Investigating the influence of ultrasound parameters on ibuprofen drug release from hydrogels.

Hydrogels are promising ultrasound-responsive drug delivery systems. In this study, we investigated how different ultrasound parameters affected drug release and structural integrity of self-healing hydrogels composed of alginate or poloxamers. The effects of amplitude and duty cycle at low frequency (24 kHz) ultrasound stimulation were first investigated using alginate hydrogels at 2% w/v and 2.5% w/v. Increasing ultrasound amplitude increased drug release from these gels, although high amplitudes caused large variations in release and damaged the gel structure. Increasing duty cycle also increased drug release, although a threshold was observed with the lower pulsed 50% duty cycle achieving similar levels of drug release to a continuous 100% duty cycle. Poloxamer-based hydrogels were also responsive to the optimised parameters at low frequency (24 kHz, 20% amplitude, 50% duty cycle for 30 s) and showed similar drug release results to a 2.5% w/v alginate hydrogel. Weight loss studies demonstrated that the 2% w/v alginate hydrogel underwent significant erosion following ultrasound application, whereas the 2.5% w/v alginate and the poloxamer gels were unaffected by application of the same parameters (24 kHz, 20% amplitude, 50% duty cycle for 30 s). The rheological properties of the hydrogels were also unaffected and the FTIR spectra remained unchanged after low frequency ultrasound stimulation (24 kHz, 20% amplitude, 50% duty cycle for 30 s). Finally, high-frequency ultrasound stimulation (1 MHz, 3 W.cm-2, 50% duty cycle) was also trialled; the alginate gels were less responsive to this frequency, while no statistically significant impact on drug release was observed from the poloxamer gels. This study demonstrates the importance of ultrasound parameters and polymer selection in designing ultrasound-responsive hydrogels.

Broadband-excitation-based mechanical spectroscopy of highly viscous tissue-mimicking phantoms.

Standard rheometers assess mechanical properties of viscoelastic samples up to 100 Hz, which often hinders the assessment of the local-scale dynamics. We demonstrate that high-frequency analysis can be achieved by inducing broadband waves and monitoring their media-dependent propagation using optical coherence tomography. Here, we present a new broadband wave analysis based on two-dimensional Fourier transformation. We validated this method by comparing the mechanical parameters to monochromatic excitation and a standard oscillatory test data. Our method allows for high-frequency mechanical spectroscopy, which could be used to investigate the local-scale dynamics of different biological tissues and the influence of diseases on their microstructure.

Prospective one year study of corneal biomechanical changes following high intensity, accelerated cornea cross-linking in patients with keratoconus using a non-contact tonometer.

PURPOSE: To characterize corneal biomechanical properties utilizing a dynamic ultra-high-speed Scheimpflug camera equipped with a non-contact tonometer (CorVis ST, CST) in keratoconic corneas following continuous high intensity, high irradiance corneal cross-linking. DESIGN: Prospective longitudinal single-centre study at a tertiary referral center. METHODS: Corneal biomechanical properties were measured in patients with progressive keratoconus undergoing high intensity (30 mW/cm2), high irradiance (5.4 J/cm2), accelerated corneal cross-linking with continuous exposure to ultraviolet-A for 4 min. CST was used to assess corneal biomechanical properties pre-operatively and at 1, 3, 6 and 12 months post-operatively. CST output videos were further analyzed using several previously reported algorithms. RESULTS: A total of 25 eyes of 25 participants were examined. The mean age of participants was 20.9 ± 5.3 years; 56% were male and 80% were of Maori or Pacific Island origin. Energy absorbed area (mN mm), was the only significantly changed parameter compared to baseline at all time points measuring 3.61 ± 1.19 preoperatively, 2.81 ± 1.15 at 1 month (p = 0.037), 2.79 ± 0.81 (p = 0.033) at 3 months, 2.76 ± 0.95 (p = 0.028) at 6 months and 2.71 ± 1.18 (p = 0.016) at 12 months. CONCLUSIONS: The significant difference between the pre and post-operative energy absorbed area appears to reflect changes in corneal viscous properties that occur following corneal cross-linking.

