is necessary to quantify the amount of pDNA condensed with the delivery system. There is a severe deficiency in this area, thus far no methods have been reported specifically for pDNA condensed with cationic peptide to form nanoparticles. The current study seeks to address this and describes the evaluation of a range of disruption agents to extract DNA from nanoparticles formed by condensation with cationic fusogenic peptides RALA and KALA. Only proteinase K exhibited efficient and reproducible results and compatibility with the PicoGreen reagent based quantification assay. Thus we report for the first time a simple and reliable method that can quantify the pDNA content in pDNA cationic peptide nanoparticles.

Investigation into the effect of varying l-leucine concentration on the product characteristics of spray-dried liposome powders.

OBJECTIVES: Spray-dried formulations offer an attractive delivery system for administration of drug encapsulated into liposomes to the lung, but can suffer from low encapsulation efficiency and poor aerodynamic properties. In this paper the effect of the concentration of the anti-adherent l-leucine was investigated in tandem with the protectants sucrose and trehalose. METHODS: Two manufacturing methods were compared in terms of their ability to offer small liposomal size, low polydispersity and high encapsulation of the drug indometacin. KEY FINDINGS: Unexpectedly sucrose offered the best protection to the liposomes during the spray drying process, although formulations containing trehalose formed products with the best powder characteristics for pulmonary delivery; high glass transition values, fine powder fraction and yield. It was also found that l-leucine contributed positively to the characteristics of the powders, but that it should be used with care as above the optimum concentration of 0.5% (w/w) the size and polydispersity index increased significantly for both disaccharide formulations. CONCLUSIONS: The method of liposome preparation had no effect on the stability or encapsulation efficiency of spray-dried powders containing optimal protectant and anti-adherent. Using l-leucine at concentrations higher than the optimum level caused instability in the reconstituted liposomes.