In vivo evaluation of solid lipid microparticles and hybrid polymer-lipid microparticles for sustained delivery of leuprolide.

This study aims to investigate the potential of solid lipid microparticles (MP) and hybrid polymer-lipid MPs for sustained delivery of a peptide drug, leuprolide. A peptide-phospholipid complex was prepared to increase the compatibility of the peptide with triglyceride (TG) and poly (lactide-co-glycolide) (PLGA). Peptide loaded solid lipid MPs, PLGA MPs, and hybrid MPs were prepared using a spray drying method and characterized in terms of particle size, morphology and encapsulation efficiency. The pharmacokinetics and pharmacodynamics of leuprolide after subcutaneous injection of spray-dried MPs were evaluated in rats. Spray-dried MPs were spherical ranging in size from 4 µm to 10 µm, which are suitable for injection. After subcutaneous administration of reconstituted MPs, leuprolide could be detected in plasma samples of rats for one to two months, depending on the formulation and dose. Sustained release of leuprolide from PLGA MPs and glyceryl tristearate (TG18) MPs was observed over one month, with a chemical castration effect of 25 and 30 days, respectively. The bioavailability of leuprolide from PLGA-TG18 hybrid MPs was approximately four times higher than that from TG18 MP and PLGA MP alone using the same dose of leuprolide (6 mg/kg). Chemical castration in rats was observed over 30 and 60 days after injection of the PLGA-TG18 hybrid MP with a dose of 3 mg/kg and 6 mg/kg leuprolide, respectively. Additionally, a much lower Cmax was observed for the hybrid MP group. In conclusion, spray-dried PLGA-triglyceride hybrid MPs can be used as better carriers than other MPs for subcutaneous delivery of peptide drugs due to the synergetic effect of lipids and PLGA for sustained drug release from the spray-dried MP.

Qualitative and quantitative analysis of the biophysical interaction of inhaled nanoparticles with pulmonary surfactant by using quartz crystal microbalance with dissipation monitoring.

Understanding the interaction between inhaled nanoparticles and pulmonary surfactant is a prerequisite for predicting the fate of inhaled nanoparticles. Here, we introduce a quartz crystal microbalance with dissipation monitoring (QCM-D)-based methodology to reveal the extent and nature of the biophysical interactions of polymer- and lipid-based nanoparticles with pulmonary surfactant. By fitting the QCM-D data to the Langmuir adsorption equation, we determined the kinetics and equilibrium parameters [i.e., maximal adsorption (Δmmax), equilibrium constant (Ka), adsorption rate constant (ka) and desorption rate constant (kd)] of polymeric nanoparticles adsorption onto the pulmonary surfactant (e.g., an artificial lipid mixture and an extract of porcine lung surfactant). Furthermore, our results revealed that the nature of the interactions between lipid-based nanoparticles (e.g., liposomes) and pulmonary surfactant was governed by the liposomal composition, i.e., incorporation of cholesterol and PEGylated phospholipid (DSPE-PEG2000) into DOPC-based liposomes led to the adsorption of intact liposomes onto the pulmonary surfactant layer and the mass exchange between the liposomes and pulmonary surfactant layer, respectively. In conclusion, we demonstrate the applicability of the QCM-D technique for qualitative and quantitative analysis of the biophysical interaction of inhaled nanoparticles with pulmonary surfactant, which is vital for rational design and optimization of inhalable nanomedicines.

Effect of excipients on encapsulation and release of insulin from spray-dried solid lipid microparticles.

The study aimed at investigating the potential of spray drying method for encapsulation of protein drugs into solid lipid microparticles (MP) and evaluating effects of excipients on encapsulation and release of protein from MP. After transformation of model protein insulin to insulin-phospholipid complex, it was dissolved together with lipid excipients in organic solvent, which was spray-dried to form solid lipid MP. Polymeric MP with D, L-lactic-co-glycolic acid (PLGA) were prepared similarly. Around 90% of insulin was encapsulated in glycerol monostearate MP and glycerol distearate MP, whereas the encapsulation efficiency was 60% and 35% for tristearate (TG18) MP and tribehenate (TG22) MP, respectively. The secondary structure of insulin in the spray-dried MP was substantially similar to that of the insulin control solution, suggesting that only minor alterations occurred during the spray drying process. Sustained release of insulin was observed from both TG18 MP and TG22 MP. The burst release of insulin from TG18 MP and TG22 MP was around 30% and 10%, respectively, which was significantly lower than that from PLGA MP (40%). In conclusion, spray drying a solution containing both lipids and protein-phospholipid complex is a promising method for encapsulating protein into solid lipid MP, which can be used for sustained delivery of protein drugs.