Externally triggered release of growth factors - A tissue regeneration approach.

Tissue regeneration aims to achieve functional restoration following injury by creating an environment to enable the body to self-repair. Strategies for regeneration rely on the introduction of biomaterial scaffolding, cells and bioactive molecules into the body, at or near the injury site. Of these bioactive molecules, growth factors (GFs) play a pivotal role in directing regenerative pathways for many cell populations. However, the therapeutic use of GFs has been limited by the complexity of biological injury and repair, and the properties of the GFs themselves, including their short half-life, poor tissue penetration, and off-target side effects. Externally triggered delivery systems have the potential to facilitate the delivery of GFs into the target tissues with considerations of the timing, sequence, amount, and location of GF presentation. This review briefly discusses the challenges facing the therapeutic use of GFs, then, we discuss approaches to externally trigger GF release from delivery systems categorised by stimulation type; ultrasound, temperature, light, magnetic fields and electric fields. Overall, while the use of GFs for tissue regeneration is still in its infancy, externally controlled GF delivery technologies have the potential to achieve robust and effective solutions to present GFs to injured tissues. Future technological developments must occur in conjunction with a comprehensive understanding of the biology at the injury site to ensure translation of promising technologies into real world benefit.

Ex vivo evaluation of the influence of pH on the ophthalmic safety, antibacterial efficacy and storage stability of povidone-iodine.

CLINICAL RELEVANCE: The monitoring and controlling of pH is important when preparing solutions for ophthalmic administration. In the case of povidone-iodine, dilution in an appropriate buffer is needed to improve its ophthalmic safety. BACKGROUND: Povidone-iodine is a broad-spectrum antiseptic agent that is commonly used in ophthalmic applications due to its cost-effectiveness and accessibility. However, native povidone-iodine has a pH of about 4.0 and is known to irritate the ocular surface. This study assessed whether adjusting povidone-iodine formulation pH would influence its ex vivo ophthalmic safety, alongside its impact on antibacterial efficacy and storage stability. METHODS: One per cent w/v povidone-iodine was diluted in normal saline, or 0.1-mol/l citrate or phosphate buffers to yield solutions with a pH ranging from 4.0 to 7.0. Ocular irritancy was evaluated using the bovine cornea opacity and permeability assay. Antibacterial efficacy was assessed by evaluating povidone-iodine minimum inhibitory concentration and minimum bactericidal concentration at varied pH. Storage stability of the preparations was determined over 30-days at room temperature (20-25°C). RESULTS: Combining povidone-iodine with phosphate buffer notably decreased ocular irritancy of the antiseptic. Surprisingly, combining povidone-iodine with citrate buffer potentiated irritant effects of the preparation. Antibacterial efficacy of povidone-iodine was reduced when formulation pH was increased from 4.0 to 7.0, although its general activity was retained. Finally, povidone-iodine remained stable in both normal saline and phosphate buffer over 30-days. CONCLUSION: Ophthalmic application of povidone-iodine can be optimised by adjusting the pH of the formulation to 7.0 using phosphate buffer, reducing irritancy while maintaining adequate antibacterial efficacy and storage stability.

The influence of hyperglycemia on the safety of ultrasound in retinal pigment epithelial cells.

Ultrasound (US) assisted drug delivery is receiving interest in treating posterior eye diseases, such as diabetic retinopathy due to its ability to maximize drug penetration into difficult to reach tissues. Despite its promise, the technique has only been investigated using healthy cell and tissue models, with no evidence to date about its safety in active disease. As a result, the aim of this study was to evaluate the safety of US administration in vitro in retinal pigment epithelial cells under normal and high glucose conditions. US protocols within the presently accepted safety threshold were applied and their influence on cell membrane and tight junction integrity as well as intracellular inflammation was evaluated using lactate dehydrogenase (LDH), zona occludens-1 (ZO-1), fluorescein isothiocyanate (FITC)-dextran dye leak and nuclear factor-kappaB (NF-κB) assays, respectively. Under high glucose conditions, US application increased LDH release and resulted in loss of ZO-1 labeling at 2 h; however, normal levels were restored within 24 h. US within its safety parameters did not induce any FITC-dextran dye leak or NF-κB nuclear translocation in normal or high glucose conditions. In conclusion, our results suggest that while high glucose conditions increase cell susceptibility to US-mediated stress, basal conditions can be restored within 24 h without long-lasting cell damage.

