Transpupillary collagen photocrosslinking for targeted modulation of ocular biomechanics.

The central vision-threatening event in glaucoma is dysfunction and loss of retinal ganglion cells (RGCs), thought to be promoted by local tissue deformations. Here, we sought to reduce tissue deformation near the optic nerve head by selectively stiffening the peripapillary sclera, i.e. the scleral region immediately adjacent to the optic nerve head. Previous scleral stiffening studies to treat glaucoma or myopia have used either pan-scleral stiffening (not regionally selective) or regionally selective stiffening with limited access to the posterior globe. We present a method for selectively stiffening the peripapillary sclera using a transpupillary annular light beam to activate methylene blue administered by retrobulbar injection. Unlike prior approaches to photocrosslinking in the eye, this approach avoids the damaging effects of ultraviolet light by employing red light. This targeted photocrosslinking approach successfully stiffened the peripapillary sclera at 6 weeks post-treatment, as measured by whole globe inflation testing. Specifically, strain was reduced by 47% when comparing treated vs. untreated sclera within the same eye (n = 7, p=0.0064) and by 54% when comparing the peripapillary sclera of treated vs. untreated eyes (n = 7, p<0.0001). Post-treatment characterization of RGCs (optic nerve axon counts/density, and grading), retinal function (electroretinography), and retinal histology revealed that photocrosslinking was associated with some ocular toxicity. We conclude that a transpupillary photocrosslinking approach enables selective scleral stiffening targeted to the peripapillary region that may be useful in future treatments of glaucoma.

Sustained reduction of intraocular pressure by supraciliary delivery of brimonidine-loaded poly(lactic acid) microspheres for the treatment of glaucoma.

Although effective drugs that lower intraocular pressure (IOP) in the management of glaucoma exist, their efficacy is limited by poor patient adherence to the prescribed eye drop regimen. To replace the need for eye drops, in this study we tested the hypothesis that IOP can be reduced for one month after a single targeted injection using a microneedle for administration of a glaucoma medication (i.e., brimonidine) formulated for sustained release in the supraciliary space of the eye adjacent to the drug's site of action at the ciliary body. To test this hypothesis, brimonidine-loaded microspheres were formulated using poly(lactic acid) (PLA) to release brimonidine at a constant rate for 35 days and microneedles were designed to penetrate through the sclera, without penetrating into the choroid/retina, in order to target injection into the supraciliary space. A single administration of these microspheres using a hollow microneedle was performed in the eye of New Zealand White rabbits and was found to reduce IOP initially by 6 mmHg and then by progressively smaller amounts for more than one month. All administrations were well tolerated without significant adverse events, although histological examination showed a foreign-body reaction to the microspheres. This study demonstrates, for the first time, that the highly-targeted delivery of brimonidine-loaded microspheres into the supraciliary space using a microneedle is able to reduce IOP for one month as an alternative to daily eye drops.

Pocketed microneedles for rapid delivery of a liquid-state botulinum toxin A formulation into human skin.

Botulinum toxin A (BT) is used therapeutically for the treatment of primary focal hyperhidrosis, a chronic debilitating condition characterised by over-activity of the eccrine sweat glands. Systemic toxicity concerns require BT to be administered by local injection, which in the case of hyperhidrosis means multiple painful intradermal injections by a skilled clinician at 6-monthly intervals. This study investigates the potential of a liquid-loaded pocketed microneedle device to deliver botulinum toxin A into the human dermis with the aim of reducing patient pain, improving therapeutic targeting and simplifying the administration procedure. Initially, ß-galactosidase was employed as a detectable model for BT to (i) visualise liquid loading of the microneedles, (ii) determine residence time of a liquid formulation on the device and (iii) quantify loaded doses. An array of five stainless steel pocketed microneedles was shown to possess sufficient capacity to deliver therapeutic doses of the potent BT protein. Microneedle-mediated intradermal delivery of ß-galactosidase and formaldehyde-inactivated botulinum toxoid revealed effective deposition and subsequent diffusion within the dermis. This study is the first to characterise pocketed microneedle delivery of a liquid formulation into human skin and illustrates the potential of such systems for the cutaneous administration of potent proteins such as BT. A clinically appropriate microneedle delivery system for BT could have a significant impact in both the medical and cosmetic industries.