Lipid Shell-Enveloped Polymeric Nanoparticles with High Integrity of Lipid Shells Improve Mucus Penetration and Interaction with Cystic Fibrosis-Related Bacterial Biofilms.

Nanoparticle (NP) mediated drug delivery into viscous biomatrices, e.g., mucus and bacterial biofilms, is challenging. Lipid shell-enveloped polymeric NPs (Lipid@NPs), composed of a polymeric NP core coated with a lipid shell, represent a promising alternative to the current delivery systems. Here, we describe the facile methods to prepare Lipid@NPs with high integrity of lipid shells and demonstrate the potential of Lipid@NPs in an effective mucus penetration and interaction with cystic fibrosis-related bacterial biofilms. Lipid shell-enveloped polystyrene NPs with high integrity of lipid shells ( cLipid@PSNPs) were prepared by using an electrostatically mediated layer-by-layer approach, where the model polystyrene NPs (PSNPs) were first modified with positively charged poly-l-lysine (PLL) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), followed by subsequent fusion with zwitterionic, PEGylated small unilamellar vesicles (SUVs). The interaction of the PSNPs with SUVs was significantly enhanced by modifying the PSNPs with PLL and DOTAP, which eventually resulted in the formation of cLipid@PSNPs, i.e., Lipid@PLL-PSNPs and Lipid@DOTAP-PSNPs. Improved mucus-penetrating property of cLipid@PSNPs was demonstrated by quartz crystal microbalance with dissipation monitoring measurements. Furthermore, fluorescence resonance energy transfer measurements showed that the interaction of the cLipid@PSNPs with bacterial biofilms was significantly promoted. In conclusion, we prepared cLipid@PSNPs via an electrostatically mediated layer-by-layer approach. Our results suggest that the integrity of the lipid envelopes is crucial for enabling the diffusion of Lipid@PSNPs into the mucus layer and promoting the interaction of Lipid@PSNPs with a bacterial biofilm.

Electrospinnability of Poly Lactic-co-glycolic Acid (PLGA): the Role of Solvent Type and Solvent Composition.

PURPOSE: In this study, the electrospinnability of poly(lactic-co-glycolic acid) (PLGA) solutions was investigated, with a focus on understanding the influence of molecular weight of PLGA, solvent type and solvent composition on the physical properties of electrospun nanofibers. METHOD: Various solvents were tested to dissolve two PLGA grades (50 KDa-RG755, 100 KDa-RG750). The viscoelasticity, surface tension, and evaporation rate of the PLGA solutions were characterized prior to the electrospinning process. The resulting electrospun nanofibers were characterized with respect to the morphology and mechanical properties. RESULTS: Two pairs of solvent mixtures, i.e. dimethylformamide (DMF)-tetrahydrofuran (THF) and DMF-chloroform (CHL), were identified to provide a stable cone-jet. Within the polymer concentration range studied (10-30%, w/v), RG750 solutions could be electrospun into uniform fibers at 30% (w/v) or at 20% (w/v) when modifying the solvent composition. In comparison to DMF-THF solution, fibers had larger diameter, higher stiffness and tensile strength when electrospun from DMF-CHL solution. CONCLUSION: The high molecular weight polymer could ensure sufficient intermolecular interaction to generate uniform fibers. The solvent could influence the morphology and mechanical properties of the electrospun fibers by altering the properties of PLGA solution, and drying rate of fibers in the electrospinning process.

Modulating protein release profiles by incorporating hyaluronic acid into PLGA microparticles Via a spray dryer equipped with a 3-fluid nozzle.