Characterization of Zinc Oxide Nanoparticle Cross-Linked Collagen Hydrogels.

Collagen is the most abundant protein in mammals and possesses high biocompatibility and low antigenicity. These biological properties render it one of the most useful biomaterials for medical applications. This study investigated the mechanical and physical characteristics of collagen hydrogels cross-linked with different ratios of polyvinylpyrrolidone capped zinc oxide nanoparticles (ZPVP). Fourier transform infrared spectroscopy indicated molecular interactions between collagen fibers and ZPVP. Texture analysis revealed a significant increase in gel hardness, adhesiveness, and viscosity after cross-linking with ZPVP. Rheological measurements showed that as the ratio of ZPVP increased, stronger hydrogels were formed which in turn resulted in more sustained release of the model drug, dexamethasone sodium phosphate. We can therefore conclude that the mechanical properties of collagen hydrogels can be modified by controlling the ratio of ZPVP used for cross-linking, offering the potential to develop biocompatible sustained release drug delivery systems.

Ultrasound-responsive lipid microbubbles for drug delivery: A review of preparation techniques to optimise formulation size, stability and drug loading.

Lipid-shelled microbubbles have received extensive interest to enhance ultrasound-responsive drug delivery outcomes due to their high biocompatibility. While therapeutic effectiveness of microbubbles is well established, there remain limitations in sample homogeneity, stability profile and drug loading properties which restrict these formulations from seeing widespread use in the clinical setting. In this review, we evaluate and discuss the most encouraging leads in lipid microbubble design and optimisation. We examine current applications in drug delivery for the systems and subsequently detail shell compositions and preparation strategies that improve monodispersity while retaining ultrasound responsiveness. We review how excipients and storage techniques help maximise stability and introduce different characterisation and drug loading techniques and evaluate their impact on formulation performance. The review concludes with current quality control measures in place to ensure lipid microbubbles can be reproducibly used in drug delivery.

Relationship between rheological properties and transverse relaxation time (T2) of artificial and porcine vitreous humour.

Vitreous liquefactive processes play an integral role in ocular health. Knowledge of the degree of liquefaction would allow better monitoring of ocular disease progression and enable more informed therapeutic dosing for an individual patient. Presently this process cannot be monitored in a non-invasive manner. Here, we evaluated whether magnetic resonance imaging (MRI) could predict the viscoelasticity and in turn liquefactive state of artificial and biological vitreous humour. Gels comprising identical concentrations of hyaluronic acid and agar ranging from 0.125 to 2.25 mg/ml of each polymer were prepared and their T2 was measured using a turbo-spin echo sequence via 3T clinical MRI. The gels were subsequently subjected to rheological frequency and flow sweeps and trends between T2 and rheological parameters were assessed. The relationship between T2 and vitreous humour rheology was further assessed using ex vivo porcine eyes. An optimised imaging technique improved homogeneity of obtained artificial vitreous humour T2 maps. Strong correlations were observed between T2 and various rheological parameters of the gels. Translation to porcine vitreous humour demonstrated that the T2 of biological tissue was related to its viscoelastic properties. This study shows that T2 can be correlated with various rheological parameters within gels. Future investigations will assess the translatability of these findings to live models.

Validation of hyaluronic acid-agar-based hydrogels as vitreous humor mimetics for in vitro drug and particle migration evaluations.