Delivery systems for intradermal vaccination.

Intradermal (ID) vaccination can offer improved immunity and simpler logistics of delivery, but its use in medicine is limited by the need for simple, reliable methods of ID delivery. ID injection by the Mantoux technique requires special training and may not reliably target skin, but is nonetheless used currently for BCG and rabies vaccination. Scarification using a bifurcated needle was extensively used for smallpox eradication, but provides variable and inefficient delivery into the skin. Recently, ID vaccination has been simplified by introduction of a simple-to-use hollow microneedle that has been approved for ID injection of influenza vaccine in Europe. Various designs of hollow microneedles have been studied preclinically and in humans. Vaccines can also be injected into skin using needle-free devices, such as jet injection, which is receiving renewed clinical attention for ID vaccination. Projectile delivery using powder and gold particles (i.e., gene gun) have also been used clinically for ID vaccination. Building off the scarification approach, a number of preclinical studies have examined solid microneedle patches for use with vaccine coated onto metal microneedles, encapsulated within dissolving microneedles or added topically to skin after microneedle pretreatment, as well as adapting tattoo guns for ID vaccination. Finally, technologies designed to increase skin permeability in combination with a vaccine patch have been studied through the use of skin abrasion, ultrasound, electroporation, chemical enhancers, and thermal ablation. The prospects for bringing ID vaccination into more widespread clinical practice are encouraging, given the large number of technologies for ID delivery under development.

Saving cells from ultrasound-induced apoptosis: quantification of cell death and uptake following sonication and effects of targeted calcium chelation.

Applications of ultrasound for noninvasive drug and gene delivery have been limited by associated cell death as a result of sonication. In this study, we sought to quantify the distribution of cellular bioeffects caused by low-frequency ultrasound (24 kHz) and test the hypothesis that Ca(2+) chelation after sonication can shift this distribution by saving cells from death by apoptosis. Using flow cytometry, we quantitatively categorized sonicated cells among four populations: (i) cells that appear largely unaffected, (ii) cells reversibly permeabilized, (iii) cells rendered nonviable during sonication and (iv) cells that appear to be viable shortly after sonication, but later undergo apoptosis and die. By monitoring cells for 6 h after ultrasound exposure, we found that up to 15% of intact cells fell into this final category. Those apoptotic cells initially had the highest levels of uptake of a marker compound, calcein; also had highly elevated levels of intracellular Ca(2+); and contained an estimated plasma membrane wound radius of 100-300 nm. Finally, we showed that chelation of intracellular Ca(2+) after sonication reduced apoptosis by up to 44%, thereby providing a strategy to save cells. We conclude that cells can be saved from ultrasound-induced death by appropriate selection of ultrasound conditions and Ca(2+) chelation after sonication.

Cutaneous vaccination using microneedles coated with hepatitis C DNA vaccine.

The skin is potentially an excellent organ for vaccine delivery because of accessibility and the presence of immune cells. However, no simple and inexpensive cutaneous vaccination method is available. Micron-scale needles coated with DNA were tested as a simple, inexpensive device for skin delivery. Vaccination with a plasmid encoding hepatitis C virus nonstructural 3/4A protein using microneedles effectively primed specific cytotoxic T lymphocytes (CTLs). Importantly, the minimally invasive microneedles were as efficient in priming CTLs as more complicated or invasive delivery techniques, such as gene gun and hypodermic needles. Thus, microneedles may offer a promising technology for DNA vaccination.

Prediction and optimization of gene transfection and drug delivery by electroporation.

Although electroporation is widely used for laboratory gene transfection and gaining increased importance for nonviral gene therapy, it is generally employed using trial-and-error optimization schemes for lack of methods to predict electroporation's effects on cells. Therefore, we used a statistical approach to quantitatively predict molecular uptake and cell viability following electroporation and show that it predicts both in vitro and in vivo results for a wide range of molecules, including DNA, in 60 different cell types. Mechanistically, this broad predictive ability suggests that electroporation is mediated primarily by lipid bilayer structure and only secondarily by cell-specific characteristics. For gene therapy applications, this approach should facilitate rational design of electroporation protocols.

Overcoming skin's barrier: the search for effective and user-friendly drug delivery.