PURPOSE: The purpose of this study was to modulate the release profiles of the model protein drug from spray dried poly(DL-lactic-co-glycolic acid) (PLGA) microparticles by incorporating hyaluronic acid (HA) in the formulation. METHODS: Bovine serum albumin (BSA)-loaded PLGA microparticles with or without HA were prepared using a spray dryer equipped with a 3-fluid nozzle. The effects of HA on the surface tension and the rheological behavior of the inner feed solution were investigated. The physicochemical properties of the resulting microparticles were characterized using scanning electron microscopy (SEM), laser diffraction (LD), confocal laser scanning microscopy (CLSM) and X-ray photoelectron spectroscopy (XPS). Circular dischoism (CD) was used to characterize conformational integrity of BSA released from the microparticles. RESULTS: Spherical microparticles with D50 of 5-10 µm were obtained. Addition of HA in inner feed solutions increased the feed viscosity, but with no influence on the surface tension. All inner feed solutions showed non-Newtonian shear thinning behavior and the rheological properties were not time dependent. The CLSM and XPS analyses suggested a core-shell like structure of the microparticles when HA was incorporated. The release profiles of BSA were extended and the initial burst releases were suppressed with an increase in HA in the microparticle formulations. In addition, HA seemed to protect BSA from degradation upon the spray-drying process. CONCLUSIONS: The present work demonstrates the potential of HA to modulate protein release profile from PLGA microparticle formulations produced via spray drying using 3-fluid nozzle.

Property profiling of biosimilar mucus in a novel mucus-containing in vitro model for assessment of intestinal drug absorption.

Oral delivery of drugs, including peptide and protein therapeutics, can be impeded by the presence of the mucus surface-lining the intestinal epithelium. The aim of the present project was to design and characterize biosimilar mucus compatible with Caco-2 cell monolayers cultured in vitro to establish a more representative in vitro model for the intestinal mucosa. The rheological profile of a biosimilar mucus mixture composed of purified gastric mucin, lipids and protein in buffer was optimized by supplementing with an anionic polymer to display viscoelastic properties and a microstructure comparable to freshly isolated porcine intestinal mucus (PIM). Further, this multicomponent biosimilar mucus mixture was optimized with regard to the lipid content in order to obtain cellular compatibility with well-differentiated Caco-2 cell monolayers. In contrast, PIM was found to severely disrupt the Caco-2 cell monolayer. When combined with the Caco-2 cell monolayers, the final biosimilar mucus was found to significantly affect the permeability profiles for hydrophobic and hydrophilic small and large model drug compounds in different ways. In conclusion, the present study describes an improvement of the biorelevance of the Caco-2 cell culture model by application of mucus, resulting in an in vitro model of oral mucosa suitable for future assessment of innovative drug delivery approaches.

The influence of size, structure and hydrophilicity of model surfactants on the adsorption of lysozyme to oil-water interface--interfacial shear measurements.

The flexibility and aggregation of proteins can cause adsorption to oil-water interfaces and thereby create challenges during formulation and processing. Protein adsorption is a complex process and the presence of surfactants further complicates the system, in which additional parameters need to be considered. The purpose of this study is to scrutinize the influence of surfactants on protein adsorption to interfaces, using lysozyme as a model protein and sorbitan monooleate 80 (S80), polysorbate 80 (T80), polyethylene-block-poly(ethylene glycol) (PE-PEG) and polyglycerol polyricinoleate (PG-PR) as model surfactants. Rheological properties, measured using a TA AR-G2 rheometer equipped with a double wall ring (DWR) geometry, were used to compare the efficacy of the surfactant in hindering lysozyme adsorption. The system consists of a ring and a Delrin® trough with a circular channel (interfacial area=1882.6 mm(2)). Oscillatory shear measurements were conducted at a constant frequency of 0.1 Hz, a temperature of 25°C, and with strain set to 1%. The adsorption of lysozyme to the oil-water interface results in the formation of a viscoelastic film. This can be prevented by addition of surfactants, in a manner depending on the concentration and the type of surfactant. The more hydrophilic surfactants are more effective in hindering lysozyme adsorption to oil-water interfaces. Additionally, the larger surfactants are more persistent in preventing film formation, whereas the smaller ones eventually give space for the lysozyme on the interface. The addition of a mixture of two different surfactants was only beneficial when the two hydrophilic surfactants were mixed, in which case a delay in the multilayer formation was detected. The method is able to detect the interfacial adsorption of lysozyme and thus the hindering of film formation by model surfactants. It can therefore aid in processing of any delivery systems for proteins in which the protein is introduced to oil-water interfaces.

Adsorption of proteins at the oil/water interface--observation of protein adsorption by interfacial shear stress measurements.