Artificial vitreous humor holds immense potential for use in in vitro intravitreal drug delivery assays. In this study, we investigated rheological properties and drug or nanoparticle migration in hyaluronic acid (HA) - agar based hydrogels and compared these characteristics with bovine vitreous humor. Gel compositions identified in literature containing HA (0.7-5.0 mg/ml) and agar (0.95-4.0 mg/ml) were classified as either high (VH), medium (VM) or low (VL) polymer load. Viscoelastic behavior was evaluated using oscillatory rheology, and migration of differently sized and charged polystyrene nanoparticles (NPs) through the different gels was determined via multiple particle tracking. Comparable rheological behaviour was observed between VL and bovine vitreous. Tracking evaluations revealed that increasing particle size and gel viscosity slowed NP migration. Additionally, 100 nm anionic NPs migrated slower than neutral NPs in VL and VM, while cationic NPs were immobile in all gels. Finally, distribution and clearance of sodium fluorescein was used to model drug mobility through the gels using a custom-built eye model. Flow and angular movement only influenced drug migration in VL and VM, but not VH. Finally, VL and VM demonstrated to have the most similar sodium fluorescein clearance to that of bovine vitreous humor. Together, these evaluations demonstrate that low viscosity HA-agar gels can be used to approximate nanoparticle and drug migration through biological vitreous humor.

Depot formulations to sustain periocular drug delivery to the posterior eye segment.

The periocular space is a promising alternative route for the delivery of drugs to the posterior eye segment, especially when treating conditions in the outer ocular layers. In this review, we discuss the different periocular routes as well as the physiological barriers and elimination mechanisms limiting drug bioavailability at the back of the eye. We then highlight various types of depot formulations, including particulate delivery systems, semisolid formulations, and implants, used to increase the contact time with the ocular tissues. With the additional advantage of sustaining drug release, such depot formulations could enhance periocular drug delivery to the posterior eye segment.

Ex vivo evaluation of the stability, safety and antibacterial efficacy of an extemporaneous povidone-iodine preparation for ophthalmic applications.

BACKGROUND: Povidone-iodine is used as a cost-effective broad-spectrum antiseptic in the prophylaxis and treatment of certain ocular infections. In this study, the stability, ophthalmic irritation potential and antibacterial efficacy of an extemporaneous povidone-iodine preparation was determined using established ex vivo and in vitro assays. METHODS: Extemporaneous iodine was prepared by simple dilution in normal saline. Preparation stability was evaluated by monitoring concentration and pH. Ocular safety was determined using the bovine cornea opacity and permeability assay. Efficacy was assessed by determining the minimum inhibitory and minimum bactericidal concentration of the preparation on Staphylococcus aureus and Pseudomonas aeruginosa. RESULTS: Diluted povidone-iodine maintained its stability over the 28-day evaluation. The formulation caused mild ocular irritation at the lowest prepared concentration (0.5 per cent w/v), with irritation noticeably increased at higher concentrations. The preparation showed minimum bactericidal and inhibitory concentrations of 0.078 and 0.3 per cent w/v on S. aureus and P. aeruginosa, respectively. CONCLUSIONS: This study confirms the stability and broad-spectrum antibacterial efficacy of povidone-iodine, while addressing the ocular irritation potential of this chemical.

Ultrasound-responsive nanobubbles for enhanced intravitreal drug migration: An ex vivo evaluation.