Although hypodermic needles rapidly deliver large doses of drugs such as insulin across the skin for systemic administration, the pain, local trauma, and difficulty to achieve sustained or complex delivery profiles has motivated development of novel alternative technologies. Microneedles, jet injectors, and thermal poration make micron-scale holes in skin through which drugs can be driven in a user-friendly manner. Chemical enhancers, iontophoresis, electroporation, and ultrasound increase skin permeability by making submicron alterations in skin microstructure for continuous delivery over time.

Ultrasound-mediated disruption of cell membranes. I. Quantification of molecular uptake and cell viability.

Ultrasound-mediated drug delivery is a nonchemical, nonviral, and noninvasive method for targeted transport of drugs and genes into cells. Molecules can be delivered into cells when ultrasound disrupts the cell membrane by a mechanism believed to involve cavitation. This study examined molecular uptake and cell viability in cell suspensions (DU145 prostate cancer and aortic smooth muscle cells) exposed to varying peak negative acoustic pressures (0.6-3.0 MPa), exposure times (120-2000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. With increasing pressure and exposure time, molecular uptake of a marker compound, a calcein, increased and approached equilibrium with the extra cellular solution, while cell viability decreased. Varying pulse length produced no significant effect. All viability and molecular uptake measurements collected over the broad range of ultrasound conditions studied correlated with acoustic energy exposure. This suggests that acoustic energy exposure may be predictive of ultrasound's nonthermal bioeffects.

Ultrasound-mediated disruption of cell membranes. II. Heterogeneous effects on cells.

Ultrasound has been shown to reversibly and irreversibly disrupt membranes of viable cells through a mechanism believed to involve cavitation. Because cavitation is both temporally and spatially heterogeneous, flow cytometry was used to identify and quantify heterogeneity in the effects of ultrasound on molecular uptake and cell viability on a cell-by-cell basis for suspensions of DU145 prostate cancer and aortic smooth muscle cells exposed to varying peak negative acoustic pressures (0.6-3.0 MPa). exposure times (120-2,000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. Cell-to-cell heterogeneity was observed at all conditions studied and was classified into three subpopulations: nominal uptake (NUP), low uptake (LUP), and high uptake (HUP) populations. The average number of molecules within each subpopulation was generally constant: 10(4)-10(5) molecules/cell in NUP, approximately 10(6) molecules/cell in LUP, and approximately 10(7) molecules/cell in HUP. However, the fraction of cells within each subpopulation showed a strong dependence on both acoustic pressure and exposure time. Varying pulse length produced no significant effect. The distribution of cells among the three subpopulations correlated with acoustic energy exposure, which suggests that energy exposure may govern the ability of ultrasound to induce bioeffects by a nonthermal mechanism.

Sonoluminescence as an indicator of cell membrane disruption by acoustic cavitation.

Ultrasound (US) has been shown to transiently disrupt cell membranes and, thereby, facilitate the loading of drugs and genes into viable cells. Because these effects are believed to be mediated by cavitation, we hypothesized that measured levels of cavitation-induced sonoluminescence should correlate with levels of US bioeffects. We, therefore, quantified the number of calcein molecules delivered and the loss of viability in prostate cancer cells exposed to 24-kHz US over a range of different pulse lengths (1 to 100 ms), total exposure times (0.1 to 10 s) and pressures (1.0 to 9.8 atm). Consistent with previous observations, uptake increased and viability decreased with increasing pulse length, total exposure time and pressure. As a new observation, we established correlations between the amount of light produced by sonoluminescence and both molecular uptake and cell viability. These results support a cavitation-based mechanism for these bioeffects and suggest a means to control US effects on cells using sonoluminescence-based feedback.

Lack of pain associated with microfabricated microneedles.

Microscopic needles previously shown capable of transdermal delivery of drugs and proteins are demonstrated to be painless when pressed into the skin of human subjects.

Quantitative study of electroporation-mediated molecular uptake and cell viability.

Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.

Predicted permeability of the cornea to topical drugs.