The interfacial adsorption of proteins is a problem during processing and formulation. The flexibility and aggregation of the protein cause the formation of a viscoelastic multilayer upon adsorption to the oil/water interface. Protein adsorption is a complex process and therefore it is difficult to elaborate which protein characteristics are important for the interfacial protein adsorption. From our results it seems that the molecular weight influences this the most. In this study, the adsorption of three proteins, bovine serum albumin (BSA), lysozyme and insulin, to oil/water interfaces is characterized by interfacial shear stress measurements using a sensitive rheometer equipped with a Du Noüy ring geometry. The interfacial protein adsorption is concentration dependent for the three proteins investigated, where the complex viscosity of the viscoelastic multilayer increases with increased concentration. The adsorption rate of the proteins varies, BSA reaches a plateau after 2h, insulin takes 24h to reach a plateau value and lysozyme adsorbs fast in the beginning, then levels of to a slow but steady increase in the complex viscosity, BSA and insulin were shown to have similar characteristics in the evolvement of the interfacial layer despite the rate, indicating similar mechanism of adsorption, whereas lysozyme differs. This is also seen when G' and G'' are used in order to relate the rheological measurements to the three adsorption regimes. This study shows the potentials and effectiveness of the Du Noüy ring geometry for characterization of protein adsorption to oil/water interfaces.

Rheological characterization and turbidity of riboflavin-photosensitized changes in alginate/GDL systems.

Riboflavin (RF) in combination with light, in the wavelength range of 310-800 nm, is used to induce degradation of alginic acid gels. Light irradiation of alginate solutions in the presence of RF under aerobic conditions causes scission of the polymer chains. In the development process of a new drug delivery system, RF photosensitized degradation of alginic acid gels is studied by monitoring changes in the turbidity and rheological parameters of alginate/glucono-delta-lactone (GDL) systems with different concentrations of GDL. Addition of GDL induces gel formation of the samples by gradually lowering the pH-value of the system. The turbidity is measured and the cloud point determined. The turbidity starts to increase after shorter times with enhanced concentration of GDL. Enhanced viscoelasticity is detected with increasing GDL concentration in the post-gel regime, but small differences are detected at the gel point. The incipient gel is 'soft' and has an open structure independent on the GDL concentration. In the post-gel regime solid-like behavior is observed, this is more distinct for the systems with high GDL concentrations. The effect of photosensitized RF on alginate/GDL systems decreases with increasing amount of GDL in the system. The same trend is detected whether the systems are irradiated in the pre-gel or in the post-gel regime.

Characterization of riboflavin-photosensitized changes in aqueous solutions of alginate by dynamic light scattering.

The effect of irradiation, in the wavelength range of 310-800 nm, on aqueous solutions (pH = 7.4) of alginate in the presence of the photosensitizer riboflavin (RF) has been investigated with the aid of dynamic light scattering (DLS). Under aerobic conditions light irradiation of RF causes scission of the polymer chains which affects the polymer dynamics. The time correlation data obtained from DLS experiments showed at all conditions the existence of two relaxation modes: one single exponential at short times, followed by a stretched exponential at longer times. The slow relaxation time revealed, over the whole considered concentration range, lower values for the alginate/RF system, whereas no effect of photochemical degradation was observed for the fast relaxation time in the semidilute regime. The results suggest that the photochemically induced fragmentation of alginate affects the slow relaxation mode, associated with disengagement relaxation of individual chains or cluster relaxation, in a similar way as the zero-shear viscosity. These findings provide detailed insight into the dynamics of the polymer matrix, and this knowledge can be useful in the context of controlled-release delivery of drugs. The chemical units of alginate (M = mannuronic acid and G = guluronic acid).

Riboflavin-photosensitized changes in aqueous solutions of alginate. Rheological studies.

Interactions between photoexcited riboflavin (RF), promoted by irradiation in the range of 310-800 nm, and alginate have been studied in air equilibrated aqueous solutions with the aid of rheological methods. Light irradiation of RF causes under aerobic conditions fragmentation of alginate and a decrease in the shear viscosity and other rheological parameters of its solutions. The decrease is most pronounced in concentrated polymer solutions. The photochemical degradation of alginate is inhibited in the presence of the quenchers/scavengers d-mannitol, glutathione, potassium iodide, and sodium azide and in excess oxygen. The addition of thiourea to alginate-RF solutions leads to enhanced degradation of the polymer. Significant shear-thinning effects and deviations from the Cox-Merz rule are observed at higher polymer concentrations.