The intravitreal route faces many challenges in rapidly and effectively reaching posterior eye pathology, with administered therapeutics experiencing non-specific distribution around and premature clearance from ocular tissues. Nanobubbles and ultrasound may improve outcomes of intravitreally administered drugs by influencing the directionality of drug-containing particle migration. In this study, we assessed the impact of trans-scleral or corneal ultrasound application on the distribution of intravitreally-injected nanobubbles. Rhodamine-tagged gas entrapped nanobubble formulations were prepared and injected into ex vivo bovine and porcine eyes and subjected to ultrasound (1 MHz, 0-2.5 W/cm2, 50-100% duty, 60 s). Bovine eyes were partially dissected to visualize the vitreous humor and particle migration was evaluated via optical fluorescence spectroscopy. Directional migration in porcine eyes was evaluated using a snap freezing protocol complemented by quantification of regional fluorescence. The impact on nanobubble migration following pars-plana injection and sequential ultrasound cycle application from scleral or corneal-surface positions was also assessed. Administration of ultrasound significantly enhanced the directional migration of nanobubbles in both ex vivo models, with multiple corneal ultrasound cycles promoting greater migration of dye-filled nanobubbles to posterior regions of the vitreous. Moreover, particles moved in a directional manner away from the ultrasound wave source demonstrating an ability to effectively control the rate and path of nanobubble migration. These findings establish an encouraging new and safe modality enabling rapid distribution of intravitreally-injected therapeutics where expeditious therapeutic intervention is warranted.

Evaluation of 2 ex vivo Bovine Cornea Storage Protocols for Drug Delivery Applications.

The use of corneal tissue for ex vivo therapeutic evaluations is limited due to its rapid loss of viability after excision. Optimization of storage conditions may allow prolonged retention of physical tissue properties. In this study, we evaluated how storage in optimized organ culture (OC) medium at 37°C or phosphate-buffered saline (PBS) at 2-8°C impacted physical properties of bovine corneas. Tissue hydration, permeability and histology were monitored at baseline and following 1, 4 and 7 days of storage. Corneas stored in OC demonstrated significantly higher hydration and permeability when compared to those stored in PBS. Histology revealed that storage in OC consistently caused detachment of the epithelial layer by day 4 of storage, whereas both storage conditions caused a significant increase in stromal thickness and tissue vacuolation. This study highlights the limitations of currently available corneal tissue storage approaches for ex vivo drug permeation studies.

Phase transition of a microemulsion upon addition of cyclodextrin - applications in drug delivery.

This study reports on the impact of cyclodextrin addition on the phase behavior of microemulsion systems. Three distinct oil-in-water microemulsions were formulated and subjected to increasing concentrations of various cyclodextrins. The prepared formulations underwent visual, textural and microscopic characterization followed by the evaluation of their in vitro drug release and ex vivo tissue retention behavior. Combining microemulsions with cyclodextrins resulted in either phase separation or transition into a liquid crystalline state depending on the concentration and type of cyclodextrin utilized. Formulations combined with α-cyclodextrin consistently demonstrated transition into a liquid crystalline state as confirmed by polarized light and cryo-scanning electron microscopy. In these cases, cyclodextrin addition was also positively correlated with an increase in formulation hardness, adhesiveness and turbidity. Release and clearance studies revealed that drug diffusion from the microemulsions could be slowed and tissue retention prolonged by increasing the cyclodextrin content. These findings pave the way for the development of novel cyclodextrin-microemulsion-based liquid crystalline formulations in a variety of sustained drug delivery applications.

Development and Evaluation of Lipid Nanoparticles Containing Natural Botanical Oil for Sun Protection: Characterization and in vitro and in vivo Human Skin Permeation and Toxicity.

The use of sunscreen products is widely promoted by schools, government agencies, and health-related organizations to minimize sunburn and skin damage. In this study, we developed stable solid lipid nanoparticles (SLNs) containing the chemical UV filter octyl methoxycinnamate (OMC). In parallel, we produced similar stable SLNs in which 20% of the OMC content was replaced by the botanical urucum oil. When these SLNs were applied to the skin of human volunteers, no changes in fluorescence lifetimes or redox ratios of the endogenous skin fluorophores were seen, suggesting that the formulations did not induce toxic responses in the skin. Ex vivo (skin diffusion) tests showed no significant penetration. In vitro studies showed that when 20% of the OMC was replaced by urucum oil, there was no reduction in skin protection factor (SPF), suggesting that a decrease in the amount of chemical filter may be a viable alternative for an effective sunscreen, in combination with an antioxidant-rich vegetable oil, such as urucum. There is a strong trend towards increasing safety of sun protection products through reduction in the use of chemical UV filters. This work supports this approach by producing formulations with lower concentrations of OMC, while maintaining the SPF. Further investigations of SPF in vivo are needed to assess the suitability of these formulations for human use.