PURPOSE: To develop a theoretical model to predict the passive, steady-state permeability of cornea and its component layers (epithelium, stroma, and endothelium) as a function of drug size and distribution coefficient (phi). The parameters of the model should represent physical properties that can be independently estimated and have physically interpretable meaning. METHODS: A model was developed to predict corneal permeability using 1) a newly developed composite porous-medium approach to model transport through the transcellular and paracellular pathways across the epithelium and endothelium and 2) previous work on modeling corneal stroma using a fiber-matrix approach. RESULTS: The model, which predicts corneal permeability for molecules having a broad range of size and lipophilicity, was validated by comparison with over 150 different experimental data points and showed agreement with a mean absolute fractional error of 2.43, which is within the confidence interval of the data. In addition to overall corneal permeability, the model permitted independent analysis of transcellular and paracellular pathways in epithelium, stroma and endothelium. This yielded strategies to enhance corneal permeability by targeting epithelial paracellular pathways for hydrophilic compounds (phi < 0.1 - 1), epithelial transcellular pathways for intermediate compounds, and stromal pathways for hydrophobic compounds (phi > 10 - 100). The effects of changing corneal physical properties (e.g., to mimic disease states or animals models) were also examined. CONCLUSIONS: A model based on physicochemical properties of the cornea and drug molecules can be broadly applied to predict corneal permeability and suggest strategies to enhance that permeability.

Intracellular drug delivery using low-frequency ultrasound: quantification of molecular uptake and cell viability.

PURPOSE: To determine the dependence on acoustic parameters of molecular uptake and viability of cells exposed to low-frequency ultrasound. METHODS: DU145 prostate cancer cells bathed in a solution of calcein were exposed to ultrasound at 24 kHz over a range of different acoustic pressures. exposure times, pulse lengths, and duty cycles. Flow cytometry was employed to quantify the number of calcein molecules delivered into each cell and levels of cell viability. RESULTS: Both molecular uptake and cell viability showed a strong dependence on acoustic pressure and exposure time, weak dependence on pulse length, and no significant dependence on duty cycle. When all of the data were pooled together, they exhibited good correlation with acoustic energy exposure. Although molecular uptake showed large cell-to-cell heterogeneity, up to approximately 15% of cells achieved an intracellular calcein concentration approximately equal to its extracellular concentration. CONCLUSIONS: Large numbers of molecules can be delivered intracellularly using low-frequency ultrasound. Both uptake and viability correlate with acoustic energy, which is useful for design and control of ultrasound protocols.

Microfabricated microneedles for gene and drug delivery.

By incorporating techniques adapted from the microelectronics industry, the field of microfabrication has allowed the creation of microneedles, which have the potential to improve existing biological-laboratory and medical devices and to enable novel devices for gene and drug delivery. Dense arrays of microneedles have been used to deliver DNA into cells. Many cells are treated at once, which is much more efficient than current microinjection techniques. Microneedles have also been used to deliver drugs into local regions of tissue. Microfabricated neural probes have delivered drugs into neural tissue while simultaneously stimulating and recording neuronal activity, and microneedles have been inserted into arterial vessel walls to deliver anti-restenosis drugs. Finally, microhypodermic needles and microneedles for transdermal drug delivery have been developed to reduce needle insertion pain and tissue trauma and to provide controlled delivery across the skin. These needles have been shown to be robust enough to penetrate skin and dramatically increase skin permeability to macromolecules.

Mechanistic studies of skin electroporation using biophysical methods.

The mechanism by which high-voltage pulses transiently disrupt lipid bilayers in cell membranes has been the subject of controversy since electroporation was first observed almost three decades ago. Determining the mechanism by which such pulses permeabilize the complex, multilamellar bilayer structures in skin poses an even greater challenge. To address this issue, a range of methods have been employed to perform biophysical characterization for skin electroporation studies. In this chapter, we provide an overview of these methods and highlight representative findings which biophysical characterization has yielded.

Electrical impedance spectroscopy for rapid and noninvasive analysis of skin electroporation.

Transient disruption of skin's barrier properties using high-voltage pulses involves complex changes in skin microstructure believed to be due to electroporation. Electroporation of cell membranes is a well known phenomenon which has found extensive use as a method of DNA transfection in biological laboratories (1-3). More recently, it has been shown that the multilamellar lipid bilayer membranes found in skin can also be electroporated (4-17). The dramatic and reversible increases in skin permeability caused by electroporation indicate that drugs might be delivered transdermally at significantly enhanced rates. Especially for macromolecules, such as protein- and gene-based drugs, electroporation-mediated transdermal drug delivery could be an important pharmaceutical approach.