High-Resolution Single Particle Zeta Potential Characterisation of Biological Nanoparticles using Tunable Resistive Pulse Sensing.

Physicochemical properties of nanoparticles, such as size, shape, surface charge, density, and porosity play a central role in biological interactions and hence accurate determination of these characteristics is of utmost importance. Here we propose tunable resistive pulse sensing for simultaneous size and surface charge measurements on a particle-by-particle basis, enabling the analysis of a wide spectrum of nanoparticles and their mixtures. Existing methodologies for measuring zeta potential of nanoparticles using resistive pulse sensing are significantly improved by including convection into the theoretical model. The efficacy of this methodology is demonstrated for a range of biological case studies, including measurements of mixed anionic, cationic liposomes, extracellular vesicles in plasma, and in situ time study of DNA immobilisation on the surface of magnetic nanoparticles. The high-resolution single particle size and zeta potential characterisation will provide a better understanding of nano-bio interactions, positively impacting nanomedicine development and their regulatory approval.

Ultrasound-mediated nanoparticle delivery across ex vivo bovine retina after intravitreal injection.

Intravitreal injection is the most common administration route for the treatment of retinal diseases. However, the vitreous and some of the retinal layers themselves act as significant barriers to efficient delivery of drugs administered intravitreally. This study aimed to improve the diffusive mobility of nanoparticles (NPs) in the vitreous and enhance their permeation across the retina after intravitreal injection by application of ultrasound (US). Ex vivo posterior bovine eye cups were used and the vitreous was either left intact or removed gently from the neural retina. Hyaluronic acid coated human serum albumin NPs were administered into the eye cups and continuous US with a frequency of 1MHz, an intensity of 0.5W/cm2, and a duration of 30s was applied once or repeatedly via the transscleral route. After pre-determined time points, fluorescence intensities in the vitreous and the retina were analyzed. Short pulses of US significantly improved the diffusive mobility of NPs through the vitreous as well as their penetration across the neural retina into the retinal pigment epithelium and choroid without causing any detectable damage to the ocular tissues. Therefore, transscleral US could be a powerful and safe tool to enhance retinal delivery of intravitreally injected NPs.

Stably engineered nanobubbles and ultrasound - An effective platform for enhanced macromolecular delivery to representative cells of the retina.

Herein we showcase the potential of ultrasound-responsive nanobubbles in enhancing macromolecular permeation through layers of the retina, ultimately leading to significant and direct intracellular delivery; this being effectively demonstrated across three relevant and distinct retinal cell lines. Stably engineered nanobubbles of a highly homogenous and echogenic nature were fully characterised using dynamic light scattering, B-scan ultrasound and transmission electron microscopy (TEM). The nanobubbles appeared as spherical liposome-like structures under TEM, accompanied by an opaque luminal core and darkened corona around their periphery, with both features indicative of efficient gas entrapment and adsorption, respectively. A nanobubble +/- ultrasound sweeping study was conducted next, which determined the maximum tolerated dose for each cell line. Detection of underlying cellular stress was verified using the biomarker heat shock protein 70, measured before and after treatment with optimised ultrasound. Next, with safety to nanobubbles and optimised ultrasound demonstrated, each human or mouse-derived cell population was incubated with biotinylated rabbit-IgG in the presence and absence of ultrasound +/- nanobubbles. Intracellular delivery of antibody in each cell type was then quantified using Cy3-streptavidin. Nanobubbles and optimised ultrasound were found to be negligibly toxic across all cell lines tested. Macromolecular internalisation was achieved to significant, yet varying degrees in all three cell lines. The results of this study pave the way towards better understanding mechanisms underlying cellular responsiveness to ultrasound-triggered drug delivery in future ex vivo and in vivo models of the posterior eye.